Wildtype Glioblastoma Cells in a Co-Culture Model with Fibroblasts

Carnosine selectively inhibits migration of IDH- wildtype glioblastoma cells in a co-culture model with fibroblasts

Dissertation zur Erlangung des akademischen Grades Dr. med.

an der Medizinischen Fakultät der Universität Leipzig

eingereicht von: Johannes Andreas Dietterle

Geburtsdatum / Geburtsort: 04.04.1990/ Cottbus angefertigt an der: Universität Leipzig Klinik und Poliklinik für Neurochirurgie

Betreuer: Prof. Dr. Jürgen Meixensberger Prof. Dr. Frank Gaunitz

Beschluss über die Verleihung des Doktorgrades vom:

Table of contents

1 Introduction ...... 2

1.1 Glioblastoma ...... 2 1.1.1 Taxonomy, epidemiology and general features of GBM ...... 2 1.1.2 GBM subtypes and molecular diagnostic ...... 3 1.1.3 Therapy ...... 4 1.1.4 GBM cell migration and invasion ...... 5 1.2 Carnosine ...... 6 1.2.1 Chemistry, Biology, Distribution ...... 7 1.2.2 Carnosine homeostasis ...... 7 1.2.3 Physiological functions ...... 8 1.2.4 Therapeutic potential ...... 9 1.2.5 Carnosine and cancer ...... 9 1.3 Objective of the study ...... 10 2 Publication ...... 12

2.1 General information ...... 12 2.2 Carnosine selectively inhibits migration of IDH-wildtype glioblastoma cells in a co-culture model with fibroblasts ...... 13 3 Summary ...... 23

4 References ...... 27

5 Appendix ...... 34

5.1 Supplemental material ...... 34 5.2 Author’s contribution ...... 36 5.3 Erklärung über die eigenständige Abfassung der Arbeit ...... 37 5.4 Curriculum vitae ...... 38 5.5 List of publications ...... 39 5.6 Acknowledgements ...... 41

1 Introduction

1.1 Glioblastoma

In 1858 Rudolf Virchow described glia cells for the first time in medical history [1]. Seven years later he pathologically characterized gliomas, separating them into low- and high-grade glioma [2].

1.1.1 Taxonomy, epidemiology and general features of GBM Today, Glioblastoma (GBM) terms a tumor entity of the central nervous system (CNS) that arises from normal brain parenchyma and is classified as WHO grade IV. Therefore it belongs, next to Gliosarcoma, Pineoblastoma or all embryonal tumors of the central nervous system (CNS), to the subgroup of the most malignant CNS tumors which are normally associated with poor prognosis [3]. On cellular basis, it is characterized by a high level of mitotic activity, endothelial proliferation, necrotic areas and wide ranged invasion of adjacent brain parenchyma. Patients, if not asymptomatic, suffer from typical symptoms of raised intracranial pressure, like (tension) headache, nausea and vomiting, as well as focal neurological deficits, e.g. epileptic episodes, seizure or personality changes [3, 4]. According to the Central Brain Tumor Registry of the United States (CBTRUS; presenting data collected between 2010 and 2014), GBM had an incidence rate of 3.2 per 100,000 population in the US [5]. According to the report of Ostrom et al ., it accounted for 14.9% of all primary brain and other CNS tumors and 47.1% of primary malignant brain tumors. It was 1.58 times more common in male than in female patients and was diagnosed at a median age of 64 years. Despite all treatment efforts, Glioblastoma comes with a poor prognosis of an estimated 5.5% survival rate 5 years post diagnosis [6]. Here, highly infiltrative and migratory behavior of GBM cells within the brain are considered as important factors for short survival rates and will be discussed further down. On the other hand, formation of extra-CNS metastasis of GBM (ECMGBM) is a rarely seen phenomenon in GBM patients and has been found in the lungs, lymphatic nodes, liver, bones or the pancreas [7]. Ogungbo et al. also reported a case in which a metastasis had been found in the patient’s parotid gland [8]. In a study performed by Müller et al. [9], GFAP positive circulating tumor cells were found in the peripheral blood from 29 of 141 (20.6%) GBM patients.

1.1.2 GBM subtypes and molecular diagnostic To understand the nature of GBM and to individualize treatment for a better outcome, a major task is to distinguish between its subtypes. A general difference is made between primary and secondary GBM. Primary GBM arise de novo, are commonly diagnosed in patients older than 50 years and show a small-cell type; in contrast, secondary glioblastoma are the terminus of the malignant transformation originating from astrocytoma and occur more frequently in younger to middle-aged patients and present a giant-cell type phenotype [4, 10, 11]. Additionally, 5% of glioblastoma cases are associated with family history, mostly without detectable genetic cause and rarely due to genetic syndromes, e.g. Li-Fraumeni-syndrome, Turcot's syndrome or neurofibromatosis types 1 and 2 [4]. On the other hand, Kleihues and Oghaki suggested already twenty years ago to supplement clinical and histologic features by genetic data to separate primary from secondary GBM [12]. Up today, classification on a molecular level is a well- established method [13, 14], because diagnostic, prognostic or predictive information is available [15, 16]. The impact of molecular markers went so far that whole therapy algorithm, i.e. whether temozolomide should be applied or not, are based on different expression patterns. The important role of molecular markers finally found its representation within the actual WHO classification of CNS tumors [3]. For GBM, the isoenzymes of isocitrate dehydrogenase (IDH1 and IDH2) and the methylation of the O6-methylguanin-DNA methyltransferase (MGMT) promoter are the most important markers and hence were integrated into neuropathological routine assessment guidelines for glioma diagnostics [13]. A third well established molecular marker is loss of heterozygosity of 1p and 19q, the molecular signature of oligodendroglial tumours which provides prognostic value for those entities [15]. Five genes encode three isoforms of IDH, but only IDH1 and 2 are known to play a role for cancer research [14]. Being either located in the cytosol and peroxisomes (IDH1) or in mitochondria (IDH2) [14], these enzymes physiologically catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate and carbon dioxide with reduction of the cofactor nicotinamide adenine dinucleotide phosphate (NADP +) [13]. Point mutations in codon 132 of the IDH1 gene (over 85-90% of detected alterations [13, 15] or in codon 172 of the IDH2 gene (only around 3% of the cases [15]) appear to occur at an early stage of glioma transformation and are more frequents in younger patients [14]. As demonstrated by a meta-analysis published

3 by Cheng et al ., including over 1600 patients with glioblastoma, IDH1 mutation is associated with prolonged overall survival and therefore of prognostic value [17]. This is reflected within the recent WHO classification distinguishing GBM subtypes based on the mutational status of IDH. We now differentiate between three GBM subtypes: GBM with IDH wildtype (primary GBM), with mutated IDH (secondary GBM) and GBM not otherwise specified (NOS) [3]. The second routine molecular marker for GBM is the MGMT promoter methylation status [18]. The importance of MGMT derives from its function as a repair enzyme that reverses DNA cross-links at the O6 position of guanine [19] and, in a clinical context, thus opposes the effects of DNA alkylating substances, such as temozolomide (TMZ) and nitrosoureas. [16]. In 2000 the predictive value of MGMT gene silencing via hypermethylation for WHO grade III and IV entities was shown by Esteller et al . [20]. Patients with a methylated MGMT promotor phenotype responded significantly better to carmustine treatment (12 of 19 vs. 1 of 28) and their median progression free survival was prolonged by 13 months. Later, Hegi et al . [21] published studies which revealed a positive correlation between MGMT gene silencing and temozolomide therapy. Therefore, the decision whether an adjuvant therapy with alkylating substances is beneficial for GBM patients is highly dependent on the MGMT promoter methylation status of the tumor (see further down).

1.1.3 Therapy Today, treatment of GBM starts with navigated (ultrasound or MRI based guidance) microsurgical resection, aiming for gross total resection [18]. According to guidelines for glioma therapy, surgery using 5-aminolevulinic acid [22] should be the first choice for patients with newly diagnosed glioblastoma. Tumor exstirpation should then be followed by adjuvant radio-chemotherapy (RCx) [18]. Conformal radiation up to 60 Gy in 2 Gy fractions remains the standard for primary GBM treatment [23]. Depending on MGMT status and the patient’s general condition, X-irradiation is accompanied by treatment with temozolomide (TMZ), called concomitant RCx. Then, prolonged adjuvant therapy with TMZ can be applied. Whether prolonged chemotherapy with TMZ should extent 6 cycles is controversial and still under investigation [24]. TMZ was approved by the Food and Drug Administration, United States, [25] in reaction to a phase-III-study performed by Stupp et al . [26],

4 demonstrating significantly prolonged survival of patients combining radiotherapy with TMZ treatment. Tumor-treating fields, using alternating electric fields in order to inhibit mitosis through spindle apparatus disruption, showed promising results [27], yet remain controversial [28]. Treatment guidelines of recurring GBM are more unprecise and there is no standardized therapy regimen [28]. In general, treatment includes re-surgery, re- irradiation, second-line monotherapy or combination chemotherapy [29].

1.1.4 GBM cell migration and invasion As mentioned before, highly infiltrative behavior is a hallmark of GBM cell biology playing a crucial role for patients' prognosis [3]. In fact, as demonstrated by Sahm et al ., diffuse glioma cells can be found throughout the whole brain [30]. Hence, R0 resection of GBM, or high-grade glioma in general, is virtually impossible and malignant gliomas should be treated as systemic brain diseases. Cancer cell infiltration depends on the cancer cell’s specific physiology as well as a complex interplay with extra-cellular matrix (ECM) and tumor cell microenvironment [31]. In the case of gliomas, ECM and tumor microenvironment include specific macroscopic brain structures as glioma cells follow white matter tracts and the perivascular space to diffusely infiltrate the whole brain, as already shown in 1940 [32]. The following part will illustrate some characteristics how GBM cells interact with ECM and tumor microenvironment within the CNS. The first phenomenon specific to glioma cell migration and invasion is the so called ‘go and grow’ theory. It has first been described by Giese et al ., defining proliferation and migration as mutually exclusive events in high-grade astrocytoma cells in vitro [33]. Later, it has been assumed in bio-mathematical models that this dichotomy can not only be driven by mutational heterogeneity within GBM but must also be triggered by microenvironmental factors such as hypoxia [34], and there is indeed evidence for both. Several factors have been identified to either stimulate GBM cell proliferation by inhibiting migration and vice versa . This, for example, includes microRNA-451 [35] or EbBH 2 receptor [36], but also better-known targets such as vascular endothelial growth factor (VEGF) [37]. Additionally, GBM cells within the tumor mass or the tumor rim, respectively, show different overexpression patterns for transcriptional factors such as c-myc or NF-κB, which are either involved in GBM cell migration (NF-κB, cells of the tumor rim) or proliferation (c-Myc, cells of tumor

5 center) [38]. Moreover, a work published in 2016 proofed that the regulation of the pentose phosphate pathway and glycolysis in glioblastoma cells by oxygen levels of the tumor environment strongly influence whether a cell migrates or proliferates [39]. The second principle which is used by GBM cells to disseminate is chemotaxis. It defines the mechanism when the direction of cell movement is driven by an extracellular chemo gradient [40]. Here, bradykinin and oxygen play important roles for perivascular infiltration of glioma cells. It is a common fact that bradykinin is produced by vascular endothelium through enzymatic cleavage to regulate vascular permeability. In a series of experiments, it has been demonstrated by Montana and Sontheimer that glioma cells express bradykinin 2 receptors which allow them to follow a chemo-gradient towards the bradykinin-rich perivascular space in the brain [41]. GBM is also characterized by central necrosis caused by oxygen deprivation, resulting in a constant urge to move towards the more oxygen-rich perivascular space [42]. Here, hypoxia-induced factor 1 (HIF-1) plays a pivotal role and has been shown to regulate pathways involved in cell migration and infiltration, such as production of matrix-metalloproteases, ECM remodeling and angiogenesis via VEGF secretion [43]. Although ECM only comprises approximately 20% of brain parenchyma, which renders the brain to be overall a rather soft organ [44], perivascular basement membranes composed of collagen IV, laminin, heparan sulphate proteoglycans and others [45] are rigid. Interestingly, a work published in 1999 by Mahesparan et al . [46] demonstrates that migration of GBM cells obtained from glioma biopsy specimen is induced by laminin, fibronectin, collagen type IV and vibronectin in vitro . This phenomenon has been termed mechanotaxis or durotaxis [47] and appears to be important for glioma cell migration and invasion following white matter tracts [48].

1.2 Carnosine

The high malignancy of GBM, which is reflected by the low progression free and overall survival of patients, is an ongoing challenge, and new treatment strategies and new compounds are investigated by many research teams world-wide. In our group, we focus on the potential use of the naturally occurring dipeptide to evaluate whether its anti-neoplastic effect could be employed for the treatment of GBM patients. 6

1.2.1 Chemistry, Biology, Distribution The dipeptide β-alanyl-L-histidine, better known as L-carnosine, has been isolated for the first time in 1900 by Gulewitsch and Amiradzibi from Liebig's meat extract [49]. It is a member of the family of naturally occurring imidazole-containing dipeptides, which also includes homocarnosine ( γ-Aminobutyryl-L-histidine) and carcinine (N-(2-(1H-Imidazol-5-yl)ethyl)-3-aminopropanamide; β-Alanylhistamine) as well as the naturally most common variants ophidine ( β-alanyl-3-N-methyl-histidine) and anserine ( β-alanyl-1-N-methyl-L-histidine) [50]. Histamine-containing dipeptides can be found throughout the vertebrate kingdom in kidney, brain and skeletal muscle [51]. Interestingly, anserine and ophidine cannot be found in human tissue [50]. Carnosine was detected in the olfactory system of various mammalian species and in a particular fraction of astrocytes known as Bergmann glia in the cerebellum of rodents [52]. In human tissues, highest concentrations of carnosine have been measured in skeletal muscle (21.3±4.2 mmol/kg dry mass in males) [53]. In comparison, the carnosine concentration of human brain tissue (3.0 µmol/100g) is considerably lower than that of homocarnosine (33.4µmol/100g) [54].

