Evaluation of the neurotensin receptor-1 as target for molecular imaging and radiotherapy of pancreatic and prostate cancer

Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg

zur

Erlangung des Doktorgrades Dr. rer. nat.

vorgelegt von Sandra Geer aus Bad Windsheim

Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 24.09.2018

Vorsitzende/r des Promotionsorgans: Prof. Dr. Georg Kreimer Gutachter: Prof. Dr. Peter Gmeiner Gutachter: Prof. Dr. Torsten Kuwert

Acknowledgements

The present work was carried out at the Department of Chemistry and Pharmacy in cooperation with the Professor of Molecular Imaging and Radiochemistry of the Friedrich-Alexander-University Erlangen-Nürnberg, Prof. Dr. Olaf Prante and supervised by Prof. Dr. Peter Gmeiner. This dissertation could not have been completed without the support that I have received over the last three years. I would like to thank all the people who made this thesis possible and a formative experience for me. First, I thank my advisor Prof. Dr. Olaf Prante for accepting me into his group. Thank you for the support and the possibility to give my project my own direction according to my interests. Special thanks go to Dr. Simone Maschauer who always kept her office door open and was always available for advice. I want to express my deep gratitude to Manuel Geisthoff, who has always given me a hand to deal with the practicalities of working and without whom this thesis would not have been possible in its entirety. Ulrike Ittstein performed tireless cell culture work. Both of you spent a lot of time and effort on my experiments. My colleagues Ulrike Herrmann, Natascha Nebel, Julian Ott and Johannes Toms made our lab to a comfortable place to work. Your support made the times of struggling a lot easier. For giving me the possibility of gaining knowledge in our GMP Radiopharmacy I would like to thank Prof. Dr. Torsten Kuwert, Dr. Carsten Hocke, Peter Hennig and Benedikt Morgenroth. Thank you for your support. Furthermore, my thanks go to a number of people who contributed to different parts of my work. Regarding this, I am thankful to Barbara Happich (Department of Internal Medicine 3), Michael Aigner and Stefanie Schaffer (Department of Internal Medicine 5) and Natascha Leicht (Department of Pathology and Anatomy). Harald Hübner and Jürgen Einsiedel (Department of Chemistry and Pharmacy) did precursor synthesis and first functional assays, Philipp Ritt (Imaging and Physics, Clinic of Nuclear Medicine) provided his knowledge about dosimetry and Jonathan Schiller, Svenja Zihsler, Hannah Vogt and Moritz Macht helped a lot with practical work. Finally, I must express my very profound gratitude to my family who was always there cheering me up and encouraging me. Beyond, my thanks go to all friends who had to bear my moaning throughout the years and always gave fresh courage to me.

I

This thesis and proceeding work has been published in parts:

Poster presentations:

S. Geer, U. Reigl, S. Maschauer, P. Ritt, P. Gmeiner, E. Grill, T. Kuwert, O. Prante. The Neurotensin Receptor Subtype 1 as a Target for Radiotherapy in Prostate Cancer. GRK1910, Regensburg, Germany.

S. Geer, U. Reigl, S. Maschauer, P. Ritt, C. Lang, A. Banerjee, P. Gmeiner, E. Grill, O. Prante. The neurotensin receptor subtype 1 as a molecular target for endoradiotherapy in prostate cancer. Emil Fischer Graduate School (EFS) Research Day 2017, Erlangen, Germany.

Short lectures:

S. Geer, U. Reigl, S. Maschauer, P. Gmeiner, E. Grill, O. Prante. The neurotensin receptor subtype 1 as target for radiotherapy in prostate cancer. 24. Jahrestagung der Arbeitsgemeinschaft Radiochemie/Radiopharmazie 2016, Morschach, Switzerland.

S. Geer, U. Reigl, Dr. S. Maschauer, Dr. P. Ritt, Prof. P. Gmeiner, Prof. E. Grill, Prof. O. Prante. The neurotensin receptor subtype 1 as target for molecular imaging and radiotherapy in prostate cancer. International Symposium on radiopharmaceutical sciences 2017, Dresden, Germany.

S. Geer, U. Reigl, S. Maschauer, A. Banerjee, P. Gmeiner, O. Prante. Der Neurotensinrezeptor-1 als Target für die Radiotherapie und molekulare Bildgebung des Prostatakarzinoms. 25. Jahrestagung der Arbeitsgemeinschaft Radiochemie/Radiopharmazie 2017, Starnberg, Germany.

II

III

Table of Contents

1 INTRODUCTION...... 1

1.1 Theranostics – Radiochemistry with Gallium-68 and Lutetium-177 ...... 1 1.1.1 Positron Emission Tomography ...... 2 1.1.2 Gallium-68 ...... 4 1.1.3 Lutetium-177 and the Concept of Endoradiotherapy ...... 5

1.2 Pancreatic cancer ...... 8 1.2.1 Epidemiology ...... 8 1.2.2 Pathophysiology ...... 9 1.2.3 Diagnosis and Therapy ...... 9

1.3 Prostate Cancer ...... 10 1.3.1 Epidemiology and Diagnosis ...... 10 1.3.2 Molecular Imaging of Prostate Cancer ...... 12 1.3.3 Therapy of Prostate Cancer ...... 13

1.4 Neurotensin and its Receptor Subtypes ...... 15 1.4.1 Neurotensin ...... 15 1.4.2 Function and Signaling of Neurotensin Receptor Subtype 1 ...... 17 1.4.3 Neurotensin Receptor Subtypes 2, 3 and 4 ...... 21

1.5 The Neurotensin Receptor-1 in Pancreatic and Prostate Cancer ...... 22

1.6 Non-Peptide Neurotensin Receptor Antagonists ...... 24

2 OBJECTIVE AND AIM ...... 27

3 MATERIAL AND METHODS ...... 29

3.1 Precursors and Radioligands ...... 29

3.2 Cell Biology ...... 31 3.2.1 Cell Culture ...... 31 3.2.2 Description of Selected Cancer Cell Lines ...... 33 3.2.3 Freezing Cells...... 33 3.2.4 Determination of Proliferation Rates ...... 34 IV

3.2.5 WST-1 Cell Proliferation Assay ...... 34 3.2.6 Neurotensin Stimulation ...... 34 3.2.7 Colony Formation Assay ...... 35 3.2.8 Wound Healing/Scratch Assay ...... 36 3.2.9 Resazurin Cell Viability Assay ...... 36 3.2.10 Immunocytochemistry ...... 36 3.2.11 Flow Cytometry Analysis ...... 37

3.3 Radiochemistry and Radiochemical Experiments ...... 37 3.3.1 Radiolabeling of FAUC 468 and CL 156 ...... 37 3.3.2 Preparation of [68Ga]Ga-NT118195 ...... 38 3.3.3 Preparation of [68Ga]Ga-PSMA-11 ...... 39 3.3.4 Preparation of [68Ga]Ga-NODAGA-RGD ...... 40 3.3.5 Receptor Binding Assay ...... 40 3.3.6 Radioligand Binding Assay ...... 41 3.3.7 Cell Uptake and Internalization Studies ...... 41 3.3.8 Efflux Studies...... 42

3.3.9 Saturation Binding Studies (Determination of Kd and Bmax Values) ...... 43 3.3.10 NTS1 In Vitro Receptor Autoradiography ...... 45

3.4 Animal Studies ...... 46 3.4.1 Biodistribution Studies ...... 46 3.4.2 Ex Vivo Autoradiography ...... 48 3.4.3 Tumor Therapy Study ...... 48 3.4.4 Therapy Monitoring with Small-Animal PET Imaging ...... 50

3.5 Quantitative Real-Time PCR ...... 51 3.5.1 RNA Isolation...... 51 3.5.2 Reverse Transcription ...... 51 3.5.3 Quantitative Real-Time PCR ...... 52

3.6 Western Blot ...... 53 3.6.1 Protein Isolation ...... 53 3.6.2 Protein Quantification ...... 54 3.6.3 Western Blot ...... 54

V

3.7 Immunohistochemical Methods ...... 56 3.7.1 Immunohistochemistry ...... 56 3.7.2 HE Staining ...... 58 3.7.3 Masson-Goldner Staining ...... 58 3.7.4 PAS Staining ...... 58

3.8 Primary Tissue Samples from Patients ...... 59

4 RESULTS AND DISCUSSION ...... 60

4.1 Prostate and Pancreatic Cancer Cell Lines Showed Endogenous Expression of Neurotensin Receptor mRNA and Protein ...... 60

4.2 Proliferative Response to NT ...... 72

4.3 Effects of FAUC 468 and CL 156 on Colony Formation, Migration, Viability and Proliferation of PC-3 Cells ...... 74

4.4 Radiolabeling with Gallium-68 and Lutetium-177 ...... 76

4.5 Uptake, Internalization and Efflux Studies ...... 78 4.5.1 NTS1 Uptake, Internalization and Efflux Studies ...... 78 4.5.2 Uptake, Internalization and Efflux Studies Comparing NTS1 and PSMA ... 80 4.5.3 Discussion of Uptake, Internalization and Efflux Studies ...... 81

4.6 Saturation Binding Experiments ...... 83 4.6.1 Saturation Binding Experiments with the NTS1 antagonists ...... 83 4.6.2 Saturation Binding Experiments Comparing the NTS1 Agonist [68Ga]Ga- NT118 and NTS1 Antagonist [68Ga]Ga-ABN 468 ...... 86 4.6.3 Saturation Binding Experiments addressing PSMA ...... 92

4.7 Biodistribution ...... 93 4.7.1 [68Ga]Ga-ABN 468 and [68Ga]Ga-CL 157 Biodistribution Studies ...... 93 4.7.2 Small-animal PET imaging ...... 94 4.7.3 Biodistribution Studies of [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162 ...... 95 4.7.4 Displacement studies ...... 100 4.7.5 Dosimetry Calculation ...... 101 4.7.6 Ex Vivo Autoradiography ...... 102

VI

4.8 NTS1-targeted Endoradiotherapy ...... 104 4.8.1 Therapy Study with [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162 in PC-3 Tumor-Bearing Nude Mice ...... 104 4.8.2 Toxicity evaluation ...... 106 4.8.3 PET Imaging with [68Ga]Ga-NODAGA-RGD ...... 108

4.9 NTS1 as Therapeutic and Potential Imaging Target in Prostate Cancer ...... 111 4.9.1 Therapy Study with [177Lu]Lu-FAUC 469 in PC3-PIP Tumor-Bearing Nude Mice ...... 111 4.9.2 PET Imaging with [68Ga]Ga-PSMA-11 and [68Ga]Ga-NT118 ...... 113 4.9.3 Biodistribution of [68Ga]Ga-PSMA-11 and [68Ga]Ga-NT118 ...... 117

4.10 Quantification of NTS1 Density on Tumor Cells and Xenograft Tissue ...... 122

4.11 Quantification of NTS1 Density in Primary Patient Tissue ...... 125 4.11.1 qRT-PCR Results of NTS1, NTS2 and PSMA in Prostate Cancer Patients Tissue ...... 125 4.11.2 Autoradiography Results of NTS1 in Prostate Cancer Patients ...... 127 4.11.3 qRT-PCR Results of NTS1 and NTS2 in Pancreatic Cancer Patient Tissues 131 4.11.4 Autoradiography Results of NTS1 in Pancreatic Cancer Patients ...... 133

5 CONCLUSION ...... 137

6 SUMMARY ...... 139

7 ZUSAMMENFASSUNG ...... 145

I REFERENCES ...... 151

II LIST OF ABBREVIATIONS ...... 175

III LIST OF FIGURES ...... 179

IV LIST OF TABLES ...... 183

VII

Introduction

1 Introduction

1.1 Theranostics – Radiochemistry with Gallium-68 and Lutetium- 177

“Theranostics” describes the concept of the combined use of one tracer for diagnosis and therapy of a disease. This principle means to be able to radiolabel a radiopharmaceutical agent with a therapeutic radionuclide, such as lutetium-177, and a diagnostic radionuclide, such as gallium-68 (Figure 1). The reason for using one molecular target for imaging and therapy for individualized treatment is that pre-/post-therapeutic imaging for diagnosis, dose estimations and imaging of therapy success is combined with endoradiotherapy with the same tracer. This concept is well established for molecular imaging and therapy of neuroendocrine tumors using the theranostic pair of 68Ga- and 177Lu-labeled DOTA-D-Phe1-Tyr3-octreotide (DOTATOC) addressing somatostatin receptors.1,2

Figure 1. Concept of “theranostics“. One agent is used either for imaging/diagnosis if radiolabeled with gallium-68 or for targeted endoradiotherapy if radiolabeled with lutetium-177. (Modified after http://www.nirs.qst.go.jp/ENG/core/mit/index.html)

1 Introduction

1.1.1 Positron Emission Tomography The positron emission tomography (PET) technique is a non-invasive diagnostic imaging technique with high spatial resolution, high sensitivity and accurate quantification of up to 1 pM in vivo.3,4 interactions between physiological targets like receptors and ligands, for example neurotransmitters, can be visualized with this radionuclide-based imaging tool.5 PET is used in the fields of oncology,6 cardiology,7 neurology and during drug development and evaluation.8,9

Radiopharmaceuticals used in PET need to show a high level of radiochemical purity (typically > 95 %) and sterility as they are mainly administered intravenously.5 The radiolabeled compounds are only introduced in pico- to nanomolar quantities into the imaging subject. Common examples of positron-emitters are fluorine-18, oxygen-15, nitrogen-13, carbon-11 and gallium-68 (Table 1).3,10

Table 1. Clinical relevant positron-emitting radionuclides for PET imaging with their ß+ energy and half-lives.3,10

Nuclide F-18 O-15 N-13 C-11 Ga-68

ß+ energy [MeV] 0.64 1.70 1.20 0.97 1.90

Half-life [min] 110 2 10 20 68

Imaging tracers are radiolabeled with positron-emitting radionuclides that decay by emission of a positively charged particle, the positron (ß+). After emission of the positron from the nucleus, it collides after a short distance with an electron of the surrounding tissue. This event is called annihilation. During annihilation two 511 keV γ-rays are produced that correspond to the rest masses of the positron and the electron which are separated simultaneously by 180 °. These two γ-rays are detected by an array of surrounding scintillation detectors of inorganic scintillation crystals which generate visible light and are detected by photomultiplier tubes. The light is converted into an electronic signal which is proportional to the incident photon energy and can be used for spatial and time information (Figure 2). Although the exact site of annihilation is unknown, the acquisition of a large number of coincidence events can be used to reconstruct a three-dimensional image, representing the spatial distribution of the radioactive source as a function of time. The measured radioactivity

2 Introduction can be quantified in absolute units [Bq/mL].4,5 The energy of the emitted positrons is radionuclide-dependent and determines the path length before annihilation. Low positron energy implicates short range in tissue resulting in high resolution in PET imaging.4,5 For selection of the optimal radionuclide for PET imaging, many different parameters, such as nuclear properties, production, availability, costs, the ease and reliability of molecule labeling, in vivo behavior, dosimetry and potential effects of the radionuclide on the intrinsic spatial PET resolution should be considered.3

Figure 2. Schematic illustration of the principle of PET. (A) A positron from the nucleus and an electron annihilate producing two 511 keV γ-rays travelling in opposite directions by 180 °. (B) These two γ-rays are detected by an array of surrounding scintillation detectors which generate visible light and are detected by photomultiplier tubes. The light is converted into an electronic signal which is proportional to the incident photon energy and can be used for spatial and time information. Picture was adapted from Irina Velikyan, 2014.11

The most common used diagnostic PET radiopharmaceutical for staging and re- staging various types of cancer is [18F]fluorodeoxyglucose ([18F]FDG; Figure 3). However, [18F]FDG has limitations: Firstly, cancers with low growth rates like prostate carcinoma, neuroendocrine tumors and hepatocellular carcinoma may not take up enough [18F]FDG. Secondly, lesions that are located in or near tissues with high metabolic activity or physiological accumulation cannot be assessed. Thirdly, the differentiation of malignant tissues from an infective or inflammatory process with [18F]FDG is challenging.12

3 Introduction

Figure 3. Structures of D-glucose and the 18F- labeled analogue [18F]FDG.

1.1.2 Gallium-68 Naturally, gallium-68 can only be found in trace amounts on earth.13 For gallium-68 chemistry, a 68Ge/68Ga generator represents an in-house source for the radionuclide on demand at any time and independent from the operation of a cyclotron.3 The parent nuclide germanium-68 is produced on Ga2O3 targets by a (p,2n)-reaction in an accelerator (Figure 4) and absorbed on a solid support, such as titanium dioxide.12,14 Germanium-68 has a half-life of 271 days and decays by electron capture which is 12,14 part of the beta decay. The daughter radionuclide gallium-68 (t1/2 = 68 min) can be efficiently and selectively eluted by a suitable solvent.13 Each elution washes a small amount of germanium-68 from the column that does not exceed the value of 0.001 % of the total radioactivity, allowing the manufacturing of 68Ga-labeled radiopharmaceuticals with high radionuclide purity.13 For an efficient purity and concentration of gallium-68, the pre-extraction of gallium-68 by ion exchange chromatography is frequently applied. Gallium-68 decays to 89 % by positron emission and to 11 % via electron capture during beta decay.3

Figure 4. Production of germanium-68 by the (p, 2n)-reaction of gallium-69. The parent germanium-68 has a half-life of 271 days and decays by beta decay to the daughter radionuclide gallium-68. The scheme was taken from Fani et al. (2007).3

4 Introduction

The Ga(III) ion is in aqueous solution stable only under acidic conditions. Under raising pH conditions, hydrolysis occurs and insoluble gallium trihydroxide is built.13 Several suitable chelators have been developed and coupled to biomolecules for 68Ga-labeling allowing kit production and easy availability of various 68Ga-labeled radiopharmaceuticals (Table 2).14

Table 2. Examples of 68Ga-labeled radiopharmaceuticals.

Radiopharmaceutical Target

[68Ga]Ga-DOTATOC2 Somatostatin receptor – Neuroendocrine tumors

[68Ga]Ga-DOTATATE15 Somatostatin receptor – Neuroendocrine tumors

[68Ga]Ga-PSMA-1116 PSMA – Prostate cancer

Therefore, gallium-68 is a convenient PET imaging nuclide as it is available from an in-house 68Ge/68Ga generator and thus is independent of an on-site cyclotron. The combination of this radionuclide with a pharmaceutical such as antibody fragments, peptides, aptamers and oligonucleotides leads to goal-driven localization of the target in vivo.3,12,13

1.1.3 Lutetium-177 and the Concept of Endoradiotherapy Lutetium-177 has emerged as promising radionuclide for targeted molecular endoradiotherapy that addresses peptides and cell surface receptors which are overexpressed on the surface of tumor cells. The effect of applied 177Lu-labeled radiopharmaceuticals can be shown in different kinds of cancers, including colon cancer,17 non-Hodgkin's lymphoma,18 lung,19 ovarian,20 and prostate cancer,21,22 and neuroendocrine tumors.23 The half-life of 6.71 d (Table 3) allows more elaborated synthesis and purification steps of radiopharmaceuticals and further allows good logistic due to the ability to be transported over long distances.24,25 Lutetium-177 emits beta radiation with a maximum energy of 498 keV and therefore is applicable for endoradiotherapy of small tumors and metastases (optimal size 1.2 – 3.0 mm, mean penetration range of β− particles emitted by lutetium-177 in soft tissue is 670 μm), while avoiding high doses in normal tissue and minimization of the kidney dose.26 In addition, lower radiation for staff and contact persons has been observed compared to the alternative yttrium-90.24,25 The additional emission of low energetic 5 Introduction gamma rays (208 and 113 keV with 10 % and 6 % abundance, respectively) makes it suitable for direct tissue distribution monitoring of the radioactivity with a γ-camera (single-photon emission computed tomography, SPECT). The ability to obtain scintigraphic images provides the opportunity to evaluate the targeting, pharmacokinetics and excretion behavior of 177Lu-labeled radiopharmaceuticals which, in turn, allows subsequent estimation of dosimetry.24

Table 3. Characteristics of the radionuclide lutetium-177.

Property Lutetium-177 Atomic number 71 Gamma energy [keV] (%) 113 (6); 208 (10)

Eßmax [MeV] 0.5 Half-life [days] 6.71 Production method Reactor Production mode Lutetium-176 (n, γ), Yterbium-176 (n, γ) Decay mode ß, γ Daughter Hafnium-177

The production of lutetium-177 can be realized in two possible ways in a nuclear reactor. Either it can be produced by a direct or by an indirect route (Figure 5). Within the direct route, lutetium-177 is produced with a high specific activity by neutron activation of lutetium-176 on enriched target material since the natural abundance of lutetium-176 is only 2.6 %.27 Lutetium-176 undergoes neutron capture to provide lutetium-177 by the 176Lu(n, γ)177Lu nuclear reaction.25 As an alternative production route, lutetium-177 can be obtained carrier-free from beta decay of ytterbium-177 produced by neutron activation of ytterbium-176 by the indirect route.28 The needed enriched target material can be recycled since the burn-up of ytterbium-177 is negligible. Lutetium-177 can be separated from ytterbium-177 by radiochemical processes.29 The quality of lutetium-177 of the indirect route depends on the extent to which lutetium-177 can be separated from the neighboring ytterbium lanthanide.29-32

6 Introduction

Figure 5. Two different production routes for lutetium-177. Production can be performed by either the direct route with lutetium-176 (n, γ) or the indirect route by neutron activation of yterbium-176 (n, γ).

Lutetium is the smallest atom in the family of lanthanides and the only common oxidation state is +3.25 Lutetium salts and their aqueous solutions are colorless and form white crystalline/solids upon drying (with the exception of the iodide). Lutetium forms stable complexes with several ligands.33 Among the chelators, the macrocyclic DOTA (1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid)-chelator (Figure 6) is widely used for radiometals in +3 oxidation state as it forms sufficiently stable complexes to be used in clinical practice.25,34,35 Due to the slow rate of complexation of DOTA-based ligands with lutetium-177, heating at 80 - 95 °C is essential in order to achieve near quantitative radiolabeling yields for the preparation of 177Lu−DOTA-conjugates.36

Figure 6. Structure of the [177Lu]Lu-DOTA (1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid) complex.

7 Introduction

These radiopharmaceuticals are developed for targeted endoradiotherapy. In targeted therapy differences between “normal” healthy and cancers cells, such as overexpression of receptors, are used. For this issue drugs that are able to differentiate between receptors, and thus between healthy and cancer tissue, are developed. These therapeutics target cancer cells so that radioactivity is delivered selectively to the disease sites. With the development of [177Lu]Lu−DOTATATE that targets neuroendocrine tumors, lutetium-177 has gained routine attention as a therapeutic radionuclide (Table 4).37

Table 4. Examples of 177Lu-labeled radiopharmaceuticals.

Radiopharmaceutical Target [177Lu]Lu-DOTATATE37 Somatostatin receptor – Neuroendocrine tumors [177Lu]Lu-DOTATOC1,2 Somatostatin receptor – Neuroendocrine tumors [177Lu]Lu-DOTA-Trastuzumab38 HER2 – Breast cancer [177Lu]Lu-PSMA-61721 PSMA – Prostate cancer

1.2 Pancreatic cancer

1.2.1 Epidemiology Pancreatic cancer is the fourth leading cause of cancer-related deaths in Western countries.39 This highly malignant neoplasm leads to estimated 213,000 deaths each year worldwide.40 The majority of pancreatic neoplasms are pancreatic ductal adenocarcinomas (about 90 %) of which 70 % are located in the pancreatic head, 20 % in the body and 10 % in the tail of the pancreas (Figure 7).41 This cancer grows extremely rapid and aggressive and has a poor prognosis with less than 8.2 % 5-year survival rate.42 Despite advances in medicinal research the poor prognosis is not improving because of low early detection rates due to the lack of suitable screening tests for diagnosis.43,44 Family history, chronic pancreatitis, male sex, smoking and advanced age are major risk factors, with smoking and family history being the dominant ones.45 46

8 Introduction

Figure 7. Anatomy of the pancreas (left) and 5-year survival rate of pancreatic cancer patients (right). The pancreas is a gland which is located in the abdomen between duodenum and spleen. The organ is segmented into the three areas head, body and tail. The majority (about 70 %) of pancreatic neoplasms are located in the pancreatic head, 20 % in the body and 10 % in the tail of the pancreas (left, figure is adapted from https://www.cancer.gov/types/pancreatic/patient/pancreatic-treatment- pdq). The right part shows the 5-year survival rate of patients suffering from pancreatic adenocarcinomas. Data are from the Surveillance, Epidemiology, and End Results (SEER) Program resulting from data of the years 2007 - 2013. Grey figures represent patients who died from pancreas cancer. Green figures represent patients surviving at least five years (right, figure is adapted from https://seer.cancer.gov/statfacts/html/pancreas.html).

1.2.2 Pathophysiology Pancreatic cancer derives from non-invasive precursor lesions, in which neoplastic cells interact with cancer-associated fibroblasts. This neoplasm represents a molecular, pathologically and clinically heterogeneous disease resulting from the accumulation of different mutations which for example lead to the inactivation of tumor suppressor genes,47 activation of oncogenes,41 chromosomal losses,48 gene amplifications,49 or epigenetic dysregulation.50 A subgroup of pancreatic cancer cells (1 – 5 % of the tumors) has been identified to maintain stem cell-like properties meaning that they are capable of unlimited cell division and show a severe drug resistance.39,51

1.2.3 Diagnosis and Therapy At the time of diagnosis, 80 – 85 % of pancreatic cancer patients have an advanced unresectable state of the disease.52 Apart from missing biomarkers for early diagnosis, early stages of the disease are clinically silent and blood tests generally show nonspecific results, such as mild abnormalities in liver-function, hyperglycemia

9 Introduction and anemia.42 Severe physical symptoms emerge with invasive disease progression and can be abdominal pain, jaundice and weight loss.42 For people with suspicion of pancreatic cancer, computed tomography (CT) is the commonly used initial test for diagnosis and tumor staging.45 From this step, pancreatic cancer is categorized into three stages: Firstly, resectable disease that occurs without infiltrating regional vasculature (stages T1 - T3). Secondly, locally advanced/unresectable disease which infiltrates vascular structures, but does not spread by metastases (T4). Thirdly, metastatic disease that grows aggressively and without any option for treatment.42 Patients with localized disease show a mean survival of 9 - 15 months from diagnosis, whereas patients with metastatic disease only have a mean survival of 3 - 6 months.45 The state-of-the-art treatment options which include surgery, radiation, chemotherapy or various combinations of these, do not demonstrate any true chances of total recovery.42 For patients presenting with resectable disease, surgery offers the only possible treatment of cure with a 5-year survival of 20 - 25 %.45,53 Patients suffering from advanced or metastatic disease are treated with fluorouracil- based chemoradiation (Gemcitabine, Gemzar®, Lilly Deutschland GmbH, Germany) for pain control and prolonged survival.42,54 Therefore, there is urgent need for new targets for molecular imaging and therapy of pancreatic adenocarcinoma.

1.3 Prostate Cancer

1.3.1 Epidemiology and Diagnosis Considering the group of European men, prostate cancer is with 22.2 % the most common of all diagnosed cancers.55 Following lung and colorectal carcinoma, prostate cancer causes every third cancer-related death in this group of men.55 The 5-year survival rate for men with localized tumor burden approximates 100 % but decreases to 30 % in the cases of advanced and metastatic disease (Figure 8).56 Hallmarks of prostate cancer include slow growth rates of the tumors at the beginning of the disease,57 increasing levels of prostate specific antigen (PSA) and multiple disease foci.58-60 Numbers of new cases increased in the last years due to PSA screenings, which is the most commonly used screening marker for early detection and monitoring of recurrent prostate cancer.61 However, only a minority of 30 % of all cancer patients has elevated PSA values and 25 % of patients with benign prostatic hypertrophy show elevated levels of PSA.62 Nevertheless, in general, PSA has been

10 Introduction turned out to correlate with tumor burden, tumor progression and metastatic disease, but also for observation of treatment success with surgery, irradiation or androgen ablation therapy.63 In case of relapse, PSA levels increase before clinically detectable recurrence,64 but provide no hint to differentiate between local, regional and systemic disease.63

Figure 8. 5-year survival rate of patients suffering from prostate cancer. Data are from SEER resulting from data of the years 2007 - 2013. Grey figures represent patients who died from prostate cancer. Almost 100 % of patients survive at least five years after diagnosis. Cancer stage at diagnosis has a strong influence on the length of survival. For prostate cancer, 79 % are diagnosed at local stage for whom the 5- year survival is 100 %, for metastasized cancer it drops down to 30 %. (figure is adapted from https://seer.cancer.gov/statfacts/html/prost.html)

About 92 % of the patients with metastatic prostate cancer have prostate specific membrane antigen (PSMA) positive cancer cells.62,65 PSMA expression can be detected in 81 % of benign prostate hyperplasia, 59 % of prostatic intraepithelial neoplasia and 53 % of primary prostate carcinomas.62 PSMA is a type II transmembrane protein with 100 - 120 kDa.66 This protein, consisting of one intracellular domain, one transmembrane domain and an extracellular domain, 66 is present as noncovalent homodimer and exhibits folate hydrolase activity and neuropeptidase function (NAALADase) (Figure 9).67-69

11 Introduction

Figure 9. Crystal structure of the prostate-specific membrane antigen (PSMA). PSMA is a symmetric homodimer with each polypeptide chain containing three domains: one protease domain (blue), one apical domain (green) and one helical domain (red) (figure is adapted from Davis et al. (2005).70

PSMA can be detected on single non-prostate tissues including brain, salivary gland, duodenum and kidney.71-73 Overexpression of PSMA was shown in primary and metastatic prostate cancer and on a small number of tumor-associated neovasculature of solid malignant tumors, such as neuroendocrine tumors, mesenchymal tumors, melanoma and glioma.62,74,75 In prostate cancer, high PSMA expression is observed in poorly differentiated and advanced tumors and in hormone- insensitive cancers after androgen-deprivation therapy.62,76 PSMA is involved in the regulation of tumor cell invasion and angiogenesis by influencing cytoskeletal dynamics via integrin signal transduction.77 After binding of antibodies or inhibitors, PSMA is internalized accumulating in endosomes in a juxtanuclear location.78 As that transmembrane protein can be detected on the cell membrane of cancer cells and is overexpressed in aggressive prostate cancer, the antigen is used as target for molecular imaging and targeted endoradiotherapy.66,79

1.3.2 Molecular Imaging of Prostate Cancer Various efforts have been made to screen for and visualize potential prostate cancer burden with PET, while the tumor is localized and the disease potentially gets cured by surgery due to the fact that once metastatic prostate cancer has developed, the disease is incurable.80 Diagnostic PET imaging provides information about lesions and TNM staging that are relevant for prognosis and cancer treatment.64,80 In the past, numerous radiotracers for PET imaging of human prostate cancer have been developed. However, the detection rates were 34 - 88 % for [11C]choline,81 43 – 79 % for [18F]F-fluorocholine,82 and 59 – 80 % for [11C]acetate derivatives.83 Beside these, the group of M. Pomper developed several strategies for prostate cancer imaging by

12 Introduction targeting PSMA with fluorescent molecules or radioligands radiolabeled with different isotopes like 111In, 68Ga, 64Cu, and 86Y.73,84-87 The newly developed extracellular PSMA inhibitor [68Ga]Ga-PSMA-11 shows high and specific tumor uptake in a LNCaP mouse model.16 In patient studies, [68Ga]Ga-PSMA-11 proves high detection rates even with low PSA values independent of the localized area (97 % for values > 2 ng/mL, 93 % for 1 to < 2 ng/mL, 73 % for 0.5 to < 1 ng/mL and 60 % for 0.2 to < 0.5 ng/mL). 64 PET imaging shows positive results in 86.7 % of patients with Gleason Score ≤ 7 and in 96.8 % with Gleason Score ≥ 8.64 Previous anti-androgen therapy does not influence the detection efficacy.64 Although [68Ga]Ga-PSMA-11 shows high uptake in kidney, spleen and lung, it is an attractive PET tracer for diagnosis, because of its rapid clearance from blood circulation and PSMA-negative organs and high uptake in primary tumor tissue (7.7 % ID/g) as well as in relapses and metastases.16,64,88 The detection rate of 82.8 % of patients with suspicion of prostate cancer causes a highly frequent and increasing clinical use of [68Ga]Ga- PSMA-11 for PET across many countries.88 Thus, [68Ga]Ga-PSMA-11 PET/CT represents a new technology to differentiate between localized, advanced and metastatic disease and could facilitate the planning of individualized targeted endoradiotherapy.64

1.3.3 Therapy of Prostate Cancer Standard treatments for localized prostate cancer are radical prostatectomy, external- beam radiotherapy and brachytherapy.89 Prostate cancer represents an androgen- dependent cancer and most of the tumors express the androgen receptor (AR).90 Due to this fact, advanced prostate cancer is treated by androgen-deprivation: either by reducing endogenous androgen levels or by competing AR activity with the receptor antagonist enzalutamide (Xtandi, second-generation androgen receptor antagonist) and the CYP17A1-inhibitor abiraterone (Zytiga, inhibitor of steroidogenesis).91,92 These substances reduce cell proliferation and induce programmed cell death of androgen-dependent cancer cells.93,94 Initially, patients respond well to anti-androgen therapy with suppression of PSA levels in 80 – 90 % of patients.91 However, in the long term, prostate carcinomas lose their androgen- dependency, because the AR is hypersensitive and can be activated by alternative molecules like estrogens, progesterone or corticosteroids.95 Subsequently, PSA levels usually increase again in the majority of patients.91 This observation is paralleled by the progression of disease characterized by an androgen-dependent

13 Introduction treatable to an incurable androgen-independent tumor. This effect occurs mainly due to the heterogeneity of prostate cancers that are composed of a mixture of both androgen-dependent and -independent cell clones.96 The latter are of neuroendocrine phenotype, which mediate androgen-independence by increasing their cell density and number during androgen-deprivation therapy.97

For this reason, endoradiotherapy emerges as a therapeutic approach in prostate cancer treatment. Different basic approaches have been developed: Since 2013, Xofigo is approved for metastatic castrate-resistant prostate cancer.98 The drug product is the α-emitter [223Ra]radium dichloride, which is commercialized by Bayer AG (Germany). Xofigo improves the overall survival of patients by 3.6 months.98 Another attempt is [177Lu]Lu-PSMA I&T that passed first proof-of-concept investigation: endoradiotherapy is well tolerated in two patients without side effects or clinically detectable pharmacologic effect.99 A new therapy option for metastatic prostate cancer is [177Lu]Lu-PSMA-617.21 This radiotracer is a DOTA-derivative of the Glu-urea-Lys motif of [68Ga]Ga-PSMA-11 100 that binds specifically with high affinity (Ki = 2.34 nM) to PSMA (Figure 10).

Figure 10. Structures of the imaging agent [68Ga]Ga-PSMA-11 (left) and the therapeutic agent [177Lu]Lu-PSMA-617 (right).

14 Introduction

In vitro studies reveal that following binding, [177Lu]Lu-PSMA-617 is internalized with a rate of 17.51 percent of injected radioactivity.100 The radiotracer shows high specific tumor and kidney uptake with high tumor-to-background ratios (tumor-to-blood, 1,058; tumor-to-muscle, 529) in LNCaP xenografted nude mice.100 Kidney clearance is fast within 24 h, whereas tumor uptake stays constant (10.58 ± 4.50 % ID/g) for 24 h post-injection.100 Co-injection of the PSMA inhibitor 2- (phosphonomethyl)pentane-1,5-dioic acid (PMPA) in patients improves tumor-to- kidney ratio in targeted endoradiotherapy by reducing off-target radiation.101 Therapy with [177Lu]Lu-PSMA-617 results in a decrease of 70 % of PSA levels without showing any toxicity.21 These results suggest the [177Lu]Lu-PSMA-617 as effective therapeutic radioligand to improve the quality of life and to increase the overall survival of patients suffering from advanced and metastatic prostate cancer.

1.4 Neurotensin and its Receptor Subtypes

1.4.1 Neurotensin Neurotensin (NT) is a tridecapeptide (Glu1-Leu2-Tyr3-Glu4-Asn5-Lys6-Pro7-Arg8-Arg9- Pro10-Tyr11-Ile12-Leu13) (Figure 11) originally isolated from bovine hypothalamus by Carraway and Leeman in 1973.102 NT is mainly present in the central nervous system where it induces hypothermia and analgesia and acts as neuromodulator of dopamine transmission.103-105 In the periphery, NT acts as local hormone and modulates the digestive tract.106 There, NT is secreted from enteroendocrine N cells of the small intestine.107

15 Introduction

Figure 11. Chemical structure of the tridecapeptide neurotensin.

NT is involved in the stimulation of pancreatic and biliary secretion,108 stimulation of colonic motility,109 inhibition of small bowel and gastric motility,110 the ease of fatty acid absorption and growth stimulation of various gastrointestinal tissues.111,112 Furthermore, NT exerts protective anti-inflammatory, anti-oxidant, mitogenic and anti- apoptotic action on the intestinal barrier.112 With a half-life of 0.5 min, NT is rapidly degraded by endopeptidases and angiotensin-converting enzyme in close proximity to the site of secretion.113 NT is synthesized from the precursor preproneurotensin in all gastrointestinal and neural tissues.114 Studies show the involvement of the mature NT in some mental disorders like Huntington’s chorea, schizophrenics and Parkinson’s disease.115-117 NT also acts as growth factor on different kinds of cancer cells. The physiological and pathophysiological processes of NT are mediated through the binding of the peptide to two G-protein coupled receptors (GPCRs), namely neurotensin receptor-1 (NTS1) and neurotensin receptor-2 (NTS2) and to one nonspecific sorting receptor sortillin/neurotensin receptor-3 (NTS3).118,119 The binding to these receptors occurs via the carboxyl-terminal end of NT between amino acids 8 and 13 (Arg8-Arg9-Pro10-Tyr11-Ile12-Leu13) (Figure 12), whereas its amino- terminal end does not bind to the receptor and is responsible for full pharmacological potency.120 Therefore, the N-terminus is amenable to chemical modifications and radioactive labeling, resulting in radioligands for the neurotensin receptor with nearly retained affinity.

16 Introduction

Figure 12. NT(8–13) - the pharmacologically active C-fragment of neurotensin.

1.4.2 Function and Signaling of Neurotensin Receptor Subtype 1 The first cloned neurotensin receptor that has been cloned is the rat NTS1 from rat brain in 1990.121 In 1993, Caput and his group cloned the human NTS1 from the colonic adenocarcinoma cell line HT-29.122 The NTS1 encoding gene is translated into a protein composed of 418 amino acids and a molecular weight of 46 kDa.123 The receptor forms seven transmembrane domains and shares 84 % homology with 121-123 124 rat NTS1. NTS1, the high affinity receptor for NT (Kd = 0.1 - 0.4 nM), has a known crystal structure (Figure 13), and is insensitive for levocabastine, a selective histamine H1 receptor antagonist (marketed as Livostin by Novartis).125

17 Introduction

Figure 13. Crystal structure of the rat NTS1 with the bound peptide agonist NT(8-13) from White et al. Figure is adapted from White, J. F., et al. (2012).126

Binding of the natural ligand NT to its receptor on the cell surface causes the replacement of Guanosine 5'-diphosphate (GDP) by Guanosine 5'-triphosphates (GTP) in the G-protein through conformational changes and the α-subunit dissociates from the βγ heterodimer.127 This process starts intracellular signaling pathways by triggering effector molecules like adenylate cyclase (AC) or phospholipase C (PLC) or by directly regulating ion channels or kinase functions (reviewed in 128) with activation or production of cyclic adenosine 3':5'-monophosphate (AMP) and inositol 2+ 1, 4, 5-trisphosphate (IP3) which triggers Ca release and diacylglycerol (DAG) that activates protein kinase C (PKC).129-131 All these steps end up in increased gene transcription activity (Figure 14).

Deregulation of NTS1 pathway is mostly mediated through the Gαq/11 subunit that activates PLC and hydrolysis of phosphatidylinositol bisphosphate into DAG and 131,132 2+ 131 IP3. These stimulate PKC, Ca mobilization, mitogen-activated protein kinase (MAPK) pathway and epidermal growth factor receptor (EGFR) transactivation.132 MAPK has prominent impact on Wnt (acronym of homologous wingless (wg) and Int-1 gene)/ß-catenin pathway resulting in enhancement of DNA synthesis (Figure 14).132,133 One of the downstream targets of the Wnt/ß-catenin

18 Introduction signaling pathway is the NTS1 gene.134 Beside the influence on cell signaling, NT helps to maintain an inflamed tumor microenvironment by interacting with immune cells and induces anti-apoptotic effects.135,136

Figure 14. Scheme of activation and signaling cascade of the GPCR NTS1. After binding of the ligand, there are conformational changes in the receptor leading to its activation. Activation occurs through exchange of Guanosine 5'-diphosphate (GDP) by Guanosine 5'-triphosphates (GTP). As consequence the G-protein dissociates in the α- and ßγ-subunit for further signal transduction. For intracellular signaling, effector molecules such as adenylate cyclase (AC) or phospholipase C (PLC) are activated. NT = neurotensin; MAPK = mitogen-activated protein kinase; cAMP = cyclic adenosine 3':5'-monophosphate; PKA = protein kinase A; IP3 = inositol 1, 4, 5-trisphosphate; DAG = diacylglycerol; ER = endoplasmatic reticulum; PKC = protein kinase C

Following ligand binding to NTS1, 60 - 70 % of NTS1 is internalized by clathrin vesicles in a time- and temperature-dependent manner.127,137 The NTS1-ligand complex accumulates in endosomes and ligand and receptor dissociate. Then, two options are given by principle: First, the receptor is degraded in lysosomes and cells re-sensitize by de novo synthesis, or second, the receptor is recycled to the cell membranes, whereas NT is transferred to the trans-Golgi network.127,138-140 Dividing

19 Introduction cells retain the sensitization to chronic ligand exposure by recycling of the receptor to the cell membrane. In this way, cells remain sensitive to NT and intracellular pathways like MAPK are constitutively active.138 NTS1-positive HT-29 human colonic adenocarcinoma cells show a differentiated regulation of the NTS1 during agonist exposition. Incubation for 6 h with the NT agonist JMV 499 results in elevated mRNA levels up to 270 % due to change of transcription rates. The longer agonist stimulation for up to 96 h leads to decreased mRNA levels of 70 %, because mRNA half-life is shortened by 50 %.141 During chronic exposure of cells to NT, NTS1 agonists or antagonists an increase in mRNA levels of NTS1 can be observed in rat brain.142,143 This regulation of NTS1 expression offers the opportunity of oncogenic mechanisms and supports the role for NT in carcinogenesis. Various research groups show that stimulation of NTS1 through NT in different types of cancer cells is involved in tumor growth, proliferation, migration, invasion and metastasis. Growth promoting effects of NT are proven on prostate cancer cells,144 human pancreatic cancer cells,131 lung cancer,145 breast cancer,135 colon cancer,146 astrocytic tumors,147 glioblastomas and head and neck squamous cell carcinomas (HNSCC).147,148 Beside the proof of NTS1 mRNA and protein expression in different cell lines, the resulting xenografts are also positive for NTS1.149 In patient tissues, overexpression of NTS1 is associated with a higher rate of metastases and poor prognosis.145,150 NT and NTS1 expression can be detected in human tissues of different kind of cancers, such as pancreatic ductal carcinomas,151 prostate cancer,144 colon cancer,152 non-small cell lung cancer (NSCLC) or breast carcinomas,145,150 whereas related healthy tissues are NTS1-negative (Table 5).153

Table 5. NTS1 mRNA and protein expression in different types of cancers.

Cancer type % positive mRNA protein in cancer cells % positive patient tissue (detection method) (detection method) (detection method) Pancreatic cancer 83 % 154 1.2 × 105 sites/cell 155 75 % 151 (Northern blot) (binding experiments) (autoradiography) Prostate cancer 89 % 156 42 fmol/mg 157 14 % 156 (RT-PCR) (binding experiments) (Western Blot) Colon cancer 40 % 158 85 – 1000 fmol/mg 158 76 % 159 (RT-PCR) (binding experiments) (immunohistochemistry) NSCLC - - 60 % 145 (immunohistochemistry) Breast carcinoma 82 % 150 96 fmol/mg 135 76 % 150 (RT-PCR) (binding experiments) (immunohistochemistry)

20 Introduction

1.4.3 Neurotensin Receptor Subtypes 2, 3 and 4

The low affinity receptor NTS2 (Kd (NT) = 2.6 nM) was cloned from rat (416 aa), mouse (417 aa) and human brain (410 aa) and has an apparent molecular mass of 45 kDa.118,160,161 NTS2 is, as well as NTS1, a member of the GPCR family and contains the typical 7-transmembrane structure.160 NTS2 is not as efficiently coupled 161 162 to G-proteins as NTS1 in signaling, but also mediates IP3 formation, stimulation of Ca2+ mobilization and MAPK.132,163 This receptor is mostly expressed in cerebral structures,160 involved in pain control and its distribution differs from NTS1 with only a few common regions.164 In overlapping regions, NTS2 forms heterodimers with NTS1, resulting in downregulation of the cell surface density of NTS1. However, the affinity to NT, receptor internalization or signaling is not affected.165

The third neurotensin receptor, NTS3, was isolated from mouse and human brain.119,166 This receptor is a nonspecific single transmembranous sorting receptor belonging to the family of Vps10p receptors which binds numerous ligands.167,168 NTS3 contains a cysteine-rich extracellular region, one transmembrane domain and a short intracellular tail bearing signals for rapid internalization after NT binding.169 The mature protein is expressed at high levels in brain, spinal cord, heart and skeletal muscle, thyroid, placenta, and testis with binding affinity to NT of 0.3 nM.119 NTS3 is insensitive to SR 48692, a potent selective non-peptide NTS1 antagonist and to levocabastine.170,171 The physiological functions of the receptor are still not fully enlightened. Only 20 % of the protein is expressed on the cell surface.171 After binding to NTS3, NT is transported to the trans-Golgi network by internalization.169 Furthermore, NTS3 is expressed in intracellular vesicles containing the glucose transporter GLUT4 and is translocated to the cell membrane in response to insulin.172,173 In a variety of human cancer cells such as prostate, colonic and pancreatic cancer, NTS3 potentially modulates NT induced growth response.157 In adeno- and squamous cell carcinoma and in colon cancer NTS3 mRNA is expressed at even higher levels than NTS1 and NTS2.149 This fact may also be a hint that the receptor may have an essential role in cell survival and function.156

The existence of a fourth neurotensin receptor sorLA/LR11 which contains a structure similar to that of NTS3 and represents a mosaic protein, combining the families of Vps10p domain receptor family and low density lipoprotein receptor family, is under discussion.174

21 Introduction

1.5 The Neurotensin Receptor-1 in Pancreatic and Prostate Cancer

During hormone-deprivation therapy, the NTS1 provides an alternative growth mechanism in androgen-sensitive prostate cancer (Figure 15).175 Anti-androgen treatment induces neuroendocrine differentiation leading to neurotensin receptor overexpression, NT production and secretion increasing cell proliferation and invasion.175 Some particularly aggressive prostate tumors are complete neuroendocrine neoplasm.176 In healthy prostate tissue, NT is not expressed.177 This observation can be shown in vitro and in patient material.156 The NTS1 expression is shown to be dependent on malignancy in different prostatic cancer cell lines. NTS1 expression levels positively correlate with the grade of malignancy.156 Besides NTS1, which shows uniform distribution in the cells, NTS2 is highly concentrated around the apical cell surface.156 NTS3 is concentrated within the luminal cell layer, particularly at the extremity of the apical cell surface.156 In primary patient material, NTS1 expression can be detected in the majority of malignant samples, whereas NTS2 is detectable in only a small number of samples. In contrast, all benign samples express low or undetectable levels of NTS1 and NTS2, whereas NTS3 is expressed in both types of samples.156 Furthermore, NTS1 can also be responsible for radio-resistance of prostate cancer. For this reason, inhibition of the NTS1 can be a possibility to enhance the sensitivity of prostate cancer to radiotherapy.178

22 Introduction

Figure 15. NTS1 as an alternative growth mechanism during androgen-deprivation therapy. Anti-androgen treatment induces neuroendocrine differentiation leading to neurotensin receptor overexpression, NT production and secretion increasing cell proliferation, migration, survival and invasion.

The NTS1 protein is expressed in 75 % of primary pancreatic ductal adenocarcinomas and in 63 % of liver metastases, whereas NTS1 cannot be detected in healthy pancreatic tissue, chronic pancreatitis or endocrine pancreatic cancers.151,179 However, NTS1 mRNA levels can be detected in all kinds of these tissues. Here, pancreatic cancer and chronic pancreatitis show 4.4-fold higher expression levels than healthy controls.154 Within the group of pancreatic cancers, mRNA levels show an increase from poorly differentiated cancer towards early and advanced tumor stages.154 Pancreatic intraepithelial neoplasia can also express NTS1 but don’t have to. If they express NTS1, expression increases with staging, if NTS1 is absent, resultant invasive ductal adenocarcinomas are also NTS1- negative.179 There is no expression of neurotensin receptor subtypes 2 and 3 in these carcinomas.179

23 Introduction

1.6 Non-Peptide Neurotensin Receptor Antagonists

The expression of NTS1 in pancreatic ductal adenocarcinomas and prostate cancers triggers the development of radioactive neurotensin analogs and neurotensin receptor antagonists for in vivo targeting of the receptor for visualization and endoradiotherapy. The majority of designed neurotensin analogs that can be radiolabeled are NT agonists.180-183 Working with such compounds is challenging, because of rapid peptidase-mediated degradation of the radiotracers in blood.184-186 The development of neurotensin receptor antagonists as new drugs and radiotracers has the advantage of greater metabolic stability and increased tumor uptake.187,188

As a promising lead structure for the development of NTS1-targeting radioligands, the non-peptide NTS1 antagonists previously described by Sanofi, is chosen. The first member of a potent selective non-peptide NTS1 antagonist is SR 48692 (Meclinertant; Sanofi-Aventis, France; Figure 17A).170 SR 48692 can selectively and efficiently inhibit NTS1-mediated tumoral proliferation in colon,189 pancreatic,155 head and neck,148 and prostate cancer.190 In vivo, SR 48692 is unable to inhibit neurotensin-mediated hypothermia and analgesia in mice and rats.191 SR 48692 exhibits higher affinity for the high-affinity receptor NTS1 (Ki = 12 nM) than for the 170 low-affinity site NTS2 (Ki = 200 nM). Insertion of SR 48692 combined with radioactivity efficiently decreases selectively survival in prostate cancer cells in vitro and lowers tumor burden in vivo.178

The second-generation non-peptide NTS1 antagonist SR 142948A (Figure 17B) (Sanofi-Aventis, France) has oral bioavailability and crosses the blood-brain barrier with long lasting effects.192 The biochemical and pharmacological properties are still chemically related to the lead structure of SR 48692.192 The new compound cannot distinguish between NTS1 and NTS2 and binds to neurotensin receptors with high affinity (Ki = 0.28 nM), very similar to the endogenous ligand NT. In addition, SR 142948A has been found to antagonize the effect of NTS1-induced hypothermia and analgesia in mice and rats in a dose-dependent manner.192 Furthermore, SR 142948A blocks neurotensin receptor-mediated growth stimulations.157 At NTS2, 2+ SR 142948A acts as agonist triggering Ca mobilization following IP3 formation, arachidonic acid release and stimulation of MAPK.118 In summary, SR 142948A represents a potent NTS1-selective non-peptide antagonist which serves as a lead

24 Introduction structure for the development of radioligands for PET and endoradiotherapy of NTS1- positive tumors.193 Based on the lead structure of SR 142948A, Lang et al. synthesized the first non-peptide radioligand with high affinity to NTS1 for PET, which reveals excellence blood clearance and high tumor retention in vivo and thus provides a favorable tracer for imaging of NTS1-positive cancers.187 Aiming at the development of 177Lu-labeled analogs, Schulz et al. present new NTS1-selective theranostic compounds as promising radioligands for endoradiotherapy and translation into clinics.194 These are derivatives with short linker-DOTA-chelator moieties for radiolabeling with a variety of different diagnostic and therapeutic radionuclides. One of these structures (3BP-227) can be used as theranostic agent in a model of colon carcinoma.194

Figure 16. Chemical structure of 3BP-227 published by Schulz et al.188

An alternative set of compounds, structurally related to SR 142948A, has been developed in cooperation with Prof. Dr. Peter Gmeiner from the Department of Chemistry and Pharmacy, Friedrich-Alexander University Erlangen-Nürnberg, namely FAUC 468 (European patent application EP16182597, August 3, 2016; Figure 17C) and CL 156 (Figure 17D).187 The set of the Erlangen FAU compounds consists of a triazolyl-conjugated linker after the NTS1-binding site followed by a DOTA-chelator for stable coordination of the radionuclide, such as gallium-68 ([68Ga]Ga-ABN 468 and [68Ga]Ga-CL 157) or lutetium-177 ([177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162), in order to use both compounds for theranostics applications.

25 Introduction

Figure 17. Chemical structures of the first members of potent selective non-peptide NTS1 antagonist (A) SR 48692 (Meclinertant; Sanofi-Aventis, France) and the second-generation non-peptide NTS1 antagonist (B) SR 142948A (Sanofi-Aventis, France). Chemical structures of the non-peptide NTS1 antagonist radiotracers (C) FAUC 468 (with the free chelator) and its radiolabeled derivatives [177Lu]Lu- FAUC 469 and [68Ga]Ga-ABN 468 and (D) CL 156 (with the free chelator) and its radiolabeled derivatives [177Lu]Lu-CL 162 and [68Ga]Ga-CL 157. M = 68Ga or 177Lu.

26 Objective and Aim

2 Objective and Aim

The NTS1 has emerged as an interesting target for molecular imaging and targeted endoradiotherapy (“theranostics”) due to its overexpression in a variety of tumors, such as prostate and pancreatic cancer.151,156 For PSMA-negative prostate cancers and the highly malignant pancreatic adenocarcinoma it would be of utmost importance to find a tool for imaging and endoradiotherapy of the carcinoma. Overexpression of NTS1 was proven for pancreatic cancer in a large number of tumors. Therefore, in the present work, NTS1 was evaluated as target for molecular imaging and targeted endoradiotherapy in prostate and pancreatic cancer (Figure 18).

Figure 18. Objective and Aim

Various cell lines were studied for NTS1 expression via quantitative real-time PCR, Western Blot and radioligand binding assay in order to assess the potential of NTS1 as therapeutic target in prostate and pancreatic cancer in appropriate cell lines for the establishment of a xenotransplantation mouse model. Based on these results, suitable human cell lines were chosen for in vitro uptake and efflux studies and endoradiotherapy studies using the NTS1 antagonist [177Lu]Lu-FAUC 469 in two prostate cancer models in vivo. The observation of therapy success was determined during therapy by monitoring tumor growth and determination of the effects of endoradiotherapy on the receptor expression state. Analysis of NTS1 expression on 27 Objective and Aim the tumor was assessed by autoradiography, immunohistochemistry, qRT-PCR and Western Blot. These results were compared with the receptor expression of cells during cell culture to determine potential changes of receptor expression between the situations in cell culture and in vivo conditions. This finding could be used to estimate the application of the NTS1 as target for endoradiotherapy in multiple therapeutic cycles.

Preclinical results were verified in primary patient material, evaluating the NTS1 expression on human tumor tissues. Therefore, the major aim of this study was to assess whether the NTS1 receptor could emerge as a highly attractive theranostic target in nuclear medicine for accurate and rapid visualization of tumor burden, improved staging and highly effective endoradiotherapy of receptor-positive lesions with radioligands in a specific and tumor-selective manner.

28 Material and Methods

3 Material and Methods

3.1 Precursors and Radioligands

Radiolabeled FAUC 468, CL 156 and NT118,195 as shown in Figure 19, were chosen for in vitro and in vivo evaluation of NTS1 as target for molecular imaging and endoradiotherapy. FAUC 468 was synthesized by Ashutosh Banerjee and CL 156 by Christopher Lang at the Department of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer Center – Erlangen, Friedrich Alexander University Erlangen-

Nürnberg (FAU). Respective Ki values were determined at the department of chemistry and pharmacy (Table 6). Furthermore, [68Ga]Ga-PSMA-1116 and [68Ga]Ga- NODAGA-RGD were chosen for in vivo imaging studies. Precursor syntheses of FAUC 468, CL 156 and NT118195 were performed at the department of pharmaceutical chemistry in Erlangen. Precursor of [68Ga]Ga-PSMA-1116 was purchased from ABX advanced biochemical compounds GmbH, Germany. NODAGA-RGD was kindly provided by Dr. Roland Haubner, University of Innsbruck, Austria.

Table 6. Binding data of compounds to their target used during the present study (see structures in Figure 19).

Compound Ki Target FAUC 468 1.9 nM NTS1 [68Ga]Ga-ABN 468 n.d. NTS1 [177Lu]Lu-FAUC 469 0.19 nM NTS1 CL 156 1.0 nM NTS1 [68Ga]Ga-CL 157 0.46 nM NTS1 [177Lu]Lu-CL 162 0.18 nM NTS1 [68Ga]Ga-NT118 20 nM NTS1 [68Ga]Ga-PSMA-11 12 nM PSMA 68 [ Ga]Ga-NODAGA-RGD n.d. αVß3

Ki = Inhibitory constant; n.d. = not detected

29 Material and Methods

Figure 19. Structures of radiotracers included in this study. FAUC 468 and CL 156 represented non-peptide NTS1 antagonists, the peptide NTS1 agonist NT118195, the 16 PSMA-binding inhibitor PSMA-11 , and NODAGA-RGD for imaging integrin αVß3 during angiogenesis. M = 68Ga or 177Lu.

30 Material and Methods

3.2 Cell Biology

3.2.1 Cell Culture Cell lines included in this work are listed in Table 7. Cell lines were cultured continuously in flasks as monolayers at 37 °C in a humidified 5 % CO2 atmosphere in specific media (Table 7). Routine cell culture was performed twice a week. Standard procedure for passaging cells was employed. Briefly, cells in flasks were washed with sterile PBS (Sigma-Aldrich, USA) and incubated with 0.05 % trypsin/EDTA (Biochrom, Germany) for 3 - 5 min at +37 °C. Detached cells were diluted in complete culture medium. Finally, cells were split into culture flasks. Before seeding for cell based assays, cells were counted using a Neubauer counting chamber (Carl Roth GmbH, Germany).

Table 7. Cell lines and their respective culture conditions.

Cell Line and Supplier Cancer Type Medium Seeding Density PC-3196 Human Prostate Ham’s F12/RPMI 1640 2 × 106 (Dr. Sven Wach, Molecular Adenocarcinoma + 10 % FCS cells/T75 Urology, FAU Erlangen, + 1 % L-Glutamine Germany) PC3-PIP75 (PC-3 cells Human Prostate RPMI 1640 2 × 106 transfected with PSMA)62 Adenocarcinoma + 10 % FCS cells/T75 (Prof. Catherine Foss, Johns + 1 % L-Glutamine Hopkins University, + 1 % Sodium Pyruvate Baltimore, USA) LNCaP197 Human Prostate RPMI 1640 4 × 106 (Prof. Uwe Haberkorn, Carcinoma + 10 % FCS cells/T75 DKFZ, Heidelberg, + 1 % L-Glutamine Germany) + 1 % Sodium Pyruvate JHU-LNCaP-SM198 Human Prostate RPMI 1640 4 × 106 (Prof. Catherine Foss, Johns Carcinoma + 10 % FCS cells/T75 Hopkins University, + 1 % L-Glutamine Baltimore, USA) + 1 % Sodium Pyruvate DU-145199 Human Prostate MEM Earles 1.5 × 106 (CLS Cell Lines Service Carcinoma + 10 % FCS cells/T75 GmbH, Eppelheim, + 1 % L-Glutamine Germany) + 1 % NEAA + 1 % Sodium Pyruvate

31 Material and Methods

HEK293T200 Human Embryonic DMEM 1.5 × 106 (Prof. Peter Gmeiner, Kidney Cells + 10 % FCS cells/T75 Pharmaceutical Chemistry, + 1 % L-Glutamine FAU Erlangen, Germany) HT-29201 Human Colon MEM Earles 2 × 106 (CLS Cell Lines Service Adenocarcinoma + 10 % FCS cells/T75 GmbH, Eppelheim, + 1 % L-Glutamine Germany) + 1 % NEAA + 1 % Sodium Pyruvate U87MG202 Human DMEM 1 × 106 (Prof. Udo Gaipl, Radiation Glioblastoma + 10 % FCS cells/T75 Clinics, Erlangen, Germany) + 1 % L-Glutamine + 1 % NEAA + 1 % Sodium Pyruvate AsPC-1203 Human Pancreatic RPMI 1640 4 × 106 (Prof. Florian Haller, Institute Adenocarcinoma + 1 % L-Glutamine cells/T75 of Pathology, FAU Erlangen, + 10 % FCS Germany) Panc-1204 Human Pancreatic DMEM 2 × 106 (CLS Cell Lines Service Epithelioid + 10 % FCS cells/T75 GmbH, Eppelheim, Carcinoma + 1 % L-Glutamine Germany) CFPac-1205 Human Ductal IMDM 3.3 × 106 (Prof. Florian Haller, Institute Pancreatic + 10 % FCS cells/T75 of Pathology, FAU Erlangen, Adenocarcinoma + 2 % L-Glutamine Germany) BxPC3206 Human Primary RPMI 1640 2 × 106 (Prof. Florian Haller, Institute Pancreatic + 10 % FCS cells/T75 of Pathology, FAU Erlangen, Adenocarcinoma + 1 % L-Glutamine Germany) + 1 % Sodium Pyruvate HPAF-II207 Human Pancreatic MEM Earles 4 × 106 (Prof. Florian Haller, Institute Adenocarcinoma + 10 % FCS cells/T75 of Pathology, FAU Erlangen, + 1 % L-Glutamine Germany) + 1 % Sodium Pyruvate JoPaCa208 Human Ductal IMDM 4 × 106 (Prof. Florian Haller, Institute Pancreatic + 10 % FCS cells/T75 of Pathology, FAU Erlangen, Adenocarcinoma + 1 % Sodium Pyruvate Germany)

32 Material and Methods

MiaPaCa2209 Human Pancreatic DMEM 1.9 × 106 (Prof. Florian Haller, Institute Adenocarcinoma + 10 % FCS cells/T75 of Pathology, FAU Erlangen, + 1 % L-Glutamine Germany) + 1 % Sodium Pyruvate PSC156/200/107/57/34 Pancreatic stromal DMEM - (Prof. Christian Pilarsky, cells + 20 % FCS Clinics for Surgery, + 1 % L-Glutamine University hospital Erlangen) + 1 % Sodium Pyruvate

3.2.2 Description of Selected Cancer Cell Lines PC-3 and PC3-PIP cells: PC-3 (androgen independent, PSA negative, androgen receptor negative) and PC3-PIP (PC-3 cells stably transfected with PSMA62) cell lines represented human prostate adenocarcinoma cell lines. PC-3 cells were isolated from a lumbar vertebral metastasis in 1979.196 NTS1 expression in PC-3 cells has been reported.157 Taylor et al. (2012) proved that both PC-3 cells and PC-3 xenograft showed NTS1 expression whereas both were devoid of PSMA.210

AsPC-1 and Panc-1 cells: AsPC-1 cells originated from a human pancreatic adenocarcinoma. The cell line has been established from the ascites of a female patient with adenocarcinoma of the head of the pancreas in 1982.211 Panc-1 cell line came from a human pancreatic epithelioid carcinoma of ductal cell origin.204 NTS1 expression has been shown for pancreatic carcinomas in literature.151,154,179

HT-29 cells: HT-29 cell line was isolated in 1977 from a primary human colon adenocarcinoma.201 These cells were reported to express NTS1.146,212

3.2.3 Freezing Cells Cells in culture were treated with trypsin and centrifuged as described in 3.2.1. Cell pellets were re-suspended in complete culture medium supplemented with 20 % FCS (Biochrom, Germany) and 10 % DMSO (Carl Roth GmbH, Germany). Finally, 1.8 mL aliquots of the cell suspensions were gradually cooled down in cryo tubes (Sigma- Aldrich, USA) to -80 °C. For long term storage, cells were transferred to liquid nitrogen.

33 Material and Methods

3.2.4 Determination of Proliferation Rates In order to determine proliferation rate of cells, 2.5 × 104 cells were seeded in a 24- well plate. The following 8 days, cells were counted daily. Therefore, cells were washed with PBS (Sigma-Aldrich, USA), 200 μL of 0.05 % trypsin/EDTA (Biochrom, Germany) were added and cells incubated for 3 min in the incubator to detach. The solution was transferred into reaction tubes, mixed in volume 1:1 with 0.2 % trypan blue (Sigma-Aldrich, USA) and counted in a Neubauer counting chamber (Carl Roth GmbH, Germany). Experiments were performed in three independent replicates including triplicate sample measurements. Growth curve was evaluated using GraphPad Prism5 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com. Proliferation rate was calculated using the following formula (Roth V. 2006 Doubling Time Computing):

3.2.5 WST-1 Cell Proliferation Assay Proliferation assay was performed by using cell proliferation reagent WST-1 (Roche, Germany) according to the manufacturer’s protocol. Cells were seeded at a density 3 of 3 × 10 cells in 100 μL in each well of a 96-well plate. 10 µL of WST-1 reagent was added after 1, 2 and 7 days. The plate was incubated for two hours at 37 °C, and luminescence at 450 nm was measured with an iMark Microplate Absorbance Reader (Bio-Rad Laboratories, Inc., USA). Untreated cells were used as controls. Assays were performed in three independent experiments including fivefold sample measurements.

3.2.6 Neurotensin Stimulation PC-3 cells were cultured under serum withdrawal before neurotensin stimulation. Therefore, cells were cultured 3 weeks under serum reduced conditions in medium containing 5 % FCS, following 3 weeks more with 2.5 % FCS. During this time, twice the number of cells as usually was needed for seeding in routine cell culture. For neurotensin stimulation, 3 × 103 cells were seeded into 96-well plates in serum- depleted medium supplemented with 1 g/L BSA (Sigma-Aldrich, USA), 2.38 g/L

34 Material and Methods

HEPES (Sigma-Aldrich, USA), 0.02 mM chymostatin (Calbiochem, Merck, Germany) and 1 mM trypsin inhibitor from Glycine max (soybean) (Sigma-Aldrich, USA) and grown for 48 h in the presence of 1 nM NT (Bachem, Germany). After this time, proliferative response of PC-3 cells was assessed using WST-1 reagent as described above (3.2.5). In order to antagonize the NT-mediated proliferative response of PC-3 cells, 10 µL of the NTS1 antagonists FAUC 468, CL 156 and SR 142948A (Sigma- Aldrich, USA) in Ultrapure water were added 15 min prior to addition of NT to a final concentration of 1 µM in a total volume of 100 µL. Proliferation was measured by means of two experiments of WST-1 assay including fivefold sample measurements. Results were expressed in relation to cells without neurotensin treatment (= 100 %). Data are presented as mean ± SD of measurements.

3.2.7 Colony Formation Assay Colony formation assay was performed as described by Yan et al.213 This assay was an in vitro cell survival assay to assess the ability of a single cell to grow into a colony by division. With this method cell survival was analyzed in presence of precursors in order to exclude cytotoxic effects of the latter. For the assay, 100 PC-3 cells in 10 mL of growth medium were seeded into 10-cm dishes and incubated with 20 pmol of FAUC 468 or CL 156 for 10 days in duplicates. Colonies were washed with PBS (Sigma-Aldrich, USA), stained with 0.5 % crystal violet (Sigma-Aldrich, USA) in PBS for 2 h at room temperature (rt), air dried and photographed. Colonies were counted using Adobe Photoshop CS5 (Adobe Systems Software Ireland Limited, UK). In the end, the plating efficiency (PE) and the surviving fraction (SF) were calculated as described by Franken et al.:214

35 Material and Methods

3.2.8 Wound Healing/Scratch Assay To evaluate migration rate, cells were seeded in triplicates in 24-well plates in culture–inserts for self-insertion (Ibidi GmbH, Germany) at a density of 5 × 104 cells/70 μL and grown overnight. The next day, medium was changed to serum-free medium containing 20 pmol of FAUC 468 or CL 156. Self-inserts were removed 24 h later. Detached cells were removed by washing twice with PBS (Sigma-Aldrich, USA). Scratches were photographed after 0, 6, 24, 30 and 48 h. To determine the differences in migration between distinct conditions, open image area was calculated at each time point on the consecutive pictures at the same place with Adobe Photoshop CS5 (Adobe Systems Incorporated, USA). Experiments were performed in three independent replicates including triplicate sample measurements.

3.2.9 Resazurin Cell Viability Assay Cell viability was assessed by the resazurin assay (Sigma-Aldrich, USA). 2.5 × 104 cells were seeded in 24-well plates and incubated at 37 °C for 24 h. Resazurin was added to a final concentration of 10 μg/mL after 1, 2 and 7 days and incubated for 4 h at 37 °C. Fluorescence was measured with an iMark Microplate Absorbance Reader (Bio-Rad Laboratories, Inc., USA) with extinction/emission of 530/590 nm. The experiments were performed in three independent replicates including triplicate sample measurements.

3.2.10 Immunocytochemistry 2 × 105 cells of PC3-PIP cells were seeded on glass cover slips in a 24-well plate in 2 mL culture medium per well. One day later, medium was removed and cells were fixed for 10 min with ice cold methanol at -20 °C. All following steps were performed at rt. Unspecific binding was blocked with 5 % goat serum in PBS (Sigma-Aldrich, USA) for 1 h. After incubation with the primary antibody mouse anti-PSMA (1:100 in PBS, Abcam, UK) for 1 h, cells were washed three times with PBS and incubated with the fluorescein-coupled secondary antibody goat anti-mouse (1:100 in PBS, Calbiochem, Germany) for 1 h in the dark. After repeating the washing step once more, cells were incubated for 3 min with DAPI (Carl Roth GmbH, Germany) in the dark, washed and covered with fluorescence mounting medium (Dako Deutschland GmbH, Germany). Evaluation was performed with the fluorescence microscope Leica DM6000B (LAS X Software, Leica, Germany).

36 Material and Methods

3.2.11 Flow Cytometry Analysis Flow cytometry analysis was performed with a FACS Canto II (BD, Germany). 5 × 105 cells were used for antibody staining. Cells have been washed with 1 mL PBS (Sigma-Aldrich, USA) and pelleted by centrifugation at 500 × g for 4 min. For identification of PC-3 and PC3-PIP cells by specific surface markers, the following fluorochrome-conjugated Antibodies were used: CD71-FITC (Beckman Coulter, Germany), HLA-ABC-APC (BioLegend, USA). Staining of cell surface integrin on PC-

3 and U87MG cells was performed with Integrin αVß3 (mouse IgG1 anti-human, Merck, Germany) and a secondary FITC-labeled antibody (goat anti-mouse IgG1; BD, Germany). IgG isotypes were used as negative controls. All antibodies were used according to the manufacturer’s instructions. Flow cytometry data were analyzed with Kaluza v1.5a (Beckman Coulter, Germany). FACS analysis was performed in the Department of Internal Medicine 5, Hematology and Oncology, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Erlangen, Germany.

3.3 Radiochemistry and Radiochemical Experiments

3.3.1 Radiolabeling of FAUC 468 and CL 156 68Ga-labeling: For cell culture experiments, the 68Ge/68Ga-generator (Eckert & Ziegler, Germany) eluate (0.1 M HCl, pure, 37 %; Carl Roth GmbH, Germany) was subjected to a cation exchanger cartridge (Chromafix PS-H+, 230 mg, Macherey- Nagel GmbH, Germany) and the trapped 68Ga(III)-cations were eluted with 1 mL of 5 M NaCl (Carl Roth GmbH, Germany). 100 μL of gallium-68 eluate were added to 2 nmol of FAUC 468 or CL 156 (in 2 μL Water Ultrapur; Merck, Germany) in 50 μL 2.5 M HEPES buffer (Sigma-Aldrich, USA) pH 5, respectively. The solution was heated at 98 °C for 10 min. Labeling efficiency and radiochemical purity were determined using reversed-phase high-pressure liquid chromatography (RP-HPLC, Series 1100, Agilent, USA) on a C18 reversed-phase column (Chromolith-RP18e, 4.6 × 100 mm; 5 μm) with a linear acetonitrile/water gradient (0.1 % TFA) 10 - 100 % acetonitrile (0.1 % TFA) in water (0.1 % TFA) over 5 min (flow rate 4 mL/min). The 68 68 radiochemical yield of [ Ga]Ga-ABN 468 (tR = 2.43 min) and [ Ga]Ga-CL 157

(tR = 2.43 min) after 10 min reached > 95 %. For saturation binding studies, a 10- times molar excess of Ga(III)-nitrate (Sigma-Aldrich, USA) in 10 μL metal free water (Water Ultrapur, Merck, Germany) was added to the reaction for 5 min at 95 °C in

37 Material and Methods order to achieve complete complexation of non-radioactive precursor to adjust receptor affinities.

For animal studies, the radiolabeled product was trapped on a Sep-Pak® Light C18 Cartridges (Waters, USA), washed with NaCl (0.9 %) and eluted with 2 mL ethanol. The latter was evaporated and the radiotracer was re-dissolved in 500 µL NaCl (0.9 %). The radiochemical purity was determined using reversed-phase high- pressure liquid chromatography (RP-HPLC, Series 1100, Agilent, USA) on a C18 reversed-phase column (Chromolith-RP18e, 4.6 × 100 mm; 5 μm) with a linear acetonitrile/water gradient (0.1 % TFA) 10 - 100 % acetonitrile (0.1 % TFA) in water (0.1 % TFA) over 5 min (flow rate 4 mL/min). Radiochemical purity of [68Ga]Ga- 68 ABN 468 (tR = 2.44 min) and [ Ga]Ga-CL 157 (tR = 2.27 min) was > 98 %. 177 177 Lu-labeling: For cell culture and animal studies, about 200 MBq [ Lu]LuCl3 (approximately 20 µL; 120 GBq/ μmol, ITG Garching, Germany) in 0.04 M HCl were added to 4 nmol of FAUC 468 or CL 156 (in 4 μL Water Ultrapur; Merck, Germany) in 200 µL 0.5 M HEPES (Sigma-Aldrich, USA). The solution was incubated at 98 °C for 10 min. Labeling efficiency and radiochemical purity were determined using reversed- phase high-pressure liquid chromatography (RP-HPLC, Series 1100, Agilent, USA) on a C18 reversed-phase column (Chromolith-RP18e, 4.6 × 100 mm; 5 μm) with a linear acetonitrile/water gradient (0.1 % TFA) 10 - 100 % acetonitrile (0.1 % TFA) in water (0.1 % TFA) over 5 min (flow rate 4 mL/min). The radiochemical yield of 177 177 [ Lu]Lu-FAUC 469 (tR = 2.44 min) and [ Lu]Lu-CL 162 (tR = 2.37 min) after 10 min reached > 98 %. For cell culture experiments the product was used without further purification, before injection in animals the solution was diluted in 0.9 % NaCl.