1.2.2 Carnosine homeostasis Human carnosine homeostasis is well regulated by rather unspecialized enzymes. Carnosine synthetase (CS; EC 6.3.2.11), a member of the ATP-grasp family of ATPases, catalyzes the synthesis of carnosine from β-alanine and L-histidine and also catalyzes the formation of homocarnosine from L-histidine and γ-aminobutyrate with a 14 to 26-fold lower efficiency. Its activity is 98% cytosolic [55]. CS is related to ATP-grasp domain containing protein-1 that can be found in skeletal and heart muscle as well as in certain brain regions [56]. Within the CNS only oligodendrocytes contain the ability to form carnosine while carnosine uptake is restricted to astrocytes [57]. Carnosine degradation is catalyzed by two hydrolytic enzymes that are specific for carnosine and its related compounds. A carnosinase (CN) was first described in 1949 and found in swine kidney [58]. Carnosinase 1 (CN1, EC 3.4.13.20), also called human serum carnosinase, exists extra-cellular and specifically degrades carnosine with a high activity and contains activity against homocarnosine. Interestingly, CN1 is undetectable in the blood of neonates and shows gradually increasing serum concentrations until the age of 13-15, whereas relatively higher

7 concentrations of CN1 can be found in human brain tissue [59, 60]. Carnosinase 2 (CN2, EC 3.4.13.3), also called tissue carnosinase or cytosolic carnosinase, can be found in nearly every tissue of the human body. Having a broad spectrum of possible substrates in the presence of magnesium or cadmium, but not against homocarnosine, it should be referred to as non-specific cytosolic dipeptidase [60– 62]. Another difference to CN1 is that CN2 activity can be inhibited by bestatin [63]. Despite metabolic characteristics, reduced levels of CN1 protein have been found in GBM patients' serum [64] and a decreased CN1 activity was found in patients with systemic neurological diseases, such as Parkinson’s disease, epilepsy, motor neuron disease, multiple sclerosis, cerebrovascular incidents and dementia [65], [66]. Reduced CN1 plasma concentrations also seem to be linked to a bad prognosis in patients with malignancies, such as GBM or metastatic prostate cancer [67]. Overall, CN1 activity seems to be connected to levels of oxidative stress and cellular stress response which also lead to typical age-related brain changes [68]. Generally, cellular cross-membrane uptake of carnosine in humans is realized through four transporters of the proton-coupled oligopeptide transporter family, namely: PEPT1, PEPT2, PHT1 and PHT2 [50]. PEPT1 can mainly be found on the luminal side of enterocytes, there being responsible for the absorption of peptides across the brush-border membrane [50, 69]. Its renal isoform, PEPT2, reabsorbs carnosine with a low capacity but high affinity from filtered urine back into the cytosol of epithelial cells [70]. Other studies indicate the crucial role of PEPT2 for carnosine homeostasis as it was found in the choroid plexus, astrocytes, human nasal epithelium and cardiomyocytes [71]. The physiological role of PHT1 and PHT2 with regard to carnosine transport still remains unclear, as does their direction of transport. Evidence shows that these transporters can be found in skeleton muscle where they might be responsible for carnosine homeostasis (for further information see [50]).

1.2.3 Physiological functions Many physiological properties are attributed to carnosine, which can be explained by its chemical and biochemical properties, such as its pH buffering capacity, its antioxidant effects and its metal chelating function. Therefore, tissues of most interest are muscle and CNS. Already in 1938 the pH buffering ability of carnosine was described [72, 73], proposing the idea that carnosine has a proton snatching

8 effect during the anaerobic glycolysis of contracting muscles. Because of its relatively high abundance in the olfactory bulb, where it is found in the same presynaptic vesicles as glutamate, it was also discussed whether carnosine may be involved in glutamatergic neurotransmission [74]. This idea is also supported by the fact that carnosine and other peptides appear to work as cell-specific glia-to-neuron neurotransmitters in the brain [75], and there is evidence that carnosine stimulates glutamate release of rat oligodendrocytes in vitro [76].

1.2.4 Therapeutic potential Since Holliday and McFarland demonstrated an anti-senescence and rejuvenating effect of carnosine [77, 78] and Shao et al . proved a reduction of telomere damage and shortening [79] (both in cultured human fibroblasts), carnosine is considered to have anti-aging effects [80]. A more recent work also showed carnosine’s effect on reducing oxidative stress and apoptosis in a galactose aging model of the brain [81]. Even in vivo carnosine demonstrated anti-aging effects, e.g. in Drosophila [82] or senescent-accelerated mice [83], showing a connection between a lifeform’s ability to respond to reactive oxygen species (ROS) and age. With regard to clinical investigations with patients, positive effects of carnosine have been assumed for autistic spectrum disorders [84], cataract and open-angle glaucoma [85], gulf war disease [86], schizophrenia [87, 88] or attention-deficit/hyperactivity disorder in children [89]. Another interesting aspect is carnosine’s involvement in glycemic control and therefore diabetes and its consequences. Nagai et. al demonstrated that blood glucose concentrations can be lowered by carnosine [90]. In addition, oral carnosine supplementation proved to prevent diabetic deterioration of liver and kidney in diabetic Balb/cA mice [91]. A first clinical trial published in 2016 [92] demonstrated an influence of carnosine on postprandial blood glucose and insulin levels in patients with impaired insulin tolerance.

1.2.5 Carnosine and cancer A potential use of carnosine for the treatment of cancer was discovered by Nagi and Suda [93] demonstrating an anti-neoplastic effect of carnosine in vivo on ddY mice with implanted sarcoma-180 cells. Later on, Holliday and McFarland confirmed these findings in vitro and were even able to show that HeLa cells can be selectively eliminated in co-cultures with fibroblasts, using a medium supplemented with 20 mM carnosine [94]. Because these results could be inhibited by supplementing pyruvate 9 and because carnosine has previously been shown to react with aldehyde and keto groups of sugars by an Amadori reaction [95, 96], they suggested an inhibitory effect of carnosine on glycolysis. This idea also took into account that tumor cells are highly dependent on glycolysis – known as the Warburg effect [97], which could be proven true for many different tumor entities [98]. Until today, the anti-neoplastic effect of carnosine has been shown for various types of cancer, such as colorectal cancer [99–101], human gastric cancer cells [102] or human renal carcinoma cells [103]. In a series of experiments performed at the Department for Neurosurgery of the Medical Faculty at the University of Leipzig, this anti-tumor activity was investigated for malignant gliomas [104–106], demonstrating a reduced ATP production of T98G cells in vitro and a link between carnosine treatment and alterations in protein folding. Additionally, we recently were able to present the influence of carnosine on epigenetic regulation in GBM cells [107]. To get a deeper understanding of how carnosine might influence cell tumor signaling, some possible mechanisms will be discussed briefly (for a more detailed presentation see [108]). Due to its ability of buffering ROS, carnosine was shown to inhibit the activation of Extracellular-signal Regulated Kinases 1/2 (ERK1/2) in neurons [109]. An earlier investigation already demonstrated that carnosine strongly inhibits the phosphorylation of ERK 1/2, mitogen-activated protein kinase p38 (p38 MAPK) and Akt in Caco-2 cells, an intestinal epithelial cell line, thus inhibiting the phosphorylation of Eukaryotic translation initiation factor 4E (eIF4E) [110]. This indicates a possible effect of carnosine on the MAPK/ERK-pathway, which is involved in cell proliferation, migration, survival and angiogenesis [111]. Another possible mechanism by which carnosine might affect tumor cell growth could be downregulation of HIF’s (a detailed summary about HIF is given in [112]), as demonstrated by experiments with human colon cancer cells [100, 101].

1.3 Objective of the study

Due to its poor prognosis caused by high tumor cell proliferation, invasiveness and the localization within the central nervous system [3], the diagnosis of glioblastoma is always devastating news for patients and remains challenging for neurosurgeons, neurologists and (radio-)oncologists. Even an aggressive therapy regimen,

10 consisting of gross total resection and adjuvant chemo-radiotherapy, leads to a short overall survival of patients [5]. Therefore, the need for new therapeutic drugs is evident and the key point in glioblastoma research. One strategy could be the inhibition of glioblastoma cell proliferation and migration into healthy tissue. Interestingly, it has been discussed before that high cell proliferation and migration are mutually exclusive events in tumor cells, called the ‘go and grow’ principle [34]. Hence, a new cancer drug should not inhibit one while promoting the other. In the past, we already proved that carnosine reduces the viability and metabolic activity of glioblastoma cells [104–106]. This work aimed at investigating, whether carnosine, while reducing glioblastoma cell proliferation, is also impairing the ability of tumor cells to migrate and consecutively to invade into surrounding tissue. To do so, we compared the metabolic effects of carnosine on primary glioblastoma cells and fibroblasts and established a new co-culture model to evaluate the effect of carnosine on glioblastoma cell invasion.

2 Publication

2.1 General information

Title: Carnosine selectively inhibits migration of IDH-wildtype glioblastoma cells in a co-culture model with fibroblasts

Authors: Oppermann, Henry*; Dietterle, Johannes*; Purcz, Katharina; Morawski, Markus; Eisenlöffel, Christian; Müller, Wolf, Meixensberger Jürgen; Gaunitz, Frank *: contributed equally

Journal: Cancer Cell International

Impact factor: 3.96

Received: 17 April 2018

Accepted: 4 August 2018

Published: 13 August 2018

References: 30

Language: English

Publishing house: BioMed Central Ltd, Springer GmbH

PubMed ID: 30123089

DOI: 10.1186/s12935-018-0611-2

Oppermann et al. Cancer Cell Int (2018) 18:111 https://doi.org/10.1186/s12935-018-0611-2 Cancer Cell International

PRIMARY RESEARCH Open Access Carnosine selectively inhibits migration of IDH‑wildtype glioblastoma cells in a co‑culture model with fbroblasts Henry Oppermann1†, Johannes Dietterle1†, Katharina Purcz1, Markus Morawski2, Christian Eisenlöfel3, Wolf Müller3, Jürgen Meixensberger1 and Frank Gaunitz1*

Abstract Background: Glioblastoma (GBM) is a tumor of the central nervous system. After surgical removal and standard therapy, recurrence of tumors is observed within 6–9 months because of the high migratory behavior and the infltrative growth of cells. Here, we investigated whether carnosine (β-alanine-l-histidine), which has an inhibitory efect on glioblastoma proliferation, may on the opposite promote invasion as proposed by the so-called “go-or-grow concept”. Methods: Cell viability of nine patient derived primary (isocitrate dehydrogenase wildtype; IDH1R132H non mutant) glioblastoma cell cultures and of eleven patient derived fbroblast cultures was determined by measuring ATP in cell lysates and dehydrogenase activity after incubation with 0, 50 or 75 mM carnosine for 48 h. Using the glioblastoma cell line T98G, patient derived glioblastoma cells and fbroblasts, a co-culture model was developed using 12 well plates and cloning rings, placing glioblastoma cells inside and fbroblasts outside the ring. After cultivation in the presence of carnosine, the number of colonies and the size of the tumor cell occupied area were determined. Results: In 48 h single cultures of fbroblasts and tumor cells, 50 and 75 mM carnosine reduced ATP in cell lysates and dehydrogenase activity when compared to the corresponding untreated control cells. Co-culture experiments revealed that after 4 week exposure to carnosine the number of T98G tumor cell colonies within the fbroblast layer and the area occupied by tumor cells was reduced with increasing concentrations of carnosine. Although primary cultured tumor cells did not form colonies in the absence of carnosine, they were eliminated from the co-culture by cell death and did not build colonies under the infuence of carnosine, whereas fbroblasts survived and were healthy. Conclusions: Our results demonstrate that the anti-proliferative efect of carnosine is not accompanied by an induction of cell migration. Instead, the dipeptide is able to prevent colony formation and selectively eliminates tumor cells in a co-culture with fbroblasts. Keywords: Glioblastoma, Migration assay, Fibroblast ring co-culture, Carnosine

*Correspondence: [email protected]‑leipzig.de †Henry Oppermann and Johannes Dietterle contributed equally to this work 1 Department of Neurosurgery, University Hospital Leipzig, Liebigstraße 20, 04103 Leipzig, Germany Full list of author information is available at the end of the article

© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

13 Oppermann et al. Cancer Cell Int (2018) 18:111 Page 2 of 10

Background recently published experiments performed with HCT- Isocitrate dehydrogenase (IDH)-wildtype glioblastoma 116 human colon cancer cells which also indicated that is the most malignant brain tumor of the adult brain the invasion ability of these cells is signifcantly inhibited and designated as Grade IV tumor by the World Health already at a concentration 0.5 mM carnosine [17]. Tere- Organization (WHO) [1]. All tumors used in this study fore, we analyzed the infltrative capacity of IDH-wildtype were IDH1R132H-non-mutant glioblastoma of elderly glioblastoma cells in the presence of carnosine in a newly patients and, for reasons of simplicity, will further be developed co-culture model. In our model glioblastoma referred to as GBM. Aside from a high mitotic activity cells were seeded inside a cloning ring placed in the well and its ability to vascularize, GBM, as all difuse glioma, of a 12 well plate, seeding patient-derived fbroblasts out- has a high potential to infltrate into intact brain tis- side the cloning rings. Te rings were removed after the sue which makes it virtually impossible for the surgeon cells had attached to the culture dishes and the cells were to completely remove the tumor. Cells able to migrate incubated for diferent periods of time in the absence and within intact tissue are considered to be the main cause presence of diferent concentrations of carnosine. Finally, of tumor recurrence which is generally observed within the infltrative potential of the tumor cells was analyzed 6–9 month after surgery and standard therapy [2]. Tere- by determining the number of colonies formed within fore, any therapeutic approach has to consider that it the fbroblast layer and the area they covered. may not be enough to inhibit the proliferation of cells, but should although prevent their spreading into intact Materials and methods tissue. Moreover, as Giese et al. [3] pointed out already Reagents more than 20 years ago, proliferation and migration Unless stated otherwise, all chemicals were purchased appear to be mutually exclusive behaviors. Te con- from Sigma Aldrich (Taufkirchen, Germany). Carnosine cept of a dichotomy of proliferation/migration has been was kindly provided by Flamma (Flamma s.p.a. Chignolo observed by many groups and has coined the term “go d’Isola, Italy). or grow” [4]. Having this dichotomy in mind it is important that a substance that inhibits proliferation does not Cell lines and primary cell cultures at the same time trigger migration and invasive behavior. Te GBM cell line T98G, negative for IDH1R132H-muta- Tis is the case for the dipeptide l-carnosine (β-alanyl- tion and O-6-methylguanine-DNA methyltransferase l-histidine). Tis naturally occurring dipeptide has been (MGMT) promoter methylation, was obtained from the discovered in 1900 by Gulewitsch and Amiradzibi [5]. ATCC, genotyped using a PowerPlex 21 System (Pro- Aside from a number of physiological roles attributed mega; Mannheim; Germany) by the Genolytic GmbH to it, such as pH-bufering or the chelation of metal ions (Leipzig, Germany) and authenticated by comparison to (for review see [6]), it is discussed as a potential drug for data at the ATCC and the DSMZ. T98G cells were used the treatment of tumors (for reviews see [7, 8]). After the at passage 5–7 after genotyping and authentication. Both, frst observations made by Nagai and Suda [9] and the primary GBM cultures and primary fbroblast cultures rediscovery of its anti-neoplastic efect by Holliday and were established from tissue samples obtained during McFarland [10], carnosine’s anti-tumor efect has been standard surgery performed at the Neurosurgery Depart- shown in vitro for a variety of cells derived from diferent ment of the University Hospital Leipzig during 2015 and tumors. Tis, for instance, includes gastric cancer cells 2016. All patients provided written informed consent [11], colon cancer cells [12] and, with special emphasis according to the German laws as confrmed by the local to this work, cells derived from glioblastoma [13]. Unfor- committee. When possible, one primary GBM cell cul- tunately, the exact mechanisms by which the dipeptide ture and one primary fbroblast culture was established exerts its anti-neoplastic efect are still unknown but from tissue samples obtained from each patient (Addi- appear to be pleiotropic and dependent on the tumor tional fle 1: Table S1). All GBM samples were diagnosed cells investigated (for review see [14]). and have been approved by the Neuropathology Depart- Although previous experiments pointed towards the ment of the Leipzig University Hospital. IDH 1 status possibility that carnosine also reduces migration and has been determined using immunohistochemistry and infltration via inhibition of Matrix Metalloproteinase-9 pyrosequencing, MGMT promoter methylation status in SK-Hep-1 hepatoma cells [15] and in oxygen–glucose was determined using nucleic acid amplifcation followed deprived reactive rat astrocytes [16] tumor cell inva- by pyrosequencing. sion in these experiments was determined using trans For cultivation, tissue specimens from the tumor, well chamber assays, which cannot answer the question from galea or from periost were cut into approximately whether migration into tissue or a layer of cells will also 1 mm3 large pieces and then separately placed into be inhibited by the dipeptide. Te same is the case with 25 mm2 culture flasks (TPP, Trasadingen, Switzerland)

14 Oppermann et al. Cancer Cell Int (2018) 18:111 Page 3 of 10

until tumor cells or fibroblasts grew out. When more concentrations of carnosine. On the following days than 90% confluence was reached specimens were medium was exchanged twice a week. removed and primary cell cultures were transferred into 75 mm2 culture flasks (TPP) for further cultiva- Carnosine co‑culture experiments tion. Cell cultures were maintained in high glucose Carnosine was diluted in 0.7% NaCl solution and car- DMEM (4.5 g glucose/ml) supplemented with 2 mM nosine experiments were performed with concentra- Glutamax™, 1% penicillin/streptomycin (all from tions of 0 mM, 10 mM, 25 mM, 50 mM and 75 mM. All Gibco Life Technologies, now Thermo Fisher Scien- ring-culture experiments were prepared as described tific, Darmstadt, Germany) and 10% FBS (Biochrom above. Control experiments with T98G cells inside the GmbH, Berlin, Germany), further referred to as ring and without fbroblasts in the outer part were kept “standard medium”, and kept in incubators (37 °C, 5% for over 2 weeks. Ring co-cultures with T98G and fbro- CO 2/95% air). blasts (P0385) and with GBM cells and fbroblasts of the same patient (P0383 with P0385 and P0431 with P0433) were cultivated for 4 weeks. Troughout cultivation, cell Cell viability assays growth and dissemination were monitored by bright feld For cell viability assays, cells were counted and seeded microscopy. After 4 weeks all co-cultures were fxated in into sterile 96-well plates (µClear, Greiner Bio One, 4.5% paraformaldehyde and stored in 1% sodium azide Frickenhausen, Germany) at a density of 5000 cells/well solution at 4 °C until microscopic analysis. in 200 µl standard medium. After 24 h of cultivation (37 °C, 5% CO2/95% air) the medium was aspirated and Immunostaining fresh medium supplemented with or without carnosine Immunofuorescent staining was carried out to discrimi- was added (100 µl/well) and the cells were incubated for nate between tumor cells and fbroblasts using anti- additional 48 h. Ten, the CellTiter-Glo Luminescent fbroblast TE-7 (CBL271, Merck Millipore, Darmstadt, Cell Viability Assay (CTG, Promega, Mannheim, Ger- Germany), anti-nestin (AB5922, Merck Millipore) and many) was employed to determine viable cells by meas- secondary antibodies (ab6563, ab150081, abcam, Cam- uring ATP in cell lysates and the CellTiter-Blue Cell bridge, UK). Briefy, for the detection of TE-7 the fxated Viability Assay (CTB, Promega) was used to quantify co-cultures were permeabilized with 0.1% TritonX-100 the cell’s metabolic capacity in living cells. All assays at room temperature (RT) for 5 min. After blocking with were carried out according to manufacturer’s protocols. 10% goat serum for 15 min, samples were incubated Luminescence and fuorescence were measured using a with anti-fbroblast TE-7 primary antibodies (dilution SpectraMax M5 multilabel reader (Molecular Devices, 1:100) at 5 °C overnight, washed with TBS (20 mM Tris, Biberach, Germany). 134 mM NaCl) and subsequently incubated with the secondary antibody (1:250; ab6563) for 45 min at RT. For the detection of nestin, fxated cell cultures were per- Co‑cultivation of GBM cells and fbroblasts (ring‑cultures) meabilized with 0.1% TritonX-100 in TBS for 1 h at RT, When cells reached more than 90% confuence in blocked for 15 min with 10% goat serum and then incu- 2 75 cm cell culture fasks they were detached using bated with an anti-nestin antibody (dilution 1:250) for 1 h accutase (Termo Fisher), counted and diluted for at RT. Afterwards, cultures were washed with TBS and co-cultivation. Te ring-cultures were established in incubated with a dilution of secondary antibody (1:250; 12-well plates. Terefore, sterile cloning rings (steel, ab150081) for 45 min at RT. Finally, nuclei were coun- 6 mm inner; 8 mm outer diameter, Hartenstein, Wür- terstained with DAPI (4 µg/ml) and cell cultures were zburg, Germany) usually used for the isolation of preserved in 10% sodium azide solution at 4 °C until clones, were placed in the middle of each well divid- microscopy. ing it into an inner-ring and an outer-ring part. Ten, 2500 tumor cells suspended in standard medium Microscopy (112 µl) were seeded inside the ring. Afterwards, 50,000 fbroblasts (in 658 µl standard medium) were seeded For microscopic analysis a Zeiss Axiovert 200M micro- outside of the ring (Additional fle 2: Figure S1). Co- scope (Zeiss, Oberkochen, Germany) equipped with a cultures with cloning rings were incubated for 4 h motorized stage (Märzhäuser, Wetzlar, Germany) with MosaiX software and by means of a CCD camera (Zeiss (37 °C, 5% CO2/95% air) before the rings were carefully removed using sterile forceps. Medium was exchanged MRC) connected to an AxioVision 4.8.2 image analysis immediately after ring removal containing various system (Zeiss) was used to create tile pictures. Each tile picture is composed of 285 single microscopic images

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taken at a magnifcation of 50 and represents a whole well of a 12-well plate. As denoted in the fgure legend to Fig. 2 ImageJ images in this fgure have been graphically enhanced for representation purposes using the Corel Draw Graphics Suite 2017 (Corel Corporation, Ottawa, Canada).

Quantitative and statistical analysis Te number of colonies in ring co-culture experiments was determined using ImageJ after a color threshold and a common pixel size were defned. All pictures used for the analysis were taken at the same magnifcation and Fig. 1 Viability of patient derived fbroblasts and primary had the same size. Statistical analysis was performed glioblastoma cells under the infuence of carnosine. Fibroblast cell cultures (11) and primary GBM cell cultures (9) were exposed using the algorithm for t-test implemented in Microsoft for 48 h to diferent concentrations of carnosine (0, 50 or 75 mM). Excel 2010. Viability was determined by measuring the amount of ATP in cell lysates (a) and by assessing metabolic activity (b). Each dot within the boxplots represents the mean obtained from six measurements Results of an individual cell culture. For comparison, the grey-colored dots Viability of patient derived fbroblasts and GBM cells (depicted by arrows) indicate the results obtained from experiments with the cell line T98G which was used for co-culture experiments. under the infuence of carnosine Statistical signifcance using data obtained from 11 fbroblast cultures In order to fgure out how fbroblasts isolated from and 9 primary GBM cultures was determined using Student’s t-test patients who underwent surgery for GBM do respond to with: *p < 0.05; **p < 0.005; ***p < 0.0005 the presence of carnosine, 11 primary fbroblast cultures derived from galea or periost were exposed to diferent 75 mM carnosine, viability of primary GBM cells was sig- concentrations of carnosine. Viability was determined by nifcantly stronger reduced than in fbroblasts (metabolic measuring the amount of ATP in cell lysates and by the activity: p < 0.05; ATP: p < 0.05). analysis of metabolic activity, as refected by dehydroge- Please also note, that for statistical analysis data nase activity. In order to compare the efect of the dipep- obtained from experiments with the cell line T98G were tide on fbroblasts, we performed the same experiment excluded. with 9 primary GBM cell cultures derived from patients and cells from the GBM cell line T98G. All cells were Outgrowth of cells from a ring culture seeded at a density of 5000 cells into the wells of 96-well without co‑cultivated fbroblasts plates and exposed to carnosine for 48 h. Te result of Next, we asked how carnosine infuences the growth of the experiment is presented in Fig. 1. At a concentration glioblastoma cells over a longer period of time when they of 50 mM carnosine, the amounts of ATP and the meta- start to grow after being seeded inside of a cloning ring bolic activity in fbroblasts were signifcantly reduced and removal of it in the absence of other cells. Tere- compared to untreated control cells (metabolic activity: fore, 2500 cells from the glioblastoma cell line T98G 89.4% ± 7.53, p < 0.005; ATP: 96.6% ± 5.08%. p < 0.05). were seeded inside a cloning ring placed in a 12-well We also observed a signifcant reduction of metabolic plate. Four hours later the rings were removed and the activity in primary GBM cells treated with 50 mM car- cells were allowed to grow for 2 weeks in the absence nosine (compared to untreated control: 79.7% ± 18.51%, and presence of carnosine (10 mM, 25 mM, 50 mM and p < 0.05). However, although the amounts of ATP were 75 mM). Medium was exchanged twice a week. After decreased at 50 mM carnosine (87.8% ± 16.35% com- fxation of cells, 285 tiled images were taken from each pared to the untreated control) this efect was not sig- well for each concentration employed (in quadruplicate) nifcant (p = 0.056). Increasing the concentration of and the area covered by tumor cells was determined carnosine to 75 mM leads to a signifcant reduction using ImageJ. Te result of the experiment is presented of metabolic activity and a decrease in the amounts of in Fig. 2. As can be seen, the tumor cell covered area is ATP in both, primary GBM cells (metabolic activity: continuously decreasing with increasing concentrations 61.2% ± 18.96, p < 0.0005; ATP: 71.5% ± 19.68%. p < 0.005) of carnosine although the efect becomes signifcant only and fbroblasts (metabolic activity: 81.5% ± 12.03, at a concentration of 50 mM carnosine. At a concentra- p < 0.0005; ATP: 88.4% ± 5.07%. p < 0.005) compared to tion of 75 mM carnosine, no tumor cells were remain- the untreated control. Furthermore, in the presence of ing on the plates, demonstrating that this concentration

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do survive for 2 weeks when seeded at a density of 2500 cells inside a cloning ring and are incubated at carnosine concentrations of 50 mM or below.