3.3.2 Preparation of [68Ga]Ga-NT118195 For cell culture experiments 68Ge/68Ga-generator (Eckert & Ziegler, Germany) eluate (0.1 M HCl, pure, 37 %; Carl Roth GmbH, Germany) was subjected to a cation exchanger cartridge (Chromafix PS-H+, 230 mg, Macherey-Nagel GmbH, Germany) and eluted with 1 mL of 5 M NaCl (Carl Roth GmbH, Germany). 100 μL of gallium-68 eluate were added to 20 nmol of NT118 (in 20 μL Water Ultrapur; Merck, Germany) in 80 μL 2.5 M HEPES buffer (Sigma-Aldrich, USA) pH 5. The solution was incubated at rt for 10 min. Labeling efficiency and radiochemical purity were determined using reversed-phase high-pressure liquid chromatography (RP-HPLC, Series 1100, Agilent, USA) on a C18 reversed-phase column (Chromolith-RP18e, 4.6 × 100 mm;

38 Material and Methods

5 μm) with a linear acetonitrile/water gradient (0.1 % TFA) 10 - 50 % acetonitrile (0.1 % TFA) in water (0.1 % TFA) over 5 min (flow rate 4 mL/min). Radiochemical 68 yield of [ Ga]Ga-NT118 (tR = 2.13 min) after 10 min incubation time reached > 95 %. For saturation binding assays a 10-times molar excess of Ga(III)-nitrate (Sigma- Aldrich, USA) was added to the reaction for 5 min at rt in order to achieve complete complexation of non-radioactive precursor to adjust receptor affinities. For animal studies the radiotracer was trapped on a Sep-Pak® Light C18 cartridge (Waters, USA) and eluted with 2 mL ethanol which was evaporated. The dried product was solved in 500 μL 0.9 % NaCl (Carl Roth GmbH, Germany). Radiochemical purity was determined using reversed-phase high-pressure liquid chromatography (RP-HPLC, Series 1100, Agilent, USA) on a C18 reversed-phase column (Chromolith-RP18e, 4.6 × 100 mm; 5 μm) with a linear acetonitrile/water gradient (0.1 % TFA) 10 - 100 % acetonitrile (0.1 % TFA) in water (0.1 % TFA) in 5 min (flow rate 4 mL/min). The radiochemical purity of [68Ga]Ga-NT118

(tR = 2.13 min) was > 98 %.

3.3.3 Preparation of [68Ga]Ga-PSMA-11 For cell culture experiments 68Ge/68Ga-generator (Eckert & Ziegler, Germany) eluate (0.1 M HCl, pure, 37 %; Carl Roth GmbH, Germany) was subjected to a cation exchanger cartridge (Chromafix PS-H+, 230 mg, Macherey-Nagel GmbH, Germany) and eluted with 1 mL of 5 M NaCl (Carl Roth GmbH, Germany). 100 μL of gallium-68 eluate were added to 1 nmol of PSMA-11 (in 100 μl sodium acetate buffer) and 60 μl 2.5 M HEPES buffer (Sigma-Aldrich, USA) pH 5. The solution was heated at 98 °C for 20 min. Labeling efficiency and radiochemical purity were determined using reversed-phase high-pressure liquid chromatography (RP-HPLC, Series 1100, Agilent, USA) on a C18 reversed-phase column (Chromolith-RP18e, 4.6 × 100 mm; 5 μm) with an acetonitrile/water gradient (0.1 % TFA) 0 - 1 min 10 % acetonitrile, 1 – 5 min 10 - 50 % acetonitrile and 50 - 100 % acetonitrile (0.1 % TFA) in water (0.1 % TFA) 5 - 10 min (flow rate 3 mL/min). The radiochemical yield of [68Ga]Ga-PSMA-11

(tR = 2.78 min) reached > 99 %. For saturation binding assays a 10-times molar excess of Ga(III)-nitrate (Sigma-Aldrich, USA) was added to the reaction for 10 min at 98 °C in order to achieve complete complexation of non-radioactive precursor to adjust receptor affinities. For animal studies the radiotracer was trapped on a Sep-Pak® Light C18 cartridge (Waters, USA) and eluted with 2 mL ethanol which was evaporated. The

39 Material and Methods dried product was solved in 500 μL 0.9 % NaCl (Carl Roth GmbH, Germany). Labeling efficiency and radiochemical purity were determined using reversed-phase high-pressure liquid chromatography (RP-HPLC, Series 1100, Agilent, USA) on a C18 reversed-phase column (Chromolith-RP18e, 4.6 × 100 mm; 5 μm) with an acetonitrile/water gradient (0.1 % TFA) 0 - 1 min 10 % acetonitrile, 1 – 5 min 10 - 50 % acetonitrile and 50 - 100 % acetonitrile (0.1 % TFA) in water (0.1 % TFA) 5 - 10 min (flow rate 3 mL/min). The radiochemical yield of [68Ga]Ga-PSMA-11

(tR = 2.78 min) reached > 98 %.

3.3.4 Preparation of [68Ga]Ga-NODAGA-RGD For 68Ga-labeling of NODAGA-RGD, 68Ge/68Ga-generator (Eckert & Ziegler, Germany) eluate (0.1 M HCl, pure, 37 %; Carl Roth GmbH, Germany) was subjected to a cation exchanger cartridge (Chromafix PS-H+, 230 mg, Macherey-Nagel GmbH, Germany) and eluted with 1 mL of 5 M NaCl. 500 μL of gallium-68 eluate were added to 10 nmol of NODAGA-RGD (in 10 μL water ultrapure) in 250 µL 2.5 M HEPES buffer (pH 5; Sigma-Aldrich, USA). The solution was incubated for 15 min at 98 °C. The product was trapped on a Sep-Pak® Light C18 cartridge (Waters, USA) and eluted with 2 mL ethanol which was evaporated using a Rotavapor R-205 (Büchi, Germany). The dried product was solved in 500 μL 0.9 % NaCl (Carl Roth GmbH, Germany). Labeling efficiency and radiochemical purity were determined using reversed-phase high-pressure liquid chromatography (RP-HPLC, Series 1100, Agilent, USA) on a C18 reversed-phase column (Chromolith-RP18e, 4.6 × 100 mm; 5 μm) with a linear acetonitrile/water gradient (0.1 % TFA) 10 - 100 % acetonitrile (0.1 % TFA) in water (0.1 % TFA) over 5 min (flow rate 4 mL/min). The radiochemical 68 yield and purity of [ Ga]Ga-NODAGA-RGD (tR = 1.57 min) was > 98 %.

3.3.5 Receptor Binding Assay Determination of NTS1 receptor binding was performed by Harald Hübner, Department of Chemistry and Pharmacy, Chair of Medicinal Chemistry, Friedrich- Alexander-University Erlangen-Nürnberg (FAU) as described by Lang et al.187 In short, NTS1 binding was measured in competition studies using homogenates of Chinese hamster ovary (CHO) cell membranes (2 – 4 µg/well). These cells expressed the human NTS1 after stable transfection. The radioligand [3H]neurotensin (specific activity 101 Ci/mmol, Perkin Elmer, Germany) was applied at the concentration of 0.3 – 0.5 nM. Nonspecific binding was determined in the presence of

40 Material and Methods

10 µM NT. [3H]NT(8-13) (specific activity 136 Ci/mmol, custom synthesis by GE Healthcare, Germany) was used at a concentration of 0.5 nM for investigation of NTS2 binding using homogenates of membranes from HEK293 cells transfected with the human NTS2 gene. Membranes were incubated at a final concentration of 4 – 6 µg/well. Nonspecific binding was determined in the presence of 10 µM NT(8-13).

3.3.6 Radioligand Binding Assay In vitro radioligand binding assay was performed using [125I]Tyr3-NT (Perkin Elmer, USA). Assays were performed with 2 × 105 cells in quadruplicates in 24-well plates. Cells were washed with binding buffer (respective cell medium containing 0.1 % BSA (Sigma-Aldrich, USA), 10 mM HEPES (Sigma-Aldrich, USA), 2 mg/L chymostatin (Merck, Germany), 100 mg/L trypsin inhibitor from Glycine max (soybean) (Sigma- Aldrich, USA) and incubated with 45 pM [125I]Tyr3-NT at 37 °C for 60 min. Additional cells were incubated in the presence of 1 μM NT (for nonspecific binding; Bachem, Germany) or 1 μM CX107 (NTS1-specific binding).215 After 1 h, medium was removed, cells washed twice with cold PBS and lysed with 0.1 M NaOH. The amount of radioactivity in the cells was quantified in a γ-counter (Wallac Wizard 1470, Perkin Elmer, USA). Protein determination was performed using Bradford reagent (Sigma- Aldrich, USA). For analysis GraphPad Prism5 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com was used.

3.3.7 Cell Uptake and Internalization Studies Cell uptake and internalization studies were performed using PC-3 and PC3-PIP cells with 1 × 106 cells per well in 6-well plates. Cells were washed with ice cold PBS (Sigma-Aldrich, USA) twice before they were incubated with 100 μL of the respective radiotracer (41 fmol, 3 kBq [177Lu]Lu-FAUC 469/[68Ga]Ga-ABN 468 and [177Lu]Lu- CL 162/[68Ga]Ga-CL 157, 12 nM [68Ga]Ga-PSMA-11) per well in a final volume of 1 mL of RPMI 1640 basal medium supplemented with 0.1 % BSA (Sigma-Aldrich, USA). For the definition of nonspecific binding, the experiment was performed in the presence of an excess of a known receptor ligand (1 µM FAUC 468 to determine nonspecific binding of [177Lu]Lu-FAUC 469/[68Ga]Ga-ABN 468, 1 µM CL 156 to determine nonspecific binding of [177Lu]Lu-CL 162/[68Ga]Ga-CL 157 to NTS1 and 50 µM PMPA (Tocris Bioscience, UK) to determine nonspecific binding of [68Ga]Ga- PSMA-11 to PSMA) was added in 100 μL to a final volume of 1 mL of RPMI 1640 basal medium (0.1 % BSA; Sigma-Aldrich, USA). At specific time points, t = 0, 5, 15,

41 Material and Methods

30, 60, 120 and 240 min for 177Lu-labeled tracers and t = 0, 5, 15, 30, 45, 60 and 90 min for 68Ga-labeled tracers, supernatant containing the unbound tracer was collected. Cells were washed twice with PBS (Sigma-Aldrich, USA), twice with glycine buffer (pH 2.8; 50 mM glycine (Sigma-Aldrich, USA), 150 mM NaCl (Carl Roth GmbH, Germany)) for 5 min and lysed in 1 M NaOH (Carl Roth GmbH, Germany). The amount of radioactivity in the supernatant, NTS1-specifically surface bound (in glycine buffer), and in the cells (lysed in NaOH) was measured using a γ-counter (Wallac Wizard 1470, Perkin Elmer, USA). Experiments were performed in three independent replicates including triplicate sample measurements. Results were calculated as internalization rate (related to the sum of cell surface bound and internalized fraction as 100 %) and as cell uptake rate (related to total added radioactivity as 100 %).

3.3.8 Efflux Studies Efflux kinetic was determined using PC-3 and PC3-PIP cells with 1 × 106 cells per well in 6-well plates. Cells were washed with ice cold PBS (Sigma-Aldrich, USA) twice before they were incubated with 100 μL of the respective 177Lu- or 68Ga-labeled tracer (41 fmol, 3 kBq [177Lu]Lu-FAUC 469/[68Ga]Ga-ABN 468 and [177Lu]Lu- CL 162/[68Ga]Ga-CL 157, 12 nM [68Ga]Ga-PSMA-11) per well in a final volume of 1 mL of RPMI 1640 basal medium supplemented with 0.1 % BSA (Sigma-Aldrich, USA) for 45 min at 37 °C. The incubation buffer was removed, cells were washed twice with PBS and glycine buffer (pH 2.8; 50 mM glycine (Sigma-Aldrich, USA), 150 mM NaCl (Carl Roth GmbH, Germany)) was added twice for 1 min to remove radioactivity from cell surface. Finally, 1 mL of RPMI 1640, supplemented with 0.1 % BSA (Sigma-Aldrich, USA) was added to each well for incubation at 37 °C for different time points. These were t = 0, 5, 15, 30, 60, 120 and 240 min for assays using 177Lu-labeled tracers and t = 0, 5, 15, 30, 45, 60 and 90 min for assays using 68Ga-labeled tracers. At these time points, the supernatant was removed for measuring the efflux fraction. Cells were solubilized with 1 M NaOH (Carl Roth GmbH, Germany) to determine internalized/cell-bound fraction. Fractions were collected in tubes and the amount of radioactivity in the supernatant and in cells was measured using a γ-counter (Wallac Wizard 1470, Perkin Elmer, USA). Experiments

42 Material and Methods were performed in three independent replicates including triplicate sample measurements.

3.3.9 Saturation Binding Studies (Determination of Kd and Bmax Values)

Saturation binding studies to determine Kd and Bmax were performed using the respective cell lines according to the following procedure: Experiments were performed with 2 × 105 cells per well in 24-well plates. Cells were washed with ice cold PBS (Sigma-Aldrich, USA) twice before they were incubated with seven different concentrations (see Table 8) each added in 100 μL of the respective radiotracer in a final volume of 1 mL of incubation medium. For the determination of nonspecific binding, the experiment was performed in the presence of an excess of a selective receptor ligand with high affinity to the respective receptor (1 µM SR 142948A (Sigma-Aldrich, USA) to determine nonspecific binding of [177Lu]Lu- FAUC 469/[68Ga]Ga-ABN 468 and [177Lu]Lu-CL 162/[68Ga]Ga-CL 157 to NTS1 and 50 µM PMPA (Tocris Bioscience, UK) to determine nonspecific binding of [68Ga]Ga- PSMA-11 to PSMA) was added in a volume of 100 μL to a final volume of 1 mL of incubation medium for 60 min at 4 °C or 37 °C (see Table 8). Subsequently, the medium was withdrawn, cells were washed twice with ice cold PBS and solubilized in 1 M NaOH (Carl Roth GmbH, Germany). The amount of radioactivity in the cells was measured using a γ-counter (Wallac Wizard 1470, Perkin Elmer, USA). Protein determination was performed using Bradford reagent (Sigma-Aldrich, USA). Three independent experiments were conducted, each performed in triplicate. The specific binding was calculated by subtraction of the nonspecific binding values from the total binding. Kd and Bmax were determined using the software GraphPad Prism (Prism 5.0 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com), applying the one-site specific binding model. The receptor number per cell was calculated by multiplying Bmax with Avogadro constant and divided by the seeded cell number.

43 Material and Methods

Table 8. Radiotracer, their dilution series in medium and incubation temperature that were used in saturation binding studies (determination of Kd and Bmax Values).

Radiotracer Concentrations Incubation Medium per well temperature [177Lu]Lu-FAUC 469/ 2.4 × 10-9 down to 4 °C, RPMI [68Ga]Ga-ABN 468, 3.75 × 10-11 M with 37 °C + 0.1 % BSA [177Lu]Lu-CL 162/ six 1:1 dilution [68Ga]Ga-CL 157 steps [68Ga]Ga-NT118 150 × 10-9 down to 37 °C RPMI 2.34 × 10-9 M with + 0.1 % BSA six 1:1 dilution + 10 mM HEPES steps + 2 mg/L chymostatin + 100 mg/L trypsin inhibitor from Glycine max (soybean) [68Ga]Ga-PSMA-11 45 × 10-9 down to 4 °C RPMI 0.703 × 10-9 M with + 0.1 % BSA six 1:1 dilution steps

PC-3 and PC3-PIP cells were treated with 1 nM CL 156 or 200 nM NT100195 (Figure 20) for 6 h, 1, 2 and 4 days before saturation binding experiments In order to evaluate the potential influence of the NTS1 antagonists and agonist on NTS1 expression. During this time, medium was changed every day. The concentrations were chosen following the example set by Souazé et al.141 For incubation with NTS1 agonist incubation medium was completed with 1 g/L BSA (Sigma-Aldrich, USA), 2.38 g/L HEPES (Sigma-Aldrich, USA), 2 mg/L chymostatin (Calbiochem, Merck, Germany) and 100 mg/L trypsin inhibitor from Glycine max (soybean) (Sigma-Aldrich, USA). Afterwards, medium was filtered sterile through an injection filter (Sterifix, 0.2 µm, B. Braun Melsungen AG, Germany).

44 Material and Methods

Figure 20. Chemical structure of NT100.195

3.3.10 NTS1 In Vitro Receptor Autoradiography In vitro receptor autoradiography was performed with mouse tumor slices obtained from tissues out of biodistribution and therapy studies and primary patient tissue of prostate and pancreatic cancer. Tumors from sacrificed mice were quickly removed and frozen in hexane cooled to -60 °C by addition of dry ice nuggets. Tissues were fixed in Tissue Tek O.C.T. Compound (Sakura Finetek USA Inc., USA) and 20 µm tumor sections were cut on cryostat microtome (Microm GmbH, Germany). Tumor slices were mounted in pairs on glass slides (Marienfeld, Germany) and stored at - 20 °C. Patient tissue sections (18 µm) were obtained from the Gewebebank des UCC Erlangen at the institute of pathology in Erlangen. Neurotensin receptor measurements were performed using [125I]Tyr3-NT (81.4 TBq/mmol, Perkin Elmer, USA) according to the protocol established by Reubi et al.151 Thawed slices were dried at rt, edged with liquid blocker (Science Services GmbH, Germany) and pre-incubated for 15 min at rt in a solution of 50 mM Tris-HCl

(Carl Roth GmbH, Germany) and 1 mM MgCl2 (Roth, Germany), pH 7.4. Slides were transferred to horizontal position and incubated for 2 h at rt with pre-incubation buffer, modified by addition of 0.02 mM chymostatin (Calbiochem, Germany), 1 mM trypsin inhibitor from Glycine max (soybean) (Sigma-Aldrich, USA) and 90 pM [125I]Tyr3-NT. Additional sections were incubated with 1 µM NT for determination of nonspecific binding. Glass slides were washed three times for 5 min in pre-incubation buffer at 4 °C, dipped in 4 °C cold water and dried at rt. Sections were exposed for 14 days to a Storage Phosphor Screen BAS-IP MS 2040 E Multipurpose Standard (20 × 40 cm, GE Healthcare Life Sciences, UK) together with a standard slide, containing different volumes of [125I]Tyr3-NT in order to generate a standard curve. Screens were analyzed with CR 35 Bio imager (Dürr Medical, Germany) using the Aida Image

45 Material and Methods

Analyzer v4.27 software (Raytest Isotopenmeßgeräte GmbH, Germany) for radioligand binding quantification. Values were calculated in fmol per mg tissue according to the standards. Specific binding was generated by subtraction of corresponding nonspecific binding values. At the end, receptor densities were calculated using Microsoft Excel 2010 (Microsoft Corporation, USA).

3.4 Animal Studies

All mouse experiments were approved by the local animal protection authorities (Government of Central Franconia, Germany, No. 54-2532.1-22/10 and 55.2 2532-2- 279). Mice were maintained in groups in an IVC Recovery Unit – Blue Line (25 °C ± 1 °C, Tecniplast S.p.A, Italy) with autoclaved bedding, food and water on a daily 12-hour light/dark cycle.

3.4.1 Biodistribution Studies

3.4.1.1 Biodistribution of 68Ga- and 177Lu-labeled FAUC 468 and CL 156 Biodistribution studies were performed with eight-week-old female, athymic nude mice (NMRI FOXn1/nu), obtained from Envigo (former Harlan), Netherlands. For tumor inoculation, 1.5 × 106 PC-3 or PC3-PIP cells (in 50 μL PBS and 50 μL Matrigel®) were administered in the left and 1.5 × 106 HT-29 cells in 100 μL PBS in the right shoulder of mice. Two weeks later, when tumors reached approximately 9 mm diameter, about 1 MBq [177Lu]Lu-FAUC 469 or [177Lu]Lu-CL 162 (in 100 μL NaCl 0.9 %, pH 7.4) or about 5 MBq [68Ga]Ga-ABN 468 or [68Ga]Ga-CL 157 (in 100 μL NaCl 0.9 %, pH 7.4) were randomized injected via the tail vein. During the experiment, the mice were checked for signs of unease and survival five times a week and the tumor size was measured. For 177Lu-labeled radioligands, mice were euthanized by cervical dislocation under deep isoflurane anesthesia 1 h (n = 2 - 3), 4 h (n = 3), 1 day (n = 2 - 3), 2 days (n = 2 - 3), 4 days (n = 2) and 7 days (n = 2) p.i. The organs of mice were removed, weighed and radioactivity was counted in a γ- counter (Wallac Wizard 1470, Perkin Elmer). Displacement studies for [177Lu]Lu- FAUC 469 were performed by pre-injection of 100 µg of CL 156 15 min before the injection of radioactivity. For 68Ga-labeled radioligands, organs were removed at 1 h

46 Material and Methods p.i. ([68Ga]Ga-ABN 468 n = 2; [68Ga]Ga-CL 157 n = 2). The results were presented as percentage injected dose per gram organ (% ID/g) and tumor-to-organ ratios were calculated thereof.

3.4.1.2 Dosimetry Calculations Dosimetry calculations were done by Dr. Ing. Philipp Ritt, Imaging and Physics, Clinic of Nuclear Medicine, University Hospital Erlangen, Erlangen, Germany.

As stated above, uptake was measured as percent of injected dose per mass of tissue (% ID/g) for each animal group in ten types of normal tissue (blood, lungs, liver, kidneys, heart, spleen, brain, muscle, femur, small intestine) and in the tumor. Dosimetry was carried out on the basis of the average uptake of each individual group at t = 1 h, 24 h, 48 h and 168 h p.i. The uptake at t = 0 h was assumed to be 0. Time points > 168 h were extrapolated by fitting a mono- exponential decay function to the points belonging to t = 48 h and 168 h p.i. If resulting half-lives were longer than physical half-life of 177Lu (159.5 h), decay with physical half-life was assumed. Areas under the time-activity curves of each tissue type were obtained by numerical integration using the trapezoidal rule for 0 h ≤ t ≤ 168 h p.i. and by direct integration of the respective mono-exponential decay function for t > 168 h p.i. This resulted in time-integrated activity coefficients (TIAC in [MBq × h/MBq/g]). For lung, liver, kidneys, heart, spleen, small intestine and brain, the absorbed dose per gram of tissue per injected radioactivity (in [Gy/MBq/g) was calculated by multiplying TIACs with S-values reported from Larsson et al.,216 which had been rescaled to the proper units (Gy × g/MBq/h). Since S-values for blood, muscle, femur and tumor were not directly available from that publication, the respective values of bone marrow, liver, skull and liver from Larsson et al. were used as a surrogate.216 Due to the low energy electron spectrum of 177Lu, only self-irradiation of tissues was taken into account.

47 Material and Methods

3.4.1.3 Biodistribution of [68Ga]Ga-NT118 and [68Ga]Ga-PSMA-11 Biodistribution studies with [68Ga]Ga-NT118 and [68Ga]Ga-PSMA-11 were performed with athymic female nude mice (CD1-Foxn1/nu, homozygous), obtained from Charles River Laboratories, Germany. For tumor inoculation, 1.5 × 106 PC3-PIP cells (in 50 μL PBS and 50 μL Matrigel®) were administered in the left and 1.5 × 106 PC-3 cells (in 50 μL PBS and 50 μL Matrigel®) in the right shoulder of mice. Two and four weeks later, about 5 MBq [68Ga]Ga-NT118 or [68Ga]Ga-PSMA-11 (each in 130 μL NaCl 0.9 %, pH 7.4) were injected via tail vein. During the experiment five times a week, mice were checked for signs of unease and survival and tumor size was measured. Mice were euthanized by cervical dislocation under deep isoflurane anesthesia and after cervical dislocation 1 h (n = 3 - 4) later organs were removed, weighed and radioactivity was counted in a γ-counter (Wallac Wizard 1470, Perkin Elmer, USA). The results were represented as percentage injected dose per gram organ (% ID/g) and tumor-to-organ ratio.

3.4.2 Ex Vivo Autoradiography Anesthetized tumor-bearing female mice were injected with about 1 MBq of 177Lu- labeled or 5 MBq of 68Ga-labeled radiotracers via the tail vein. For displacement studies, 100 µg of CL 156 (in 50 µL Water Ultrapur; Merck, Germany) was injected 15 min before radioligand injection. Mice were sacrificed by cervical dislocation under deep isoflurane anesthesia at specific time points and tumors and excretion organs were removed, frozen in -70 °C cooled n-hexane and slices of organs were fixed on silanized glass slides (Histobond®, Marienfeld) after being cut on a cryostat microtome (HM550, Microm, Germany). Glass slides were exposed to a phosphor screen (Fujitsu, Germany) overnight, the screens analyzed using CR35 Bio Imager (Dürr Medical, Germany) and evaluated with the AIDA Image Analyzer 4.27 (Elysia- raytest GmbH, Germany). Subsequently, slices were HE stained (see 3.7.2).

3.4.3 Tumor Therapy Study Four-week-old female, athymic nude mice (CD1-Foxn1/nu, homozygous) were purchased from Charles River Laboratories, Germany. To induce tumor growth, mice were anesthetized with 3.5 % isoflurane and inoculated with 1.5 × 106 PC-3 or PC3- PIP cells (in 50 μL PBS and 50 μL Matrigel®) in the left shoulder. One week later, when tumors reached approximately 5 mm in diameter, mice were randomized into groups. For the therapy study with PC-3-xenografted nude mice, three groups were

48 Material and Methods formed. The first group was injected with 25 - 30 MBq of [177Lu]Lu-FAUC 469 (n = 5), the second group with 25 - 30 MBq of [177Lu]Lu-CL 162 (n = 5) (each in 130 μL NaCl 0.9 %, pH 7.4) via the tail vein. The third group represented the control group and received no treatment (n = 7). For the therapy study with PC3-PIP-xenografted nude mice, two groups were formed. One treatment group which received 25 - 30 MBq of [177Lu]Lu-FAUC 469 (n = 6) and one control group (n = 6) During the experiment, mice were checked for signs of unease and body weight and tumor size was measured (Table 9) five times a week. Termination criteria of the study were reached if tumor diameter reached > 15 mm, loss of body weight was > 15 % of initial value and when active ulceration of the tumor or obvious signs of unease were observed. After sacrifice of animals with cervical dislocation under deep isoflurane anesthesia, liver, spleen, kidney, heart and tumor tissue was removed and paraffin embedded. Possible tissue damages were assessed by HE (3.7.2), Masson-Goldner (3.7.3) and PAS (3.7.4) staining of 3 µm paraffin slices. Survival curves and in vivo data were expressed as mean ± SD. Tumor volumes were calculated with the formula V = 4/3 × π × r³ with r being the tumor radius. For therapy study with PC-3 cells a statistical permutation test using a two-sample t-statistic was performed to compare growth curves of each therapy to control group by Prof. Eva Grill (Ludwig-Maximilians University Munich (LMU), Institute for Medical Information Processing, Biometry and Epidemiology, Munich, Germany).

Table 9. Parameters monitored in tumor therapy studies.

Parameter Determination Body weight Five times a week Tumor volume Five times a week PET imaging One time a week Morphology of tumor Final Liver toxicity Final Kidney toxicity Final Blood plasma parameters Final NTS1 expression Final Final meant after euthanasia when an endpoint criterion was reached or at the end of the study.

49 Material and Methods

3.4.4 Therapy Monitoring with Small-Animal PET Imaging Small animal PET imaging with [68Ga]Ga-NT118, [68Ga]Ga-PSMA-11 and [68Ga]Ga- NODAGA-RGD was performed for 15 min on the Inveon microPET scanner (Siemens Healthcare, Germany) once before injection of [177Lu]Lu-FAUC 469 or [177Lu]Lu-CL 162 as baseline measurement following imaging every 7 days with [68Ga]Ga-NODAGA-RGD until day 28 after [177Lu]Lu-FAUC 469 or [177Lu]Lu-CL 162 injection and on days 7 and 14 and then every 2 weeks with [68Ga]Ga-NT118 and [68Ga]Ga-PSMA-11. Radiotracers were synthesized as described above. Approximately 5 MBq of the respective 68Ga-labeled radiotracer were injected into the tail vein of anesthetized mice at 55 min for [68Ga]Ga-NODAGA-RGD and 45 min for [68Ga]Ga-NT118 and [68Ga]Ga-PSMA-11 prior to the start of data acquisition by PET. Competitive binding experiments were performed by co-injection of about 5 MBq [68Ga]Ga-ABN 468 or [68Ga]Ga-CL 157 (in 100 μL NaCl 0.9 %, pH 7.4) together with 100 µg SR 142948A (in 50 µL Water Ultrapur; Merck, Germany) via the tail vein. For the reconstruction of images, the Inveon Acquisition Workplace software (Siemens Healthcare, Germany) was used. Imaging analysis was performed with Amide Software (Andreas M. Loening, Sanjiv S. Gambhir, Crump Institute for Molecular Imaging, UCLA School of Medicine, Department of Radiology and the Bio- X Program, Stanford University, USA).

50 Material and Methods

3.5 Quantitative Real-Time PCR

3.5.1 RNA Isolation Total RNA isolation from cell lines and tumor tissues was performed with peqGOLD MicroSpin Total RNA Kit (Peqlab Biotechnologie GmbH, Germany) according to the manufacturer’s recommendations. 5 × 105 cells or 5 mg of tissue were lysed in RNA Lysis Buffer T by pipetting and storage at -20 °C overnight. The lysate was transferred to a DNA Removing Column and centrifuged for 1 min at 10,000 × g. The filtrate was thoroughly mixed with 70 % ethanol, loaded on a PerfectBind MS RNA Column and centrifuged at 10,000 × g for 1 min at rt. PerfectBind MS RNA Column was washed once with RNA Wash Buffer I and two times with RNA Wash Buffer II by centrifuging for 1 min at 10,000 × g. After the last washing step, PerfectBind MS RNA Column was dried by centrifugation for 2 min at 10,000 × g. RNA was eluted in RNase-free water by centrifugation for 1 min at 6,000 × g.

3.5.2 Reverse Transcription Reverse transcription was performed with QuantiTect Reverse Transcription Kit (Qiagen GmbH, Germany) according to the manufacturer’s protocol. Genomic DNA was eliminated first as described in Table 10. The mixture was incubated for 5 min at 42 °C and placed on ice.

Table 10. Components for genomic DNA elimination reaction.

Component Volume/reaction gDNA Wipeout Buffer, 7 x 2 μL Template RNA Variable RNase-free water Variable Total reaction volume 14 μL

The master mix for reverse transcription was prepared on ice according to Table 11 and added to the genomic DNA elimination reaction.

51 Material and Methods

Table 11. Reverse transcription master mix.

Component Volume/reaction Reverse-transcription master mix 1 μL Quantiscript Reverse Transcriptase Quantiscript RT Buffer, 5 × 4 μL RT Primer Mix 1 μL Template RNA 14 μL Entire genomic DNA elimination reaction Total reaction volume 20 μL

The whole reaction was incubated for 30 min at 42 °C and then for 3 min at 95 °C. The reaction was kept on ice and diluted four-fold prior to analysis by qRT-PCR.

3.5.3 Quantitative Real-Time PCR Quantitative RT-PCR was performed using QuantiFast SYBR Green PCR Kit from Qiagen GmbH (Germany) according to the manufacturer’s protocol. PCR primers were tested first by analyzing two-fold serial dilution series of cDNA. Primer pairs that were capable of accurate discrimination between cDNA dilutions were used for quantification of transcript abundance in biological samples. Furthermore, the different primer pairs had to have equal amplification efficiencies (R² > 0.9; efficiency of 2 ± 0.2). The composition of qRT-PCR reactions was summarized in Table 12.

Table 12. qRT-PCR master mix with QuantiFast SYBR Green PCR kit. qRT-PCR Master Mix Volume/reaction 2 × QuantiFast SYBR Green PCR Master Mix 6 μL Primer Mix (each final 300 nM) 0.36 μL

H2O 3.64 μL Total Volume 10 μL

The reaction was dispensed in the wells of a 96-well plate. A total of 2 μL of cDNA were added to every well. PCR primers are shown in Table 13. mRNA levels were normalized to abundance of the housekeeping control gene GAPDH. qPCR was performed according to the following amplification program: the amplification program for the two step quantitative real-time PCR encompassed 40 cycles of denaturation at 94 °C for 15 s, annealing at 50 °C for 30 s and extension at 72 °C for 30 s. Melting curve analysis was performed subsequently.

52 Material and Methods

Table 13. qRT-PCR primers.

Gene Primer Amplicon length [bp] Source Hs_NTSR1_1_SG Qiagen GmbH, NTS1 104 QuantiTect Primer Assay Germany Hs_NTSR2_1_SG Qiagen GmbH, NTS2 133 QuantiTect Primer Assay Germany Hs_FOLH1_1_SG Qiagen GmbH, PSMA 92 QuantiTect Primer Assay Germany Hs_GAPDH_1_SG Qiagen GmbH, GAPDH 95 QuantiTect Primer Assay Germany

Each cDNA was amplified by real-time PCR using GAPDH specific primers in order to test if the internal control gene GAPDH was consistent between all samples. This reference allowed the comparison of the different samples.

3.6 Western Blot

3.6.1 Protein Isolation For protein isolation, cells were washed with PBS (Sigma-Aldrich, USA) and harvested by trypsinization with trypsin/EDTA (Biochrom, Germany) 0.05 %. Cells were collected in a final volume of 10 mL growth medium and pelleted by centrifugation at 1000 × g for 5 min, the supernatant was discarded. This step was repeated after re-suspending the pellet in 10 mL PBS. Cell pellet was re-suspended in 250 µL RIPA buffer (composition Table 14). The solution was incubated at 4 °C for 30 min and mixed every 10 min. For ultrasonic fragmentation (amplitude 32 %, 3 cycles, 4 impulses) Sonoplus HD 2070 (Bandelin electronic GmbH, Germany) was used. The lysate was centrifuged at 4 °C for 10 min at 25,000 × g. Protein lysates were kept at -20 °C for long term storage.

53 Material and Methods

Table 14. Composition of lysis RIPA buffer.

RIPA buffer Stock Solution Concentration in 10 mL 10 mL

Tris HCl pH 7,4 1 M 50 mM 500 µL NaCl 5 M 150 mM 300 µL NP40 - 1 % 100 µL Na-Deoxycholate 10 % 0,5 % 500 µL SDS 1 % 0,1 % 1 mL Protease Inhibitor - - 1 Tablet

dH2O - - 7,6 mL

3.6.2 Protein Quantification The protein amount was quantified with the QuantiPro BCA Assay Kit (Sigma-Aldrich, USA). Therefore, 100 µL reagent QA was mixed with 100 μL reagent QB and 4 µL QC per sample. Standard protein solutions and samples were measured in duplicates in 96-well plates. After adding 100 μL of BCA-solution to the samples, the mixture was incubated for 2 h at 37 °C followed by measurement of optical density at 562 nm using an iMark Microplate Absorbance Reader (Bio-Rad Laboratories, Inc., USA).

3.6.3 Western Blot Polyacrylamide gels – each consisting of a 5 % stacking and an 8 % separating gel - were prepared according to Table 15.

Table 15. Composition of separating (8 %) and stacking (5 %) gel. Given volumes for separating and stacking gels were sufficient for 4 gels.

Separating gel Stacking gel Aqua dest. (mL) 9.3 6.8 30 % acrylamide mix (mL) 5.3 1.66 Tris buffer (1.5 M, pH 8.8) (mL) 5 1.26 SDS (10 % solution) (μL) 200 100 APS (10 % solution) (μL) 200 100 TEMED (μL) 20 10

54 Material and Methods

A total of 15 µg protein was separated in each lane. Protein samples were mixed with 7 μL of 4 × reducing dye (composition see Table 16) and RIPA buffer in a total volume of 20 μL for a 10-well gel and denatured by boiling at 95 °C for 5 min.

Table 16. Composition of the 4 × reducing dye used for sample preparation.

Compound Amount Concentration in 10 mL Tris-HCl 0,666 g 63 mM Tris-Base 0,682 g 0.6 M SDS 0,800 g 2 % EDTA 0,00874 g 0.02 % Glycerin 4,0 g 10 % (v/v) 2 % Bromophenol blue solution 0,2 mL 0.1 % dH2O Ad 10 mL -

Electrophoresis was started at 60 V for 15 min to concentrate the sample followed by separation at 200 V for 40 min in 1 × running buffer (100 mL of 10 × stock and 900 mL deionized water, composition see Table 17). After electrophoretic separation, membrane (Amersham Hybond LFP 0.2 PVDF, GE Healthcare, USA) was equilibrated for 2 min in methanol and then together with gels and filters (Extra Thick Blot paper, Bio-Rad Laboratories, USA) for 10 min in 1 × blotting buffer (100 mL of 10 × stock (composition see Table 17) and 900 mL deionized water with 0.04 % SDS).

Table 17. Compositions of 10 × running buffer, 10 × blotting buffer and TBS-T.