Fluorescent discrimination of tumor cells and fbroblasts in co‑culture In order to perform co-culture experiments with GBM cells and fbroblasts, we had to establish a method by which both types of cells can be discriminated. Using patient derived fbroblasts (P0385) and primary glioblastoma cells (P0383) we were able to discriminate both types of cells using an antibody directed against nestin (Fig. 3). Although both types of cells were detected by the anti-nestin specifc antibody, staining was signifcantly more intense on GBM cells allowing discrimination between both types of cells as demonstrated by the images presented in Fig. 3. Unfortunately, discrimination between fbroblasts and cells from the glioblastoma cell line T98G was not possible using the nestin-specifc antibody. We found a solu- Fig. 2 Outgrowth of T98G cells under the infuence of carnosine. T98G cells were seeded inside a cloning ring placed in a 12-well tion, using an anti-body directed against TE-7 which plate. Rings were removed after 4 h and cells were allowed to stains an unknown antigen specifc for fbroblasts [18]. grow in the presence of 0, 10, 25, 50 and 75 mM carnosine. After As demonstrated in Fig. 4 the TE-7 specifc anti-body 14 days, images were taken and the area covered by tumor cells was did also stain the nuclei of T98G cells, but did not detect determined. The mean and standard deviation of the covered area antigen in their cytoplasm. Terefore, counterstaining from four independent wells for each concentration are presented in a. A statistical signifcant reduction was only seen at 50 mM with DAPI (color-coded in red) and TE-7 (color-coded in carnosine (*) and at a concentration of 75 mM carnosine no tumor green) results in a yellow color of T98G nuclei and fbro- cells were left on the plates. In the lower part of the panel examples blasts appear in green (cytosol) with red nuclei. At this of images are presented that were used for data analysis (b without point it should also be noted that the TE-7 specifc anti- carnosine; c 10 mM; d 25 mM and e 50 mM carnosine). #2 presents body did also stain cells in primary GBM cultures which the corresponding ImageJ processed pictures (Note: for presentation purposes the pictures shown have been vectorized and image is the reason why this antibody had not been used for co- enhanced using Corel Draw Graphic Suite 2017) cultures with primary cells.

Colony formation of T98G tumor cells in ring culture does not only inhibit proliferation but also eliminates the with fbroblasts under the infuence of carnosine tumor cells when exposed to the dipeptide for 2 weeks. Next we asked whether T98G cells seeded inside a In conclusion, the experiment demonstrates that the cells cloning ring can migrate into a surrounding layer of

Fig. 3 Nestin staining of patient derived GBM cells and fbroblasts in ring co-culture. The fgure shows staining of a co-culture of fbroblasts (P0385) with GBM cells (P0383) with DAPI (a), a nestin-specifc antibody (b) and the merged image (c). GBM cells are presented in bright green at the right side of the image. Scale bar: 300 µm

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Fig. 4 TE-7 staining of T98G cells and fbroblasts in mono-, co- and in ring co-culture. The fgure shows staining in mono cultures of fbroblasts (P0375) with DAPI (a), with a TE-7-specifc antibody (e) and the merged image (i) and the corresponding images of staining of T98G cells (b, f, j). Images c, g and k were taken from a co-culture of both cells and d, h and f shows an image of a ring culture at low magnifcation. Fibroblasts appear with green staining in the cytoplasm and red nuclei, whereas T98G cells are presented by their yellow stained nuclei in the merged images. Scale bars a–c, e–g, i–k 30 µm, d, h, i 120 µm

fbroblasts and whether migration and development of tumor cells was analyses using ImageJ. In Fig. 6a example tumor cell colonies inside the fbroblast layer is infu- fuorescence images of cultures and their corresponding enced by carnosine. Terefore, ring co-cultures were ImageJ derived images are presented as well as the result established with T98G cells inside the cloning ring and of the determination of area occupancy (Fig. 6b) and the fbroblasts (P0375) outside of it. After removal of the measurement of the number of colonies formed (Fig. 6c). ring, cells frst have to migrate towards each other flling As can be seen, the size of the area occupied by tumor the gap left after ring removal. As presented in Fig. 5 cells cells and the number of colonies are decreasing with are migrating to fll the gap within 4–11 days. As can be increasing concentrations of carnosine. In the absence seen, both, fbroblasts (right side) and T98G cells are able of carnosine tumor cells occupied 13.5% ± 3.5% of the to migrate towards each other, flling the gap between plate. Already in the presence of 10 mM carnosine area day 4 and day 11 in the absence of carnosine. In the pres- occupancy was signifcantly decreased to 7.7% ± 2.5% ence of 50 mM carnosine migration is impaired in both (p < 0.05) and diminished further by increasing con- types of cells as shown in the image taken after 4 days. centrations of carnosine (25 mM: 6.0% ± 3.0%, p < 0.05; At day 11 the gap gets flled, too, but it seems that mainly 50 mM: 3.1% ± 3.3%, p < 0.0005). In addition, the num- fbroblasts are located in the previous gap (Note: Tis ber of colonies decreased with increasing concentrations experiment resembles a classical scratch assay). In a fol- from 80.8 ± 46.8 colonies in the absence of carnosine to lowing series of experiments ring co-cultures of T98G 46.6 ± 24.4 colonies (10 mM; not signifcant), 28.8 ± 24.0 cells and fbroblasts (P0375) incubated for 4 weeks in dif- (25 mM, p < 0.05) and to 1.5 ± 0.8 colonies (50 mM, ferent concentrations of carnosine (0, 10, 25 and 50 mM) p < 0.05). Tis clearly demonstrates that carnosine inhib- were fxated and stained with DAPI and the TE-7 specifc its the potential of T98G cells to infltrate the fbroblast antibody. After fuorescence microscopy the number of layer. colonies formed by T98G cells and the area occupied by

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Fig. 5 Cells migrating into the gap after removal of the cloning ring in a co-culture of T98G cells and fbroblasts. T98G cells (2500) were placed inside the cloning ring and fbroblasts (P0375; 50.000) outside of it. Migration of cells into the gap left after removal of the cloning ring 3, 4 and 11 days after ring removal with fbroblasts on the right side and T98G cells to the left as detected by bright feld microscopy is presented. In the upper panel cultures were incubated in medium without carnosine and in the lower panel in medium containing 50 mM carnosine. Scale bar: 30 µm

Fig. 6 Colony formation of T98G cells in a fbroblast layer in the presence of diferent concentrations of carnosine. T98G cells (2500) were placed inside the cloning ring and fbroblasts (P0375; 50.000) outside of it. The ring was removed 4 h later and after 4 weeks of cultivation in the presence of diferent concentrations of carnosine the cultures were fxated and stained with DAPI and a TE-7 specifc antibody. Fluorescence images are presented in the upper part of panel a and pictures generated by ImageJ for the determination of area occupancy and the number of colonies formed is presented in the lower part. On the right side of the graph, area occupancy (b 100% equals the whole well area) and the number of tumor cell colonies formed (c) is presented. Statistical signifcance using data obtained from 8 to 10 independently treated cultures for each concentration of carnosine was determined using Student’s t-test with: *p < 0.05; ***p < 0.0005

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Colony formation of primary glioblastoma cells in ring from adults or even senescent patients may behave difer- culture with fbroblasts under the infuence of carnosine ent to fbroblasts isolated from fetal or newborn human In order to verify that the efect of carnosine is not tissue. In our experiments presented in Fig. 1 we did not restricted to a cell line but can also be seen with tumor see a measurable benefcial efect of carnosine on fbro- cells derived from a patient, we also performed ring co- blast viability, although it is interesting to note, that culture experiments with two patient-derived tumor fbroblasts cultivated in 50 mM carnosine appeared to cell cultures (P0383 and P0431) and fbroblasts from the be rejuvenated compared to fbroblasts cultivated in the same patient (P0385 and P0433, respectively). Again, absence of carnosine in accordance to the observations tumor cells were seeded inside the cloning ring and of McFarland and Holiday [21]. We also did not see any fbroblasts outside of it. After 4 weeks of cultivation correlation between age of the patient, the tissue of ori- cells were stained with DAPI and a nestin-specifc anti- gin or gender, though it has to be realized that the num- body and analyzed using ImageJ. Surprisingly, we did ber of samples may be too low for such an analysis and not detect colony formation inside the fbroblast layer most of our patients have been of comparable age. More even in the absence of carnosine. On the other hand, the importantly, a prolonged cultivation of fbroblasts as in tumor occupied area was signifcantly reduced already the co-culture experiments demonstrates that the fbro- at a concentration of 10 mM carnosine. As the two pri- blasts are alive and able to occupy the space left by dying mary cultured glioblastoma cells occupied diferent areas glioblastoma cells even under the highest concentration in the absence of carnosine, we set the area occupied in of carnosine employed (50 mM). It is also very interest- the absence of carnosine for each cell culture as 100% in ing to note that we observed complete cell death of T98G order to compare the efect of carnosine and to deter- tumor cells in long term culture incubating the cells at mine an average. We found a reduction of the occupied a concentration of 75 mM carnosine (Fig. 2). In previ- area to 68.1% ± 11.3% (p < 0.05) at a concentration of ous experiments, cells were usually kept in the presence 10 mM carnosine, to 70.6% ± 3.1% (p < 0.0005) at 25 mM of carnosine for 24, 48 or 96 h [13] but not for 2 weeks. and to 18.7% ± 3.0% (p < 0.0005) at 50 mM. Comparable Tis is interesting, as shorter exposure times in previous to the experiments with T98G cells and fbroblasts pre- experiments resulted in reduced proliferation but not sented in Fig. 6, the space previously occupied by tumor complete elimination of tumor cells. Which processes cells becomes occupied by the fbroblasts. (For compari- are responsible for reduced tumor cell proliferation are son: setting the occupied area to 100% in the experiment under the infuence of carnosine are still unknown [14]. with T98G cells the reduction is 57.0% ± 18.5% at 10 mM, However, it is an interesting question whether the pro- 44.4% ± 22.2% at 25 mM and 23.0% ± 24.4% at 50 mM cesses which reduce proliferation after short term expo- carnosine). sure may fnally lead to cell death when the tumor cells are exposed to carnosine for a longer period of time. Discussion In order to discriminate tumor cells from fbroblasts in As outlined in the introduction, we and others have ring co-culture experiments several markers were tested shown that the naturally occurring dipeptide carnosine including glial fbrillary acidic protein (GFAP) which did inhibits the growth of cancer cells in vitro and in vivo also stained fbroblasts in accordance with observations [14], whereas benefcial efects have been observed in made by others [22]. We fnally identifed that nestin- cultured human fbroblasts [19]. Using a colorimetric staining was suitable to discriminate primary cultured assay (tetrazolium compound [3-(4,5-dimethylthiazol- tumor cells from patient-derived fbroblasts. Nestin is a 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- class VI intermediate flament protein and a marker for 2H-tetrazolium, inner salt; MTS]) and human dermal neural stem cells. In addition, it has been reported as a fbroblasts, Ansurudeen et al. also demonstrated a greater cancer stem cell-specifc marker [23] and a recent meta- number of viable cells in the presence of 50 mM carno- analysis performed by Lv et al. [24] demonstrated that sine after 24 h incubation [20]. Unfortunately, it is not increased expression of nestin is positively associated traceable whether the fbroblasts used by Ansurudeen with higher histological grade in glioma patients. Tis et al. were from juvenile foreskin or from adult skin. In analysis also indicated that patients with higher nes- addition, the experiments performed by Holliday and tin expression are prone to recurrence and glioma cell McFarland were done by using a fbroblast cell line estab- infltration into intact brain tissue. Surprisingly, in our lished from human foreskin of a newborn male (HFF-1) ring co-culture experiments T98G cells, which did not and a second cell line established from lung tissue of a express nestin, as previously reported by other investiga- male at 14 weeks gestation (MRC-5). Considering using tors [25, 26], gave rise to many colonies in the surround- carnosine for the treatment of elderly patients the ques- ing fbroblast layer which was not the case when primary tion had to be answered whether fbroblasts isolated cultures with a high expression of nestin were cultivated