10 × running buffer 10 × blotting buffer (pH 8.6) 1 × TBS-T

250 mM Tris-Base 250 mM Tris-Base 120 mM NaCl

1.92 M Glycine 1.92 M Glycine 50 mM Tris-Base

1 % SDS 1 % Tween20

55 Material and Methods

Semidry blotting was performed by using Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories, USA) at 400 mA for 30 min. The membrane was blocked in 5 % dry milk (Carl Roth GmbH, Germany) in TBS-T (composition see Table 17) for 1 h. The protein specific antibody mouse anti-PSMA (1:5000, Abcam, UK) or rabbit anti-NTS1 (1:2000, Abgent, USA) was added next in 0.5 % dry milk in 1 × TBS-T and incubated overnight at +4 °C on a rolling shaker. The membrane was washed three times for 5 min with 1 × TBS-T before adding the peroxidase- conjugated secondary antibody goat anti-mouse (1:20000, Calbiochem, Germany) for PSMA or goat anti-rabbit (1:1000, Calbiochem, Germany) for NTS1 in 0.5 % dry milk in 1 × TBS-T for 1 h at rt the next day. Protein was detected by chemiluminescence at the Amersham Imager 600 (GE Healthcare, UK) with Amersham ECL Prime Western blotting Detection Reagent (GE Healthcare, UK). Rabbit anti-ß-actin (1:500, Abcam, UK) or mouse anti-GAPDH (1:2000, Merck, Germany) was used as loading control. For incubation with these antibodies, membranes were washed three times for 5 min with 1 × TBS-T after chemiluminescence detection and then incubated overnight at +4 °C on a rolling shaker. Secondary antibody incubation and chemiluminescence detection were performed as described above.

3.7 Immunohistochemical Methods

3.7.1 Immunohistochemistry Tissues were fixed in 4 % buffered formalin (Carl Roth GmbH, Germany) for 6 h at rt and then transferred to 70 % ethanol. Fixed tissues were transferred to embedding cassettes (Carl Roth GmbH, Germany) and dehydrated for embedding in paraffin by use of an alcohol series of 80 % (2.5 h) / 96 % / 96 % (2 × 2.5 h) ethanol / 100 % 2- propanol / 100 % 2-propanol (2 × 3 h) and xylene for 1.5 h, followed by incubation in xylene for 2 h. Dehydration was followed by incubation in paraffin for 3 h and 24 h. Tissues were embedded in cassettes in 60 °C hot paraffin and cooled down before cutting on a sledge microtome. 3 µm slices were cut using a microtome (PFM, Germany). The slices were placed in a 60 °C water bath, fixed on glass slides and dried at rt. After 1 h at 60 °C, slices were rehydrated using xylene twice for 10 min. This step was followed by descending alcohol series of 100 % / 100 % / 100 % / 96 % / 96 % / 80 % / 70 % ethanol, each for 3 min. Glass slides were rinsed twice for 5 min in distilled water, before boiling in citrate buffer (pH 6, 10 mM in distilled water).

56 Material and Methods

The cooled slides were washed with distilled water three times for 5 min, dried and edged with liquid blocker (Science Services GmbH, Germany). This step was followed by incubation in 3 % H2O2 (Sigma-Aldrich, USA) for 10 min, two times distilled water for 5 min, 5 min TBS-T and 1 h blocking in TBS-T containing 5 % serum of the animal of the secondary antibody. The incubation with the primary antibody in blocking solution was performed first for 1 h at rt followed by incubation at 4 °C overnight. The next day, glass slides were rinsed three times for 5 min with TBS-T, followed by incubation with the HRP-coupled secondary antibody (in blocking solution) for 1 at rt (description of antibodies and their dilutions: see Table 18). After washing three times for 5 min, slices were stained with DAB substrate (DCS, Germany). DAB represents a HRP substrate which reacts to an insoluble brown end product. Time of staining varied dependent on intensity. Reaction was stopped in distilled water, followed by nuclear staining with hematoxylin (1:1 in distilled water) for 40 s, dipped in 0.1 % HCl (in distilled water) and blued for 3 min in running water. Until embedding of the sections in Aquatex (Merck, Germany), glass slides were kept in distilled water. TBS-T was used to substitute the primary antibody for negative control. Evaluation was made using light microscopy with Leica DM6000B microscope and LAS X Software (Leica Microsystems GmbH, Germany).

Table 18. Antibodies and their dilutions used for immunohistochemistry.

Primary Ab Dilution Company Secondary Ab Dilution Company Calbiochem, Rabbit anti- 1:100 Abcam, UK Goat anti-rabbit 1:5000 Merck, CD34 Germany Calbiochem, Rabbit anti- 1:100 Abcam, UK Goat anti-rabbit 1:5000 Merck, Ki67 Germany Calbiochem, Mouse anti- Santa Cruz, 1:100 Goat anti-mouse 1:20000 Merck, Integrin ß3 USA Germany

57 Material and Methods

3.7.2 HE Staining Paraffin slices (3 µm) were prepared and rehydrated as described above (3.7.1). After that, glass slides were transferred to hematoxylin (Merck, Germany) for 4 min and dipped in 0.1 % HCl (in distilled water) for 2 s. Slices were differentiated in running water for 3 min. Incubation in eosin (Merck, Germany) was performed for 3 min followed by repeated differentiation in running water for 30 s. In order to embed slices in Entellan® (Merck, Germany), glass slides were dehydrated using an ascending ethanol series 70 % (4 s) / 70 % ethanol (4 s) / 96 % (2 × 2 min) and xylene twice for 5 min. Images were taken by the Leica DM6000B microscope and LAS X Software (Leica Microsystems GmbH, Germany).

3.7.3 Masson-Goldner Staining Masson-Goldner staining was performed using 3 μm paraffin slices prepared and rehydrated as described above (3.7.1). Reagents were purchased from Carl Roth GmbH (Germany). Glass slides were placed in hematoxylin for 1 min and put in running water for 7 min before transferring them into Goldner I solution for 7 min. Before incubation in Goldner II solution (incubation time dependent on the respective organ; tumor 5 min, kidney 5 min, liver 10 min, heart 8 min, spleen 20 min) slides were put into 1 % acetic acid for 30 s. This step was repeated before putting the slides into Goldner III solution for 7 min and again in 1 % acetic acid for 3 min. Slices were dehydrated, embedded and images were taken as described above (3.7.2).

3.7.4 PAS Staining PAS staining was performed with 3 μm paraffin slices, prepared and rehydrated as described above (3.7.1). Reagents were purchased from Morphisto (Germany). Glass slides were put in 0.5 % periodic acid for 7 min and subsequently into running water for 5 min. This step was followed by incubation in distilled water for 1 min, in Schiff reagent for 15 min, twice in distilled water for 1 min and again in running water for 10 min. Before placing the slides into hematoxylin for 3 min they were put in distilled water for 1 min followed by running water for 10 min. Slices were dehydrated, embedded and images were taken as described above (3.7.2).

58 Material and Methods

3.8 Primary Tissue Samples from Patients

Primary tissues from a total of 49 patients suffering from different stages of prostate cancer and 38 tissues of pancreatic cancer patients were included in this study. Patient material was collected at the institute of pathology at the University of Erlangen. The patient characteristics were presented in Table 19 and Table 20. All tissues were frozen after surgical resection and cut into 18 µm sections for autoradiography which were stored at -80 °C. HE staining of all samples was prepared by the institute of pathology in Erlangen. The study was approved by Comprehensive Cancer Center (CCC) Erlangen-EMN (application number: 2015- 1310-03).

Table 19. Prostate Cancer Patient Characteristics.

Prostate Cancer Patient Characteristics n

No. of patients 49 Median: 67 Age [y] (Range: 52 – 80) Median: 15 PSA [ng/mL] (Range: 6 – 70) Median: 7 Gleason score (Range: 6 – 9) Median: 7 Tumor volume (Range: 0.1 – 53) Grading pT2a 10 pT2b 8 pT2c 10 pT3a 10 pT3b 9 pT4 2 No. of patients with prostatectomy 22 (10.8 %)

Table 20. Pancreatic Cancer Patient Characteristics.

Pancreatic Cancer Patient Characteristics n

No. of patients 38 Median: 2 Histological grading (Range: 1 - 3) Grading pT3 38

59 Results and Discussion

4 Results and Discussion

4.1 Prostate and Pancreatic Cancer Cell Lines Showed Endogenous Expression of Neurotensin Receptor mRNA and Protein

The transcript abundance of the neurotensin receptor subtypes 1 and 2 was quantified by qRT-PCR in a panel of cancer cell lines that included the colon carcinoma cell line HT-29 as accepted positive control cell line (green) with known NTS1 expression (Figure 21) in order to assess different NTS1 positive human cancer cell lines and to choose an appropriate cell model for functional experiments addressing the NTS1.146,212 Furthermore, human embryonic kidney cells HEK293T (red) were carried along as negative control.217 Beside these, four prostate cancer, eight pancreatic cancer and five pancreatic stromal cell lines were tested for NTS1/2 expression. The results were expressed relative to the housekeeping gene GAPDH. This analysis revealed an endogenous expression of only NTS1 in some of the tested cell lines. Among prostate cancer cell lines, NTS1 mRNA was found in PC-3 and PC3-PIP cells with comparable amounts, but not in DU-145 and LNCaP cells. Among the pancreatic cancer cell lines, all cell lines except CFPac-1 and BxPC3 (two out of eight) revealed NTS1 expression. Panc-1 and AsPC-1 showed the highest, JoPaCa the lowest expression level among the pancreatic cancer cell lines. Pancreatic stromal cells from healthy pancreatic tissue did not show any NTS1 mRNA expression. Only LNCaP and Panc-1 cells showed NTS2 mRNA expression (Figure 21).

60 Results and Discussion

Figure 21. mRNA expression of NTS1 and NTS2 in prostate and pancreatic cancer cell lines. The human embryonic kidney cell line HEK293T was used as negative control (red), colon carcinoma cell line HT-29 as positive control (green). Five pancreatic stromal cell lines were tested in order to validate exclusive NTS1 expression only on pancreatic cancer cells and not on healthy pancreatic tissue. Relative NTS1 (black bars, left axis, relative to HT-29) and NTS2 (white bars, right axis, relative to LNCaP) abundance (vertical axis) ± SD (n = 3) quantified by qRT- PCR was plotted for four prostate, five pancreatic stromal and eight pancreatic cancer cell lines. Results were shown relative to internal control GAPDH.

Sehgal et al. proved in Northern Blot mRNA analysis of different prostate cancer cell lines that the LNCaP cells as well as DU-145 and PC-3 cells expressed NTS1 mRNA. This observation could be shown in LNCaP cells independent of the androgen status. They produced and secreted and were sensitive to NT. This group suggested that NT provided an alternative growth mechanism in prostate cancer during hormone-deprivation therapy. They did not test for NTS2 expression in the same study.144 Swift et al. also demonstrated NTS1 mRNA expression in PC-3 and DU-145 cell lines but not in LNCaP cell line.156 That group was the only one that could prove NTS2 mRNA expression in the LNCaP cell line. Dal Farra et al. provided evidence that no NTS1 mRNA could be detected in LNCaP cells.157 Furthermore, they could confirm NTS1 mRNA expression in androgen-independent human prostate cancer cell line PC-3. During the present study, NTS1 mRNA expression in PC-3 and the transfected PC3-PIP cells could be confirmed. Although having no

61 Results and Discussion explanation for the discrepancy within the LNCaP and DU-145 cell lines, these two prostate cancer cell lines were NTS1-negative in the present study. Dal Farra et al. also proved that pancreatic carcinoma cell line MIA PaCa2 expressed NTS1 mRNA PCR product.157 Yin et al. studied Panc-1 and AsPC-1 cell lines and could confirm NTS1 mRNA expression.218 All these findings were in accordance with the results obtained in the present study. Information about CFPac- 1, HPAF-II, BxPC-3, JoPaCa and Capan2 cell lines were hard to find in the literature. Thus, the present work represented the first report with the comparative screening of various pancreatic cancer cell lines. The present study of pancreatic stromal cell lines (PSC) for NTS1 mRNA expression has not been performed before. These stromal cells play a crucial role for the tumor micro-environment. Stromal cells sustain and nourish the tumor stem cells by producing and secretion of growth factors, but do not represent cancer cells.219

After NTS1 screening, the two NTS1 mRNA expressing prostate cancer cell lines PC-3 and PC3-PIP were additionally tested for PSMA mRNA expression together with the two pancreatic cancer cell lines with the highest NTS1 expression, Panc-1 and AsPC-1. The prostate cancer cell line LNCaP was carried along as positive control for PSMA,16 HT-29 for NTS1.146,212 The values were given relative to the internal control GAPDH. HEK293T cells were carried along as negative control which did not show any expression for NTS1, NTS2 and PSMA meaning that qPCR signal did not exceed background levels. PC-3, AsPC-1 and Panc-1 showed again only NTS1 mRNA expression, whereas PC3-PIP cells expressed mRNA of NTS1 and PSMA (Figure 22). The expression of PSMA mRNA in PC3-PIP cells resulted from transfection of PSMA in the otherwise PSMA-negative PC-3 cells.75

62 Results and Discussion

Figure 22. mRNA expression of NTS1, NTS2 and PSMA in prostate and pancreatic cancer cell lines. Human embryonic kidney cell line HEK293T was used as negative control (red), colon carcinoma cell line HT-29 as positive control for NTS1 and prostate cancer cell line LNCaP for PSMA (both green). Relative NTS1 (black bars, left axis, HT-29 as reference), NTS2 (white bars, right axis) and PSMA (blue bars, left axis, LNCaP as reference) abundance (vertical axis) ± SD (n = 3) quantified by qRT- PCR were plotted for two prostate and two pancreatic cancer cell lines. Results were shown relative to internal control GAPDH.

As there was knowledge about the fact that mRNA expression did not necessarily correspond to the actual protein levels,220 for receptor expression studies on protein level, radioligand binding studies were performed (Figure 23). 45 pM of [125I]Tyr3-NT was added to a number of different prostate (n = 6) and pancreatic (n = 7) cancer and pancreatic stromal (n = 2) cell lines, including the colon carcinoma cell line HT- 29 as positive control (yellow bars on the right) and the human embryonic kidney cells HEK293T (grey bars on the left) have been carried along as negative control.146,212,217 An excess of nonradioactive NT was added for the determination of nonspecific binding and the NTS2-selective peptide-peptoid CX107 (Pra(6FGlc)-N- Me-Arg-Arg-Pro-N-homo-Tyr-Ile-Leu-OH) was used to determine NTS1-specific binding, allowing the differentiation between binding to the neurotensin receptor

63 Results and Discussion subtypes 1 and 2.215 Results of ligand binding were calculated from binding data in fmol/mg as shown in Figure 23. This analysis revealed the expression of NTS1 in PC-3 (4.4 ± 0.6 fmol/mg) and PC3-PIP cells (2.3 ± 0.1 fmol/mg) among the prostate cancer cell lines (2 out of 5, 40 %, Figure 23A) and in AsPC-1 (10 ± 0.1 fmol/mg), JoPaCa (2.1 ± 0.2 fmol/mg) and Panc-1 (1.6 ± 0.3 fmol/mg) cells among pancreatic cancer cell lines (3 out of 7, 43 %). Pancreatic stromal cells did not show any NTS1 expression. Radioligand binding assay was performed exposing the cell lines to 45 pM of [125I]Tyr3-NT. This concentration allowed providing evidence for the existence of the neurotensin receptor subtypes but was not sufficient to saturate receptors and to determine receptor density. None of the tested cell lines showed significant NTS2 expression levels. Differences between total binding and NTS1- specific binding in HT-29 and AsPC-1 cells show no statistical significance (t-test: p- values 0.123 and 0.497, respectively) (Figure 23B). In literature only little information about NTS2 in cancer could be found. A new NTS2-targeting radioligand was published in 2015 by Maschauer et al.215 They could prove the selective binding of their radioligand in a mouse model bearing HT-29 and PC-3 tumors in small-animal PET imaging. Additionally, NTS2 expression was demonstrated by Western blotting. At least, this could be an explanation for the discrepancy between bars 1 and 3 in HT-29 cells shown in Figure 23. Nevertheless, both cell lines showed no NTS2 mRNA abundance during qPCR experiments in the present study (Figure 22).

64 Results and Discussion

Figure 23. Uptake of [125I]Tyr3-NT into prostate (A) and pancreatic (B) cancer or pancreatic stromal cell lines determined by radioligand binding assay referred to total activity added. Bars represented total binding (1), nonspecific binding (2) which was assessed by adding 1 µM of NT and NTS1-specific binding (3) that was determined by adding 1 µM of the NTS2-selective peptide-peptoid CX107 (Pra(6FGlc)-N-Me-Arg- Arg-Pro-N-homo-Tyr-Ile-Leu-OH).215 Shown were mean values [fmol/mg] ± SD of two independent experiments performed in quadruplicates (n = 8). The negative control HEK293T (Figure 23, grey bars, left) did not show any NTS1 abundance on the protein level in agreement with the literature – neither for NTS1 nor for NTS2.217 HT-29 cells were used as positive control (10.2 ± 2.21 fmol/mg), because of the proven NTS1 expression in the literature (Figure 23, yellow bars, right).146,212 In these experiments, NTS1-specific binding in bar 3 showed a small difference compared to total binding. The signal of NTS2 mRNA for HT-29 and AsPC-1 cells did not exceed background signal during qPCR (Figure 21A) and therefore no protein could be transcribed. Regarding prostate cancer cell lines, expression of NTS1 in PC-3 (and thus in PC3-PIP) cells in the present work was in

65 Results and Discussion accordance with the literature. Different groups could prove the existence of NTS1 in their PC-3 cells.156,157,178 LNCaP cells were found to be NTS1-negative during the present study. NTS1 expression on these cells was discussed controversial in the literature. Two research groups showed NTS1 expression in LNCaP cells.144,156 However, Sehgal et al. detected NTS1 mRNA by Northern Blot analysis without showing direct evidence for NTS1 protein on the cell surface.144 Contrary, Swift et al. revealed NTS1 expression on protein level in Western Blot but could not prove NTS1 mRNA abundance in RT-PCR analysis from which protein could be translated.156

The results of the present study were confirmed by the observations made by Dal Farra et al. who could not detect any NTS1 expression in LNCaP cells.157 JHU- LNCaP-SM cells represented a new LNCaP derivative which was no longer androgen-dependent and was generated by long term passage in cell culture.198 However, radioligand binding assay showed that androgen-independence did not change receptor expression concerning NTS1 and NTS2. Two research groups published NTS1 mRNA expression in DU-145 cells.144,156 These findings were in contrast to the results found in the present study. However, none of the two groups provided evidence for NTS1 protein expression on the membrane of DU-145 cells. During the present study no discrepancy between mRNA and protein expression on the cells could be observed among prostate cancer cell lines. Considering the pancreatic cancer cell lines, in this study, AsPC-1, JoPaCa, HPAF-II and Panc-1 cell lines expressed NTS1 on the cell surface. NTS1 expression on Panc-1 and AsPC-1 cells was confirmed by Yin et al.218 However, the positive result of Panc-1 cells by Yin et al. was in contrast to the findings of Gradiz et al. who could not detect any NTS1 expression on Panc-1 cells by immunohistochemistry.221 NTS1 expression on BxPC3 cells was proven by Olszewski and Hamilton,222,223 who showed that NT stimulation of these cells induced calcium release. This result was in contrast to the present findings, in which neither NTS1 mRNA nor NTS1 protein expression on the cell surface could be observed. NTS1 protein expression on HPAF-II, JoPaCa and Panc-1 cells was confirmed by a variety of research groups.224,225 MIA PaCa2 and CFPac1 cells have been shown to react to NT stimulation with calcium mobilization, not being in accordance with the present findings.222,224-227 Explanations for differences in expression profiles among the various cell lines were hard to find. Pancreatic stromal cells (PSC57, PSC156, PSC34, PSC107 and PSC200) did not show any receptor expression as was

66 Results and Discussion expected from the results of the quantitative real-time PCR experiment. Capan2 cells were not included into the binding assay experiments as this cell line was not easy to cultivate. Nevertheless, according to literature, receptor expression could be expected what would be in agreement with qPCR findings in Figure 21.225 NTS2 protein expression was not found in one single included cell line. In general, comparing mRNA data of Figure 21 with radioligand binding data of Figure 23, the discrepancy between mRNA levels and protein levels concerning the GPCR NTS1 became clear. Wang et al. have already shown that although NTS1 mRNA was expressed in patient samples of chronic pancreatitis and pancreatic cancer, the mRNA seemed to be translated into protein only in cancer samples.154 In conclusion, NTS1 mRNA was not a reliable and suitable tumor marker. A similar observation was made by Fisher et al., who showed that only a subset of cells that express mRNA of the somatostatin receptor had functional receptors on the cell membrane.228

In addition to the radioligand binding studies, Western Blot analysis of whole cell lysates was performed, in order to investigate NTS1 and PSMA protein expression on cells. After separating proteins during gel electrophoresis and blotting on PVDF membranes the latter were incubated with the respective primary antibodies. Chemiluminescence detection revealed false-positive bands in Western Blot for NTS1. This result became clear by comparing Western Blot results with mRNA and radioligand binding data. HT-29 and/or AsPC-1 cells were carried along as positive control and cells being tested negative during qPCR and radioligand binding experiments were used as negative controls. Unfortunately, none of the cell lines being suitable as negative control samples showed a negative result in Western blotting. Therefore, the applied Western Blot method frequently resulted in false- positive bands for NTS1 (Figure 24).

67 Results and Discussion

Figure 24. Exemplary Western Blot analyses of 15 µg protein for proving endogenous NTS1 protein expression on different prostate and pancreatic cancer cell lines. Blots did not show any specific bands independent of antibody and Western Blot conditions. (A) showed an unspecific band in NTS1-negative LNCaP cells (Alomone Labs ANT-015 1:500 4 °C o.n.) despite positive result for blocking with respective blocking peptide (1 µg NTS1 Peptide, Control Antigen, 2 h rt pre- incubation with antibody). (B) showed two membranes treated with different blocking conditions (10 % milk 2 h rt vs. 5 % milk / 1 % BSA 1 h rt). (C) Different antibody

68 Results and Discussion dilutions (Santa Cruz sc-376958; 1:200, 1:500, 1:1000). (D) False positive signals in negative control HEK293T cells (Abgent AG1174; 1:500 4 °C o.n., red box). (E) bands with false size (correct band size in the red box; NPE-Buffer; AAT Bioquest #G413; 1:5000 4 °C o.n.) and (F) showed membranes incubated with different antibodies (Santa Cruz sc-15311 1:100 4 °C o.n. vs. Abgent AG1174 4 °C o.n.). AsPC-1 and HT-29 cells were carried along as positive controls. As loading control GAPDH was used (exemplary result is shown in C and D).

During the time of the present work, six different antibodies have been tested in combination with variations in blotting and blocking conditions and antibody dilutions (Table 21). All these attempts suggested that specific antibodies for this receptor were currently not available on the market.154

Table 21. Antibodies and conditions for Western blotting.

Blotting Blocking Antibody Lysis Buffers Primary Ab conditions conditions 5 % milk Abgent AG1174 RIPA-Buffer Semi dry 30 min o.n. 4 °C o.n. 4 °C 5 % milk AAT Bioquest #G413 NPE-Buffer Semi dry 60 min 1 h rt 1 h rt 5 % milk w/ or w/o 0.5 % Santa Cruz sc-15311 RIPA-Buffer Tank blot 4 h rt milk 2.5 – 10 % milk Dilutions 1:100 – Santa Cruz sc-376958 RIPA-Buffer Semi dry 30 min 4 h rt 1:5000 Milk ± BSA Abcam, Cambridge RIPA-Buffer Semi dry 30 min 24 h, 4 °C (0.1 – 1 %) 5 % milk Alomone Labs ANT-015 RIPA-Buffer Semi dry 30 min o.n. 4 °C 4 h rt o.n. = overnight, rt = room temperature

Different groups published Western Blot results of NTS1 in the last years. However, the specificity of the antibodies was doubtful. Swift et al. conducted Western blotting with cell lysates and patient tissue samples.156 Results of the chemiluminescence detection of the blot showed bands for NTS1 in the four tested cell lines LNCaP, PC- 3, PNT1a and PNT2.C2 (two malignant and two non-malignant prostate cell lines, respectively). However, LNCaP cells did not show any NTS1 mRNA expression when comparing protein bands with PCR results shown above in the published blot. Thus, no protein could be translated and the result of the specific detection of NTS1 was therefore questionable. The same could be observed in the present study. False positive band were found in cells lines that were shown not to express NTS1 on mRNA level by qPCR (Figure 21) and protein level during radioligand binding assay (Figure 23) (for example HEK293T). Haase et al. reported congruent results of PCR 69 Results and Discussion and Western Blot. Nevertheless, in that work for protein detection corresponding negative control was missing so that regarding antibody specificity no point could be made.149 Recently, Yin et al. published specific immunohistochemical staining of the NTS1 receptor in pancreatic ductal adenocarcinoma tissue.218 Pancreatic adenocarcinoma was found positive for NTS1 protein in the staining while insulinoma and pancreatitis were negative. Although the described protocol in that publication agreed with the one used in our working group, these results could not be reproduced. No tissues of pancreatitis or insulinoma were available, but even cells from other species like from rat always were positive for NTS1 in our experiments (data not shown). An alternative method for protein detection was published by Seethalakshmi et al. in 1997.190 They crosslinked 125I-labeled NT to the mature NTS1 in vitro in PC-3 cells, solubilized them, performed gel electrophoresis and detected the radiolabeled protein by autoradiography. The specificity of binding of the radioligand was proven by co-incubation with 1 µM cold NT. Unfortunately, this method could not be successfully reproduced during the current thesis. PC-3 cells were incubated once with 125I-labeled NT and one time with [177Lu]Lu-FAUC 469. Thereafter, crosslinking and gel electrophoresis with solubilized proteins was performed. SDS-Page was visualized by autoradiography. Crosslinking of 125I-labeled NT and [177Lu]Lu-FAUC 469 to NTS1 in PC-3 cells did not show any band on the 10 % acrylamide gel (data not shown).

In contrast to NTS1 Western blotting, the analysis of PSMA expression on PC3-PIP cells proceeded without any problem at all (Figure 25). Chemiluminescence detection revealed only one specific band at about 100 kDa, even though the predicted band size was 84 kDa. GAPDH was used as loading control (46 kDa). Furthermore, PSMA was detected by immunofluorescence staining of PC3-PIP cells (Figure 25). PC-3 cells were used as negative control and LNCaP cells were used as positive control for both experiments as these cells were known to express PSMA.16

70 Results and Discussion

Figure 25. Representative Western Blot analysis for elucidating PSMA expression on PC3-PIP cells (left). LNCaP cells with endogenous PSMA expression was used as positive, PC-3 cells as negative control. GAPDH (46 kDa) was shown as loading control. Each lane was loaded with 15 µg of protein. Immunofluorescence staining of PSMA on PC3-PIP cells (right). PSMA was shown in green; nuclei were stained in blue with DAPI.

PC3-PIP and LNCaP cells were shown in different publications to express PSMA. PSMA expression was proven directly in vivo with PSMA-binding radiolabeled ligands.73,84,85,87,99 Differences in predicted and actual band sizes in Western Blot were a common problem even mentioned by the manufacturer. The same observation was made by two more groups who also detected PSMA with a molecular weight of 100 kDa.66,229 The shift of the PSMA band in Western Blot could be explained by the fact that PSMA contains a number of glycosylation sites.230

The NTS1-positive prostate cancer cell lines PC-3 and PC3-PIP, the colon cancer cell line HT-29 and the two pancreatic cancer cell lines Panc-1 and AsPC-1 were chosen for further experiments out of the tested human cancer cell lines. Experiments with the pancreatic cancer cell line AsPC-1 will be described in another ongoing doctoral thesis performed by Ulrike Herrmann also being a member of the working group of Prof. Olaf Prante.

71 Results and Discussion

4.2 Proliferative Response to NT

PC-3 cells were exposed to exogenous NT to test the ability of NT to stimulate cell growth. Addition of NT to PC-3 cells grown in serum-depleted medium stimulated cell growth in a dose-dependent manner from 0.01 nM up to 10 nM (Figure 26A). The growth promoting effect could not be inhibited by the new non-peptide antagonists FAUC 468 and CL 156 (Figure 26B).

Figure 26. (A) Effect of NT on growth stimulation of PC-3 cells. 3 × 103 PC-3 cells in 96-well plates were incubated in serum-free medium containing the indicated concentrations neurotensin for 2 days at 37 °C. Proliferation was determined by WST-1 assay. The values represented mean ± SD of two independent experiments with fivefold measurements (n = 10). T-test; *p < 0.05, **p < 0.01 compared to basal level of cell proliferation. (B) Effect of the addition of antagonists (1 µM) and NT on growth stimulation of PC-3 cells. 3 × 103 PC-3 cells in 96-well plates were incubated in serum-free medium containing 1 nM NT and the respective antagonist 2 days at 37 °C. The values represented mean ± SD of two independent experiments with six- fold measurements (n = 12).

These findings were confirmed by former experiments of Valerie et al. who showed that in transfected, serum-depleted PC-3 cells neurotensin stimulated cell proliferation could be reversed by adding SR 48692.178 They also could show NT- mediated cell proliferation in serum-depleted medium. Souazé et al. proved the same concentration-dependent growth promoting effect of NT and inhibiting effect of SR 48642 on HT-29 cells.134 In this context, they could elucidate the link between NTS1 and cancer triggering Wnt signaling pathway being crucial for human colon carcinogenesis. This fact was undergird with another publication by the same group,141 demonstrating that a NTS1 agonist was capable to stimulate NTS1-

72 Results and Discussion mediated effects. mRNA synthesis of NTS1 was influenced dependent on time of treatment (1 – 96 h) and concentration (0.3 nM; 100 nM). Increases in NTS1 mRNA synthesis rates could be inhibited by addition of SR 48692. Brun et al. elucidated the influence of NT on the migration potential of colon cancer cells, when the migration was enhanced in a dose-dependent manner, in the same tumor model.112 Again, addition of SR 48692 blocked the NT-mediated effects. It was assumed that this migration effect may have a crucial role in mucosal healing in healthy individuals. In MCF-7 breast cancer cells, NT and the NT agonist JMV 449 inhibited apoptosis and stimulated cell proliferation.135 Cell numbers were increased by 60 % and a decrease in apoptosis of 66 % could be observed after NT exposure. This result was explained by the influence of NT on the MAPK pathway. Increased MAPK activation after NT stimulation prevented cells from entering the cell death program. Shimizu et al. described a critical role for NTS1 in head and neck squamous cell carcinoma.148 In this tumor entity they proved influences of NT stimulation on cellular invasion and migration and mRNA synthesis. These affected genes played a role in tumor metastasis, cell motility, proliferation, migration, invasion, adhesion and apoptosis.148 Servotte et al. investigated the influences of NT on the migration abilities of U373 glioblastoma cells.147 Treatment of the serum-starved cells with NT modified the organization of the actin cytoskeleton and increased their motility. All these effects could be inhibited by NTS1 siRNA knock down and supported the role of NT on tumor cell growth. These observations could not be confirmed in cells cultured with serum. The authors assumed that the missing stimulation of proliferation was due to either the serum did already contain NT or that other growth factors in the serum activated the same signaling pathways as NT does. An alternative reason for the missing inhibitory effect of the non-peptide antagonists on cell proliferation could be explained by comparison with the results of Schulz et al.188 This group published a series of non-peptide NTS1 antagonists which were derivatives of SR 142948A, containing a DOTA-chelator that was connected to the core of the molecule by a linker. These substances have been described as “particularly potent competitive inhibitors”, as shown by the results of calcium flux assays. During these assays, their panel of NTS1 antagonists inhibited the calcium flux which was caused by the binding of NT or a NT agonist to NTS1. Botto et al. described SR 48692 not being able to antagonize NT induced cell proliferation in an artificial system of NTS1 expressing Xenopus oocytes.231 Even though the non-peptide NTS1 antagonists

73 Results and Discussion

FAUC 468 and CL 156 represented compounds that were structurally related to SR 142948A, the inhibitory effect of SR 142948A that was shown in the literature, could not be reproduced. Maybe a similar set up as described by Schulz et al. could prove a similar result as “particularly potent competitive inhibitors”.188

4.3 Effects of FAUC 468 and CL 156 on Colony Formation, Migration, Viability and Proliferation of PC-3 Cells

Colony formation assay (CFA), wound healing/scratch assay, growth curve, viability and proliferation assays were performed in order to determine whether the NTS1 antagonists FAUC 468 and CL 156 exerted any effects on prostate cancer PC-3 cells in vitro. Exposure to 2 nM of the NTS1-selective non-peptide antagonists over the period of 10 days did not influence the number of colonies of PC-3 cells during colony formation assay. Images of colonies in the absence or presence of FAUC 468 or CL 156 were shown in Figure 27A. For the assay, 100 PC-3 cells in 10-cm dishes were incubated with 2 nM of FAUC 468 or CL 156 for 10 days in duplicates. Plating efficiency and surviving fraction were calculated from CFA data (Chapter 3.2.7). Different cell lines showed different plating efficiencies (PE). PE measured the ability of single cells to grow to colonies. The plating efficiency in the given experiment was 68 %. The survival fraction (SF) expressed the number of colonies that grew after treatment. SF for FAUC 468 was 1.091 and for CL 156 0.904. The measured PE of 68 % for PC-3 cells was in agreement with the PE of 50 – 60 % given in the literature.214 In wound healing/scratch assays, the effect of NTS1 antagonists on cell migration behavior was analyzed. Scratch assays revealed no changed migration rate of PC-3 cells upon antagonist exposure when compared to untreated control cells (Figure 27B). Scratch assays indicated that NTS1 antagonist precursors were not associated with migration capability in PC-3 cells. Subsequently, the possible effect of NTS1 antagonists FAUC 468 and CL 156 on cell division (growth curve), viability (resazurin assay) and proliferation (WST-1 assay) compared to control cells was determined. Figure 27C – E showed that exposure of PC-3 cells to NTS1 antagonist precursors did neither significantly influence cell viability nor proliferation capability.

74 Results and Discussion

The NTS1 antagonists SR 48692 and SR 142948A were shown to be able to inhibit NT-mediated effects in vitro and in vivo by different research groups.112,134,141,178 The both DOTA-precursors FAUC 468 and CL 156 did not influence colony formation, migration, viability or proliferation of PC-3 cells. This finding could be explained by the fact that they as well as their lead structures SR 48692 and SR 142948A represent NTS1 antagonists.170,192 These experiments provided evidence that all effects in the following in vitro and in vivo experiments were not due to the precursors of the non-peptide antagonists but due to the radioactivity and the deposited dose in the cells or tissue caused by the radiotracer.

Figure 27. (A) Colony formation assay performed in 10-cm dishes (n = 3) with colonies produced by PC-3 prostate cancer cells during 10 days: colony images (left) and numbers of colonies (right). Untreated control cells with 68 colonies, after treatment with FAUC 468, 77 colonies are formed. 76 colonies are formed after treatment with CL 156. (B) Results of in vitro migration assay (n = 9). Wound healing/scratch assays of PC-3 cells did not show any inhibitory effects of FAUC 468 (2 nM) and CL 156 (2 nM) on cell migration. Relative open image area was plotted as

75 Results and Discussion a function of time (horizontal axis) in control and treated PC-3 cells. (C) Results of growth curves using PC-3 cells comparing growth rates between untreated control cells and cells exposed to 2 nM FAUC 468 or 2 nM CL 156 (n = 9). PC-3 cells revealed that NTS1 antagonists could not influence the proliferation rate of PC-3 cells. Results of (D) viability (resazurin) and (E) proliferation (WST-1) assays of PC-3 cells with and without exposure of FAUC 468 (0 – 1 µM) and CL 156 (0 – 1 µM). Both assays were assessed after incubation of the cells for 1, 2 and 7 days. The data were shown as mean values of three replications each measured in triplicates (n = 9). The error bars represented the standard deviation (SD) of the mean in each graph. During all experiments no NT was added for growth stimulation.

4.4 Radiolabeling with Gallium-68 and Lutetium-177

All compounds used were radiolabeled with gallium-68 in radiochemical yields of > 98 %. Radiolabeling of the compounds with lutetium-177 was carried out in radiochemical yields of > 98 %.

Figure 28. Radiosynthesis of 177Lu- and 68Ga-labeled NTS1 non-peptide antagonists FAUC 468 and CL 156.

High-performance liquid chromatography (HPLC) confirmed the high radiochemical purity of > 98 % for all radioligands as exemplified in Figure 29. The retention time for the non-peptide NTS1 antagonists [68Ga]Ga-ABN 468 and [177Lu]Lu-FAUC 469

76 Results and Discussion was 2.44 min, for [68Ga]Ga-CL 157 2.27 min and for [177Lu]Lu-CL 162 2.37 min (Chapter 3.3.1), whereas the peptide [68Ga]Ga-NT118 had a retention time of 68 tR = 2.13 min (Chapter 3.3.2), retention time for [ Ga]Ga-PSMA-11 was 2.78 min 68 (Chapter 3.3.3) and for [ Ga]Ga-NODAGA-RGD tR = 1.57 min (Chapter 3.3.4).

Figure 29. Representative radio-HPLC chromatograms of 68Ga- and 177Lu-labeled compounds. Radiolabeling yields and radiochemical purity were determined using a C18 reversed-phase column (Chromolith-RP18e, 4.6 × 100 mm; 5 μm) with a flow 68 177 rate of 4 mL/min. (A) [ Ga]Ga-ABN 468 (tR = 2.44 min), (B) [ Lu]Lu-FAUC 469 68 177 (tR = 2.44 min), (C) [ Ga]Ga-CL 157 (tR = 2.27 min), (D) [ Lu]Lu-CL 162 68 68 (tR = 2.37 min), (E) [ Ga]Ga-NT118 (tR = 2.13 min), (F) [ Ga]Ga-PSMA-11 68 (tR = 2.78 min), [ Ga]Ga-NODAGA-RGD (tR = 1.57 min).

NTS1 binding was measured in competition binding studies using homogenates of Chinese hamster ovary (CHO) cell membranes at the Department of Chemistry and Pharmacy (Dr. H. Hübner). These cells expressed the human NTS1 after stable transfection. The radioligand [3H]neurotensin was applied at the concentration of 0.3 – 0.5 nM. [3H]NT(8-13) was used at a concentration of 0.5 nM for investigation of NTS2 binding using homogenates of membranes from HEK293 cells transfected with the human NTS2 gene. Respective Ki values were listed in Table 22.

77 Results and Discussion

Table 22. In vitro binding affinities (Ki ± SEM) of the two non-peptide NTS1 antagonists FAUC 468 and CL 156 to human NTS1 and NTS2 of three individual experiments. Determination of NTS1 receptor binding was performed by Dr. Harald Hübner, Department of Chemistry and Pharmacy, Chair of Medicinal Chemistry, Friedrich-Alexander-University Erlangen-Nürnberg (FAU). NTS1 binding was measured in competition studies using homogenates of Chinese hamster ovary (CHO) cell membranes stably transfected with human NTS1. NTS2 binding was measured using homogenates of membranes from HEK293 cells transiently transfected with the human NTS2 gene.

Compound Ki (hNTS1) Ki (hNTS2) (see Figure 28 for structures) [3H]neurotensin [3H]NT(8-13) FAUC 468 1.9 nM 100 nM Ga-ABN 468 n.d. n.d. Lu-FAUC 469 0.19 nM 13 nM CL 156 1.0 nM 160 nM Ga-CL 157 0.46 nM 25 nM Lu-CL 162 0.18 nM 7.7 nM

Ki = Inhibitory constant; n.d. = not detected

4.5 Uptake, Internalization and Efflux Studies

4.5.1 NTS1 Uptake, Internalization and Efflux Studies After agonist binding, internalization of the NT analog-receptor complex has been shown.137,161,180,232 Only few reports proved evidence on the behavior of the antagonist-receptor complex.233,234 Pheng et al. demonstrated internalization of the pseudopeptide Y1 receptor antagonist GR231118 (Ile-Glu-Pro-Dpr-Tyr-Arg-Leu- Arg-

Tyr-CONH2) in transfected HEK293 cells by internalization assays using [125I] GR231118 and confocal microscopy.233 Internalization of the G-protein coupled cholecystokinin receptor was shown by Röttger et al. using the antagonist analogue 28,31 30 of cholecystokinin (D-Tyr-Gly-[(Nle ,D-Trp )cholecystokinin-26–32]-phenethyl ester) that was either tagged with the fluorescent rhodamine or radioiodinated.234

Comparative uptake, internalization and efflux studies of 68Ga- and 177Lu-labeled antagonists FAUC 468 and CL 156 were performed in vitro using NTS1 expressing PC-3 cells in order to measure the rate and extent of receptor-mediated radioligand uptake and subsequently release from the cells. These data provided insight into cellular uptake and retention of the radioligand. The obtained results were depicted in Figure 30.

78 Results and Discussion

Figure 30. Internalization, efflux and cell binding studies of 177Lu- and 68Ga-labeled FAUC 468 and CL 156 in human PC-3 cells. Experiments were performed with 1 ×106 cells per well. (A, C) Specific cell uptake/efflux were shown for [177Lu]Lu- FAUC 469 and [177Lu]Lu-CL 162 (A) and [68Ga]Ga-ABN 468 and [68Ga]Ga-CL 157 (C). (B, D) Internalization rates were shown for [177Lu]Lu-FAUC 469 and [177Lu]Lu- CL 162 (B) and [68Ga]Ga-ABN 468 and [68Ga]Ga-CL 157 (D). 41 pmol (2 nM) radiolabeled non-peptide NTS1 antagonist was used as tracer. Results were calculated as internalization (related to the sum of cell surface bound and internalized fraction as 100 %) and as cell uptake (related to total added radioactivity as 100 %). Data were expressed as mean ± SD of three independent experiments performed in triplicates (n = 9).

Uptake values of radiolabeled compounds increased rapidly during the first 60 min with rates of 48 ± 1 % for [177Lu]Lu-FAUC 469 and 35 ± 3 % for [177Lu]Lu-CL 162 (Figure 30B, D). The cell surface receptor-bound fraction was about 15 – 17 % for both compounds. The internalized fractions of [177Lu]Lu-FAUC 469 and [68Ga]Ga- ABN 468 were higher (75 ± 5 % and 69 ± 10 %, respectively) in comparison with the corresponding amide derivatives [177Lu]Lu-CL 162 and [68Ga]Ga-CL 157 (59 ± 7 % and 55 ± 5 %, respectively, Figure 30A, C), which could be decreased to less than 5 % in the presence of SR 142948A (1 µM). Externalization was determined at different time points over a period of 90 min for 68Ga- and 240 min for 177Lu-labeled

79 Results and Discussion compounds at 37 °C (Figure 30A,C). Only 15 - 20 % of the internalized radioactivity was released from the cells after internalization of both tracers for 45 min. The efflux rate for [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162 in PC-3 cells revealed values of 17 ± 5 % and 15 ± 4 % after 240 min, respectively. The results of the conducted in vitro experiments with [177Lu]Lu-FAUC 469, [68Ga]Ga-ABN 468, [177Lu]Lu-CL 162 and [68Ga]Ga-CL 157 were summarized in Table 23.

Table 23. In vitro characteristics of the radiolabeled NTS1 antagonists FAUC 468 and CL 156 obtained in studies using PC-3 cells. Listed were the mean results of cell uptake, internalization and efflux studies in percent (%) ± SD of three independent experiments performed in triplicates (n = 9) at 37 °C. Results were calculated as internalization rate (related to the sum of cell surface bound and internalized fraction as 100 %) and as cell uptake rate (related to total added radioactivity as 100 %).

Assay/Radioligand [68Ga]Ga- [177Lu]Lu- [68Ga]Ga- [177Lu]Lu- ABN 468* FAUC 469** CL 157* CL 162** Cell Uptake [%] 27 ± 6 48 ± 1 29 ± 9 35 ± 3 Internalization [%] 66 ± 9 75 ± 5 55 ± 5 59 ± 7 Efflux [%] 15 ± 4 23 ± 5 15 ± 3 23 ± 4 * after 90 min; ** after 240 min

In conclusion, due to high receptor-mediated cell uptake of the radiolabeled NTS1 antagonists in these NTS1-positive prostate cancer cell lines, both compounds may be useful tools for the preclinical evaluation of NTS1 as a molecular target in nuclear medicine. NTS1 may provide a highly specific theranostic target for accurate and rapid visualization, improved staging and highly effective endoradiotherapy of receptor-positive lesions with radioligands in a specific and NTS1-selective manner.

4.5.2 Uptake, Internalization and Efflux Studies Comparing NTS1 and PSMA Internalization and externalization studies on PC3-PIP cells were performed with [177Lu]Lu-FAUC 469, [68Ga]Ga-ABN 468, [68Ga]Ga-CL 157 and [68Ga]Ga-PSMA-11 (Figure 31). This cell line represented a good model for comparing the gold standard PSMA in prostate cancer with the new promising theranostic target NTS1, although it had to be taken into account that the receptor densities of NTS1 and PSMA of PC3- PIP cells were significantly different. Internalization experiments with [177Lu]Lu- FAUC 469 revealed values of 63 ± 4 % (n = 9) after 60 min. In comparison, 68Ga- labeled FAUC 468 and CL 156 showed values of 49 ± 6 % and 42 ± 6 %, respectively. Internalization rates of [68Ga]Ga-PSMA-11 were considerably lower

80 Results and Discussion reaching values of only 20 ± 2 %. Uptake of radiolabeled FAUC 468 and CL 156 could be blocked by co-incubation of 1 µM of their respective DOTA-precursors, uptake of [68Ga]Ga-PSMA-11 by co-incubation of 50 µM of PMPA (2- (phosphonomethyl)pentane-1,5-dioic acid). Efflux rates for [68Ga]Ga-ABN 468 and [68Ga]Ga-CL 157 were 48 ± 10 % and 34 ± 10 % after 60 min. [177Lu]Lu-FAUC 469 showed values of 44 ± 7 % after 60 min and 50 ± 6 % after 240 min. In contrast, [68Ga]Ga-PSMA-11 revealed an externalization of 59 ± 6 % already after 60 min.

Figure 31. Internalization and efflux studies in PC3-PIP cells performed at 37 °C. Internalization of radioligands expressed as the percent of internalized radioactivity from totally cell-associated radioligand (surface bound + internalized) shown for [68Ga]Ga-ABN 468, [68Ga]Ga-CL 157 and [68Ga]Ga-PSMA-11 (A) and [177Lu]Lu- FAUC 469 (B). Efflux of [68Ga]Ga-ABN 468, [68Ga]Ga-CL 157 and [68Ga]Ga-PSMA- 11 (C) and [177Lu]Lu-FAUC 469 (D). Data were expressed as mean ± SD of three independent experiments each performed in triplicates (n = 9).

4.5.3 Discussion of Uptake, Internalization and Efflux Studies The expression of NTS1 in pancreatic and prostate cancers and the suitability of that receptor as possible tumor marker promoted the development of radioactive neurotensin analogs and neurotensin receptor antagonists. The majority of neurotensin analogs that were radiolabeled were agonists.180-183 Working with such compounds was challenging, because of rapid proteolytic degradation in the blood.184-186 The development of neurotensin receptor antagonists that could be

81 Results and Discussion radiolabeled as new drugs had the advantage of higher radiotracer stability in blood serum and increased tumor uptake.187,188 The published radiolabeled agonist ligand [177Lu]NT127 revealed high affinity to the human NTS1 and rather high internalization of up to 82 % but efflux rate accounted for 42 % after 90 min.235 Another series of 68Ga- and 18F-labeled NT peptide derivatives presented by Maschauer et al. showed internalization rates between 36 – 78 %.195 Differences between these peptides were explained by distinct affinities for NTS1. Efflux rates were still low after 60 min with values of only about 20 %. The non-peptide NTS1 antagonists presented in this work came up with rather high internalization rates between 55 – 75 % and a simultaneous low externalization of only 15 – 20 %. Internalization was measured by lysing the cells after removing cell bound radioactivity by an acid wash with glycine buffer. In summary, during the in vitro studies, both antagonists revealed high uptake in the cells together with low efflux, meaning that the majority of radioactivity remained in the cells. As internalization rates were determined using glycine buffer before lysing the cells, the stable binding of the antagonists to the NTS1 which was maybe not separated by the acid wash could have suggested internalization even though radioligands were membrane-bound. Furthermore, the nonpolar structures with high affinity could diffuse and be stabilized in the lipophilic environment of the receptor. The internalization of receptor antagonists was discussed in the literature. For somatostatin receptors Dalm et al. showed that the in vitro uptake of their receptor antagonist [177Lu]Lu-DOTA-JR11 was five-fold higher compared to the receptor agonist [177Lu]Lu-DOTA-octreotate although the agonist was internalized and the antagonist remained membrane-bound.236 Similar results were reported by Nicolas et al.237 The somatostatin receptor antagonist [177Lu]Lu-OPS201 showed a higher tumor uptake and tumor retention compared to the agonist [177Lu]Lu-DOTATATE (15.6 h vs. 6.4 h). Ginj et al. showed a higher cell uptake of somatostatin receptor antagonists compared to agonists even though antagonists did not internalize. Thus, they concluded that at least in vivo antagonists were preferable over agonists due to higher cell uptake.238 Internalization of GPCR antagonists has been published by Pheng et al. and 233,234 Röttger et al. Pheng et al. showed internalization of the neuropeptide Y Y1 receptor following agonist and also antagonist binding in transfected human embryonic kidney cells. They proved receptor-mediated internalization of the ligand- receptor complex in clathrin-coated pits but without activating the Gi subunit.

82 Results and Discussion

Differences between agonist and antagonist binding were observed as receptors recovered at the cell surface by receptor recycling after agonist binding, while antagonist binding did not lead to receptor recovery. Furthermore, agonist-receptor and antagonist-receptor complexes were directed towards different intracellular courses. Röttger et al. proved specific clathrin-dependent receptor internalization of the G-protein coupled cholecystokinin receptor after antagonist binding, even though antagonist binding did not induce the same cellular response compared to agonist binding.234 Concerning the NTS1, the present study was the first one to show the internalization of the NTS1 after binding of a receptor antagonist under the given conditions. Based on the findings from Pheng et al. and Röttger et al., a similar mechanism for internalization of the antagonist-receptor complex could be assumed.233,234 Therefore, additional experiments, for example live cell imaging with fluorescence-labeled NTS1 antagonists, have to be performed to elucidate the way of the antagonist-receptor internalization into and inside the cell in more detail. Internalization of PSMA has been shown on LNCaP cells in 1998 by Liu et al. with the monoclonal antibody mAb J591.78 During the first 20 min of incubation with the antibody, 15 % of surface bound antibody was internalized via clathrin coated pits increasing up to 60 % after 1 h. In the present study, cells showed a shallow increase in internalization over time. [68Ga]Ga-PSMA-11 reached internalization values of 20 ± 2 % after 60 min. Unfortunately, internalization data of [68Ga]Ga-PSMA-11 on PC3-PIP cells were not available in the literature. Comparing the present data with internalization experiments on LNCaP cells (about 15 %) by Eder et al.,16 the findings of both studies were in accordance with each other at 60 min after start of incubation with the radioligand. Specific uptake of [68Ga]Ga-PSMA-11 into LNCaP cells was shown by displacement studies using 100 µM PMPA.

4.6 Saturation Binding Experiments

4.6.1 Saturation Binding Experiments with the NTS1 antagonists

The receptor density (Bmax, maximal binding capacity) and the radioligand equilibrium dissociation constant (Kd) were determined by saturation binding studies. Determination of the receptor density was necessary in order to analyze if NTS1 represented a suitable imaging and therapy study also for multiple therapy cycles.

83 Results and Discussion

Saturation binding analysis of the NTS1 was compared by using either the NT analogue [68Ga]Ga-NT118 or one of the two 68Ga- or 177Lu-labeled NTS1 selective antagonists FAUC 468 or CL 156 (Figure 17C, D) on different prostate and pancreatic cancer cell lines. HT-29 cell line was included as accepted reference in the literature.146,212

The equilibrium dissociation constants Kd and the total number of receptors expressed on cells Bmax obtained for the different radioligands at 4 °C are shown in Figure 32. Saturation binding demonstrated a high binding affinity of 68Ga- and 177Lu- labeled NTS1 selective antagonists FAUC 468 and CL 156 to all used cell lines in the subnanomolar range with Kd values of 0.1 – 0.5 nM. Bmax and Kd values were summarized in Table 24. During the experiments, nonspecific binding rose with increasing ligand concentrations in a linear manner. Specific binding was calculated by subtracting nonspecific binding from total binding. During all experiments, specific binding was saturable and could be displaced in competitive binding experiments. For these experiments 1 µM SR 142948A was used for competing 68Ga-/177Lu- labeled CL 156/FAUC 469 and 1 µM NT for [68Ga]Ga-NT118.

Figure 32. Saturation binding curves of 68Ga- and 177Lu-labeled non-peptide NTS1 antagonists on (A) HT-29, (B) Panc-1, (C) PC3-PIP and (D) PC-3 cells. Determination of saturation binding affinity to NTS1 revealed values between 0.1 and 0.5 nM. Shown were means of specific binding ± SD of three individual experiments performed in triplicates (n = 9).

84 Results and Discussion

Among these four cell lines, the pancreatic cancer cell line Panc-1 expressed the highest receptor density (Bmax) of 263 ± 50 fmol/mg, a 1.7-fold higher NTS1 expression than the prostate cancer cell line PC-3. PC-3 cells showed a maximal binding capacity of 158 ± 22 fmol/mg corresponding to about 60,000 receptors per cell (Chapter 3.3.9). HT-29 (143 ± 25 fmol/mg) showed similar expression profile than the PC-3 cells did. PC3-PIP cells had the lowest NTS1 expression of 49 ± 4 fmol/mg (Table 24).

85 Results and Discussion

Table 24. Saturation binding data of 68Ga- and 177Lu-labeled FAUC 468 and CL 156 obtained using the four human cancer cell lines HT-29 (colon cancer), PC-3 and PC3-PIP (prostate cancer) and Panc-1 (pancreatic cancer). Listed were Kd [nM] and Bmax [fmol/mg] values ± SD of three independent experiments performed in triplicates (n = 9) at 4 °C.

Radioligand/ [68Ga]Ga- [177Lu]Lu– [68Ga]Ga- [177Lu]Lu-

Cell line ABN 468 FAUC 469 CL 157 CL 162

Kd [nM] 0.13 ± 0.08 0.24 ± 0.11 HT-29 Bmax [fmol/mg] 143 ± 25 - 146 ± 25 - Binding sites/cell 40,000 40,000 Kd [nM] 0.27 ± 0.07 0.07 ± 0.01 0.09 ± 0.03 0.05 ± 0.02 PC-3 Bmax [fmol/mg] 158 ± 22 165 ± 9 97 ± 10 136 ± 13 Binding sites/cell 60,000 60,000 60,000 60,000 Kd [nM] 0.15 ± 0.03 0.11 ± 0.03 0.15 ± 0.04 PC3-PIP Bmax [fmol/mg] 49 ± 4 59 ± 6 60 ± 6 - Binding sites/cell 10,000 10,000 10,000 Kd [nM] 0.49 ± 0.25 0.38 ± 0.05 Panc-1 Bmax [fmol/mg] 263 ± 50 - 238 ± 11 - Binding sites/cell 100,000 100,000

Kd = dissociation constant, Bmax = maximal binding capacity

4.6.2 Saturation Binding Experiments Comparing the NTS1 Agonist [68Ga]Ga- NT118 and NTS1 Antagonist [68Ga]Ga-ABN 468 Furthermore, saturation binding experiments were performed at 37 °C with the 68Ga- labeled NTS1 agonist [68Ga]Ga-NT118 and were compared with values from experiments with the 68Ga-labeled non-peptide NTS1 antagonist FAUC 468 (Table 25). These experiments were performed at 37 °C as [68Ga]Ga-NT118 showed temperature-dependent binding properties and did not show sufficient binding at lower temperatures. Saturation binding experiments with [68Ga]Ga-NT118 revealed substantially higher total numbers (3 - 14 ×) of receptors expressed on the cells for three of the four tested cell lines. The only exception was the PC-3 cell line which showed the same amount of receptors for agonistic and antagonistic ligand. 68 Equilibrium dissociation constant Kd for [ Ga]Ga-NT118 was 25 ± 7 nM. Published Ki value for [68Ga]Ga-NT118 was 20 ± 1 nM.195

86 Results and Discussion

Table 25. Saturation binding data of the NTS1-adressing 68Ga-labeled FAUC 468 and NT118 obtained using the four human cancer cell lines HT-29 (colon cancer), PC-3 and PC3-PIP (prostate cancer) and Panc-1 (pancreatic cancer). Listed were Kd [nM] and Bmax [fmol/mg] values ± SD of 2 - 3 independent experiments performed in triplicates (n = 6 - 9) at 37 °C.

Cell line/Radiotracer [68Ga]Ga-ABN 468 [68Ga]Ga-NT118

Kd [nM] 0.17 ± 0.12 15.86 ± 5.19 HT-29 Bmax [fmol/mg] 203 ± 57 675 ± 237 Binding sites/cell 60,000 130,000 Kd [nM] 0.09 ± 0.02 27.44 ± 19.05 PC-3 Bmax [fmol/mg] 198 ± 12 159 ± 130 Binding sites/cell 60,000 33,000 Kd [nM] 0.21 ± 0.06 23.11 ± 8.88 PC3-PIP Bmax [fmol/mg] 49 ± 4 381 ± 173 Binding sites/cell 15,000 60,000 Kd [nM] 0.49 ± 0.25 33.29 ± 0.47 Panc-1 Bmax [fmol/mg] 263 ± 50 1487 ± 520 Binding sites/cell 100,000 240,000

Kd = dissociation constant, Bmax = maximal binding capacity

Exemplary curves of saturation binding assays in HT-29 cells comparing the NTS1 antagonist radioligand [68Ga]Ga-ABN 468 with the agonist radioligand [68Ga]Ga- NT118 were shown in Figure 33.

Figure 33. Exemplary presentation of saturation binding curves of the (A) non- peptide NTS1 antagonist [68Ga]Ga-ABN 468 and (B) NTS1 agonist [68Ga]Ga-NT118 68 on HT-29 cells. Kd value for [ Ga]Ga-NT118 was 25 ± 7 nM. Data were shown as mean values ± SEM of one representative experiment performed in triplicate (n = 3). In total, experiments were repeated for reaching n = 9.

87 Results and Discussion

The present study has shown that NTS1 agonist and antagonists specifically bound to different human cancer cell lines. Binding was specific and saturable. The affinity of radiolabeled antagonists was high (Kd ≈ 0.2 nM). The binding of antagonists was measured using concentrations in the range of 0.04 to 2.4 nM and agonist binding from 2.3 to 150 nM corresponding to about ± 5 × Ki. The curves demonstrated that NTS1 agonist revealed higher amounts of the NTS1 protein on the cells. Concerning these results, it had to be mentioned that saturation binding curves performed with the NTS1 agonist [68Ga]Ga-NT118 were flat and exhibited large error bars (± 35 – 82 %). More likely was that the receptor densities determined with the NTS1 agonist and antagonist kept in the same range. Moreover, the binding of the NTS1 antagonist seemed to be more stable than that of the NTS1 agonist. Chabry et al. stably expressed the rat NTS1 in fibroblasts and showed a maximal binding capacity of 1270 fmol/mg protein using an 125I-labeled NT derivative as the agonist radioligand.124 The receptor density in this artificial cell model corresponded to the endogenous expression of the human NTS1 in Panc-1 cells measured with the NTS1 agonist [68Ga]Ga-NT118 (Table 25). Another group expressed the human NTS1 in COS-7 (monkey kidney) cells.122 Experiments of this group with 125I-labeled [monoiodo-Tyr3]neurotensin resulted in receptor densities of 30000 binding sites per cell, which was within the range of the density of PC-3 and PC3-PIP cells (Table 25). In 1994 Maoret et al. measured a receptor density of 85 ± 28 fmol/mg protein in HT- 29 colon carcinoma cell line in experiments performed with 125I-labeled neurotensin.158 This value was about half as low when compared to the results of the present thesis (Table 25). Dal Farra et al. performed saturation binding experiments on membranes prepared from PC-3 cells using 125I-Tyr3-NT, demonstrating a maximal binding capacity of 42 fmol/mg protein.157 Again, this result was quite low compared to the results of the present work. However, due to the fact that the cells used in the different studies were not from the same origin and passage, a quantitative comparison should only be done with utmost caution. Labbé-Jullié et al. described saturation binding experiments comparing Kd and Bmax values between the NTS1 antagonist [3H]SR 48692 and the agonist [125I]NT on the same membrane preparations of LTK- cells (mouse fibroblasts) expressing the rat NTS1.239 They revealed a significantly lower receptor density (- 32 %) for experiments performed with the NTS1 agonist compared to the ones with NTS1 antagonist [3H]SR 48692. This group concluded that there was a fraction of receptors existing being

88 Results and Discussion inaccessible to the agonist and corroborated this hypothesis by concrete experiments with the detergent digitonin: these experiments proved that some receptors (about 20 %) were stored in vesicles and were again accessible by destroying the vesicles with a small amount of the detergent. Antagonists revealed a more hydrophobic structure compared to the agonist, enabling them to enter the NTS1-containing vesicles. One more explanation for the lower receptor density detected with the NTS1 agonist given by this group was that agonist and antagonist may address different but overlapping binding regions of receptor. Experiments for comparing receptor densities using [68Ga]Ga-ABN 468 and [68Ga]Ga-NT118 (Table 25) were performed at 37 °C in contrast to results obtained in Table 24 which were done at 4 °C.

As shown in section 4.5 the receptor-ligand complex was very fast internalized after binding of the non-peptide NTS1 antagonist. The following in vitro experiments were performed in order to analyze if internalization after ligand binding had an influence on the regulation of NTS1 expression as shown by Souazé et al.141 Saturation binding experiments with [68Ga]Ga-ABN 468 were performed at different selected time points (t = 0, 6, 24, 48 and 96 h) after exposition to 1 nM of the non-peptide antagonist CL 156 or to 200 nM of the NTS1 agonist NT100 in three independent experiments each performed in triplicates. The saturation binding results of the single experiments were averaged and plotted against the time of exposure (Figure 34).

Figure 34. Presentation of receptor availability (vertical axis Bmax in fmol/mg) against time (horizontal axis in h) on (A) PC-3 and (B) PC3-PIP cells after 0, 6, 24, 48 and 96 h exposition time to 1 nM NTS1 antagonist CL 156 or 200 nM NTS1 agonist 68 NT100 (≈ 10 × Ki) measured with the non-peptide antagonist [ Ga]Ga-ABN 468. Shown were data of three independent experiments performed in triplicates (n = 9).

89 Results and Discussion

The receptor availability was decreasing over the exposition time of 96 h with NTS1 antagonist and agonist for both PC-3 and PC3-PIP cells. The availability of free receptor in PC-3 cells rapidly decreased from 147 fmol/mg to 98 fmol/mg during the first 6 h of exposition. The available receptor density reached about 50 fmol/mg after 1 day of exposition and was constant for 2 days after the beginning of exposition. In PC3-PIP cells levels decreased from 114 fmol/mg to 87 fmol/mg during the exposition with the NTS1 antagonist CL 156 and to 72 fmol/mg during the exposition with the agonist NT100 for 6 h. These values further decreased to 44 fmol/mg during CL 156 exposition and to 25 fmol/mg during agonist incubation until the next day and were constant until 48 h later. After 96 h of incubation receptor levels decreased to 5 % of the initial level in PC-3 cells for both the NTS1 antagonist (9 fmol/mg) and the NTS1 agonist (6 fmol/mg). In PC3-PIP cells after exposition with the NTS1 agonist NT100 receptor availability decreased to 25 % and with the NTS1 antagonist CL 156 (24 fmol/mg) to 5 % (5 fmol/mg) of the initial level. Reappearance of the NTS1 at the cell membranes was not tested during the study. The results presented in Figure 34 showed that the NTS1 density on the cell membranes was regulated independent of the downstream signaling because receptor density decreased in the same range when incubated with NTS1 agonist or antagonist. In conclusion, this experiments showed that during potential NTS1-targeted endoradiotherapies receptors were not only occupied by the 177Lu-labeled radiotracer but also by the residual precursors. In the cases of a therapeutic dose of [177Lu]Lu-FAUC 469, the concentration of the unlabeled DOTA-precursor in the blood (about 7 nM) easily exceeds the Kd (~ 0.2 nM) so that a receptor occupancy of more than 50 % could be assumed.

Souazé et al. have shown that incubation with high doses of the NT agonist JMV 449 induced a biphasic response in the regulation of NTS1 mRNA in HT-29 cells.141 Initially, exposure to agonist generated a +270 % increase in NTS1 mRNA after 6 h by transcriptional activation. During this time, amounts of NT binding to NTS1 decreased due to receptor internalization. 24 h of exposition led to recovery of receptor density compared to control cells. After 72 h of continuous exposure there has been a decrease of 70 % in NTS1 mRNA quantities mediated through changes in mRNA half-life by post-transcriptional mechanisms. This event was already shown 240 for ß2-adrenergic receptor mRNA. The described biphasic response could not be shown in the present work on PC-3 and PC3-PIP cells. Receptor density decreased to about 70 % in the first 6 h. In literature values down to 20 % have been described

90 Results and Discussion during the same time followed by recovery beginning 24 h after the start of exposition reaching 60 % free receptors. This increase could be explained by de novo synthesis of receptors. In the present work the corresponding measurements showed only 20 – 40 % receptor density compared to untreated cells. The applied concentrations of 1 nM NTS1 antagonist CL 156 and 200 nM NTS1 agonist NT100 corresponded to concentrations that saturate receptors on the cells. Therefore, the sharp decrease in free receptors on the cell surface could be explained. One possible explanation for the missing recovery in the receptor density could be that NTS1 antagonist CL 156 did not induce de novo synthesis of NTS1 mRNA. Comparing the time-dependency of the receptor density in the graphs of Figure 34 of antagonist and agonist exposition, receptor density did not show any differences. Furthermore, one reason could be the daily change of medium containing the agonist or antagonist in the defined high receptor saturating concentrations, such that NTS1 receptor occupation and internalization occurred. Due to this fact, a significant receptor occupation could be observed, independent of agonist or antagonist exposition. These findings were comparable with the published results from García-Garayoa et al.241 This group reported a reduction of NTS1 density of 80 % after 60 min in consequence of agonist-exposition. Time-dependent receptor recovery could be observed by removing the NT agonist by washing and an acid wash in order to remove cell surface bound radioactivity. Lépée-Lorgeoux and coworkers examined the in vitro effects of the NTS1 agonist JMV 449 on NTS1 mRNA of cortical neurons in 2000.242 They revealed a 42 % decrease in mRNA after 48 h of incubation. This effect was reversible by treatment with the antagonist SR 48692. Regarding the results of the present study, a time-dependent receptor recovery of NTS1 could be expected by removing the NTS1 antagonist comparable to the results shown by García-Garayoa et al.241

Even if these in vitro results in NTS1-positive cancer cells did not necessarily predict corresponding good in vivo tracer uptake in xenografts, the cell uptake, internalization, efflux and saturation binding studies provided a solid basis for the following preclinical animal studies.

91 Results and Discussion

4.6.3 Saturation Binding Experiments addressing PSMA Using PC3-PIP cells, the receptor density of NTS1 and PSMA was determined in the same cell line. The equilibrium dissociation constants Kd and the total number of receptors expressed on cells Bmax obtained for PSMA at 4 °C were shown in Figure 35. During the experiments, nonspecific binding rose with increasing ligand concentrations in a linear manner. Specific binding was calculated by subtracting nonspecific binding from total binding. During all experiments, specific binding was saturable and could be displaced in competitive binding experiments. For these experiments 50 µM PMPA was used for competing [68Ga]Ga-PSMA-11.

Using PC3-PIP cells PSMA expression was analyzed on one cell line with the PSMA- binding [68Ga]Ga-PSMA-11. Saturation binding experiments revealed that compared with NTS1, PSMA is expressed 190-fold higher (49 ± 4 fmol/mg vs. 9382 ± 476 fmol/mg). This great overexpression of PSMA became clear considering that PSMA was stably transfected into PC-3 cells in order to generate PC3-PIP cell 75 line. Equilibrium dissociation constant Kd in these experiments was 7.5 ± 1.1 nM 68 which was in good accordance with the published Ki value for [ Ga]Ga-PSMA-11 of 12.0 ± 2.8 nM.16

Figure 35. Saturation binding curve of [68Ga]Ga-PSMA-11 on PC3-PIP cells. Determination of the saturation binding curve in PSMA-expressing cells revealed a Kd value for [68Ga]Ga-PSMA-11 of 7.5 ± 1.1 nM. Data were shown as mean values ± SD of three individual experiments performed in triplicates (n = 9) at 4 °C.

92 Results and Discussion

4.7 Biodistribution

4.7.1 [68Ga]Ga-ABN 468 and [68Ga]Ga-CL 157 Biodistribution Studies Biodistribution studies of [68Ga]Ga-ABN 468 and [68Ga]Ga-CL 157 were performed at 1 h after intravenous injection of 5 MBq of the respective radiotracer per animal (each n = 2 – 3) in PC-3 and PC3-PIP tumor-bearing NMRI FOXn1nu mice. Results for biodistribution data of 68Ga-labeled radiotracers were shown in Figure 36A. [68Ga]Ga-ABN 468 and [68Ga]Ga-CL 157 showed prominent radioactivity in the blood circulation with values of 2.89 ± 0.09 % ID/g and 2.22 ± 0.07 % ID/g. There was moderate uptake in the excretion organs with values of 1.17 ± 0.08 % ID/g and 0.72 ± 0.04 % ID/g for liver and 1.02 ± 0.09 % ID/g and 0.89 ± 0.07 % ID/g for kidneys. The tumor uptake was 0.7 % and 1.1 % for [68Ga]Ga-ABN 468 and [68Ga]Ga-CL 157, respectively. Comparable accumulation of radioactivity was also found in the lung (1.73 ± 0.08 % ID/g for [68Ga]Ga-ABN 468 and 1.16 ± 0.34 % ID/g for [68Ga]Ga-CL 157) and spleen (0.60 ± 0.05 % ID/g for [68Ga]Ga-ABN 468 and 0.43 ± 0.08 % ID/g for [68Ga]Ga-CL 157).

Figure 36. (A) Biodistribution of [68Ga]Ga-ABN 468 and [68Ga]Ga-CL 157 in PC-3 xenografted NMRI Foxn1nu mice at 1 h after tracer injection (approx. 5 MBq). Data were expressed as mean values in percent (%) of the injected dose per mass (g) of tissue (% ID/g) ± standard deviation (SD) from two to three mice. (B) Tumor-to-organ ratios as calculated from the biodistribution data. The high radioactivity in blood at 1 h p.i. resulted in high background accumulation and a low tumor-to-blood ratio of 0.25 and 0.5 for [68Ga]Ga-ABN 468 and [68Ga]Ga- CL 157 (Figure 36B). The fact that tumor-to-blood ratio for [68Ga]Ga-CL 157 was twice as high as the ratio for [68Ga]Ga-ABN 468 was due to the higher tumor and lower blood uptake of [68Ga]Ga-CL 157 compared to [68Ga]Ga-ABN 468. The low tumor-to-blood ratios implicated binding of the radiotracers to blood during the first

93 Results and Discussion

60 min after injection. Both radiotracers showed good tumor uptake (0.70 ± 0.11 % ID/g for [68Ga]Ga-ABN 468 and 1.09 ± 0.21 % ID/g for [68Ga]Ga- CL 157) whereas [68Ga]Ga-ABN 468 showed higher uptake into the lung (1.73 ± 0.08 % ID/g vs. 1.16 ± 0.34 % ID/g) and the liver (1.17 ± 0.08 % ID/g vs. 0.72 ± 0.04 % ID/g) compared to [68Ga]Ga-CL 157. Table 26 showed the radioactivity of the two radiotracers accumulated in the organs in percentage injected dose per gram tissue (% ID/g) and selected tumor-to-organ ratios at 1 h p.i.

Table 26. Biodistribution of [68Ga]Ga-ABN 468 and [68Ga]Ga-CL 157 in PC-3 tumor- bearing nude mice 1 h p.i. The values were given as mean % ID/g ± SD, n = 2)

Organ [68Ga]Ga-ABN 468 [68Ga]Ga-CL 157 Blood 2.89 ± 0.09 2.22 ± 0.07 Lung 1.73 ± 0.08 1.16 ± 0.34 Liver 1.17 ± 0.08 0.72 ± 0.04 Kidney 1.02 ± 0.09 0.89 ± 0.07 Heart 0.81 ± 0.12 0.63 ± 0.09 Spleen 0.60 ± 0.05 0.43 ± 0.08 Brain 0.09 ± 0.02 0.10 ± 0.05 Muscle 0.26 ± 0.01 0.22 ± 0.01 Bone 0.36 ± 0.03 0.27 ± 0.03 Intestine 0.42 ± 0.04 0.39 ± 0.09 Tumor 0.70 ± 0.11 1.09 ± 0.21 Tumor-to-blood ratio 0.25 ± 0.01 0.50 ± 0.06 Tumor-to-kidney ratio 0.69 ± 0.02 1.24 ± 0.27 Tumor-to-muscle ratio 2.73 ±0.25 5.06 ± 0.27

4.7.2 Small-animal PET imaging The results of accompanying small-animal PET images in addition to the biodistribution studies obtained from mice injected with 5 MBq of [68Ga]Ga-ABN 468 or [68Ga]Ga-CL 157 at 55 min p.i. for 15 min were shown in Figure 37. The PET images confirmed tumor uptake, however, a very high background signal due to the remaining radioactivity in the blood was clearly visible as predicted by the biodistribution study.

94 Results and Discussion

Figure 37. Representative PET scans at 55 – 70 min p.i. of [68Ga]Ga-ABN 468 (n = 3, left) and [68Ga]Ga-CL 157 (n = 3, right) obtained using PC-3 tumor-bearing nude mice. Both images were adjusted to the same standard uptake value (SUVmean). Red arrows indicated PC-3 tumors. Li = liver

Small-animal PET images with [68Ga]Ga-ABN 468 and [68Ga]Ga-CL 157 were performed because NTS1 should represent a tumor-specific target for both molecular imaging and therapy (“theranostics”). The high background signal in the images obtained at 55 – 70 min p.i. could be explained by the low tumor-to-blood ratios of 0.25 and 0.5 for [68Ga]Ga-ABN 468 and [68Ga]Ga-CL 157 (Figure 36B). Nevertheless, the tumor was definable and PET imaging at later time-points could lead to improved tumor-to-blood ratios and therefore to better PET images. The present results suggested that the NTS1 represented a suitable imaging target for NTS1-positive tumors. Specific binding of the radiotracers with displacement studies were shown using the 177Lu-labeled analogs in Chapter 4.7.3.