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with fbroblasts. Although speculative, one interpretation Abbreviations CTB: CellTiter-Blue Cell Viability Assay; CTG: CellTiter-Glo Luminescent Cell Via- could be that the primary cultures we used were of very bility Assay; GBM: IDH1R132H-non-mutant glioblastoma; GFAP: glial fbrillary early passages (Passage 1 and 5) and not as rapidly grow- acidic protein; HOXC9: Homeobox Protein C9; IDH: isocitrate dehydrogenase; ing as T98G cells which is also refected by their higher MGMT: O-6-methylguanine-DNA methyltransferase; MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner resistance towards carnosine (Fig. 1) as carnosine exerts salt; YB-1: Y-box binding protein-1; WHO: World Health Organization. its action mainly on metabolic highly active tumor cells [27]. Nonetheless, the results presented in Fig. 6 clearly Authors’ contributions JD performed most of the experiments with contributions of HO and KP. MM demonstrate that colony formation is signifcantly inhib- and CE assisted at microscopy and immuno-histochemistry. WM and CE per- ited when invasively grown glioblastoma cells are treated formed the pathologic classifcation of tumors. JM did the surgery and revised with carnosine. In the last years, a so-called “go or grow the manuscript. HO, JD and FG designed the experiments and contributed conceptually. HO and FG coordinated the experiments. JD and FG mainly concept” has been discussed, assuming that prolifera- wrote the manuscript but all authors contributed to its writing. All authors tion and migration are mutually exclusive phenomena read and approved the fnal manuscript. in cancer cells [4]. Studies confrming this hypothesis Author details are for example observations made in breast cancer cell 1 Department of Neurosurgery, University Hospital Leipzig, Liebigstraße 20, lines in which overexpression of Homeobox Protein C9 04103 Leipzig, Germany. 2 Medical Faculty, Paul-Flechsig-Institute of Brain 3 (HOXC9) resulted in increased invasiveness but at the Research, University of Leipzig, Leipzig, Germany. Department of Neuropa- thology, University Hospital Leipzig, Leipzig, Germany. same time inhibited proliferation [28]. Another example is the observation that enforced expression of Y-box Acknowledgements binding protein-1 (YB-1) in non-invasive breast epithe- We like to thank Flamma [Flamma s.p.a. Chignolo d’Isola, Italy (http://www. fammagroup.com)] for the generous supply with very high quality carnosine lial cells induces an epithelial–mesenchymal transition for all of our experiments. In addition, we like to thank Dr. Hans-Heinrich (EMT) resulting in an enhanced metastatic potential Foerster from the Genolytic GmbH (Leipzig, Germany) for genotyping and but at the same time reduces proliferation [29]. Unfortu- confrmation of cell identity and last not least Mr. Rainer Baran-Schmidt for technical assistance. nately, the exact mechanisms and down-stream targets responsible for either proliferation or invasion mediated Competing interests by HOXC9 or YB-1 signaling are still unknown. More The authors declare that they have no competing interests. importantly, our results demonstrate that carnosine does Availability of data and materials not inversely infuence proliferation and invasion. Up to All data generated or analyzed during this study are included in this published now, the mechanisms responsible for the dipeptides anti- article. proliferative efect, which has been confrmed in several Consent for publication studies with diferent types of cancer cells [11–13, 30] All authors have read and approved of its submission to Cancer Cell are still not understood. With regard to carnosine’s efect International. on migration and invasion it has been discussed that it Ethics approval and consent to participate involves regulation of matrix metalloproteinases (MMPs) All patients provided written informed consent to participate according to the [15], but more experiments are certainly needed to prop- German laws as confrmed by the local committee. erly address this question. Funding No funding has been received. We acknowledge support from the German Conclusions Research Foundation (DFG) and Leipzig University within the program of Open Access Publishing. Tis study demonstrates that carnosine’s anti-proliferative efect is not accompanied by increased invasion as Publisher’s Note suggested by the so-called “go-or-grow” concept. In fact, Springer Nature remains neutral with regard to jurisdictional claims in pub- the dipeptide can inhibit tumor cell migration, which is lished maps and institutional afliations. especially important for the treatment of highly infltrat- Received: 17 April 2018 Accepted: 4 August 2018 ing and metastasizing tumors such as IDH-wildtype glioblastoma. In addition, the co-culture model presented is a valuable alternative to the commonly used scratch or Boyden chamber assays. References 1. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK. WHO classifcation of tumours of the central nervous system. 4th ed. Lyon: International Agency for Research on Cancer; 2016. Additional fles 2. Mallick S, Benson R, Hakim A, Rath GK. Management of glioblastoma after recurrence: a changing paradigm. J Egypt Natl Cancer Inst. 2016;28:199– Additional fle 1: Table S1. Patients and patient derived cell cultures. 210. https://doi.org/10.1016/j.jnci.2016.07.001. 3. Giese A, Loo MA, Tran N, Haskett D, Coons SW, Berens ME. Dichotomy of Additional fle 2. Setup of ring-cultures. astrocytoma migration and proliferation. Int J Cancer. 1996;67:275–82.

21 Oppermann et al. Cancer Cell Int (2018) 18:111 Page 10 of 10

https://doi.org/10.1002/(SICI)1097-0215(19960717)67:2%3c275:AID-IJC20 18. Goodpaster T, Legesse-Miller A, Hameed MR, Aisner SC, Randolph- %3e3.0.CO;2-9. Habecker J, Coller HA. An immunohistochemical method for identifying 4. Hatzikirou H, Basanta D, Simon M, Schaller K, Deutsch A. ‘Go or grow’: the fbroblasts in formalin-fxed, parafn-embedded tissue. J Histochem key to the emergence of invasion in tumour progression? Math Med Biol. Cytochem. 2008;56:347–58. https://doi.org/10.1369/jhc.7A7287.2007. 2012;29:49–65. https://doi.org/10.1093/imammb/dqq011. 19. Holliday R, McFarland GA. A role for carnosine in cellular maintenance. 5. Gulewitsch W, Amiradzibi S. Ueber das Carnosin, eine neue organische Biochemistry (Mosc). 2000;65:843–8. Base des Fleischextraktes. Ber Dtsch Chem Ges. 1900;33:1902–3. 20. Ansurudeen I, Sunkari VG, Grünler J, Peters V, Schmitt CP, Catrina S-B, et al. 6. Boldyrev AA, Aldini G, Derave W. Physiology and pathophysiology of Carnosine enhances diabetic wound healing in the db/db mouse model carnosine. Physiol Rev. 2013;93:1803–45. https://doi.org/10.1152/physr of type 2 diabetes. Amino Acids. 2012;43:127–34. https://doi.org/10.1007/ ev.00039.2012. s00726-012-1269-z. 7. Gaunitz F, Hipkiss AR. Carnosine and cancer: a perspective. Amino Acids. 21. McFarland GA, Holliday R. Further evidence for the rejuvenating efects 2012;43:135–42. https://doi.org/10.1007/s00726-012-1271-5. of the dipeptide l-carnosine on cultured human diploid fbroblasts. Exp 8. Hipkiss AR, Gaunitz F. Inhibition of tumour cell growth by carnosine: Gerontol. 1999;34:35–45. some possible mechanisms. Amino Acids. 2014;46:327–37. 22. Hainfellner JA, Voigtländer T, Ströbel T, Mazal PR, Maddalena AS, Aguzzi 9. Nagai K, Suda T. Antineoplastic efects of carnosine and beta-alanine— A, Budka H. Fibroblasts can express glial fbrillary acidic protein (GFAP) physiological considerations of its antineoplastic efects. J Physiol Soc in vivo. J Neuropathol Exp Neurol. 2001;60:449–61. Jpn. 1986;48:741–7. 23. Neradil J, Veselska R. Nestin as a marker of cancer stem cells. Cancer Sci. 10. Holliday R, McFarland GA. Inhibition of the growth of transformed and 2015;106:803–11. https://doi.org/10.1111/cas.12691. neoplastic cells by the dipeptide carnosine. Br J Cancer. 1996;73:966–71. 24. Lv D, Lu L, Hu Z, Fei Z, Liu M, Wei L, Xu J. Nestin expression is associated 11. Shen Y, Yang J, Li J, Shi X, Ouyang L, Tian Y, Lu J. Carnosine inhibits the with poor clinicopathological features and prognosis in glioma patients: proliferation of human gastric cancer SGC-7901 cells through both an association study and meta-analysis. Mol Neurobiol. 2017;54:727–35. of the mitochondrial respiration and glycolysis pathways. PLoS ONE. https://doi.org/10.1007/s12035-016-9689-5. 2014;9:e104632. https://doi.org/10.1371/journal.pone.0104632. 25. Kurihara H, Zama A, Tamura M, Takeda J, Sasaki T, Takeuchi T. Glioma/ 12. Iovine B, Oliviero G, Garofalo M, Orefce M, Nocella F, Borbone N, et al. The glioblastoma-specifc adenoviral gene expression using the nestin gene anti-proliferative efect of l-carnosine correlates with a decreased expres- regulator. Gene Ther. 2000;7:686–93. https://doi.org/10.1038/sj.gt.33011 sion of hypoxia inducible factor 1 alpha in human colon cancer cells. 29. PLoS ONE. 2014;9:e96755. https://doi.org/10.1371/journal.pone.0096755. 26. Hong X, Chedid K, Kalkanis SN. Glioblastoma cell line-derived spheres in 13. Renner C, Seyfarth A, de Arriba S, Meixensberger J, Gebhardt R, Gaunitz serum-containing medium versus serum-free medium: a comparison of F. Carnosine inhibits growth of cells isolated from human glioblastoma cancer stem cell properties. Int J Oncol. 2012;41:1693–700. https://doi. multiforme. Int J Pept Res Ther. 2008;14:127–35. https://doi.org/10.1007/ org/10.3892/ijo.2012.1592. s10989-007-9121-0. 27. Oppermann H, Schnabel L, Meixensberger J, Gaunitz F. Pyruvate attenu- 14. Gaunitz F, Oppermann H, Hipkiss AR. Carnosine and cancer. In: Preedy VR, ates the anti-neoplastic efect of carnosine independently from oxidative editor. Imidazole dipeptides. Cambridge: The Royal Society of Chemistry; phosphorylation. Oncotarget. 2016;7:85848–60. https://doi.org/10.18632/ 2015. p. 372–92. oncotarget.13039. 15. Chuang C-H, Hu M-L. l-Carnosine inhibits metastasis of SK-Hep-1 cells by 28. Hur H, Lee J-Y, Yang S, Kim JM, Park AE, Kim MH. HOXC9 induces pheno- inhibition of matrix metaoproteinase-9 expression and induction of an typic switching between proliferation and invasion in breast cancer cells. antimetastatic gene, nm23-H1. Nutr Cancer. 2008;60:526–33. https://doi. J Cancer. 2016;7:768–73. https://doi.org/10.7150/jca.13894. org/10.1080/01635580801911787. 29. Evdokimova V, Tognon C, Ng T, Ruzanov P, Melnyk N, Fink D, et al. Transla- 16. Ou-Yang L, Liu Y, Wang B-Y, Cao P, Zhang J-J, Huang Y-Y, et al. Carnosine tional activation of snail1 and other developmentally regulated transcrip- suppresses oxygen–glucose deprivation/recovery-induced proliferation tion factors by YB-1 promotes an epithelial–mesenchymal transition. and migration of reactive astrocytes of rats in vitro. Acta Pharmacol Sin. Cancer Cell. 2009;15:402–15. https://doi.org/10.1016/j.ccr.2009.03.017. 2018;39:24–34. https://doi.org/10.1038/aps.2017.126. 30. Zhang Z, Miao L, Wu X, Liu G, Peng Y, Xin X, et al. Carnosine inhibits the 17. Lai P-Y, Hsieh S-C, Wu C-C, Hsieh S-L. ID: 1029 Efects of carnosine on proliferation of human gastric carcinoma cells by retarding Akt/mTOR/ regulation of migration and invasion in human colorectal cancer cells. p70S6K signaling. J Cancer. 2014;5:382–9. https://doi.org/10.7150/ Biomed Res Ther. 2017;4:104. https://doi.org/10.15419/bmrat.v4iS.305. jca.8024.

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22 3 Summary

Dissertation zur Erlangung des akademischen Grades Dr. med.