4.7.3 Biodistribution Studies of [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162 Biodistribution data of [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162 were collected at 1 h, 1, 2 , 4 and 7 days after injection of 1 MBq radiotracer per animal in PC-3 xenografted NMRI FOXn1nu mice. Displacement studies were performed 4 h after co- injection of [177Lu]Lu-FAUC 469 with 100 µg CL 156 in order to prove NTS1-specific uptake (control and competitive binding group each n = 3). The highest radioactivity uptake in NTS1-positive PC-3 tumors was observed at 1 day p.i. for [177Lu]Lu-FAUC 469 (15.6 ± 2.42 % ID/g) and at 2 days p.i. for 177Lu]Lu-CL 162 (19.5 ± 4.96 % ID/g) (Figure 38). Following this high and rapid accumulation of radioactivity in the tumor, slow radioactivity washout and an excellent retention of both radiotracers could be observed until day 7 post-injection

95 Results and Discussion

(6.40 ± 0.87 % ID/g for [177Lu]Lu-FAUC 469 and 8.62 ± 2.20 % ID/g for [177Lu]Lu- CL 162). Both [177Lu]Lu-FAUC 469 (Figure 38A) and [177Lu]Lu-CL 162 (Figure 38B) showed clearance from blood pool decreasing from 2.2 ± 0.9 % and 9.2 ± 0.5 %, respectively, after 1 h p.i. to 0.1 % after 48 h p.i. For [177Lu]Lu-FAUC 469 lung, liver, kidney and spleen showed unspecific radioactivity uptake with values of 6.78 ± 7.18, 13.8 ± 6.74, 4.33 ± 0.17 and 2.67 ± 0.92 % ID/g, respectively, at 1 day p.i. High nonspecific accumulation of [177Lu]Lu-CL 162 could be detected only in the liver showing a value of 1.06 ± 0.15 % ID/g. An increasing tumor-to-blood ratio was calculated for [177Lu]Lu-FAUC 469 from 1.55 ± 1.39 after 1 h p.i. over 300 ± 90 after 4 days to 557 ± 172 after 7 days (Figure 38C) and for [177Lu]Lu-CL 162 from 1.2 after 1 h over 944 after 4 days up to 2407 after 7 days (Figure 38C) from the biodistribution data. All tumor-to-tissue ratios were ≥ 1 for non-target tissues for time points > 24 h (Table 27) for [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162. Tumor uptake was higher than in all other selected organs at all points after [177Lu]Lu-CL 162 injection.

96 Results and Discussion

Figure 38. Data of biodistribution of (A) [177Lu]Lu-FAUC 469 and (B) [177Lu]Lu- CL 162 (1 MBq) in NMRI FOXn1nu mice bearing NTS1-positive PC-3 xenografts were expressed as mean values in percent (%) of the injected dose per mass (g) of tissue (% ID/g) ± SD. n (1 h, 1 day, 2 days) = 2 – 3, n (4 and 7 days) = 2. (C) Tumor-to- organ ratios of [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162 were calculated from biodistribution data at different time points.

97 Results and Discussion

Table 27. Biodistribution data of [177Lu]Lu-FAUC 469 in PC-3 tumor-bearing nude mice and selected tumor-to-organ ratios. Values (% ID/g) represented the mean ± SD of data from two animals. p.i. = post-injection

[177Lu]Lu-FAUC 469

1 h 24 h 48 h 96 h 7 d Organ/Time p.i. Blood 2.23 ± 0.92 0.33 ± 0.01 0.13 ± 0.04 0.02 ± 0.01 0.01 ± 0.00 Lung 1.94 ± 0.36 10.6 ± 1.46 1.15 ± 0.33 0.31 ± 0.08 0.20 ± 0.01 Liver 6.43 ± 2.81 16.6 ± 2.94 4.65 ± 0.75 0.76 ± 0.17 0.60 ± 0.03 Kidney 1.67 ± 1.03 3.98 ± 0.00 2.02 ± 0.47 0.48 ± 0.19 0.31 ± 0.03 Heart 1.39 ± 0.62 1.58 ± 0.82 0.47 ± 0.12 0.22 ± 0.09 0.16 ± 0.04 Spleen 2.22 ± 1.05 2.97 ± 1.92 1.27 ± 0.29 0.32 ± 0.08 0.24 ± 0.02 Brain 0.13 ± 0.03 0.05 ± 0.01 0.03 ± 0.01 0.02 ± 0.01 0.02 ± 0.00 Muscle 0.33 ± 0.14 0.27 ± 0.02 0.16 ± 0.05 0.06 ± 0.02 0.03 ± 0.00 Bone 0.45 ± 0.08 0.91 ± 0.11 0.60 ± 0.19 0.13 ± 0.03 0.08 ± 0.02 Intestine 1.01 ± 0.23 1.26 ± 0.12 0.62 ± 0.20 0.53 ± 0.17 0.49 ± 0.06 Tumor 7.30 ± 0.00 15.47 ± 0.00 8.77 ± 0.79 7.74 ± 1.39 6.40 ± 0.87 Tumor-to-blood 1.55 ± 1.39 46.96 ± 0.00 74.8 ± 36.2 299.7 ± 90.27 556.5 ± 172.4 Tumor-to-kidney 2.00 ± 1.49 3.89 ± 0.00 4.59 ± 1.70 13.60 ± 5.19 20.80 ± 1.33 Tumor-to-liver 0.52 ± 0.43 0.93 ± 0.00 1.94 ± 0.52 10.30 ± 0.79 10.80 ± 2.31 Tumor-to-muscle 10.23 ± 8.55 57.19 ± 0.00 61.2 ± 26.9 139.0 ± 17.2 226.3 ± 4.9

98 Results and Discussion

Table 28. Biodistribution data of [177Lu]Lu-CL 162 in PC-3 tumor-bearing nude mice and selected tumor-to-organ ratios. Values (% ID/g) represented the mean ± SD of data from 2-3 animals. p.i. = post-injection

[177Lu]Lu-CL 162

Organ/Time p.i. 1 h 24 h 48 h 96 h 7 d Blood 9.72 ± 0.50 0.11 ± 0.02 0.03 ± 0.01 0.01 ± 0.01 0.00 ± 0.00 Lung 3.99 ± 0.50 0.59 ± 0.68 0.25 ± 0.02 0.59 ± 0.30 0.46 ± 0.15 Liver 2.67 ± 0.19 1.06 ± 0.15 1.06 ± 0.06 2.10 ± 0.10 2.58 ± 0.08 Kidney 3.98 ± 0.62 0.47 ± 0.06 0.25 ± 0.05 0.34 ± 0.05 0.14 ± 0.00 Heart 2.75 ± 0.30 0.16 ± 0.02 0.13 ± 0.01 0.10 ± 0.02 0.05 ± 0.00 Spleen 1.91 ± 0.27 0.42 ± 0.02 0.47 ± 0.06 0.54 ± 0.27 1.09 ± 0.09 Brain 0.36 ± 0.07 0.02 ± 0.01 0.03 ± 0.00 0.03 ± 0.01 0.02 ± 0.01 Muscle 1.00 ± 0.18 0.06 ± 0.01 0.07 ± 0.04 0.03 ± 0.02 0.01 ± 0.01 Bone 1.18 ± 0.10 0.21 ± 0.02 0.23 ± 0.03 0.65 ± 0.28 0.55 ± 0.16 Intestine 3.21 ± 0.80 0.94 ± 0.18 1.02 ± 0.13 0.35 ± 0.04 0.30 ± 0.05 Tumor 11.77 ± 1.63 17.55 ± 2.42 19.50 ± 4.96 8.33 ± 1.38 10.18 ± 0.00 Tumor-to-blood 1.21 ± 0.11 169.9 ± 27.9 576.2 ± 19.0 944.4 ± 416.1 2407.3 ± 0.0 Tumor-to-kidney 3.03 ± 0.70 37.28 ± 4.69 78.50 ± 9.59 24.88 ± 7.39 49.9 ± 31.4 Tumor-to-liver 4.43 ± 0.61 16.79 ± 2.92 18.42 ± 2.33 3.97 ± 0.73 2.79 ± 1.63 Tumor-to-Muscle 12.0 ± 2.4 291.0 ± 51.9 306.9 ± 92.5 233.2 ± 94.8 180.3 ± 22.6

99 Results and Discussion

4.7.4 Displacement studies

In competition studies, pre-injection of 100 µg CL 156 15 min before [177Lu]Lu- FAUC 469 caused an inhibition of the tumor uptake from 5.43 ± 2.46 % ID/g down to 0.42 ± 0.36 % ID/g at 4 h p.i (-93 %). The inhibition of binding by co-injection of CL 156 confirmed the receptor-specific uptake of the radiotracer in the NTS1-positive PC-3 tumors. Furthermore, a specific inhibitory effect of 32 % could be observed in the intestine (Figure 39). In contrast, the uptake in all the other selected organs was not influenced by pre-injection of CL 156, showing the specific uptake of the radiotracer in the PC-3 tumor and intestine.

Figure 39. Data of biodistribution of [177Lu]Lu-FAUC 469 in NMRI FOXn1nu mice bearing NTS1-positive PC-3 xenografts 4 h after p.i. Displacement experiment was performed with pre-injection of 100 µg CL 156 (“Blocking”). Data were expressed as mean values in percent (%) of the injected dose per mass (g) of tissue (% ID/g) ± SD from three mice per group.

100 Results and Discussion

4.7.5 Dosimetry Calculation Dosimetry calculations were performed (Chapter 3.4.1.2) out of the defined time points of the time-dependent biodistribution study by Dr.-Ing. Philipp Ritt (Imaging and Physics, Clinic of Nuclear Medicine, University Hospital Erlangen, Erlangen, Germany). An overview of the absorbed doses of [177Lu]Lu-FAUC 469 and [177Lu]Lu- CL 162 was given in Table 29. Dosimetry calculations were performed using defined time points (t = 1 h, 24 h, 48 h and 168 h p.i.) of the biodistribution experiments. The high uptake and long effective half-lives in the tumor accounted for 1.43 Gy/g/MBq for [177Lu]Lu-FAUC 469 and for 1.66 Gy/g/MBq for [177Lu]Lu-CL 162.

Table 29. Calculated absorbed doses of [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162 in PC-3 tumor-bearing nude mice, after injection of 1 MBq of the respective radiotracer. Dosimetry calculations were performed using defined time points (t = 1 h, 24 h, 48 h and 168 h post-injection) of the biodistribution experiments.

[Gy/g/MBq] [177Lu]Lu-FAUC 469 [177Lu]Lu-CL 162 Blood 0.03 0.08 Lungs 0.17 0.09 Liver 0.64 0.52 Kidneys 0.21 0.08 Heart 0.06 0.03 Spleen 0.15 0.25 Brain < 0.01 0.01 Muscle 0.02 0.02 Femur 0.05 0.08 PC-3 Tumor 1.43 1.66 Small Intestine 0.10 0.11

In comparison with other NTS1-targeting peptide radioligands that have been described previously, tumor uptake of the two non-peptide antagonists [177Lu]Lu- FAUC 469 and [177Lu]Lu-CL 162 was very high. García-Garayoa et al. published different 99mTc-labeled compounds with values of 3.9 % ID/g after 1.5 h,241 0.9 – 6.3 % ID/g,243 and 6 % ID/g in HT-29 tumors each measured after 1.5 h.180 The previously published NTS1 antagonist 177Lu-3BP-227 revealed a similar biodistribution profile like [177Lu]Lu-CL 162.194 A high and tumor-specific persistent uptake of the radiotracer could be proved and a comparable tumor radiation dose of 830 mGy/MBq was calculated.

101 Results and Discussion

4.7.6 Ex Vivo Autoradiography Tracer accumulation and corresponding competition experiments in intestines during the biodistribution study could be confirmed by ex vivo autoradiography. Therefore, intestines had been removed, defecated and rolled up in order to freeze and prepare 20 µm cryo slices for exposition to a phosphor screen. In these experiments, radioactivity accumulation could be shown in the intestines that could be displaced by pre-injection of 100 µg CL 156 (Figure 40).

Figure 40. Representative images of ex vivo autoradiographies from tissue slices of small intestine and corresponding HE staining from mice injected with 1 MBq of [177Lu]Lu-FAUC 469 (“control”) or mice pre-injected with 100 µg CL 156 (“blocking”) at 4 h p.i. Experiments were performed in PC-3 xenografted nude mice with three animals per group.

NTS1 expression in the intestines has been discussed in a controversial manner in the literature. As NT is secreted from enteroendocrine N cells of the small intestine and modulates the digestive tract, NTS1 expression in close proximity to its site of release would be reasonable.106,107 Competition experiments, as shown above, that proved specific displacement of NTS1-selective radiotracers in biodistribution and ex vivo autoradiography indicated NTS1 expression in the intestines. Uptake in the third NTS1-expressing organ, the brain,105 could not be observed, indicating that the NTS1 antagonists did not cross the blood-brain barrier. Preclinical displacement animal studies of García-Garayoa et al. using PC-3 and HT-29 tumor-bearing nude mice

102 Results and Discussion with a 99mTc-labeled NT pseudopeptide showed displacement of the tracer by NT in tumor and intestine.241 According to García-Garayoa et al., the intestine contained high amounts of NTS1. This finding was strengthened by the presence of NTS1 mRNA in the adult small intestines of both rat and humans.244 Specific uptake of the NT analog 99mTc-NT-XI in the intestinal tissue of patients was described by Buchegger et al. in 2003.183 This group examined four patients suffering from ductal pancreatic adenocarcinoma. Two of these had NTS1-positive tumors and the same two patients showed intestinal uptake of radioactivity at tumor-to-intestine ratios of 1:2.6 and 1.2:1. Further studies should be performed in order to analyze the relevance of radiotracer uptake into gastrointestinal tissues.

Both radiotracers, [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162, were investigated in selected organs under the same conditions over a period of 7 days (Table 27, Table 28, Figure 41). Tumor accumulation was fast and tumor retention excellent for both tracers (15 – 20 % ID/g 24 h p.i., Figure 41A). The tumor-to-background ratios were good (> 40) combined with fast clearance from blood. However, the accumulation in the liver of [177Lu]Lu-FAUC 469 was markedly increased compared to [177Lu]Lu- CL 162 at 1 h p.i. (Figure 41B). This finding resulted in reduced tumor-to-liver ratios of [177Lu]Lu-FAUC 469 as compared to [177Lu]Lu-CL 162. Over the period of 7 days liver uptake of both radiotracers converged. The constant retention of radioactivity inside the tumors in vivo was in accordance to in vitro results shown in Chapter 4.5.232-234 High liver uptake of [177Lu]Lu-FAUC 469 indicated its hepatobiliary excretion and a higher dose for the liver in comparison to [177Lu]Lu-CL 162 (Figure 41B). This fact might be useful in cases of liver metastases as the liver could receive a significant radiation dose during the time of therapy due to the extraction through the liver. In summary, the high specific uptake of both radiotracers in the NTS1- positive tumors of 16 % ID/g (24 h p.i.) and the excellent tumor retention suggested that these radioligands represented suitable therapeutic agents in NTS1-positive cancers.

103 Results and Discussion

Figure 41. Time-dependent radiotracer uptake and retention of [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162 in NTS1-expressing PC-3 tumors and liver over the period of 7 days p.i. Data were expressed as mean values in percent (%) of the injected dose per mass (g) of tissue (% ID/g) ± standard deviation (SD) from two to three mice.

4.8 NTS1-targeted Endoradiotherapy

4.8.1 Therapy Study with [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162 in PC-3 Tumor-Bearing Nude Mice In this study, NTS1 was evaluated as molecular target for endoradiotherapy of NTS1- positive prostate cancers. The NTS1-mediated therapeutic effects of the two non- peptide antagonists [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162 were investigated in PC-3 tumor-bearing nude mice over the period of two months (Figure 42).

Figure 42. (A) Relative tumor volume and (B) survival curves of the respective PC-3 tumor-bearing nude mice after treatment with a single dose of 25 MBq of [177Lu]Lu- FAUC 469 (n = 5) and [177Lu]Lu-CL 162 (n = 5) in comparison with untreated control animals (n = 7). Day 0 represented the day of radiotracer injection. Data were expressed as mean ± SD. Drops of the curve of the control mice () were due to the sacrifice of animals because of fulfilling termination criteria at these days.During the

104 Results and Discussion whole time of the study, body weight of the treated animals did not show any decrease indicating the wellbeing of the mice (data not shown). The tumor volume steadily increased in the untreated control group (Figure 42A, black). The first control mouse had to be sacrificed after 15 days due to a tumor size of > 15 mm in diameter. The two groups each injected with 25 MBq/mouse (day 0) of the respective 177Lu- labeled radiotracer showed a delayed tumor growth with a significant difference to the control group with beginning of day 10 (adjusted p=0.009 for [177Lu]Lu-CL 162 and adjusted p=0.044 for [177Lu]Lu-FAUC 469; permutation test using a two-sample t- statistic). Tumor growth inhibition (TGI) at day 15 (death of the first control mouse) was 51 % for [177Lu]Lu-CL 162 and 64 % for [177Lu]Lu-FAUC 469. Tumor doubling time increased from 1.92 days in the control group to 2.06 and 2.08 days in the [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162 treated groups. Mice of the control group had to be sacrificed between days 16 and 54 after begin of the endoradiotherapy, due to fulfilling the termination criteria of either a tumor volume of ≥ 15 mm or the tumor showing active ulceration. Euthanasia of selected mice resulted in the striking drops of the curve of the control mice indicated by the arrows () in Figure 42A. A difference of the effect between the two treatment groups could not be proven. The study was terminated after 56 days when the control group lost the last animal. Animals of the two treatment groups with either 25 MBq [177Lu]Lu-FAUC 469 or 25 MBq [177Lu]Lu-CL 162 did not reach termination criteria until the last day of the study (Figure 42B, day 56). Thus, the average survival time remained undefined for both groups. Control and treated mice increased in weight about 13 % over the total observation period of eight weeks.

Table 30. Analysis of the data obtained from endoradiotherapy study with PC-3 tumor-bearing nude mice after injection of a single dose of 25 MBq of [177Lu]Lu- FAUC 469 or 25 MBq [177Lu]Lu-CL 162. TGI = tumor growth inhibition

Effective injected Mean survival Group Body weight [g] TGI [%] activity [MBq] [days] Control 25 ± 1 - - 47 ± 15 group [177Lu]Lu- 30 ± 8 26 ± 1 64 > 56 days FAUC 469 (Min. 22; Max. 41) [177Lu]Lu- 27 ± 3 25 ± 1 51 > 56 days CL 162 (Min. 24; Max. 31) Body weight is given as mean ± SD at therapy start Mean survival > 56 days of treated groups: mice were still alive at the end of the therapy study

105 Results and Discussion

Targeted radionuclide therapy was shown to be an established therapy option for a variety of cancer types. In principle, targeted endoradiotherapy provided the advantage of protecting healthy non-target tissue and pointedly deposit high radioactivity doses in the receptor-positive tumors. For example, this strategy was already used with [177Lu]Lu-PSMA-617 in PSMA-positive prostate cancers and [177Lu]Lu-DOTATOC in neuroendocrine tumors1,21. NTS1 was overexpressed in the PC-3 tumor cells representing a target for NTS1-directed endoradiotherapy. Endoradiotherapy with the non-peptide antagonists [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162 combined the advantages of high receptor affinity and high tumor uptake and long tumor retention with almost no accumulation in non-target tissues including excretion organs. The antagonist radioligands should be preferable because working with neurotensin agonists was challenging because of low tumor uptake and fast tumor wash-out.180-186 Another option would be working with antibodies such as Trastuzumab (Herceptin) in breast cancer.38 However, antibodies are large proteins with poor permeability and very slow clearance from the blood, together with slow tumor uptake. [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162 showed high internalization rates into tumor cells in vitro and excellent tumor retention in vivo, providing the possibility of targeted deposition of radioactivity. The demonstrated NTS1-mediated therapeutic effect with these two compounds in PC-3-bearing nude mice was comparable with results previously published by Schulz et al. for the SR 142948A-based NTS1 antagonist 3BP-227.194 This group worked with a HT-29 tumor mouse model and injected very high radioactivity amounts of 110 – 165 MBq of the radiotracer into tumor-bearing nude mice. Administration of the radiotracer resulted in a significant reduced tumor growth of 55 – 88 % compared to the vehicle (0.9 % NaCl)-injected control group. The few peptidic NTS1-targeting radioligands that had been applied to radiotherapy studies, could not prove this high anti-tumor effect.180,235

4.8.2 Toxicity evaluation At the end of the therapy study toxicity evaluation was performed analyzing liver and kidney enzymes (Figure 43A) combined with HE (Figure 43B), PAS and Masson- Goldner (not shown) staining of respective paraffin organ sections. Glucose and ALT (alanine aminotransferase) have been chosen to control liver and creatinine and bilirubin to control kidney function during endoradiotherapy. Increased levels of these markers would be a hint for liver/kidney damage and disease.245,246 Clinical chemistry

106 Results and Discussion and histological examination of the kidneys and liver did not show any pathological changes due to therapy compared to organs of control mice as confirmed by an independent pathologist (Prof. Dr. med. Kerstin Amann, Institute of Pathology, University Hospital Erlangen).

Figure 43. (A) Serum glucose, ALT (alanine aminotransferase), creatinine and bilirubin levels were significantly different among control (black) and treated (purple and green) groups (p > 0.05). (B) Microscopic images (20 ×) of PC-3 tumor (left column), kidney (middle column) and liver (right column) sections from therapy study groups stained with hematoxylin-eosin (HE). Tissue samples were prepared at the end of the therapy study and the staining results from untreated control mice and the two groups, each treated with 25 MBq of [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162, respectively, were shown. No pathological changes could be observed in any organ.

107 Results and Discussion

Analyzing creatinine to estimate kidney disease has been accepted also in mouse models.247 Even if creatinine levels in healthy mice were higher than in humans, increased levels among mice could be taken as signs for kidney disease. However, the non-peptide NTS1 antagonists showed almost no retention in the kidneys. Peptides used for peptide-receptor radionuclide therapy like somatostatin analogs for neuroendocrine tumors were mainly cleared through the kidneys, thereby causing kidney damages.248 Biodistribution data suggested the assumption that the non- peptide antagonists were extracted through the liver so that the liver could receive a significant radiation dose during the time of therapy. Histological Masson-Goldner staining of the liver did not reveal any damages (data not shown). Taken together, there were no side effects observed in selected organs that limit clinical use of NTS1 and the two non-peptide antagonists for therapeutic approaches.

4.8.3 PET Imaging with [68Ga]Ga-NODAGA-RGD Whole body small-animal PET images were obtained with the Inveon microPET scanner (Siemens Healthcare, Germany) using [68Ga]Ga-NODAGA-RGD (Figure

44A) addressing αVß3 in athymic nude mice bearing NTS1-positive PC-3 tumors in the left shoulder in order to monitor the effect of the endoradiotherapy in the three groups. αVß3 was shown to be expressed on new developing blood vessels. PET scanning was performed first as baseline measurement before injection of [177Lu]Lu- FAUC 469 and [177Lu]Lu-CL 162 followed by scanning at day 2, 7, 14, 21 and 28 after treatment. The 68Ga-labeled radiotracer demonstrated moderate uptake in the tumor and in non-target tissues, particularly in liver, kidneys and urinary bladder, so that the tumor could be visualized, although it was difficult to define the tumor region (Figure 44C). The tracer showed similar tumor uptake throughout the whole study.

Standard uptake values (SUVmean) in the tumor were in the range of 0.05 – 0.10. As shown in Figure 44B, no correlation of the SUVmean values with tumor growth or treatment could be found (no significant differences, p-values > 0.05). In principle, 68 imaging with the integrin αVß3-targeting [ Ga]Ga-NODAGA-RGD allowed non- invasive imaging of new developing blood vessels in PC-3 tumors. This process is called angiogenesis.249 Thus, angiogenesis allowed monitoring of the tumor burden and effects of endoradiotherapy in the present mouse model as application of therapeutic radioligands should decrease tumor growth and thus angiogenesis. Specific uptake and internalization of RGD in target cells has been proven for different RGD-peptides radiolabeled with various isotopes.250-253 Attention had to be

108 Results and Discussion

payed to the fact that αVß3 was expressed only on new developing blood vessels and not on mature ones.254

Figure 44. (A) Chemical structure of [68Ga]Ga-NODAGA-RGD. (B) Normalized 68 standard uptake values (SUVmean) of [ Ga]Ga-NODAGA-RGD in human PC-3 tumors in the three therapy study groups at day 0, 2, 7, 14, 21 and 28 after injection of a single dose of 25 MBq of [177Lu]Lu-FAUC 469. Control group (black) data obtained from seven animals, [177Lu]Lu-CL 162 (purple) and [177Lu]Lu-FAUC 469 (green) data were obtained from five animals per group. (C) Representative whole body small-animal PET scans of NTS1-positive PC-3 tumor-bearing nude mice of each therapy study group at 55 min post-injection of 5 MBq [68Ga]Ga-NODAGA-RGD at day 0, 2, 7, 14, 21 and 28 after begin of treatment. Images were decay-corrected and adjusted to the same maximum value. Red arrows indicate PC-3 tumors.

No difference in RGD tracer uptake could be observed between the three mice groups at any time. This observation indicated that endoradiotherapy with [177Lu]Lu- FAUC 469 and [177Lu]Lu-CL 162 did not influence measurably tumor vascularization even though differences in tumor growth could be detected (Figure 42A). These results were in contrast to RGD imaging results using [68Ga]DOTA-RGD published by Maschauer et al. in HT-29 tumor-bearing nude mice.235 Therein, significant differences in tumor standard uptake values (SUVmean) between control group and high dose (50 MBq) treatment group, which received the NTS1 agonist [177Lu]NT127,

109 Results and Discussion were observed. The latter showed decreased radiotracer uptake at day seven after treatment start. At later time points, no differences in the RGD tracer uptake could be observed between treated animals and untreated controls. This result was explained by regeneration of integrin expression in the HT-29 cells.255 PC-3 cells were formerly described as a radiation-resistant cell line, a fact that could explain missing response on the level of angiogenesis during endoradiotherapy and could suggest retained integrin expression.256-258 In this context it had to be mentioned that SUVs did not represent a method to quantify integrin expression. These values were only a hint for integrin expression that could explain the missing detection of a measurable effect. Furthermore, PET imaging was only performed in note form so that maybe the correct time point for detecting an effect was not included. Expression of integrin αVß3 on PC-3 cells and in tumor tissue was confirmed by FACS analysis (Figure 45A) and by immunohistochemistry on paraffin slices obtained at the end of the endoradiotherapy study (Figure 45B). PC-3 tumor cells were known to show low and 254 68 U87MG high αVß3 expression levels. Small-animal PET imaging with [ Ga]Ga- NODAGA-RGD did not reveal any differences in tumor vascularization, suggesting that anti-angiogenic effects were not significantly mediated by endoradiotherapy with [177Lu]Lu-FAUC 469.

Figure 45. (A) FACS analysis of U87MG (positive control) and PC-3 cells for integrin αVß3 expression. Levels were measures with FACS staining using integrin αVß3 (mouse IgG1 anti human, Merck, Germany) and a secondary FITC-labelled antibody (goat anti-mouse IgG1; BD, Germany). IgG isotypes were used as negative controls. Data were shown as one representative result out of two independent experiments. (B) Representative representation of immunohistochemical ß3 staining (arrow) of 3 µm paraffin slices of PC-3 tumors obtained at the end of the endoradiotherapy study.

In summary, this study demonstrated the therapeutic potential of NTS1-mediated endoradiotherapy. Injection of 25 MBq/mouse (day 0) of [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162 showed a significant inhibition in tumor growth in comparison to the control group with beginning of day 10 (adjusted p = 0.009 for [177Lu]Lu-CL 162 and

110 Results and Discussion adjusted p = 0.044 for [177Lu]Lu-FAUC 469). Tumor growth inhibition (TGI) at the day of the death of the first control mouse was 51 % for [177Lu]Lu-CL 162 and 64 % for [177Lu]Lu-FAUC 469. This effect was comparable with results recently published by Schulz et al. in 2017 for the SR 142948A-based NTS1 antagonist 3BP-227.194 The 177Lu-labeled 3BP-227 was injected in different doses into HT-29 tumor-bearing nude mice. At day 23 p.i. of the radiotracer tumor growth in the treated groups was reduced by 55 % (110 MBq; p = 0.006) and 88 % (165 MBq; p < 0.001) compared to the vehicle injected control group.

4.9 NTS1 as Therapeutic and Potential Imaging Target in Prostate Cancer

4.9.1 Therapy Study with [177Lu]Lu-FAUC 469 in PC3-PIP Tumor-Bearing Nude Mice In the present work, NTS1 has been characterized as theranostic target in NTS1- positive prostate cancers. As the detection of therapy success of [177Lu]Lu-FAUC 469 treatment by PET imaging with [68Ga]Ga-NODAGA-RGD appeared to be problematic, the NTS1- and PSMA-positive PC3-PIP75 cell line was introduced for the design of the following experiments. These cells represented PC-3 cells transfected with PSMA.62 The benefit using PC3-PIP cells was to be able to work with the well- established PET ligand [68Ga]Ga-PSMA-1188 as an imaging agent and the NTS1- targeting PET tracer [68Ga]Ga-NT118 for PET imaging of NTS1 along the course of the therapy. Therefore, the therapeutic effects of the non-peptide NTS1 antagonist [177Lu]Lu-FAUC 469 were imaged in PC3-PIP tumor-bearing nude mice over the period of two months (Figure 46) with [68Ga]Ga-NT118 in comparison to the reference [68Ga]Ga-PSMA-11.

111 Results and Discussion

Figure 46. (A) Relative tumor volume and (B) survival curves of the respective PC3- PIP tumor-bearing nude mice after treatment with a single dose of 25 MBq of [177Lu]Lu-FAUC 469 (n = 6) in comparison with untreated control animals (n = 6). Day 0 represented the day of radiotracer injection. Data were expressed as mean ± SD. p.i. = post-injection

The tumor volume steadily increased in the untreated control group (black). The first control mouse had to be sacrificed after 42 days, due to tumor size of more than 15 mm in diameter. The treated group (green) showed a delayed tumor growth between days 7 and 28 after beginning of endoradiotherapy compared to control group. However, the tumors again began to grow and assimilated to that of the control group. The tumor growth inhibition (TGI) at the day of the death of the first untreated animal was 52.5 %. The study was stopped after 54 days when 67 % of the control group reached the end point criteria. Termination criteria of the study were defined as a tumor diameter of > 15 mm, loss of body weight of > 15 % of initial value and when active ulceration of the tumor or obvious signs of unease were observed. 67 % of the animals of the treated group did also reach endpoint criteria after 54 days so that no prolonged survival of the treatment group could be observed. Average survival time was 47.5 days for control and 52.7 days for treated animals (Table 31).

112 Results and Discussion

Table 31. Analysis of the data obtained from endoradiotherapy study with PC3-PIP tumor-bearing nude mice after injection of a single dose of 25 MBq of [177Lu]Lu- FAUC 469.

Body weight Effective injected activity Mean survival Group [g] [MBq] [days] Control group 26 ± 1 - 48 [177Lu]Lu- 31 ± 4 28 ± 2 53 FAUC 469 (Min. 26; Max. 38) Body weight is given as mean ± SD at therapy start Mean survival > 56 days of treated groups: mice were still alive at the end of the therapy study

4.9.2 PET Imaging with [68Ga]Ga-PSMA-11 and [68Ga]Ga-NT118 [68Ga]Ga-PSMA-11 and [68Ga]Ga-NT118 were prepared and used during therapy study in athymic nude mice bearing NTS1-positive PC3-PIP tumors in the left shoulder in order to validate NTS1 as target for PET imaging. [68Ga]Ga-PSMA-11 represented a well-established radioligand used in PET imaging of PSMA expression.88 [68Ga]Ga-PSMA-11 has already been established in clinical routine for 88 primary diagnosis of tumors and for follow-up studies. Afshar-Oromieh et al. proved highly specific uptake of [68Ga]Ga-PSMA-11 in tumor tissues. However, they did not quantify their PET images and they mentioned that there was no need for a cut-off SUV, because PSMA-positive tissues outside the prostate should be regarded as prostate cancer lesions.88 Even though, there may be a chance with such a cut-off to differentiate between inflammatory disease and cancer. Also Sterzing et al. highlighted [68Ga]Ga-PSMA-11 as suitable imaging tool for individualized endoradiotherapy strategies in prostate cancer.80 [68Ga]Ga-PSMA-11 allowed visualization not only of primary, but also metastatic cancer even in patients with low PSA levels (> 1 ng/mL).88 In further consequence TNM (Tumor, lymph node and metastasis) staging and therapies could be adapted. The NTS1 agonist [68Ga]Ga-NT118 was used for PET imaging instead of [68Ga]Ga-ABN 468 or [68Ga]Ga-CL 157 as these two non-peptide antagonists resulted in PET images with high background radioactivity at 1-2 hours post-injection. Using [68Ga]Ga-NT118 allowed to visualize receptor occupancy by the therapeutic radiotracer (including the DOTA-precursor amount) and to visualize the results of the biodistribution studies. Whole body small-animal PET images were obtained with the Inveon microPET scanner (Siemens Healthcare. Erlangen. Germany). PET scanning was performed first as baseline measurement prior to the injection of [177Lu]Lu-

113 Results and Discussion

FAUC 469 followed by scanning every two weeks until day 54. As shown in Figure 47A, [68Ga]Ga-PSMA-11 demonstrated specific uptake in the tumor and enabled clear visualization with a good tumor-to-background (33:1; n = 3) contrast 1 h p.i. The tracer showed good tumor uptake throughout the whole study. High kidney uptake was observed during image acquisition. Standard uptake values (SUV) in the tumor were in the range of 0.1 – 0.4, when the excellent tumor-to-background (33:1; n = 3) contrast enabled tumor visualization. Specific uptake of [68Ga]Ga-PSMA-11 did not show any correlation of PET standard uptake values with tumor growth or treatment (Figure 47C). [68Ga]Ga-NT118 showed tumor uptake in NTS1-positive PC3-PIP tumors (0.26 ± 0.05). Beside the accumulation of radioactivity in the tumor, the compound revealed very high uptake in the kidneys. During the study, confines of the tumors became indistinct (Figure 47B). Standard uptake values in the tumor were between 0.06 – 0.2. A significant difference of the tumor uptake (SUV) could be proven at day 7 after [177Lu]Lu-FAUC 469 injection (p = 0.03; Figure 47D).

114 Results and Discussion

Figure 47. Whole body small-animal PET images of NTS1- and PSMA-positive PC3- PIP tumor-bearing nude mice with (A) [68Ga]Ga-PSMA-11 and (B) [68Ga]Ga-NT118 at 45 min post-injection during endoradiotherapy with [177Lu]Lu-FAUC 469. Mice were injected with 5 MBq of [68Ga]Ga-PSMA-11 or [68Ga]Ga-NT118. Beside uptake in the tumor region, the radioligand was extracted through the kidneys. Images were decay- corrected and adjusted to the same maximum value. The relative standard uptake values (SUVmean, normalized to SUV = 1 for the begin of treatment at day 0) of [68Ga]Ga-PSMA-11 (C) and [68Ga]Ga-NT118 (D) in PC3-PIP tumors at day 0, 7, 14, 28 and 42 after injection of a single dose of 25 MBq of [177Lu]Lu-FAUC 469 were shown on the right. Data were expressed as mean values ± SD from three animals per group in control (black) and treated (blue) mice.

115 Results and Discussion

The small-animal PET scans shown above were in accordance with the ones published by Eder et al.16 PET imaging of LNCaP tumor-bearing nude mice was performed during their study in 2012. Beside the tumor, kidneys and bladder were clearly visualized, reflecting the results in Figure 47A. All in all, [68Ga]Ga-PSMA-11 was a specific diagnostic tool for prostate cancer being confirmed by the clear PET images obtained during the present study. Furthermore, small-animal PET scans showed that PSMA expression was retained on the tumor cells throughout the whole endoradiotherapy study. With the beginning of day 30 a small increase in the standard uptake values could be observed. This came along with the renewed tumor growth (Figure 46A). PET image analysis of the NTS1-positive PC3-PIP tumors with [68Ga]Ga- NT118 revealed significant differences between treated and control group at day 7 after starting of endoradiotherapy. During the present study, small-animal PET imaging and endoradiotherapy were performed at the same time targeting the same receptor. This procedure raises the possibility that at day 7 after begin of treatment there could be a significant amount of receptors occupied by the therapeutic tracer [177Lu]Lu-FAUC 469 (containing 3 – 4 nmol of the DOTA-precursor) and thus were not available for binding of [68Ga]Ga-NT118. In the cases of a therapeutic dose of [177Lu]Lu-FAUC 469, the concentration of the unlabeled DOTA-precursor in the blood easily exceeds the Kd (~ 0.2 nM) so that a receptor occupancy of more than 50 % could be assumed. The in vivo specificity of the tracer and internalization has been shown in the present study (4.5) and by Maschauer et al.195 Nevertheless, PET imaging with [68Ga]Ga-NT118 throughout the whole study showed that NTS1 expression was stable during the endoradiotherapy study. This indicated that the receptor would be available for a second therapy cycle that would be a common procedure in the clinic.

All in all, small-animal PET imaging demonstrated that NTS1 represented an appropriate target for PET imaging of NTS1-positive tumors in the used animal model. Receptor specific uptake of [68Ga]Ga-NT118 into the NTS1-positive PC3-PIP tumors resulted in PET images with definable tumors. Beside the accumulation of radioactivity in the tumor, [68Ga]Ga-NT118 revealed high uptake in the kidneys. Furthermore, a significant difference of the tumor uptake (SUV) could be proved at day 7 after [177Lu]Lu-FAUC 469 injection (p = 0.03; Figure 47D). However, attention should also be payed to the fact that imaging with the small-animal PET is quite easy

116 Results and Discussion compared to PET imaging in clinics with patients because in the animal models tumors were transplanted in the shoulder in far distance from the chest and body containing all organs. Furthermore, the translation of animal PET data to humans was pointed out to be problematic,5,259 because rodents and humans have different metabolisms. One example for this fact was [18F]F-DOPA that showed uniform distribution in the brain of mice without specific accumulation in the striatum whereas the radiotracer accumulated specifically in the striatum of men.5

4.9.3 Biodistribution of [68Ga]Ga-PSMA-11 and [68Ga]Ga-NT118 In addition to small-animal PET imaging, comparison of biodistribution data between [68Ga]Ga-PSMA-11 (n = 4) and [68Ga]Ga-NT118 (n = 3) were performed in tumor- bearing nude mice with PC-3 tumor on the right and PC3-PIP tumor on the left shoulder at 1 h p.i (Figure 48). This study was performed as the detection and therapy of PSMA-negative lesions and in the case of metastatic and hormone- refractory prostate cancer remains a challenge. Therefore, there is urgent need for new diagnostic tools. NTS1 could be a target for diagnosis enabling the visualization of the tumor for estimation of tumor size and location, influencing strategies for patient treatment in the case of PSMA-negative prostate cancers. [68Ga]Ga-PSMA-11 showed specific uptake in the NTS1- and PSMA-positive PC3-PIP tumor (1.46 ± 0.32 % ID/g) whereas only low amounts of radioactivity could be detected in the PSMA-negative PC-3 tumors (0.05 ± 0.02 % ID/g). [68Ga]Ga- NT118 was measurable in both NTS1-expressing tumors reaching values of 0.46 ± 0.09 % ID/g in PC-3 and 0.26 ± 0.05 % ID/g in PC3-PIP tumor. PC3-PIP cells expressed very high amounts of PSMA (9382 fmol/mg; [68Ga]Ga-PSMA-11) compared to NTS1 (49 ± 4 fmol/mg; [68Ga]Ga-ABN 468). These numbers reflected the in vivo uptake of these two radiotracers (1.46 ± 0.32 vs. 0.26 ± 0.05 % ID/g, respectively) although the internalization studies revealed only 20 ± 2 % of internalized radioligand for [68Ga]Ga-PSMA-11 in contrast to 49 ± 6 % for [68Ga]Ga- ABN 468 after 60 min. This clearly demonstrated that the measured internalization in vitro could not predict the uptake rate in vivo. Comparing the cell lines PC3-PIP and PC-3, the higher NTS1-mediated uptake of [68Ga]Ga-ABN 468 in PC-3 cells (0.46 ± 0.09 % ID/g) compared to PC3-PIP cells (0.26 ± 0.05 % ID/g) was in accordance with the high receptor density of 126 fmol/mg ([68Ga]Ga-ABN 468) NTS1 on PC-3 cells compared to 49 ± 4 fmol/mg ([68Ga]Ga-ABN 468) NTS1 on PC3-PIP

117 Results and Discussion cells. Furthermore, NTS1-mediated internalization rate of PC-3 cells was about 20 % higher compared to internalization rate of PC3-PIP cells (66 ± 9 % vs. 49 ± 6 %). Both [68Ga]Ga-PSMA-11 and [68Ga]Ga-NT118 showed high radioactivity accumulation in the kidneys with 10.98 ± 2.74 % ID/g for [68Ga]Ga-PSMA-11 and 8.87 ± 0.78 % ID/g for [68Ga]Ga-NT118. [68Ga]Ga-PSMA-11 could not be detected in some other non-target tissues (Figure 48A) except conspicuous accumulation in the spleen (1.17 ± 0.68 % ID/g). PC3-PIP tumor-to-organ ratios of [68Ga]Ga-PSMA-11 were > 8 for all organs except kidneys and spleen as calculated from data above (Figure 48B). Specificity of radiotracer uptake could be confirmed by no accumulation in PSMA-negative PC-3 tumors. [68Ga]Ga-NT118 showed tumor uptake of 0.46 ± 0.09 % ID/g in PC-3 and 0.26 ± 0.05 % ID/g in PC3-PIP tumors. Beside the accumulation of radioactivity in the tumors, the compound revealed high uptake in the kidneys (8.87 ± 0.78 % ID/g). Uptake values in muscle and bone were comparable with that in PC-3 tumors (Figure 48C). Except for kidneys (0.05 ± 0.01 for PC-3 and 0.03 ± 0.01 for PC3-PIP), tumor-to-organ ratios were > 1 for all organs and both tumor entities (Figure 48D). Blood clearance for both radiotracers was fast leading to tumor-to-blood ratios of 33 for [68Ga]Ga-PSMA-11 and 2 and 3 for [68Ga]Ga-NT118 for PC3-PIP and PC-3 tumors, respectively. The specificity of tumor uptake of [68Ga]Ga-NT118 in vivo has previously been shown by Maschauer et al.195

118 Results and Discussion

Table 32. Biodistribution of [68Ga]Ga-PSMA-11 and [68Ga]Ga-NT118 in PC-3 (right shoulder) and PC3-PIP (left shoulder) tumor-bearing nude mice at 1 h post-injection. The values were given as mean % ID/g ± SD, n = 3 Organ [68Ga]Ga-PMSA-11 [68Ga]Ga-NT118 Blood 0.04 ± 0.01 0.15 ± 0.03 Lung 0.18 ± 0.05 0.11 ± 0.02 Liver 0.08 ± 0.02 0.11 ± 0.01 Kidney 10.98 ± 2.74 8.87 ± 0.78 Heart 0.05 ± 0.03 0.05 ± 0.02 Spleen 1.17 ± 0.68 0.08 ± 0.00 Brain 0.00 ± 0.00 0.01 ± 0.00 Muscle 0.05 ± 0.05 0.35 ± 0.31 Bone 0.07 ± 0.05 0.50 ± 0.69 PC-3 tumor 0.05 ± 0.02 0.46 ± 0.09 PC3-PIP tumor 1.46 ± 0.32 0.26 ± 0.05 Small intestine 0.10 ± 0.08 0.07 ± 0.01 Colon 0.04 ± 0.01 0.17 ± 0.09 Stomach 0.05 ± 0.02 0.04 ± 0.01 PC3-PIP-to-blood ratio 33.11 ± 7.80 2.03 ± 0.87 PC3-PIP-to-kidney ratio 0.09 ± 0.03 0.03 ± 0.01 PC3-PIP-to-Muscle ratio 27.61 ± 18.54 1.63 ± 1.95

119 Results and Discussion

Figure 48. Biodistribution and tumor-to-organ ratios of [68Ga]Ga-PSMA-11 and [68Ga]Ga-NT118 in PC-3 and PC3-PIP tumor-bearing nude mice (each n = 4) at 1 h p.i. (A) Biodistribution data of [68Ga]Ga-PSMA-11 and (B) related tumor-to-organ ratios. Data were expressed as mean values in percent (%) of the injected dose per mass (g) of tissue (% ID/g) ± standard deviation (SD) from two to three mice. (C) Biodistribution of [68Ga]Ga-NT118 and (D) related tumor-to-organ ratios.

[68Ga]Ga-PSMA-11 was shown to accumulate in endogenous expressing PSMA- positive LNCaP cells and not in PC-3 cells (7.70 ± 1.45 vs. 1.30 ± 0.12 % ID/g).16 Furthermore, fast clearance from circulation and non-target tissues could be proved at 1 h p.i. with exception of kidney, spleen and lung. These results were in accordance with the ones obtained during the present study. This observation was also in agreement with the findings obtained from patients who showed high accumulation of radioactivity in kidneys.64 The peptide [68Ga]Ga-NT118 for PET imaging of NTS1 was published by Maschauer et al. who showed specific tumor uptake (1.55 % ID/g) with good tumor-to-background ratios of 31 in HT-29 tumor- bearing nude mice and fast blood clearance.195 Furthermore, radioactivity was found to accumulate in kidneys (35 – 45 % ID/g) indicating renal excretion. Tumor uptake was 2-times higher and tumor-to-blood ratios of 31 after 60 min were 15-times higher than the PC3-PIP-tumor-to-blood ratio obtained in the present study. In contrast,

120 Results and Discussion kidney uptake was about 4-fold higher than in the present study. Difference in tumor accumulation could be explained by the higher NTS1 density on HT-29 compared to PC-3 and PC3-PIP cells (4.6). Distribution of both tracers in tumor, liver and kidneys was exemplarily shown in Figure 49. Uptake of [68Ga]Ga-PSMA-11 was in accordance with biodistribution studies and visualized by ex vivo autoradiography that revealed uptake in kidney and PC3-PIP tumors only, but not in liver and PC-3 tumors. [68Ga]Ga-NT118 showed a similar distribution profile like [68Ga]Ga-PSMA-11 with accumulation of radioactivity in the kidneys and not in liver. In addition, ex vivo slices of PC3-PIP and PC-3 tumors, kidneys and liver have been prepared of animals injected with therapeutic tracer [177Lu]Lu-FAUC 469 (Figure 49). Uptake of the radiotracer could be observed in both tumors, whereby accumulation in PC-3 tumor seemed to be higher than in PC3-PIP tumor. There was only low radioactivity in the kidneys and moderate uptake in the liver.

Figure 49. Representative ex vivo autoradiography of tissue slices after injection of mice with [68Ga]Ga-NT118, [68Ga]Ga-PSMA-11 at 1 h p.i. and [177Lu]Lu-FAUC 469 in PC3-PIP and PC-3 tumor-bearing nude mice at 14 days post-injection. Organs of mice (n = 2) were removed, frozen and cut in 20 µm slices that were exposed to a phosphor screen for 24 h.

The tissue distribution of [68Ga]Ga-PSMA-11 and [68Ga]Ga-NT118 was similar. Both tracers showed high clearance in kidneys and specific tumor uptake. [68Ga]Ga- PSMA-11 revealed better tumor-to-organ ratios, explaining the PET images with higher contrast compared to those conducted with [68Ga]Ga-NT118. The kidney uptake of [68Ga]Ga-PSMA-11 and [68Ga]Ga-NT118 was due to excretion of these tracers through the kidney.180,182,243 The definable tumor uptake of [68Ga]Ga-NT118

121 Results and Discussion throughout the preclinical study indicated that NTS1 was a suitable target not only for endoradiotherapy but also for molecular imaging and therefore diagnosis of receptor- positive cancers.

4.10 Quantification of NTS1 Density on Tumor Cells and Xenograft Tissue qPCR analysis of xenografts and saturation binding assays using tumor cells of in vitro cell culture, corresponding xenografts and resulting ex vivo cell cultures were compared in order to analyze if endoradiotherapy and PET imaging had influence on NTS1 mRNA expression or NTS1 protein density expressed on the prostate cancer cells. NTS1 mRNA analysis was performed on samples of PC-3 xenografts obtained from control and treated mice at the end point of the therapy study (Figure 50). NTS1 mRNA could be detected in all tissue samples regardless of whether mice received 177Lu-treatment or not. Within the treatment group detection levels were lower compared to control group, even though not with statistically significant differences. The NTS1 mRNA expression in the treatment group differed a lot among the samples resulting in the high values for SEM. NTS2 expression could not be detected in any sample. The NTS1 protein levels could not be analyzed by SDS-Page and Western blotting due to the lack of specific antibodies (compare 4.1).

Figure 50. qRT-PCR analysis of NTS1 expression (vertical axis) ± SEM in PC-3 tumor xenograft tissues of control (n = 6) and treated mice (n = 2) obtained from therapy study (4.8.1.). HT-29 sample was carried along as positive control. Results were shown relative to internal control GAPDH.

For in vitro and ex vivo determination of NTS1 density saturation binding studies using the two prostate cancer cell lines PC-3 (Figure 51A) and PC3-PIP (Figure 122 Results and Discussion

51B) were carried out. Receptor densities of respective xenografts were obtained by autoradiography (Figure 51C) using [125I]Tyr3-NT.

Figure 51. Saturation binding curves of [68Ga]Ga-ABN 468 using (A) PC-3 and (B) PC3-PIP cells that originated out of tumor xenografts of nude mice determined after 1 h incubation time at 4 °C (mean ± SD, n = 9). Nonspecific binding was measured in the presence of 1 µM NT. (C) Representative illustration of expression of NTS1 by in vitro receptor autoradiography in PC-3 tumor tissue. Autoradiographies of slices showed control (total binding, 90 pM [125I]Tyr3-NT), nonspecific binding (competitive binding experiment in the presence of 1 µM NT) and corresponding HE staining.

Results were summarized in Table 33. These data showed that on PC-3 cells receptor numbers in ex vivo cell culture was lower (71 ± 8 fmol/mg) compared to routine in vitro cell culture conditions (126 ± 6 fmol/mg) whereas such a tendency could not be observed on PC3-PIP cells (49 ± 5 fmol/mg vs. 48 ± 7 fmol/mg). Receptor density in xenografts was substantially lower compared to cell culture conditions (factor 100). These results were comparable with the ones of Rogers et al. from 2003 who proved that receptor numbers for PC-3 tumors in general were lower in xenografts compared to the corresponding cultured tumor cells (1:15).260 Receptor numbers did not show any correlation with treatment or tumor volume. The finding that receptor density was not influenced by the endoradiotherapy was very promising for the translation of NTS1-targeted radiotherapy into the clinic. The presence of the NTS1 independent of tumor volume and treatment means that for targeted

123 Results and Discussion endoradiotherapy in humans various treatment cycles and follow-up PET scans addressing the NTS1 would be possible.

Table 33. Bmax [fmol/mg] ± SD values for NTS1 resulting from saturation binding assays using [68Ga]Ga-ABN 468 on the two prostate cancer cell lines PC-3 and PC3- PIP. Data were obtained in three independent experiments each performed in triplicate (n = 9).

Time/Cell line PC-3 PC3-PIP In vitro cell culture 126 ± 6 fmol/mg 49 ± 5 fmol/mg Tissue slices 0.07 ± 0.07 fmol/mg 0.32 ± 0.02 fmol/mg Ex vivo cell culture 71 ± 8 fmol/mg 48 ± 7 fmol/mg

FACS analysis has been performed to confirm the purity of ex vivo cell cultures in order to ensure that values of receptor densities have not been distorted by the presence of other NTS1-positive or –negative cells. Figure 52 showed a representative result of FACS analysis performed with cells gained by ex vivo cell culture of a PC-3 or PC3-PIP tumor xenograft. Experiments revealed purities of obtained cell cultures between 85 and 98 %. Cells from mouse spleen were used as positive control for H2kb. IgG control isotypes were used as negative control.

Figure 52. Representative FACS analysis of cells gained by ex vivo cell culture of PC3-PIP tumor xenograft for human CD71 expression and mouse H2kb expression. PC-3 cells were used as positive control for CD71 and cells from mouse spleen for H2kb. IgG antibodies were used as negative control. Experiments revealed purities between 80 and 98 % (n = 3).

124 Results and Discussion

4.11 Quantification of NTS1 Density in Primary Patient Tissue

4.11.1 qRT-PCR Results of NTS1, NTS2 and PSMA in Prostate Cancer Patients Tissue For this study, expression of NTS1, NTS2 and PSMA mRNA were determined relative to GAPDH mRNA in 98 samples of normal prostate and prostate cancer tissue. Tumor stages varied between pT2a and pT4. Each cDNA was amplified by real-time PCR using GAPDH specific primers in order to test if internal control was consistent between healthy and malignant tissue samples. This was the case for the two groups so that both groups could be compared for NTS1, NTS2 and PSMA expression (Figure 53).

Figure 53. Expression of GAPDH mRNA in patient tissue. Expression of GAPDH mRNA was determined in a total of 98 patient samples of normal prostate and prostate cancer tissue using quantitative real-time PCR. Shown were data from grouped patients ± SD with tissue assignment on the y-axis.

Expression of NTS1, NTS2 and PSMA mRNA was determined in all 98 samples of normal prostate and prostate cancer tissues (Figure 54). Out of these 98 tissue samples, 53 had been tested positive for NTS1, 47 positive for NTS2 and 77 positive for PSMA. PSMA expression was 2-times higher in prostate cancer tissues (n = 41) compared to normal prostate (n = 39) being statistical significant with p = 0.0304 (Figure 54A). The expression of NTS1 (Figure 54B) was equally expressed in healthy (n = 24) and malignant tissues (n = 26), NTS2 (Figure 54C) was 1.4-fold higher in cancerous tissues (n = 22) compared to healthy tissues (n = 25). However, this difference in expression was not statistically significant. PSMA and NTS1 were

125 Results and Discussion equally expressed among the tissues independent of tumor classification, Gleason score and tumor volume. NTS2 showed very high expression in small tumors (1-2 cm³, Figure 54D) of moderate differentiation (Figure 54E) with a Gleason Score of 7 (Figure 54F).

Figure 54. Expression of (A) PSMA, (B) NTS1 and (C) NTS2 mRNAs (y-axis) in primary healthy and cancerous patient tissues (x-axis) from 49 patients determined using quantitative real-time PCR. Dependence of PSMA, NTS1 and NTS2 expression of (D) tumor volume, (E) tumor classification (TNM) and (F) Gleason- score. Patient cohorts were grouped for analysis. (A – C) showed scatter dot plots ± SD. p = 0.0304. (D – F) showed mean ± SEM.

Primary patient tissues included in this study have been collected from a total of 49 patients. qPCR showed that 52 % of all samples were NTS1 positive, including normal and cancer tissue in a ratio of 1:1. All tested samples showed expression of both NTS1 and PSMA mRNAs except one single case which revealed only NTS1 but no PSMA expression. NTS1 and NTS2 expression was shown in 37 % of the analyzed cases. Detection rates during NTS1 expression analysis performed by Swift

126 Results and Discussion et al. were substantially higher (89 %) compared to the present study.156 Rates for NTS2 were in accordance with the present findings (44 %). In the present collective statistically significant difference between healthy control and tumor tissue could only be proved for PSMA expression. Unfortunately, this was not true for NTS1 and NTS2. Tumor lesions were identified by an independent pathologist. Even if care was taken to differentiate between healthy and malignant tissue results might differ by using laser-microdissection to separate normal and tumor tissue. Schmittgen et al. reported mRNA analysis of PSMA in 107 patient tissues.79 They could not prove statistical relevant differences in PSMA expression among normal, benign and cancerous prostate tissues but showed PSMA increasing with Gleason score. Correlations between (pre-) treatment or therapy and PSMA expression as described by Wright et al. could not be assessed because relevant data were not available.76 They demonstrated correlation between pre-treatment and increasing PSMA expression after androgen-deprivation therapy. Results from the present study were in accordance with findings from Afshar-Oromieh et al. who also did not find an association between PSMA expression and Gleason-score.88

4.11.2 Autoradiography Results of NTS1 in Prostate Cancer Patients Overall, NTS1 expression was detected in 9 % (9 of 98 samples) of both human normal and tumor tissues but only 6 % of tumor tissue samples (3 of 54 cases) expressed NTS1 on protein level (Figure 55). Receptor density differed a lot among individual samples (0.01 – 0.41 fmol/mg) and no correlation could be shown to tumor staging (TNM), tumor volume or tumor grading (Gleason-score). Comparable studies were hard to find in the literature. Swift et al. showed NTS1 protein expression by Western blotting in 2 of 14 patient samples (14 %).156 However, as discussed in 4.1, specificity of the NTS1 antibodies was doubtful. In 1999, Reubi et al. described prostate tumors to be NTS1-negative. This group did not find NTS1 expression in one single tissue sample of human prostate cancer tissues out of 15.261

127 Results and Discussion

Figure 55. NTS1 density in 98 normal prostate and prostate cancer tissue samples from a total of 49 patients. Density of NTS1 on tissues [fmol/mg] was analyzed by quantitative in vitro receptor autoradiography using 90 pM [125I]Tyr3-NT. Nonspecific binding was determined by co-incubation with 1 µM NT.

In Table 34 in vitro receptor autoradiography data of NTS1 density in patients suffering from prostate cancer were summarized. Information about previous treatments was not available for all patients.

128 Results and Discussion

Table 34. NTS1 expression in primary tissue of prostate cancer patients. Values represented NTS1 receptor density in fmol/mg tissue as mean of two separate values. For the determination of nonspecific binding, the binding of [125I]Tyr3-NT (90 pM) was measured in the presence of 1 µM NT.

Patient number Receptor density [fmol/mg] Receptor density [fmol/mg] tumor tissue normal prostate tissue 1 - 7 0 0 8 0 0 9 - 18 0 0 19 0.01 0.02 20 0 0.01 21 0 0 22 0 0 23 - 30 0 0 31 0.41 0 32 - 34 0 0 35 0 0.09 36 - 44 0 0 45 0 0.02 46 - 49 0 0

Figure 56 showed a representative in vitro tissue analysis of a patient (patient no. 31) with very high NTS1 expression. Patient no. 31 was 76 years old at diagnosis, had a PSA-level of 16 ng/ml and a tumor volume of 11.5 cm³. The tumor with Gleason-Score 9 was poorly differentiated with pT3 classification, infiltrating surrounding lymph nodes. The maximal binding capacity measured in this tissue was 0.41 fmol/mg. [125I]Tyr3-NT binding could be completely displaced in the presence of 1 µM NT. In general, NTS1 displayed a dense, homogeneous distribution throughout the tumor. Positive results for NTS1 were obtained for three of the 54 tumors; the remaining 51 were NTS1-negative tumors. Unfortunately, NTS1 was equally expressed on normal prostate tissue compared to tumor tissue.

129 Results and Discussion

Figure 56. In vitro autoradiography of tissue slices of patient 31 showing NTS1 expression. The tumor showed very high NTS1 density. Autoradiograms showed total binding of [125I]Tyr3-NT (control) and nonspecific binding (“blocking”). For competitive binding studies, binding of [125I]Tyr3-NT was displaced in the presence of 1 µM NT. Corresponding sections were stained with HE.

Although NTS1 mRNA was found in 52 % (51 of 98 cases) of analyzed samples, NTS1 protein expression was only observed in 9 % (9 of 98 cases). Unfortunately, out of these, only three had been prostate cancer cases and the rest normal prostate tissue. Thus, NTS1 mRNA was no reliable and suitable marker for prostate cancer. Among the NTS1 expressing cases no correlation could be found between NTS1 levels and pathological grading, former therapies or tumor volume. In the study of Swift et al., 89 % (8 of 9 cases) of malignant patient tissues were found positive for NTS1 mRNA expression. In contrast, all three tested samples of normal prostate tissue were found negative. However, this group did not compare between mRNA findings and protein expression.156 In addition, Wang et al. confirmed that NTS1 mRNA was not a reliable and suitable marker for cancer, as they found mRNA expression of NTS1 both in the normal pancreas and in pancreatic cancer tissue, whereas NTS1 protein was only found in pancreatic carcinomas.154 Prostate cancer represents an androgen-dependent cancer. During hormone- deprivation therapy,262 NTS1 overexpression could provide an alternative growth mechanism leading to increased cell proliferation and invasion.175 The presence of high amounts of NTS1 in prostate cancer could therefore be responsible for developing androgen-independent relapses resulting in poor prognosis.56 Analysis of NTS1 expression revealed dependence of NTS1 expression state from differentiation and NTS1 was also found in the normal prostate gland in different compartments in the study of Swift et al.156

130 Results and Discussion

4.11.3 qRT-PCR Results of NTS1 and NTS2 in Pancreatic Cancer Patient Tissues Expression of NTS1 and NTS2 mRNA was determined in all 38 samples of normal pancreas and pancreatic cancer tissues originating from a total of 23 patients (Figure 57). Unfortunately, tumor and healthy control tissue was not available from every single patient. Out of the 38 tissue samples, 23 had been tested positive for NTS1 (61 %) and 18 positive for NTS2 (47 %). Among the NTS1-positive samples, only 5 (38 %) represented healthy prostate tissue and 18 (72 %) pancreatic cancer tissues. Concerning NTS2, 3 of the 18 NTS2-positive tissues (17 %) represented healthy pancreas tissue, whereas the remaining 15 samples were pancreatic cancer tissues (83 %). The relative gene expression of NTS1 (Figure 57A; C) was equally in healthy (n = 5) and malignant tissues (n = 18, t-test p = 0.7595). Nevertheless, in fact only 5 patients revealed NTS1 expression in healthy control tissue, so that it has to be kept in mind that only a minority of patients expressed NTS1 in healthy pancreatic tissue at all. That knowledge became more important when considering that all healthy tissue samples that expressed NTS1 mRNA had corresponding NTS1-positive pancreatic cancer samples. NTS2 (Figure 57B, D) was 7-fold higher in cancerous tissues (n = 15) compared to healthy tissues (n = 3) (t-test p = 0.6125). This result was probably attributed to the fact that NTS2 expression showed extreme high variations among the samples. Both NTS1 and NTS2 mRNA expression was shown in 32 % of the analyzed cases.

131 Results and Discussion

Figure 57. Expression of (A) NTS1 and (B) NTS2 mRNAs (y-axis) in primary healthy and cancerous patient tissues (x-axis) from 23 patients determined using quantitative real-time PCR. Patient cohorts were grouped for analysis. (A) and (C) showed scatter dot plots ± SD. (C) and (D) showed mean ± SEM of the patient cohorts.

Detection rates of NTS1 expression analysis performed by Wang et al. revealed similar results.154 That group did Northern Blot analysis of normal pancreatic tissue, chronic pancreatitis tissue and pancreatic cancer tissues. Their experiments showed NTS1 mRNA expression in 70 % of chronic pancreatitis and in 83 % of pancreatic cancer patients. They could prove statistical significant differences (p < 0.01) in NTS1 expression between the different groups in contrast to the present study. Comparing NTS1 mRNA levels between healthy tissues and chronic pancreatitis or pancreatic cancer, mRNA levels were increased by 3-fold and 4.4-fold, respectively. Comparing chronic pancreatitis and pancreatic cancer did not reveal differences in expression levels. Furthermore, they could prove that NTS1 mRNA showed higher levels in advanced tumor stages compared to earlier stages and that tumor differentiation had no influence on NTS1 mRNA expression levels. This last comparison could not be performed during the present study as no tissue samples of chronic pancreatitis were available and all pancreatic cancer tissues were defined as tumor stage III.

132 Results and Discussion

4.11.4 Autoradiography Results of NTS1 in Pancreatic Cancer Patients Overall, NTS1 protein expression was detected in 63 % (24 of 38 samples) of both human normal and tumor tissues. Among “normal” healthy pancreas tissue samples only 4 out of 13 expressed NTS1 protein. Receptor density differed a lot among individual samples showing values in the range of 0.004 to 0.28 fmol/mg (± 200 %) explaining the widespread scatter plot. Tumor tissue samples all derived from tissues with the staging T3. Out of tumor tissue samples 80 % (20 of 25 cases) expressed NTS1 on protein level (Figure 58).

Figure 58. NTS1 density in 38 “normal” healthy pancreas and pancreatic cancer tissue samples from a total of 23 patients. Density of NTS1 on tissues [fmol/mg] was analyzed by quantitative in vitro receptor autoradiography using 90 pM [125I]Tyr3-NT. Nonspecific binding was determined by co-incubation with 1 µM NT. In the column bar graph the one outlier among the healthy tissue samples was excluded. In Table 35 in vitro receptor autoradiography data of NTS1 density in patients suffering from pancreatic cancer have been summarized.

133 Results and Discussion

Table 35. NTS1 expression in primary tissue of pancreatic cancer patients. Values represented NTS1 receptor density in fmol/mg tissue as mean of two separate values. For nonspecific binding 90 pM [125I]Tyr3-NT was displaced by 1 µM NT.

Patient number Receptor density [fmol/mg] Receptor density [fmol/mg] tumor tissue normal prostate tissue 1 0.03 0.28 2 0.01 - 3 0.01 0.02 4 0.05 - 5 - 0.01 6 0.00 0.03 7 0.02 0.00 8 0.00 - 9 0.0005 0.0043 10 0.01 - 11 0.00 0.00 12 0.02 - 13 0.02 0.00 14 0.006 - 15 0.005 - 16 0.003 - 17 0.24 0.00 18 0.06 0.00 19 0.005 0.000 20 0.05 0.00 21 0.02 - 22 0.01 0.00 23 0.03 0.00

Figure 59 showed a representative in vitro tissue analysis of a patient (patient no. 18) with high NTS1 expression in the pancreatic carcinoma. The tumor with T3 grading was confined to the pancreas and is larger than 4 cm. The maximal binding capacity measured in this tissue was 0.06 fmol/mg. [125I]Tyr3-NT binding could be completely displaced in the presence of 1 µM NT. Corresponding healthy pancreatic tissue was proven NTS1-negative. In general, NTS1 displayed an inhomogeneous distribution throughout the tumors. Positive results for NTS1 were obtained for 20 of the 25 tumors; the remaining 5 cases were NTS1-negative tumors.

134 Results and Discussion

Figure 59. In vitro autoradiography of tissue slices of patient 18 showing NTS1 expression. The tumor showed very high NTS1 density whereas corresponding healthy pancreas tissue was NTS1-negative. Autoradiograms showed total binding of [125I]Tyr3-NT (“control”) and nonspecific binding (“blocking”). For competitive binding studies, binding of [125I]Tyr3-NT was displaced in the presence of 1 µM NT. Corresponding sections were stained with HE. Wang et al. performed NTS1 mRNA analysis and autoradiography on four tissue samples of pancreatic cancer patients.154 They revealed mRNA expression in normal pancreas, chronic pancreatitis as well as in pancreatic cancer. During autoradiography, only cancer samples revealed NTS1 protein expression: one sample was tested weakly positive for NTS1, one moderately positive and two samples strongly positive. However, this group did not provide quantitative values. In the present study, 31 % of healthy pancreatic tissues also showed translation of the NTS1 mRNA into protein. However, the patient collective of Wang et al. covered only 5 cases of normal healthy tissues, and 4 cases of both chronic pancreatitis and pancreatic cancer. J. C. Reubi suggested NTS1 as new marker for the human ductal adenocarcinoma as 75 % of all tested ductal pancreatic adenocarcinoma samples expressed NTS1 in his study, which is in accordance with the 80 % positive-tested samples of the present study.151 Among all the samples they revealed a wide spectrum in receptor densities between the lack of NTS1 expression up to 8000 dpm/mg tissue. Furthermore, they could prove that the receptor was more frequently expressed in differentiated than in undifferentiated carcinoma. NTS1 showed a heterogeneous distribution throughout the tumors. Samples of chronic pancreatitis did not show measurable levels of NTS1. Körner et al. published NTS1 protein expression cannot only be found in primary pancreatic tumors, but also in the

135 Results and Discussion corresponding liver metastases.179 They analyzed 28 samples of pancreatic cancer patients. Among these samples, 73 % of primary pancreatic cancers and 63 % of metastases expressed NTS1. Tumors showed high receptor densities above 3000 dpm/mg tissue. They could not find any relationship between former therapies and the NTS1 expression status. Though they could show that with increasing malignancy NTS1 expression was also increasing. This comparison could not be performed during the present study, as all tumors were staged as T3. In general, the results described above were in accordance with the ones found in the present study. Overexpression of NTS1 was shown in 80 % of pancreatic cancer tissues combined with absence in normal pancreas. Therefore, NTS1 represents a suitable clinical target for diagnosis and endoradiotherapy of pancreatic cancer. The differentiation between normal healthy pancreatic tissue and carcinoma was possible by targeting NTS1.

136 Conclusion

5 Conclusion

In this study, the NTS1 was evaluated for theranostic approaches as potential target for molecular imaging as well as for endoradiotherapy of prostate and pancreatic cancer. The in vitro and preclinical in vivo experiments of this study revealed promising results. The internalization assays showed high NTS1-mediated uptake of the two used 177Lu- and 68Ga-labeled non-peptide antagonists FAUC 468 and CL 156 in the PC-3 and PC3-PIP prostate cancer cells (55 -75 %). Biodistribution studies using PC-3 xenografted NMRI Foxn1nu mice showed a specific, prominent tumor uptake of 11.8 - 15.5 % ID/g at 48 h p.i. with excellent tumor retention until day seven p.i. For [177Lu]Lu-FAUC 469 lung (6.78 ± 7.18 % ID/g), liver (13.8 ± 6.74 % ID/g), kidney (4.33 ± 0.17 % ID/g) and spleen (2.67 ± 0.92 % ID/g) showed unspecific radioactivity uptake at 1 day p.i. Nonspecific accumulation of [177Lu]Lu-CL 162 could be detected only in the liver (1.06 ± 0.15 % ID/g). An increasing tumor-to-blood ratio was calculated for [177Lu]Lu-FAUC 469 from 1.55 ± 1.39 after 1 h p.i. to 557 ± 172 after 7 days and for [177Lu]Lu-CL 162 from 1.2 after 1 h up to 2407 after 7 days from the biodistribution data. All tumor-to-tissue ratios were ≥ 1 for non-target tissues for time points > 24 h for [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162. Tumor uptake was higher than in all other selected organs at all points after [177Lu]Lu-CL 162 injection. Preclinical NTS1-mediated endoradiotherapy in PC-3 tumor-bearing NMRI Foxn1nu mice revealed impressive inhibition of tumor growth of 51 % for [177Lu]Lu-CL 162 and 64 % for [177Lu]Lu-FAUC 469 and prolonged survival of treated mice. PET imaging and quantification of resulting acquisitions clearly demonstrated that NTS1 represented an appropriate target for diagnosis and monitoring of NTS-positive cancers. In conclusion, NTS1 was a suitable target for both molecular imaging and endoradiotherapy of NTS1-positive pancreatic and prostate cancers. These data suggested that appropriate radiotracers should be evaluated for clinical use. Therefore, in the meantime data regarding binding to plasma, metabolic stability have been collected. An external toxicity study in fifteen Wistar rats at hameln rds a.s. in Slovakia confirmed the results shown above. The total number of animals did not show any severe side effect after a one-dose treatment of 857 µg/kg at day 14 after the beginning of the treatment. Furthermore, the GMP-compliant validation of the radiosynthesis and quality control of [177Lu]Lu-FAUC 469 was established as this

137 Conclusion radiotracer represented a promising candidate for endoradiotherapy of NTS1-positive cancers. In future projects, more tumor entities should be studied for suitability of NTS1-targeted endoradiotherapy and an efficient patient screening needs to be developed. Due to the high expression of NTS2 in the small and moderate differentiated prostate cancers, the development of NTS2-selective radioligands could be considered.

138 Summary

6 Summary

The NTS1 has emerged as an interesting target for molecular imaging and targeted endoradiotherapy (“theranostics”) due to its overexpression in a variety of tumors, such as prostate and pancreatic cancer. PSMA represents a tumor marker being routinely used for theranostics in nuclear medicine clinics as PSMA is expressed in 92 % of prostate cancers. However, the detection and therapy of the 8 % PSMA- negative lesions and in the case of metastatic and hormone-refractory prostate cancer remains challenging. In the case of the highly malignant pancreatic adenocarcinoma, it would be of utmost importance to find additional molecular tools for imaging and endoradiotherapy of NTS1-positive carcinoma. Due to its overexpression in these tumor entities, NTS1 has emerged as an interesting target for theranostics – that is the use of this receptor for molecular imaging and targeted radiotherapy. This aim triggered the development of radiolabeled neurotensin analogs and neurotensin receptor antagonists for in vivo targeting of the receptor and for endoradiotherapy. In the present work, NTS1 was evaluated as target for molecular imaging with PET and targeted endoradiotherapy in prostate and pancreatic cancer with the two 68Ga- or 177Lu-labeled NTS1-selective non-peptide antagonists FAUC 468

(Ki (hNTS1) = 1.9 nM, Ki (hNTS2) = 100 nM) and CL 156 (Ki (hNTS1) = 1.0 nM,

Ki (hNTS2) = 160 nM) shown in Figure 60.

Figure 60. Chemical structures of the two non-peptide antagonists FAUC 468 and CL 156 which could be radiolabeled with M = 177Lu or 68Ga in the DOTA-chelator for NTS1 evaluation as target for molecular imaging and endoradiotherapy.