Carnosine selectively inhibits migration of IDH-wildtype glioblastoma cells in a co- culture model with fibroblasts eingereicht von Johannes Andreas Dietterle angefertigt an der Universität Leipzig Klinik und Poliklinik für Neurochirurgie betreut von Prof. Dr. Frank Gaunitz Prof. Dr. Jürgen Meixensberger

12/2018 Carnosine, ß-alanyl-L-histidine, is a naturally occurring dipeptide with the highest concentration in skeletal muscle and was discovered over 100 years ago [49, 53]. Research concerning carnosine mainly focuses on metabolic effects in muscle tissue [113–116] and the scavenging effect for reactive oxygen species [68], which seems to be highly related to anti-aging [83, 84] and responsible for its positive effects on neuro-degenerative diseases [65, 66, 68]. Additionally, in 1986 Nagai and Suda [93] demonstrated an anti-neoplastic effect of carnosine in a Sarcoma 180 mouse model, which was later re-discovered by Holliday and McFarland on HeLa cells in vitro [94]. In the following course, Renner et al. were able to demonstrate an anti-neoplastic effect in glioblastoma (GBM) cells and gave rise to research concerning the effect of carnosine on glioblastoma (GBM) [104–106]. GBM is the most common malignant brain-derived tumor and associated with very poor prognosis [5]. It is characterized by central necrosis, high vascularization, highly infiltrative behavior and therefore classified as WHO grade IV tumor [3]. Diagnosis,

23 prognosis and treatment of GBM are highly dependent on the mutational status of isocitrate-dehydrogenase (IDH) and methylation of the promoter of O-6- methylguanine-DNA methyltransferase (MGMT) [16, 17, 21]. This work focused on IDH-wildtype GBM, also called primary GBM. The median age of diagnosis for primary GBM is 64 years and common onset-symptoms are seizures, headache or, depending on its location, neurological deficits like paralysis or aphasia [3, 5]. Standard therapy for primary glioblastoma consists of microsurgery, aiming for gross total resection, and adjuvant radio-chemotherapy. Depending on the tumor’s MGMT promoter methylation status and the patients’ general condition (e.g. karnofsky performance score), chemotherapy with temozolomide is applied concomitantly to radiotherapy for patients with a methylated MGMT promoter and therefore of high prognostic relevance [18]. Since the discovery of temozolomide for GBM therapy in 2005 [26], no other systemic or local therapy has accomplished to be integrated as standard care into guidelines for GBM [28]. The two biggest challenges in glioblastoma therapy are its highly infiltrative behavior into adjacent brain tissue, which makes it virtually impossible to achieve a total resection, and its highly proliferative profile. Interestingly, the so called ‘go and grow’ principle assumes that a highly proliferative profile and high tumor cell migration are mutually exclusive events [34]. Thus, a new drug should address both issues in order to prevent the promotion of one by inhibiting the other. This work aimed at finding out, whether carnosine inhibits proliferation or infiltration or both characteristics of GBM to evaluate if carnosine might be a useful drug for the fight against glioblastoma. Primary cell cultures of fibroblasts and GBM cells were established from tissue samples obtained during neurosurgery performed at the University Hospital Leipzig between 2015 and 2016. The GBM cell line T98G was obtained from ATCC. In a first series of experiments, fibroblasts and GBM cells were separately seeded into 96-well plates and treated with (10 mM, 25 mM, 50 mM, 75 mM) or without carnosine. After 48 hours, cell-based assays were performed to quantify ATP in cell lysates and dehydrogenase activity in living cells to measure the metabolic activity of primary fibroblasts and GBM cells under the influence of carnosine. The results show that carnosine significantly impairs the metabolic activity of both, fibroblasts as well as GBM cells. At a concentration of 75 mM the viability in glioblastoma cells was significantly reduced when compared to fibroblasts, demonstrating a more pronounced effect of carnosine on tumor cells than on fibroblasts.

To evaluate whether carnosine also inhibits migration and proliferation of GBM cells, ring-culture experiments were performed. Therefore, sterile cloning rings were placed in the middle of 12-well plate wells, dividing them into inner and outer ring parts. In a first step T98G cells were seeded into the inner ring part and rings were removed after 4 hours. Cells were then allowed to grow over the course of 2 weeks with (10 mM, 25 mM, 50 mM and 75 mM) or without the influence of carnosine. The outgrowth of T98G cells was visualized by microscopy and the area occupied by T98G cells was analyzed with ImageJ. The experiment revealed that already the lowest carnosine concentration of 10 mM inhibits tumor cell migration and proliferation, with significant effects at 50 mM carnosine. Furthermore, when incubated in the presence of 75 mM carnosine, no viable tumor cells remained on the plates showing that carnosine not only inhibits tumor cell growth but also eliminates them. We then asked, whether carnosine also reduces the ability of GBM cells to infiltrate a fibroblast layer as a model for surrounding tissue. In order to discriminate between fibroblasts and GBM cells we firstly established a staining protocol for our co-cultures using anti-nestin and anti-TE-7 antibodies. Ring cultures were then used to establish co-cultures of GBM cells and fibroblasts by seeding T98G or primary GBM cells within the cloning ring and fibroblasts outside of it. Co- cultures were kept with (10 mM, 25 mM, 50 mM and 75 mM) or without carnosine over 4 weeks and the results were monitored using brightfield and fluorescence microscopy. Brightfield microscopy showed that carnosine reduces the migration rate of fibroblasts and T98G cells towards each other (into the space left after removing the ring) during the first days of monitoring, imitating a scratch assay. After 4 weeks, fluorescence microscopy and ImageJ were used to analyze the area occupied by tumor cells and to count new satellite lesions of T98G cells within the fibroblast monolayer as markers for tumor cell infiltration. The results revealed that all concentrations of carnosine significantly reduced the area occupied by T98G cells and that concentrations above 10 mM also significantly decreased the number of newly formed tumor cell colonies within the fibroblast monolayer. To verify these results, the co-culture experiments were repeated using primary GBM cells and fibroblasts. Despite that no colony formation was seen using primary GBM cells, carnosine again reduced the area occupied by GBM cells significantly at all concentrations compared to untreated control cultures.

In summary, our work demonstrates that carnosine not only reduces the metabolic activity of glioblastoma cells compared to fibroblasts, but also impairs the ability of GBM cells to proliferate, migrate and infiltrate surrounding tissue in vitro . Moreover, the reduction of malignant cell growth under carnosine is independent from the „go and grow“ principle. Future experiments should now be performed to understand the molecular mechanisms responsible for carnosine’s effect on proliferation and migration and whether its effect on migration can also be observed under standard therapy conditions including temozolomide and X-irradiation. The work presented has been published in Cancer Cell International, 13 August 2018 (PMID: 30123089).

4 References 1. Virchow R. Die Cellularpathologie in ihrer Begründung auf physiologische und pathologische Gewebelehre: Zwanzig Vorlesungen, gehalten während der Monate Februar, März und April 1858 im Pathologischen Institute zu Berlin. Mit 144 Holzschnitten; 1858. 2. Virchow R. Die krankhaften Geschwülste: Dreißig Vorlesungen. Berlin: Hirschwald; 1863. 3. Louis DN, Perry A, Reifenberger G, Deimling A von, Figarella-Branger D, Cavenee WK, et al. The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary. Acta Neuropathol. 2016;131:803–20. doi:10.1007/s00401- 016-1545-1. 4. Wen PY, Kesari S. Malignant gliomas in adults. N Engl J Med. 2008;359:492–507. doi:10.1056/NEJMra0708126. 5. Ostrom QT, Gittleman H, Liao P, Vecchione-Koval T, Wolinsky Y, Kruchko C, Barnholtz-Sloan JS. CBTRUS Statistical Report: Primary brain and other central nervous system tumors diagnosed in the United States in 2010-2014. Neuro-oncology. 2017;19:v1-v88. doi:10.1093/neuonc/nox158. 6. Alifieris C, Trafalis DT. Glioblastoma multiforme: Pathogenesis and treatment. Pharmacol Ther. 2015;152:63–82. doi:10.1016/j.pharmthera.2015.05.005. 7. Awan M, Liu S, Sahgal A, Das S, Chao ST, Chang EL, et al. Extra-CNS metastasis from glioblastoma: a rare clinical entity. Expert Rev Anticancer Ther. 2015;15:545–52. doi:10.1586/14737140.2015.1028374. 8. Ogungbo BI, Perry RH, Bozzino J, Mahadeva D. Report of GBM metastasis to the parotid gland. J Neurooncol. 2005;74:337–8. doi:10.1007/s11060-005-1480-9. 9. Müller C, Holtschmidt J, Auer M, Heitzer E, Lamszus K, Schulte A, et al. Hematogenous dissemination of glioblastoma multiforme. Sci Transl Med. 2014;6:247. doi:10.1126/scitranslmed.3009095. 10. Kleihues P, Burger PC, Scheithauer BW. The new WHO classification of brain tumours. Brain Pathol. 1993;3:255–68. 11. Louis DN. Molecular pathology of malignant gliomas. Annu Rev Pathol. 2006;1:97– 117. doi:10.1146/annurev.pathol.1.110304.100043. 12. Kleihues P, Ohgaki H. Primary and secondary glioblastomas: from concept to clinical diagnosis. Neuro-oncology. 1999;1:44–51. 13. Siegal T. Clinical impact of molecular biomarkers in gliomas. J Clin Neurosci. 2015;22:437–44. doi:10.1016/j.jocn.2014.10.004. 14. Weller M, Wick W, Deimling A von. Isocitrate dehydrogenase mutations: a challenge to traditional views on the genesis and malignant progression of gliomas. Glia. 2011;59:1200–4. doi:10.1002/glia.21130. 15. Eisenlöffel C, Müller W. Molekulare Marker in der Neuropathologie: heutiger Stand und Perspektiven. Neurochir Scan. 2015;03:203–25. doi:10.1055/s-0034-1392464. 16. Weller M, Pfister SM, Wick W, Hegi ME, Reifenberger G, Stupp R. Molecular neuro- oncology in clinical practice: a new horizon. Lancet Oncol. 2013;14:9. doi:10.1016/S1470-2045(13)70168-2. 17. Cheng H-B, Yue W, Xie C, Zhang R-Y, Hu S-S, Wang Z. IDH1 mutation is associated with improved overall survival in patients with glioblastoma: a meta-analysis. Tumour Biol. 2013;34:3555–9. doi:10.1007/s13277-013-0934-5. 18. Weller M, van den Bent M, Hopkins K, Tonn JC, Stupp R, Falini A, et al. EANO guideline for the diagnosis and treatment of anaplastic gliomas and glioblastoma. Lancet Oncol. 2014;15:e395-403. doi:10.1016/S1470-2045(14)70011-7.

19. Ludlum DB. DNA alkylation by the haloethylnitrosoureas: nature of modifications produced and their enzymatic repair or removal. Mutat Res. 1990;233:117–26. 20. Esteller M, Garcia-Foncillas J, Andion E, Goodman SN, Hidalgo OF, Vanaclocha V, et al. Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med. 2000;343:1350–4. doi:10.1056/NEJM200011093431901. 21. Hegi ME, Diserens A-C, Gorlia T, Hamou M-F, Tribolet N de, Weller M, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med. 2005;352:997–1003. doi:10.1056/NEJMoa043331. 22. Stummer W, Pichlmeier U, Meinel T, Wiestler OD, Zanella F, Reulen H-J. Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial. Lancet Oncol. 2006;7:392– 401. doi:10.1016/S1470-2045(06)70665-9. 23. Laperriere N, Zuraw L, Cairncross G. Radiotherapy for newly diagnosed malignant glioma in adults: a systematic review. Radiother Oncol. 2002;64:259–73. 24. Skardelly M, Dangel E, Gohde J, Noell S, Behling F, Lepski G, et al. Prolonged Temozolomide Maintenance Therapy in Newly Diagnosed Glioblastoma. Oncologist. 2017;22:570–5. doi:10.1634/theoncologist.2016-0347. 25. FDA. Temodar. 26. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJB, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352:987–96. doi:10.1056/NEJMoa043330. 27. Stupp R, Taillibert S, Kanner AA, Kesari S, Steinberg DM, Toms SA, et al. Maintenance Therapy With Tumor-Treating Fields Plus Temozolomide vs Temozolomide Alone for Glioblastoma: A Randomized Clinical Trial. JAMA. 2015;314:2535–43. doi:10.1001/jama.2015.16669. 28. Wick W, Osswald M, Wick A, Winkler F. Treatment of glioblastoma in adults. Ther Adv Neurol Disord. 2018;11:1756286418790452. doi:10.1177/1756286418790452. 29. Weller M, Cloughesy T, Perry JR, Wick W. Standards of care for treatment of recurrent glioblastoma--are we there yet? Neuro-oncology. 2013;15:4–27. doi:10.1093/neuonc/nos273. 30. Sahm F, Capper D, Jeibmann A, Habel A, Paulus W, Troost D, Deimling A von. Addressing diffuse glioma as a systemic brain disease with single-cell analysis. Arch Neurol. 2012;69:523–6. doi:10.1001/archneurol.2011.2910. 31. Armento A, Ehlers J, Schötterl S, Naumann U. Glioblastoma: Molecular Mechanisms of Glioma Cell Motility. Brisbane (AU); 2017. 32. Scherer HJ. THE FORMS OF GROWTH IN GLIOMAS AND THEIR PRACTICAL SIGNIFICANCE. Brain. 1940;63:1–35. doi:10.1093/brain/63.1.1. 33. Giese A, Loo MA, Tran N, Haskett D, Coons SW, Berens ME. Dichotomy of astrocytoma migration and proliferation. Int J Cancer. 1996;67:275–82. doi:10.1002/(SICI)1097-0215(19960717)67:2<275::AID-IJC20>3.0.CO;2-9. 34. Hatzikirou H, Basanta D, Simon M, Schaller K, Deutsch A. 'Go or grow': the key to the emergence of invasion in tumour progression? Math Med Biol. 2012;29:49–65. doi:10.1093/imammb/dqq011. 35. Godlewski J, Bronisz A, Nowicki MO, Chiocca EA, Lawler S. microRNA-451: A conditional switch controlling glioma cell proliferation and migration. Cell Cycle. 2010;9:2742–8. 36. Wang SD, Rath P, Lal B, Richard J-P, Li Y, Goodwin CR, et al. EphB2 receptor controls proliferation/migration dichotomy of glioblastoma by interacting with focal adhesion kinase. Oncogene. 2012;31:5132–43. doi:10.1038/onc.2012.16.