139 Summary

In vitro experiments and preclinical animal studies for NTS1 evaluation with the two 68Ga- or 177Lu-labeled NTS1-selective non-peptide antagonists FAUC 468 and CL 156 were performed and revealed highly promising results. After choosing PC-3, PC3-PIP, Panc-1 and HT-29 as the appropriate cell lines for studying the NTS1, first in vitro experiments showed NTS1-mediated internalization into the cells for both antagonist radioligands. Internalization was determined by lysing the cells after removing cell surface bound radioactivity by washing the cells with glycine buffer. The data analysis revealed internalization rates of 75 ± 5 % for [177Lu]Lu-FAUC 469 and 59 ± 7 % for [177Lu]Lu-CL 162 after four hours of incubation in PC-3 cells. The cellular efflux rate was low with values between 15 to 17 % for both radiotracers. With these favorable results, preclinical animal studies were performed using NTS1- positive PC-3 tumor-bearing nude mice. In biodistribution studies high specific tumor accumulation could be observed within 1 h p.i. which was continuously increasing until one day p.i. for [177Lu]Lu-FAUC 469 (15.5 % ID/g) and until day two p.i. for [177Lu]Lu-CL 162 (11.8 % ID/g). Both radiotracers showed excellent tumor retention as demonstrated by tumor uptake values of 6.7 - 8.6 % ID/g at day seven p.i. Moderate uptake of [177Lu]Lu-FAUC 469 has been observed in lung, liver, kidney and spleen until 24 h p.i. followed by almost complete radioactivity washout afterwards (Figure 61). In competition studies, the pre-injection of CL 156 (100 µg per animal) 15 min before [177Lu]Lu-FAUC 469 injection caused an inhibition of the tumor uptake of 93 %. The inhibition of binding by co-injection of CL 156 confirmed the receptor- specific uptake of the radioligand in the NTS1-positive PC-3 tumors. Furthermore, a specific inhibitory effect of 32 % could be observed in the intestine. In contrast, the uptake in all the other selected organs was not influenced by pre-injection of CL 156, demonstrating the specific uptake of the radiotracer in the PC-3 tumor and intestine.

140 Summary

Figure 61. Data of the time-dependent biodistribution of [177Lu]Lu-FAUC 469 (filled pattern) and [177Lu]Lu-CL 162 (lined pattern) in NMRI FOXn1nu mice bearing NTS1- positive PC-3 xenografts. Mice were injected with 1 MBq of the respective radioligand and organs were removed at 1 h, 1, 2 and 7 days p.i. Data were expressed as mean values of percent injected dose per gram tissue (% ID/g) ± SD (n = 2 - 3).

Biodistribution studies were followed by preclinical endoradiotherapy and imaging studies targeting NTS1. During therapy studies, mice were divided into one control and two treatment groups, of which each received 25 MBq of [177Lu]Lu-FAUC 469 or [177Lu]Lu-CL 162. The tumor growth of each treatment group compared to the untreated control group was significantly different over the whole observation period of 57 days after begin of treatment ([177Lu]Lu-CL 162: adjusted p=0.009; [177Lu]Lu- FAUC 469: adjusted p=0.044; Figure 62A). Furthermore, prolonged survival could be observed in treated mice compared to the control group (Figure 62B). There were no influences on NTS1 expression when comparing data from cell culture with data from tumor tissue after endoradiotherapy. The finding that receptor density was not influenced by the endoradiotherapy is very promising for the translation of NTS1- mediated radiotherapy into the clinic. The presence of the NTS1 independent of tumor volume and treatment means that various treatment cycles for targeted endoradiotherapy in humans and follow-up PET imaging, targeting the NTS1, would be in principle possible. The analysis of histology of kidney and liver sections at the endpoint of the preclinical endoradiotherapy studies did not show any toxic effects induced by the injected radioactive dose. Accompanying small-animal PET imaging

141 Summary with [68Ga]Ga-NODAGA-RGD during the endoradiotherapy study did not reveal any differences in tumor vascularization, suggesting that anti-angiogenic effects were not significantly mediated by the endoradiotherapy with [177Lu]Lu-FAUC 469.

Figure 62. (A) Relative tumor volume and (B) survival curves of the respective PC-3 tumor-bearing nude mice after treatment with a single dose of 25 MBq of [177Lu]Lu- FAUC 469 (n = 5) and [177Lu]Lu-CL 162 (n = 5) in comparison with untreated control animals (n = 7). Day 0 represented the day of radioligand injection. Data were expressed as mean ± SD. A sudden decline in the curve of the control mice () was due to the sacrifice of animals, because of fulfilling the termination criteria at these days.

The PET technique is a non-invasive diagnostic imaging technique with high spatial resolution, high sensitivity and accurate quantification of up to picomolar levels in vivo.3,4 The PET images obtained with the NTS1-targeting radiotracers demonstrated that NTS1 represented an appropriate target for PET imaging of NTS1-positive tumors in the used animal model. The high background signal in the images obtained with [68Ga]Ga-ABN 468 and [68Ga]Ga-CL 157 at 55 – 70 min p.i. could be explained by the slow clearance of the radioligands from the blood pool, resulting in low tumor- to-blood ratios of 0.25 and 0.5 (Figure 36B), respectively. Nevertheless, the tumor could be visualized in a defined manner and PET imaging at later time points could lead to improved tumor-to-blood ratios (> 0.25) and therefore to improved PET images. PET imaging with the peptide [68Ga]Ga-NT118 (Figure 63) showed tumor uptake in NTS1-positive PC3-PIP tumors. Using [68Ga]Ga-NT118 allowed the monitoring of the endoradiotherapy. The significant difference between control and treated mice at day 7 p.i. could be explained by the receptor occupancy by the therapeutic radiotracer [177Lu]Lu-FAUC 469.

142 Summary

Figure 63. Whole body small-animal PET images of NTS1-positive PC3-PIP tumor- bearing nude mice with [68Ga]Ga-NT118 at 45 min p.i. during endoradiotherapy with [177Lu]Lu-FAUC 469 (left). Mice were injected with 5 MBq of [68Ga]Ga-PSMA-11 or [68Ga]Ga-NT118. Beside uptake in the tumor region, the radioligand was extracted through the kidneys. Images were decay-corrected and adjusted to the same maximum value. The relative standard uptake values (SUVmean, normalized to SUV = 1 for the begin of treatment at day 0) of [68Ga]Ga-PSMA-11 (C) and [68Ga]Ga-NT118 (D) in PC3-PIP tumors at day 0, 7, 14, 28 and 42 after injection of a single dose of 25 MBq of [177Lu]Lu-FAUC 469 were shown on the right. Data were expressed as mean values ± SD from three animals per group in control (black) and treated (blue) mice.

Despite the promising preclinical NTS1-targeted endoradiotherapy and imaging results, NTS1 appeared not to be a suitable tumor marker for prostate cancer screening, due to the low rate of prevalence among prostate tumor tissue from cancer patients. Among the analyzed 98 samples of human normal and prostate cancer tumor tissues, NTS1 expression was 9 % (9 of 98 samples). In the NTS1- positive cases, NTS1 displayed a dense, homogeneous distribution throughout the tumors. Positive results for NTS1 were obtained for three of the 54 tumors; the remaining 51 were NTS1-negative tumors. Unfortunately, NTS1 was not exclusively expressed on tumor cells but also on normal prostate tissue in a ratio of 1:1. Receptor density differed a lot among the samples (0.6 – 408.1 pmol/mg) and no correlation with tumor staging (TNM classification), tumor volume or tumor grading (Gleason-score) could be shown. Nevertheless, the NTS1 expression throughout the tumors indicated that NTS1 was a suitable target for diagnostic imaging and endoradiotherapy of NTS1-positive cancers. In contrast to prostate cancer, NTS1 represented a suitable clinical target for diagnosis and endoradiotherapy of pancreatic cancer as the differentiation between normal healthy pancreatic tissue and carcinoma was possible using NTS1. Eighty percent of pancreatic cancer tissue samples expressed NTS1 on their cell surface (20 out of 24). The receptor density differed a lot among individual samples (± 200 %) showing values in the range of 0.004 to 0.28 fmol/mg with the exception of one sample showing a receptor density

143 Summary of 0.11 fmol/mg. In general, NTS1 displayed an inhomogeneous distribution throughout the tumors. Among “normal” healthy pancreas tissue samples only 4 out of 13 expressed NTS1 protein (31 %).

There is urgent need for new diagnostic and therapeutic tools for detection and therapy of metastatic and hormone-refractory prostate cancer as well as the highly malignant pancreatic adenocarcinoma. If these cancers expressed NTS1 on their tumor cells, the use of selective radiotracers allowed to target the receptor in a tumor-selective manner, very similar to the already established theranostic approach for the somatostatin receptor-mediated radiotherapy and diagnosis with [177Lu]Lu- DOTATOC and [68Ga]Ga-DOTATOC in neuroendocrine tumors. This work demonstrated that the 68Ga- and 177Lu-labeled FAUC 468 and CL 156 represented two radiolabeled non-peptide NTS1 antagonists that proved suitability as molecular tools for diagnosis and endoradiotherapy of prostate and pancreatic cancers in preclinical models. Moreover, the data on NTS1 expression in human tumor tissue suggested that 177Lu-FAUC 469 could be highly suitable for endoradiotherapy applications, especially for patients with pancreatic cancer.

144 Zusammenfassung

7 Zusammenfassung

Der Neurotensinrezeptor-1 als Target für die Radiotherapie und molekulare Bildgebung des Prostata- und Pankreaskarzinoms

Der Neurotensinrezeptor-1 (NTS1) wird in einer Vielzahl von Tumoren überexprimiert. Hierzu zählen unter anderem das Prostata- und Pankreaskarzinom. Die Diagnose, Bildgebung und Therapie von PSMA-negativen, metastasierenden und Hormon-unabhängigen Prostatakarzinomen, sowie des äußerst bösartigen Pankreaskarzinoms stellt bis heute eine Herausforderung dar. Durch die Überexpression des NTS1 in diesen beiden Tumorarten hat sich der Rezeptor in diesen Fällen zu einem überaus interessantes Zielprotein für „theranostische“ Zwecke entwickelt – dies bedeutet die Adressierung dieses einen Rezeptors sowohl für die molekulare Bildgebung mittels PET als auch für die rezeptorvermittelte Radiotherapie. Diese Möglichkeit hat dazu geführt, dass verschiedene radioaktiv- markierte Neurotensinanaloga und Neurotensinrezeptor-Antagonisten für die in-vivo- Bildgebung und Radiotherapie entwickelt wurden.

In der vorliegenden Arbeit wird daher der NTS1 als mögliches Ziel sowohl für die Bildgebung mit Hilfe der PET als auch für die rezeptorvermittelte Radiotherapie des Prostata- und Pankreaskarzinoms evaluiert. Hierfür wurden die zwei NTS1-selektiven nicht-peptidischen Antagonisten FAUC 468 (Ki (hNTS1) = 1.9 nM,

Ki (hNTS2) = 100 nM) und CL 156 (Ki (hNTS1) = 1.0 nM, Ki (hNTS2) = 160 nM) genutzt (Abbildung 1). Beide Verbindungen können sowohl mit dem kurzlebigen ß+-

Strahler Gallium-68 (t1/2 = 68 min) als auch dem langlebigeren Lutetium-177 - (t1/2 = 6,71 Tage, ß - und γ-Strahler) mit Hilfe des integrierten DOTA-Chelators markiert werden.

145 Zusammenfassung

Abbildung 1. Chemische Strukturen der zwei nicht-peptidischen NTS1-Antagonisten FAUC 468 und CL 156, die beide sowohl mit M = Lutetium-177 als auch Gallium-68 markiert werden können, um den Neurotensinrezeptor-1 als mögliches Ziel für Bildgebung und Radiotherapie des Prostata- und Pankreaskarzinoms zu evaluieren.

Die mit den beiden NTS1-Antagonisten durchgeführten In-vitro-Experimente und die anschließenden präklinischen Tierstudien führten zu vielversprechenden Ergebnissen. Beide Radioliganden zeigten eine NTS1-vermittelte Internalisierung in PC-3-, PC3-PIP-Zellen mit Raten von 75 ± 5 % für [177Lu]Lu-FAUC 469 und 59 ± 7 % für [177Lu]Lu-CL 162 nach vier stündiger Inkubation, die nach Entfernen der oberflächengebundenen Radioaktivität durch das Waschen mit Glyzinpuffer bestimmt wurde. Die Effluxrate war mit 15 -17 % für beide Radioliganden sehr gering. Nach diesen positiven In-vitro-Ergebnissen wurden präklinische Tierversuchsstudien an Nacktmäusen durchgeführt, die einen NTS1-positiven PC-3-Tumor in der linken Schulter trugen. Die Bioverteilungsstudien zeigten, dass für beide Liganden schon innerhalb der ersten Stunde nach Injektion eine spezifische Anreicherung innerhalb des Tumor zu beobachten ist, welche bei [177Lu]Lu-FAUC 469 bis nach einem Tag auf 16 % ID/g, bei [177Lu]Lu-CL 162 bis zwei Tage nach der Injektion auf 12 % ID/g ansteigt. Sogar bis sieben Tage nach Injektion zeigten beide Liganden mit 7 – 9 % ID/g eine bemerkenswerte Retention innerhalb des Tumors. Eine zusätzliche Aufnahme von [177Lu]Lu-FAUC 469 konnte in Lunge, Leber, Nieren und Milz gemessen werden, die jedoch innerhalb von 24 Stunden nach Injektion wieder ausgewaschen wurde (Abbildung 2). Die spezifische Aufnahme der Radioliganden konnte mit Hilfe von Verdrängungsstudien durch die vorherige Gabe von CL 156 (100 µg pro Tier) 15 min vor der [177Lu]Lu-FAUC 469-Injektion nachgewiesen werden. Hierbei konnte die Aufnahme in den Tumor um 93 % reduziert werden. Eine um 32 % verminderte Aufnahme des Radiopharmakons konnte im Darm der Mäuse beobachtet werden, wohingegen alle anderen Organe keine spezifische

146 Zusammenfassung

Traceraufnahme zeigten. Hierdurch konnte die spezifische Aufnahme des Radioliganden in den NTS1-positiven PC-3-Tumor und den Darm gezeigt werden.

Abbildung 2. Daten der Bioverteilungsstudien mit [177Lu]Lu-FAUC 469 (ausgefüllte Balken) und [177Lu]Lu-CL 162 (gestrichelte Balken), die mit NTS1-positiven PC-3- tumortragenden Nacktmäusen durchgeführt wurden. Die Organe wurden 1 h, 1, 2 und 7 Tage nach Injektion von 1 MBq des jeweiligen Liganden entnommen. Die dargestellten Werte entsprachen dem Mittelwert der injizierten Dosis pro Gramm Gewebe (% ID/g) ± Standardabweichung (SD). n = 2 – 3

Für die nachfolgende NTS1-vermittelte Therapiestudie wurden drei Tiergruppen gebildet. Eine Kontrollgruppe und zwei Gruppen, die mit entweder 25 MBq [177Lu]Lu- FAUC 469 oder [177Lu]Lu-CL 162 behandelt wurden. Über den gesamten Verlauf der Studie von 57 Tagen zeigte sich ein signifikant verlangsamtes Tumorwachstum innerhalb der Behandlungsgruppen im Vergleich zur Kontrollgruppe ([177Lu]Lu-CL 162: adjusted p=0.009; [177Lu]Lu-FAUC 469: adjusted p=0.044; Abbildung 3A). Weiterhin konnte eine Verlängerung der Überlebenszeit der behandelten Gruppen demonstriert werden (Abbildung 3B). Eine Veränderung der NTS1-Expression, die während der Radiotherapie auftreten könnte, konnte nicht beobachtet werden. Dies ist eine wichtige Beobachtung für die Translation der NTS1-vermittelten Rezeptorradiotherapie in die Klinik, da dies zeigt, dass die Radiotherapie die Rezeptordichte nicht beeinflusst, und daher eine Therapie in mehreren Zyklen möglich erscheint. Des Weiteren wies die histologische Analyse von Leber und Nieren am Endpunkt der präklinischen Therapiestudie darauf hin, dass keine toxischen Schäden der Ausscheidungsorgane vorhanden waren. Therapiebegleitende PET-Aufnahmen mit [68Ga]Ga-NODAGA-RGD zeigten keine 147 Zusammenfassung

Unterschiede in der Vaskularisierung der Tumore, was darauf hindeutete, dass die Radiotherapie mit [177Lu]Lu-FAUC 469 keinen entscheidenden Einfluss auf die Tumorangiogenese hatte.

Abbildung 3. (A) Relatives Tumorvolumen und (B) Überlebenskurven der verschiedenen Tiergruppen aus der Therapiestudie. PC-3-tumortragenden Nacktmäusen wurden für die Therapie mit 25 MBq [177Lu]Lu-FAUC 469 (n = 5) oder [177Lu]Lu-CL 162 (n = 5) injiziert und mit der unbehandelten Kontrollgruppe (n = 7) verglichen. Tag 0 entsprach dem Tag der Injektion des Radiopharmakons. Die Daten sind dargestellt als Mittelwert ± SD. Der Abfall der Tumorvolumina an den mit  gekennzeichneten Stellen entstanden auf Grund der Tötung von Mäusen mit großen Tumoren, die an diesen Tagen ein Kriterium für die Euthanasie erfüllten.

Die Bildgebung mittels PET stellte eine Möglichkeit dar, In-vivo-Aufnahmen mit hoher räumlicher Auflösung, hoher Sensitivität und präziser Quantifizierung bis zu 1 pM zu erstellen. Die Aufnahmen, die während einer zweiten Therapiestudie gemacht wurden, und deren Auswertung (Abbildung 4) zeigten deutlich, dass der Neurotensinrezeptor-1 ein geeignetes molekulare Target sowohl für die Bildgebung als auch für die Radiotherapie von (PSMA-negativen) Prostatatumoren darstellte. Die lange Blutbindung der Radioliganden und die daraus resultierenden niedrigen Tumor/Blut-Verhältnisse von 0,25 und 0,5 für [68Ga]Ga-ABN 468 und [68Ga]Ga- CL 157 60 min nach Injektion erklärten das hohe Hintergrundsignal, das in den PET- Bildern mit den beiden Radioliganden zu sehen war. Jedoch blieb der Tumor über die gesamte Zeit gut abgrenzbar gegenüber dem Hintergrund. PET-Bilder, die zu einem späteren Zeitpunkt aufgenommen werden, würden zu einem besseren Tumor/Blut-Verhältnis (> 0.25) und somit zu besserer Bildqualität führen. Durch die Bildgebung der NTS1-positiven PC3-PIP-Tumore mittels des Peptids [68Ga]Ga- NT118 konnte eine Verlaufskontrolle während der Radiotherapie durchgeführt werden. Der signifikante Unterschied zwischen Kontroll- und Therapiegruppe an Tag

148 Zusammenfassung

7 nach Injektion könnte durch die NTS1-Besetzung durch den Therapietracer [177Lu]Lu-FAUC 469 erklärt werden (Abbildung 4).

Abbildung 4. Kleintier-PET-Aufnahmen von NTS1-positiven PC3-PIP- tumortragenden Nacktmäusen im Verlauf der Therapiestudie mit [177Lu]Lu-FAUC 469 (links). Den Tieren wurden 45 min vor der Aufnahme 5 MBq [68Ga]Ga-NT118 injiziert. Neben der gewünschten Aufnahme des Radioliganden in den Tumor, konnte weiterhin eine Anreicherung in den Nieren beobachtet werden. Für die Quantifizierung der Aufnahmen (rechts) mit [68Ga]Ga-NT118 wurden die Standard Uptake Values (SUVmean) von je drei Tieren pro Gruppe ermittelt und auf den Tag der Tracerinjektion normalisiert (SUV = 1 für Tag 0).

Trotz dieser vielversprechenden Ergebnisse aus den präklinischen Studien zur NTS1-vermittelten Radiotherapie und Bildgebung, eignet sich der NTS1 nicht als Marker für ein Screening unter Patienten, die an einem Prostatakarzinom leiden. Für diesen Zweck ist die Rate an NTS1-exprimierenden Tumoren unter den Patienten zu gering. Von den untersuchten 98 Patientenproben von normalem, gesundem und bösartigem Gewebe lag die NTS1-Expression bei 9 % (9 aus 98 Fällen). Zwar zeigte sich eine dichte, homogene Verteilung des Rezeptors innerhalb der positiv getesteten Tumorgewebe, jedoch wurden nur drei der 54 beinhalteten Tumorgewebe positiv getestet, die restlichen 51 waren NTS1-negativ. Zudem konnte die NTS1- Expression nicht nur ausnahmslos auf Tumorgewebe, sondern auch auf einigen der 44 gesunden Gewebeproben nachgewiesen werden, ca. im Verhältnis 1:1. Unter den positiv getesteten Tumorgewebeproben schwankte die Rezeptordichte gewaltig (0.6 – 408.1 pmol/mg). Außerdem war keine Korrelation zu der Tumorklassifizierung (TNM), dem Tumorvolumen oder der Tumor-Differenzierung (Gleason-Score) zu erkennen. Nichtsdestotrotz zeigten die Expression des NTS1 im Tumor und die präklinischen Bildaufnahmen, dass dieser Rezeptor ein geeignetes Ziel sowohl für diagnostische Zwecke als auch für die Radiotherapie von NTS1-positiven Karzinomen darstellt.

149 Zusammenfassung

Im Gegensatz dazu, eignete sich der NTS1 sehr wohl als klinisches Zielprotein sowohl für die Diagnose als auch die rezeptorvermittelte Radiotherapie des Pankreaskarzinoms. Mittels des NTS1 kann in den meisten Fällen zwischen gesundem Pankreasgewebe und den bösartigen Tumoren unterschieden werden. Bei der Untersuchung von malignen Gewebeproben, die Patienten entnommen wurden, die am Pankreaskarzinom leiden, konnte die Expression des NTS1 in 80 % (20 von 24) der Fälle nachgewiesen werden. Mit einer Ausnahme bewegte sich die Rezeptordichte zwischen 0.004 und 0.28 fmol/mg. Von den untersuchten gesunden Referenzgeweben zeigten nur 4 von 13 Proben (31 %) eine Expression des NTS1- Proteins.

Bis heute stellen die Diagnose, Bildgebung und Therapie von metastasierenden und Hormon-unabhängigen Prostatakarzinomen, sowie des äußerst bösartigen Pankreaskarzinoms eine Herausforderung dar. Daher werden dringend neue Ansätze für die Diagnose und Therapie dieser Tumore benötigt. Exprimieren diese Tumorentitäten den NTS1 auf ihrer Zelloberfläche, ermöglicht dies die Karzinome mit selektiven Radioliganden zu adressieren, wie es auch schon für den theranostischen Ansatz via des Somatostatinrezeptors mit [177Lu]Lu-DOTATOC und [68Ga]Ga- DOTATOC in neuroendokrinen Tumoren gezeigt wurde. Zwei vielversprechende Radioliganden stellen FAUC 468 und CL 156 dar, die beide mittels eines DOTA- Chelators mit Gallium-68 oder Lutetium-177 radioaktiv markiert werden können. Beide nicht-peptidischen NTS1-Antagonisten stellen einen neuen molekularen Ansatz für Diagnose und Radiotherapie des Prostata- und Pankreaskarzinoms in präklinischen Modellen dar. Die Rezeptorexpression des NTS1 auf humanen Proben dieser Tumore zeigten, dass [177Lu]Lu-FAUC 469 für therapeutische Ansätze, vor allem in Fällen des Pankreaskarzinoms, geeignet wäre.

150

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II List of Abbreviations

% ID/g Percentage injected dose per gram

(m)Ab Monoclonal antibody

AC Adenylate cyclase

ALT Alanine aminotransferase

(c)AMP, ADP, ATP (cyclic) Adenosine 5'-mono, di-, and triphosphate

APC Allophycocyanin

APS Ammonium persulfate

BCA Bicinchoninic acid

Bmax Maximal binding capacity bp Base pair

Bq Becquerel (= activity of decay per second)

BSA Bovine serum albumin

CD Cluster of Differentiation

CFA Colony formation assay

CNS Central nervous system

Cpm Counts per minute

CT Computed Tomography

DAB 3,3'-Diaminobenzidine

DAG Diacylglycerol

DAPI 4'-6-diamidino-2-phenylindole

DMEM Dulbecco's modified Eagle's medium

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DMSO Dimethyl sulfoxide

(c)DNA (Copy) Deoxyribonucleic acid

DTT Dithiothreitol

ECL Enhanced chemiluminescence

EDTA Ethylenediaminetetraacetate

EGFR Epidermal growth factor receptor

FACS Fluorescence-activated cell sorter

FCS Fetal calf serum

FDG Fluorodeoxyglucose

FITC Fluorescein isothiocyanate

GAPDH Glyceraldehyde-3-phosphate dehydrogenase

(c)GMP (cyclic) Guanosine 5'-monophosphates

GPCR G-protein coupled receptor

HE Hematoxylin and eosin

HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HLA-ABC Human leukocyte antigen-A, B and C

HPLC High-performance liquid chromatography

HRP Horseradish peroxidase i.v. Intravenous(ly)

IgG Immunoglobulin G

IP3 Inosine 5'- triphosphates

Kd Dissociation constant kDa or Da Kilodalton, dalton

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keV Kiloelectron volt

Ki Inhibitory constant

MAPK Mitogen-activated protein kinase

MEM Modified eagles medium mRNA Messenger RNA

NEAA Non-essential amino acids

NPE Nucleoplasmic extract

NT Neurotensin

NTS1/2/3/4 Neurotensin receptor 1, 2, 3 or 4 o.n. Over night p.i. Post-injection

PAGE Polyacrylamide gel electrophoresis

PBS Phosphate-buffered saline

PE Plating efficiency

PET Positron emission tomography

PKC Protein kinase C

PLC Phospholipase C

PMPA 2-(Phosphonomethyl)pentane-1,5-dioic acid

PSA Prostate-specific antigen

PSMA Prostate-specific membrane antigen

PVDF Polyvinylidene fluoride q(RT-)PCR Quantitative (real-time) polymerase chain reaction

RNA Ribonucleic acid

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RP Reversed phase

Rpm Revolutions per minute

RPMI Roswell Park Memorial Institute medium

RT Reverse transcriptase rt Room temperature

SD Standard deviation

SDS Sodium dodecyl sulfate

SEER Surveillance, Epidemiology, and End Results (SEER) Program

SF Surviving fraction siRNA Small/short interfering RNA

SPECT Single photon emission computed tomography

SUV Standard uptake value t1/2 Half-life

TBS(-T) Tris-buffered saline (+ Tween20)

TEMED Tetramethylethylendiamin

TFA Trifluoroacetic acid

TGDI Tumor growth delay index

TGI Tumor growth inhibition

TNM staging Tumor, node and metastasis staging tR Retention time

Tris Tris(hydroxymethyl)-amino methane v/v Percentage by volume

Wnt Acronym of homologous wingless (wg) and Int-1 gene

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III List of Figures

Figure 1. Concept of “theranostics“...... 1 Figure 2. Schematic illustration of the principle of PET...... 3 Figure 3. Structures of D-glucose and the 18F- labeled analogue [18F]FDG...... 4 Figure 4. Production of germanium-68 by the (p, 2n)-reaction of gallium-69...... 4 Figure 5. Two different production routes for lutetium-177...... 7 Figure 6. Structure of the [177Lu]Lu-DOTA (1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid) complex...... 7 Figure 7. Anatomy of the pancreas (left) and 5-year survival rate of pancreatic cancer patients (right)...... 9 Figure 8. 5-year survival rate of patients suffering from prostate cancer...... 11 Figure 9. Crystal structure of the prostate-specific membrane antigen (PSMA)...... 12 Figure 10. Structures of the imaging agent [68Ga]Ga-PSMA-11 (left) and the therapeutic agent [177Lu]Lu-PSMA-617 (right)...... 14 Figure 11. Chemical structure of the tridecapeptide neurotensin...... 16 Figure 12. NT(8–13) - the pharmacologically active C-fragment of neurotensin...... 17 Figure 13. Crystal structure of the rat NTS1 with the bound peptide agonist NT(8-13) from White et al...... 18 Figure 14. Scheme of activation and signaling cascade of the GPCR NTS1...... 19 Figure 15. NTS1 as an alternative growth mechanism during androgen-deprivation therapy...... 23 Figure 16. Chemical structure of 3BP-227 published by Schulz et al.188 ...... 25 Figure 17. Chemical structures of the first members of potent selective non-peptide NTS1 antagonist (A) SR 48692 (Meclinertant; Sanofi-Aventis, France) and the second-generation non-peptide NTS1 antagonist (B) SR 142948A (Sanofi-Aventis, France)...... 26 Figure 18. Objective and Aim ...... 27 Figure 19. Structures of radiotracers included in this study...... 30 Figure 20. Chemical structure of NT100.195 ...... 45 Figure 21. mRNA expression of NTS1 and NTS2 in prostate and pancreatic cancer cell lines...... 61

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Figure 22. mRNA expression of NTS1, NTS2 and PSMA in prostate and pancreatic cancer cell lines...... 63 Figure 23. Uptake of [125I]Tyr3-NT into prostate (A) and pancreatic (B) cancer or pancreatic stromal cell lines ...... 65 Figure 24. Exemplary Western Blot analyses of 15 µg protein for proving endogenous NTS1 protein expression on different prostate and pancreatic cancer cell lines...... 68 Figure 25. Representative Western Blot analysis for elucidating PSMA expression on PC3-PIP cells (left)...... 71 Figure 26. (A) Effect of NT on growth stimulation of PC-3 cells...... 72 Figure 27. (A) Colony formation assay performed in 10-cm dishes (n = 3) with colonies produced by PC-3 prostate cancer cells during 10 days: colony images (left) and numbers of colonies (right)...... 75 Figure 28. Radiosynthesis of 177Lu- and 68Ga-labeled NTS1 non-peptide antagonists FAUC 468 and CL 156...... 76 Figure 29. Representative radio-HPLC chromatograms of 68Ga- and 177Lu-labeled compounds...... 77 Figure 30. Internalization, efflux and cell binding studies of 177Lu- and 68Ga-labeled FAUC 468 and CL 156 in human PC-3 cells...... 79 Figure 31. Internalization and efflux studies in PC3-PIP cells performed at 37 °C... 81 Figure 32. Saturation binding curves of 68Ga- and 177Lu-labeled non-peptide NTS1 antagonists on (A) HT-29, (B) Panc-1, (C) PC3-PIP and (D) PC-3 cells. . 84 Figure 33. Exemplary presentation of saturation binding curves of the (A) non- peptide NTS1 antagonist [68Ga]Ga-ABN 468 and (B) NTS1 agonist [68Ga]Ga-NT118 on HT-29 cells...... 87

Figure 34. Presentation of receptor availability (vertical axis Bmax in fmol/mg) against time (horizontal axis in h) on (A) PC-3 and (B) PC3-PIP cells after 0, 6, 24, 48 and 96 h exposition time to 1 nM NTS1 antagonist CL 156 or 200 nM

NTS1 agonist NT100 (≈ 10 × Ki) measured with the non-peptide antagonist [68Ga]Ga-ABN 468. Shown were data of three independent experiments performed in triplicates (n = 9)...... 89 Figure 35. Saturation binding curve of [68Ga]Ga-PSMA-11 on PC3-PIP cells...... 92 Figure 36. (A) Biodistribution of [68Ga]Ga-ABN 468 and [68Ga]Ga-CL 157 ...... 93

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Figure 37. Representative PET scans at 55 – 70 min p.i. of [68Ga]Ga-ABN 468 (n = 3, left) and [68Ga]Ga-CL 157 (n = 3, right) ...... 95 Figure 38. Data of biodistribution of (A) [177Lu]Lu-FAUC 469 and (B) [177Lu]Lu- CL 162 (1 MBq) ...... 97 Figure 39. Data of biodistribution of [177Lu]Lu-FAUC 469 ...... 100 Figure 40. Representative images of ex vivo autoradiographies ...... 102 Figure 41. Time-dependent radiotracer uptake and retention of [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162 in NTS1-expressing PC-3 tumors and liver over the period of 7 days p.i...... 104 Figure 42. (A) Relative tumor volume and (B) survival curves ...... 104 Figure 43. (A) Serum glucose, ALT (alanine aminotransferase), creatinine and bilirubin levels were significantly different among control (black) and treated (purple and green) groups (p > 0.05). (B) Microscopic images (20 ×) of PC-3 tumor (left column), kidney (middle column) and liver (right column) sections ...... 107 Figure 44. (A) Chemical structure of [68Ga]Ga-NODAGA-RGD...... 109 Figure 45. (A) FACS analysis of U87MG (positive control) and PC-3 cells for integrin

αVß3 expression...... 110 Figure 46. (A) Relative tumor volume and (B) survival curves of the respective PC3- PIP tumor-bearing nude mice ...... 112 Figure 47. Whole body small-animal PET images of NTS1- and PSMA-positive PC3- PIP tumor-bearing nude mice with (A) [68Ga]Ga-PSMA-11 and (B) [68Ga]Ga-NT118 ...... 115 Figure 48. Biodistribution and tumor-to-organ ratios of [68Ga]Ga-PSMA-11 and [68Ga]Ga-NT118 ...... 120 Figure 49. Representative ex vivo autoradiography ...... 121 Figure 50. qRT-PCR analysis of NTS1 expression (vertical axis) ± SEM in PC-3 tumor xenograft tissues ...... 122 Figure 51. Saturation binding curves of [68Ga]Ga-ABN 468 ...... 123 Figure 52. Representative FACS analysis ...... 124 Figure 53. Expression of GAPDH mRNA in patient tissue...... 125 Figure 54. Expression of (A) PSMA, (B) NTS1 and (C) NTS2 mRNAs (y-axis) in primary healthy and cancerous patient tissues ...... 126

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Figure 55. NTS1 density in 98 normal prostate and prostate cancer tissue samples from a total of 49 patients...... 128 Figure 56. In vitro autoradiography of tissue slices of patient 31 showing NTS1 expression...... 130 Figure 57. Expression of (A) NTS1 and (B) NTS2 mRNAs (y-axis) in primary healthy and cancerous patient tissues ...... 132 Figure 58. NTS1 density in 38 “normal” healthy pancreas and pancreatic cancer tissue samples from a total of 23 patients...... 133 Figure 59. In vitro autoradiography of tissue slices of patient 18 ...... 135 Figure 60. Chemical structures of the two non-peptide antagonists FAUC 468 and CL 156 ...... 139 Figure 61. Data of the time-dependent biodistribution of [177Lu]Lu-FAUC 469 (filled pattern) and [177Lu]Lu-CL 162 (lined pattern) ...... 141 Figure 62. (A) Relative tumor volume and (B) survival curves of the respective PC-3 tumor-bearing nude mice ...... 142 Figure 63. Whole body small-animal PET images of NTS1-positive PC3-PIP tumor- bearing nude mice with [68Ga]Ga-NT118 ...... 143

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IV List of Tables

Table 1. Clinical relevant positron-emitting radionuclides for PET imaging with their ß+ energy and half-lives.3,10 ...... 2 Table 2. Examples of 68Ga-labeled radiopharmaceuticals...... 5 Table 3. Characteristics of the radionuclide lutetium-177...... 6 Table 4. Examples of 177Lu-labeled radiopharmaceuticals...... 8 Table 5. NTS1 mRNA and protein expression in different types of cancers...... 20 Table 6. Binding data of compounds to their target used during the present study (see structures in Figure 19)...... 29 Table 7. Cell lines and their respective culture conditions...... 31 Table 8. Radiotracer, their dilution series in medium and incubation temperature that

were used in saturation binding studies (determination of Kd and Bmax Values)...... 44 Table 9. Parameters monitored in tumor therapy studies...... 49 Table 10. Components for genomic DNA elimination reaction...... 51 Table 11. Reverse transcription master mix...... 52 Table 12. qRT-PCR master mix with QuantiFast SYBR Green PCR kit...... 52 Table 13. qRT-PCR primers...... 53 Table 14. Composition of lysis RIPA buffer...... 54 Table 15. Composition of separating (8 %) and stacking (5 %) gel...... 54 Table 16. Composition of the 4 × reducing dye used for sample preparation...... 55 Table 17. Compositions of 10 × running buffer, 10 × blotting buffer and TBS-T...... 55 Table 18. Antibodies and their dilutions used for immunohistochemistry...... 57 Table 19. Prostate Cancer Patient Characteristics...... 59 Table 20. Pancreatic Cancer Patient Characteristics...... 59 Table 21. Antibodies and conditions for Western blotting...... 69

Table 22. In vitro binding affinities (Ki ± SEM) of the two non-peptide NTS1 antagonists FAUC 468 and CL 156 to human NTS1 and NTS2 of three individual experiments...... 78 Table 23. In vitro characteristics of the radiolabeled NTS1 antagonists FAUC 468 and CL 156 obtained in studies using PC-3 cells...... 80

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Table 24. Saturation binding data of 68Ga- and 177Lu-labeled FAUC 468 and CL 156 obtained using the four human cancer cell lines HT-29 (colon cancer), PC- 3 and PC3-PIP (prostate cancer) and Panc-1 (pancreatic cancer)...... 86 Table 25. Saturation binding data of the NTS1-adressing 68Ga-labeled FAUC 468 and NT118 ...... 87 Table 26. Biodistribution of [68Ga]Ga-ABN 468 and [68Ga]Ga-CL 157 ...... 94 Table 27. Biodistribution data of [177Lu]Lu-FAUC 469 ...... 98 Table 28. Biodistribution data of [177Lu]Lu-CL 162 ...... 99 Table 29. Calculated absorbed doses of [177Lu]Lu-FAUC 469 and [177Lu]Lu-CL 162 in PC-3 tumor-bearing nude mice ...... 101 Table 30. Analysis of the data obtained from endoradiotherapy study with PC-3 tumor-bearing nude mice ...... 105 Table 31. Analysis of the data obtained from endoradiotherapy study with PC3-PIP tumor-bearing nude mice ...... 113 Table 32. Biodistribution of [68Ga]Ga-PSMA-11 and [68Ga]Ga-NT118 ...... 119

Table 33. Bmax [fmol/mg] ± SD values for NTS1 ...... 124 Table 34. NTS1 expression in primary tissue of prostate cancer patients...... 129 Table 35. NTS1 expression in primary tissue of pancreatic cancer patients...... 134

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