37. Lu KV, Bergers G. Mechanisms of evasive resistance to anti-VEGF therapy in glioblastoma. CNS Oncol. 2013;2:49–65. doi:10.2217/cns.12.36. 38. Dhruv HD, McDonough Winslow WS, Armstrong B, Tuncali S, Eschbacher J, Kislin K, et al. Reciprocal activation of transcription factors underlies the dichotomy between proliferation and invasion of glioma cells. PLoS ONE. 2013;8:e72134. doi:10.1371/journal.pone.0072134. 39. Kathagen-Buhmann A, Schulte A, Weller J, Holz M, Herold-Mende C, Glass R, Lamszus K. Glycolysis and the pentose phosphate pathway are differentially associated with the dichotomous regulation of glioblastoma cell migration versus proliferation. Neuro-oncology. 2016;18:1219–29. doi:10.1093/neuonc/now024. 40. Roussos ET, Condeelis JS, Patsialou A. Chemotaxis in cancer. Nat Rev Cancer. 2011;11:573–87. doi:10.1038/nrc3078. 41. Montana V, Sontheimer H. Bradykinin promotes the chemotactic invasion of primary brain tumors. J Neurosci. 2011;31:4858–67. doi:10.1523/JNEUROSCI.3825-10.2011. 42. Brat DJ, van Meir EG. Vaso-occlusive and prothrombotic mechanisms associated with tumor hypoxia, necrosis, and accelerated growth in glioblastoma. Lab Invest. 2004;84:397–405. doi:10.1038/labinvest.3700070. 43. Soni S, Padwad YS. HIF-1 in cancer therapy: two decade long story of a transcription factor. Acta Oncol. 2017;56:503–15. doi:10.1080/0284186X.2017.1301680. 44. Nicholson C, Syková E. Extracellular space structure revealed by diffusion analysis. Trends Neurosci. 1998;21:207–15. 45. Yurchenco PD. Basement membranes: cell scaffoldings and signaling platforms. Cold Spring Harb Perspect Biol 2011. doi:10.1101/cshperspect.a004911. 46. Mahesparan R, Tysnes BB, Read TA, Enger PO, Bjerkvig R, Lund-Johansen M. Extracellular matrix-induced cell migration from glioblastoma biopsy specimens in vitro. Acta Neuropathol. 1999;97:231–9. 47. Lo CM, Wang HB, Dembo M, Wang YL. Cell movement is guided by the rigidity of the substrate. Biophys J. 2000;79:144–52. doi:10.1016/S0006-3495(00)76279-5. 48. Gritsenko PG, Ilina O, Friedl P. Interstitial guidance of cancer invasion. J Pathol. 2012;226:185–99. doi:10.1002/path.3031. 49. Gulewitsch W, Amiradžibi S. Ueber das Carnosin, eine neue organische Base des Fleischextractes. Ber. Dtsch. Chem. Ges. 1900;33:1902–3. doi:10.1002/cber.19000330275. 50. Boldyrev AA, Aldini G, Derave W. Physiology and pathophysiology of carnosine. Physiol Rev. 2013;93:1803–45. doi:10.1152/physrev.00039.2012. 51. Hipkiss AR, Cartwright SP, Bromley C, Gross SR, Bill RM. Carnosine: can understanding its actions on energy metabolism and protein homeostasis inform its therapeutic potential? Chem Cent J. 2013;7:38. doi:10.1186/1752-153X-7-38. 52. Bonfanti L, Peretto P, Marchis S de, Fasolo A. Carnosine-related dipeptides in the mammalian brain. Prog Neurobiol. 1999;59:333–53. 53. Mannion AF, Jakeman PM, Dunnett M, Harris RC, Willan PL. Carnosine and anserine concentrations in the quadriceps femoris muscle of healthy humans. Eur J Appl Physiol Occup Physiol. 1992;64:47–50. 54. ABRAHAM D, PISANO JJ, UDENFRIEND S. The distribution of homocarnosine in mammals. Arch Biochem Biophys. 1962;99:210–3. 55. Harding JW, O'Fallon JV. The subcellular distribution of carnosine, carnosine synthetase, and carnosinase in mouse olfactory tissues. Brain Res. 1979;173:99–109. 56. Drozak J, Veiga-da-Cunha M, Vertommen D, Stroobant V, van Schaftingen E. Molecular identification of carnosine synthase as ATP-grasp domain-containing protein 1 (ATPGD1). J Biol Chem. 2010;285:9346–56. doi:10.1074/jbc.M109.095505.

57. Hoffmann AM, Bakardjiev A, Bauer K. Carnosine-synthesis in cultures of rat glial cells is restricted to oligodendrocytes and carnosine uptake to astrocytes. Neurosci Lett. 1996;215:29–32. 58. HANSON HT, SMITH EL. Carnosinase; an enzyme of swine kidney. J Biol Chem. 1949;179:789–801. 59. Jackson MC, Kucera CM, Lenney JF. Purification and properties of human serum carnosinase. Clin Chim Acta. 1991;196:193–205. 60. Lenney JF, George RP, Weiss AM, Kucera CM, Chan PW, Rinzler GS. Human serum carnosinase: characterization, distinction from cellular carnosinase, and activation by cadmium. Clin Chim Acta. 1982;123:221–31. 61. Lenney JF, Peppers SC, Kucera-Orallo CM, George RP. Characterization of human tissue carnosinase. Biochem J. 1985;228:653–60. 62. Lenney JF. Separation and characterization of two carnosine-splitting cytosolic dipeptidases from hog kidney (carnosinase and non-specific dipeptidase). Biol Chem Hoppe-Seyler. 1990;371:433–40. 63. Peppers SC, Lenney JF. Bestatin inhibition of human tissue carnosinase, a non-specific cytosolic dipeptidase. Biol Chem Hoppe-Seyler. 1988;369:1281–6. 64. Gautam P, Nair SC, Gupta MK, Sharma R, Polisetty RV, Uppin MS, et al. Proteins with altered levels in plasma from glioblastoma patients as revealed by iTRAQ-based quantitative proteomic analysis. PLoS ONE. 2012;7:e46153. doi:10.1371/journal.pone.0046153. 65. Wassif WS, Sherwood RA, Amir A, Idowu B, Summers B, Leigh N, Peters TJ. Serum carnosinase activities in central nervous system disorders. Clin Chim Acta. 1994;225:57–64. 66. Balion CM, Benson C, Raina PS, Papaioannou A, Patterson C, Ismaila AS. Brain type carnosinase in dementia: a pilot study. BMC Neurol. 2007;7:38. doi:10.1186/1471-2377- 7-38. 67. Arner P, Henjes F, Schwenk JM, Darmanis S, Dahlman I, Iresjö B-M, et al. Circulating carnosine dipeptidase 1 associates with weight loss and poor prognosis in gastrointestinal cancer. PLoS ONE. 2015;10:e0123566. doi:10.1371/journal.pone.0123566. 68. Bellia F, Calabrese V, Guarino F, Cavallaro M, Cornelius C, Pinto V de, Rizzarelli E. Carnosinase levels in aging brain: redox state induction and cellular stress response. Antioxid Redox Signal. 2009;11:2759–75. doi:10.1089/ars.2009.2738. 69. Gardner ML, Illingworth KM, Kelleher J, Wood D. Intestinal absorption of the intact peptide carnosine in man, and comparison with intestinal permeability to lactulose. J Physiol. 1991;439:411–22. 70. Rubio-Aliaga I, Frey I, Boll M, Groneberg DA, Eichinger HM, Balling R, Daniel H. Targeted disruption of the peptide transporter Pept2 gene in mice defines its physiological role in the kidney. Mol Cell Biol. 2003;23:3247–52. 71. Yamashita T, Shimada S, Guo W, Sato K, Kohmura E, Hayakawa T, et al. Cloning and functional expression of a brain peptide/histidine transporter. J Biol Chem. 1997;272:10205–11. 72. Deutsch A, Eggleton P. The titration constants of anserine, carnosine and some related compounds. Biochem J. 1938;32:209–11. 73. Smith EC. The buffering of muscle in rigor; protein, phosphate and carnosine. J Physiol. 1938;92:336–43. 74. Sassoe-Pognetto M, Cantino D, Panzanelli P, Di Verdun Cantogno L, Giustetto M, Margolis FL, et al. Presynaptic co-localization of carnosine and glutamate in olfactory neurones. Neuroreport. 1993;5:7–10.

75. Baslow MH. A novel key-lock mechanism for inactivating amino acid neurotransmitters during transit across extracellular space. Amino Acids. 2010;38:51–5. doi:10.1007/s00726-009-0232-0. 76. Bakardjiev A. Carnosine and beta-alanine release is stimulated by glutamatergic receptors in cultured rat oligodendrocytes. Glia. 1998;24:346–51. 77. McFarland GA, Holliday R. Retardation of the senescence of cultured human diploid fibroblasts by carnosine. Exp Cell Res. 1994;212:167–75. doi:10.1006/excr.1994.1132. 78. McFarland GA, Holliday R. Further evidence for the rejuvenating effects of the dipeptide L-carnosine on cultured human diploid fibroblasts. Exp Gerontol. 1999;34:35– 45. 79. Shao L, Li Q-H, Tan Z. L-carnosine reduces telomere damage and shortening rate in cultured normal fibroblasts. Biochem Biophys Res Commun. 2004;324:931–6. doi:10.1016/j.bbrc.2004.09.136. 80. Hipkiss AR, Baye E, Courten B de. Carnosine and the processes of ageing. Maturitas. 2016;93:28–33. doi:10.1016/j.maturitas.2016.06.002. 81. Aydin AF, Coban J, Dogan-Ekici I, Betul-Kalaz E, Dogru-Abbasoglu S, Uysal M. Carnosine and taurine treatments diminished brain oxidative stress and apoptosis in D- galactose aging model. Metab Brain Dis. 2016;31:337–45. doi:10.1007/s11011-015- 9755-0. 82. Yuneva AO, Kramarenko GG, Vetreshchak TV, Gallant S, Boldyrev AA. Effect of carnosine on Drosophila melanogaster lifespan. Bull Exp Biol Med. 2002;133:559–61. 83. Boldyrev AA, Yuneva MO, Sorokina EV, Kramarenko GG, Fedorova TN, Konovalova GG, Lankin VZ. Antioxidant systems in tissues of senescence accelerated mice. Biochemistry (Mosc). 2001;66:1157–63. 84. Chez MG, Buchanan CP, Aimonovitch MC, Becker M, Schaefer K, Black C, Komen J. Double-blind, placebo-controlled study of L-carnosine supplementation in children with autistic spectrum disorders. J Child Neurol. 2002;17:833–7. doi:10.1177/08830738020170111501. 85. Babizhayev MA. Generation of reactive oxygen species in the anterior eye segment. Synergistic codrugs of N-acetylcarnosine lubricant eye drops and mitochondria-targeted antioxidant act as a powerful therapeutic platform for the treatment of cataracts and primary open-angle glaucoma. BBA Clin. 2016;6:49–68. doi:10.1016/j.bbacli.2016.04.004. 86. Baraniuk JN, El-Amin S, Corey R, Rayhan R, Timbol C. Carnosine treatment for gulf war illness: a randomized controlled trial. Glob J Health Sci. 2013;5:69–81. doi:10.5539/gjhs.v5n3p69. 87. Ghajar A, Khoaie-Ardakani M-R, Shahmoradi Z, Alavi A-R, Afarideh M, Shalbafan M- R, et al. L-carnosine as an add-on to risperidone for treatment of negative symptoms in patients with stable schizophrenia: A double-blind, randomized placebo-controlled trial. Psychiatry Res. 2018;262:94–101. doi:10.1016/j.psychres.2018.02.012. 88. Chengappa KNR, Turkin SR, DeSanti S, Bowie CR, Brar JS, Schlicht PJ, et al. A preliminary, randomized, double-blind, placebo-controlled trial of L-carnosine to improve cognition in schizophrenia. Schizophr Res. 2012;142:145–52. doi:10.1016/j.schres.2012.10.001. 89. Ghajar A, Aghajan-Nashtaei F, Afarideh M, Mohammadi M-R, Akhondzadeh S. l- Carnosine as Adjunctive Therapy in Children and Adolescents with Attention- Deficit/Hyperactivity Disorder: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial. J Child Adolesc Psychopharmacol. 2018;28:331–8. doi:10.1089/cap.2017.0157.

90. Nagai K, Niijima A, Yamano T, Otani H, Okumra N, Tsuruoka N, et al. Possible role of L-carnosine in the regulation of blood glucose through controlling autonomic nerves. Exp Biol Med (Maywood). 2003;228:1138–45. 91. Lee Y-t, Hsu C-c, Lin M-h, Liu K-s, Yin M-c. Histidine and carnosine delay diabetic deterioration in mice and protect human low density lipoprotein against oxidation and glycation. Eur J Pharmacol. 2005;513:145–50. doi:10.1016/j.ejphar.2005.02.010. 92. Courten B de, Jakubova M, Courten MP de, Kukurova IJ, Vallova S, Krumpolec P, et al. Effects of carnosine supplementation on glucose metabolism: Pilot clinical trial. Obesity (Silver Spring). 2016;24:1027–34. doi:10.1002/oby.21434. 93. Nagai K, Suda T. Antineoplastic effects of carnosine and beta-alanine--physiological considerations of its antineoplastic effects. Nihon Seirigaku Zasshi. 1986;48:741–7. 94. Holliday R, McFarland GA. Inhibition of the growth of transformed and neoplastic cells by the dipeptide carnosine. Br J Cancer. 1996;73:966–71. 95. Hipkiss AR, Michaelis J, Syrris P, Kumar S, Lam Y. Carnosine protects proteins against in vitro glycation and cross-linking. Biochem Soc Trans. 1994;22:399. 96. Hipkiss AR, Michaelis J, Syrris P. Non-enzymatic glycosylation of the dipeptide L- carnosine, a potential anti-protein-cross-linking agent. FEBS Lett. 1995;371:81–5. 97. WARBURG O. On respiratory impairment in cancer cells. Science. 1956;124:269–70. 98. Schwartz L, Seyfried T, Alfarouk KO, Da Veiga Moreira J, Fais S. Out of Warburg effect: An effective cancer treatment targeting the tumor specific metabolism and dysregulated pH. Semin Cancer Biol 2017. doi:10.1016/j.semcancer.2017.01.005. 99. Chiavarina B, Nokin M-J, Bellier J, Durieux F, Bletard N, Sherer F, et al. Methylglyoxal-Mediated Stress Correlates with High Metabolic Activity and Promotes Tumor Growth in Colorectal Cancer. Int J Mol Sci 2017. doi:10.3390/ijms18010213. 100. Iovine B, Oliviero G, Garofalo M, Orefice M, Nocella F, Borbone N, et al. The anti- proliferative effect of L-carnosine correlates with a decreased expression of hypoxia inducible factor 1 alpha in human colon cancer cells. PLoS ONE. 2014;9:e96755. doi:10.1371/journal.pone.0096755. 101. Iovine B, Guardia F, Irace C, Bevilacqua MA. l-carnosine dipeptide overcomes acquired resistance to 5-fluorouracil in HT29 human colon cancer cells via downregulation of HIF1-alpha and induction of apoptosis. Biochimie. 2016;127:196– 204. doi:10.1016/j.biochi.2016.05.010. 102. Shen Y, Yang J, Li J, Shi X, Ouyang L, Tian Y, Lu J. Carnosine inhibits the proliferation of human gastric cancer SGC-7901 cells through both of the mitochondrial respiration and glycolysis pathways. PLoS ONE. 2014;9:e104632. doi:10.1371/journal.pone.0104632. 103. Pandurangan M, Mistry B, Enkhataivan G, Kim DH. Efficacy of carnosine on activation of caspase 3 and human renal carcinoma cell inhibition. Int J Biol Macromol. 2016;92:377–82. doi:10.1016/j.ijbiomac.2016.07.044. 104. Renner C, Zemitzsch N, Fuchs B, Geiger KD, Hermes M, Hengstler J, et al. Carnosine retards tumor growth in vivo in an NIH3T3-HER2/neu mouse model. Mol Cancer. 2010;9:2. doi:10.1186/1476-4598-9-2. 105. Renner C, Asperger A, Seyffarth A, Meixensberger J, Gebhardt R, Gaunitz F. Carnosine inhibits ATP production in cells from malignant glioma. Neurol Res. 2010;32:101–5. doi:10.1179/016164109X12518779082237. 106. Asperger A, Renner C, Menzel M, Gebhardt R, Meixensberger J, Gaunitz F. Identification of factors involved in the anti-tumor activity of carnosine on glioblastomas using a proteomics approach. Cancer Invest. 2011;29:272–81. doi:10.3109/07357907.2010.550666.

107. Oppermann H, Alvanos A, Seidel C, Meixensberger J, Gaunitz F. Carnosine influences transcription via epigenetic regulation as demonstrated by enhanced histone acetylation of the pyruvate dehydrogenase kinase 4 promoter in glioblastoma cells. Amino Acids 2018. doi:10.1007/s00726-018-2619-2. 108. Gaunitz F, Hipkiss AR. Carnosine and cancer: a perspective. Amino Acids. 2012;43:135–42. doi:10.1007/s00726-012-1271-5. 109. Rybakova Y, Akkuratov E, Kulebyakin K, Brodskaya O, Dizhevskaya A, Boldyrev A. Receptor-mediated oxidative stress in murine cerebellar neurons is accompanied by phosphorylation of MAP (ERK 1/2) kinase. Curr Aging Sci. 2012;5:225–30. 110. Son DO, Satsu H, Kiso Y, Totsuka M, Shimizu M. Inhibitory effect of carnosine on interleukin-8 production in intestinal epithelial cells through translational regulation. Cytokine. 2008;42:265–76. doi:10.1016/j.cyto.2008.02.011. 111. Martinelli E, Morgillo F, Troiani T, Ciardiello F. Cancer resistance to therapies against the EGFR-RAS-RAF pathway: The role of MEK. Cancer Treat Rev. 2017;53:61–9. doi:10.1016/j.ctrv.2016.12.001. 112. Eskandani M, Vandghanooni S, Barar J, Nazemiyeh H, Omidi Y. Cell physiology regulation by hypoxia inducible factor-1: Targeting oxygen-related nanomachineries of hypoxic cells. Int J Biol Macromol. 2017;99:46–62. doi:10.1016/j.ijbiomac.2016.10.113. 113. Smith EC. The buffering of muscle in rigor; protein, phosphate and carnosine. J Physiol. 1938;92:336–43. 114. Blancquaert L, Baba SP, Kwiatkowski S, Stautemas J, Stegen S, Barbaresi S, et al. Carnosine and anserine homeostasis in skeletal muscle and heart is controlled by beta- alanine transamination. J Physiol. 2016;594:4849–63. doi:10.1113/JP272050. 115. Kaczmarek D, Lochynski D, Everaert I, Pawlak M, Derave W, Celichowski J. Role of histidyl dipeptides in contractile function of fast and slow motor units in rat skeletal muscle. J Appl Physiol (1985). 2016;121:164–72. doi:10.1152/japplphysiol.00848.2015. 116. Hannah R, Stannard RL, Minshull C, Artioli GG, Harris RC, Sale C. beta-Alanine supplementation enhances human skeletal muscle relaxation speed but not force production capacity. J Appl Physiol (1985). 2015;118:604–12. doi:10.1152/japplphysiol.00991.2014.

5 Appendix

5.1 Supplemental material

Additional File 1: Patients and patient derived cell cultures. patient cell type Label sex and MGMT status IDH status age GBM GBM 1 Fibr. (P) P0375 m, 66 - - 2 GBM P0383 f, 76 mildly positive negative Fibr. (P) P0384 Fibr. (G) P0385 3 Fibr. (G) P0408 f, 41 positive negative GBM P0410 4 Fibr. (P) P0425 f, 67 mildly positive negative GBM P0424 5 GBM P0411 m, 68 positive negative Fibr. (P) P0412 6 GBM P0431 m, 66 positive negative Fibr. (G) P0433 7 GBM P0441 f, 59 negative negative Fibr. (G) P0443 8 GBM P0446 m, 65 positive negative Fibr. (G) P0447 9 GBM P0451 f, 74 positive negative Fibr. (G) P0452 10 GBM P0454 m, 70 negative negative Fibr. (G) P0455

Primary cultures of glioblastoma cells (GBM) and fibroblast cultures either isolated from periost (Fibr. (P)) or from galea (Fibr. (G)) are shown together with patients’ age and sex and the status of promoter methylation of MGMT and whether the tumors are positive or negative for IDH mutation R132H as determined by pathology.

Additional File 2: Setup of ring-cultures.

36 5.3 Erklärung über die eigenständige Abfassung der Arbeit

Hiermit erkläre ich, dass ich die vorliegende Arbeit selbstständig und ohne unzulässige Hilfe oder Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Ich versichere, dass Dritte von mir weder unmittelbar noch mittelbar eine Vergütung oder geldwerte Leistungen für Arbeiten erhalten haben, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen, und dass die vorgelegte Arbeit weder im Inland noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde zum Zweck einer Promotion oder eines anderen Prüfungsverfahrens vorgelegt wurde. Alles aus anderen Quellen und von anderen Personen übernommene Material, das in der Arbeit verwendet wurde oder auf das direkt Bezug genommen wird, wurde als solches kenntlich gemacht. Insbesondere wurden alle Personen genannt, die direkt an der Entstehung der vorliegenden Arbeit beteiligt waren. Die aktuellen gesetzlichen Vorgaben in Bezug auf die Zulassung der klinischen Studien, die Bestimmungen des Tierschutzgesetzes, die Bestimmungen des Gentechnikgesetzes und die allgemeinen Datenschutzbestimmungen wurden eingehalten. Ich versichere, dass ich die Regelungen der Satzung der Universität Leipzig zur Sicherung guter wissenschaftlicher Praxis kenne und eingehalten habe.

...... Datum Unterschrift

5.4 Curriculum vitae

Johannes Andreas Dietterle born 4 April 1990 in Cottbus, Germany

Primary education: 1996 - 2009 Primary school and Gymnasium in Cottbus, Germany

University: October 2010 – December 2017 Studying Medicine at Leipzig University

October 2011 – September 2013 Studying Physics (IPSP) at Leipzig University

September 2013 – December 2013 Studying Mandarin at Nanjing University, China

March 2014 - June 2014 Studying Acupuncture at Nanjing University of Chinese Medicine, China

8 December 2017 Graduation with “very good” (1.5)

2 February 2018 Approbation

Employments: Since April 2018 Resident at Neurosurgery Department of Leipzig University Hospital

Honors and Scholarships: June 2009 High School Graduation Price of Deutsche Physikalische Gesellschaft (DPG, German Physical Society)

September 2013 – August 2014 Scholarship Recipient for the China Program of Studienstiftung des deutschen Volkes

September 2014 – December 2018 Scholarship Recipient of Studienstiftung des deutschen Volkes

Languages: German (mother tongue), English (C1), Chinese (HSK 5)

5.5 List of publications

Publication :

Oppermann, Henry*; Dietterle, Johannes*; Purcz, Katharina; Morawski, Markus; Eisenlöffel, Christian; Müller, Wolf et al. (2018): Carnosine selectively inhibits migration of IDH-wildtype glioblastoma cells in a co-culture model with fibroblasts. In: Cancer cell international 18, S. 111. DOI: 10.1186/s12935-018-0611-2.

*: These authors contributed equally

Congress presentations :

Frank Gaunitz, Henry Oppermann, Christiane Seidel, Katharina Purcz, Athanasios Alvanos, Johannes Dietterle , Stefanie Elsel, Sebastian Strube and Jürgen Meixensberger (2017): Carnosine and Cancer – mechanisms, physiology and specificity, International Congress on Carnosine and Anserine (ICCA), Louisville (Kentucky), USA

Johannes Dietterle , Henry Oppermann, Jürgen Meixensberger, Frank Gaunitz (2018): Carnosine increases the efficiency of treatment with temozolomide and x- irradiation as revealed by experiments with primary cell cultures derived from glioblastoma patients, Jahrestagung Sektion Neuroonkologie der DGNC, Innsbruck, Österreich

Posters :

J. Dietterle (Leipzig), H. Oppermann, J. Meixensberger, F. Gaunitz (2018): Carnosine may improve the efficiency of standard treatment of glioblastoma with temozolomide and irradiation, 69. Jahrestagung der deutschen Gesellschaft für Neurochirurgie, Münster, Deutschland

Dietterle J. ; Oppermann H.; Meixensberger J.; Gaunitz F. (2017): Carnosine inhibits the infiltrative growth of glioblastoma cells, Brain Tumor Meeting, Berlin, Deutschland

Purcz, K.; Seidel, C.; Birkemeyer, C.; Dietterle, J .; Meixensberger, J.; Gaunitz, F.; Oppermann, H. (2017): Imidazole containing compounds are potential drugs for the treatment of glioblastoma, Brain Tumor Meeting, Berlin, Deutschland

J. Dietterle (Leipzig, Deutschland), H. Oppermann, J. Meixensberger, F. Gaunitz (2017): A new model for the determination of glioblastoma cell Invasion reveals that carnosine inhibits Infiltration of tumor cells into patient derived fibroblast culture, 68. Jahrestagung der deutschen Gesellschaft für Neurochirurgie, Magdeburg, Deutschland

K. Purcz (Leipzig, Deutschland), C. Seidel, C. Birkemeyer, J. Dietterle , J. Meixensberger,F. Gaunitz, H. Oppermann (2017): Carnosine's anti-neoplastic effect

39 on glioblastoma cell growth is independent of its cleavage, 68. Jahrestagung der deutschen Gesellschaft für Neurochirurgie, Magdeburg, Deutschland

5.6 Acknowledgements

I would like to thank Prof. Dr. Jürgen Meixensberger for giving me the opportunity to research on this subject and to benefit from the facilities of the Department of Neurosurgery of Leipzig University Hospital.

I also wish to thank Prof. Dr. Frank Gaunitz for contributing the idea to this study, for his constant advice and for being a reliable and straightforward supervisor throughout the creation of this work.

I would like to thank Dr. Henry Oppermann for his never ending patience to explain, his practical supervision, his support for all the ideas I had the last 3 years and his guidance when I lost my track.

Last not least, I want to thank Mr. Baran-Schmidt for showing me how to work at a laboratory and for handling the chaos I sometimes left behind.

I owe you my joy for science.

Furthermore, I owe gratitude to my family, all my friends and Carlos Kasper. Without your support and lasting patience I would not have finished this project.