Noninvasive positron emission tomography and fluorescence imaging of CD133+ tumor stem cells

Simone Gaedickea,1, Friederike Braunb,c,1, Shruthi Prasada,c,1, Marcia Macheind, Elke Firata, Michael Hetticha,c, Ravindra Gudihale, Xuekai Zhua, Kerstin Klingnerf, Julia Schülerf, Christel C. Herold-Mendeg, Anca-Ligia Grosua,h, Martin Beheb,i, Wolfgang Weberb,h,j, Helmut Mäckeb,h, and Gabriele Niedermanna,h,2

Departments of aRadiation Oncology, bNuclear Medicine, and dNeurosurgery, University Hospital Freiburg, D-79106 Freiburg, Germany; cFaculty of Biology, University of Freiburg, D-79104 Freiburg, Germany; eAgilent Technologies India Pvt Ltd, Bangalore 560048, India; fOncotest, D-79108 Freiburg, Germany; gDepartment of Neurosurgery, University Hospital Heidelberg, D-69120 Heidelberg, Germany; hGerman Consortium for Translational Cancer Research, D-69120 Heidelberg, Germany; iCenter for Radiopharmaceutical Sciences, Swiss Federal Institute of Technology-Paul Scherrer Institute-University Hospital of Zurich, Paul Scherrer Institute, CH-5232 Villigen, Switzerland; and jMolecular Imaging and Therapy Service, Memorial Sloan-Kettering Cancer Center, New York, NY 10065

Edited by Owen N. Witte, Howard Hughes Medical Institute, University of California, Los Angeles, CA, and approved December 23, 2013 (received for review August 9, 2013) A technology that visualizes tumor stem cells with clinically relevant AC133+ tumor stem cells have been described for tracers could have a broad impact on cancer diagnosis and multiforme (the most common and most aggressive primary brain treatment. The AC133 epitope of CD133 currently is one of the tumor in adults), various pediatric brain and central nervous sys- best-characterized tumor markers for many intra- and tem tumors (medulloblastoma, ependymoma, pineoblastoma, extracranial tumor entities. Here we demonstrate the successful teratoid/rhabdoid tumors, and retinoblastoma), brain metastases, noninvasive detection of AC133+ tumor stem cells by PET and near- many different types of carcinomas including colon, pancreatic, infrared fluorescence molecular tomography in subcutaneous and lung, liver, and ovarian cancer, , sarcomas, and different − orthotopic xenografts using antibody-based tracers. Partic- types of leukemia. Although AC133 tumor stem cells also exist 64 ularly, microPET with Cu-NOTA-AC133 mAb yielded high-quality (16–18), AC133+ cells found in these and other tumor types have images with outstanding tumor-to-background contrast, clearly de- been shown to be able to self-renew, to differentiate, and to rec- lineating subcutaneous tumor stem cell-derived xenografts from sur- reate the original tumors when injected into immunocompromised – rounding tissues. Intracerebral tumors as small as 2 3mmalsowere mice (8, 17, 19–22). Both, stemness and highly agressive malignant clearly discernible, and the microPET images reflected the invasive tumors often are associated with hypoxia (23), and hypoxia can growth pattern of orthotopic -derived tumors with promote the expansion of CD133+ cells (24). Therefore the fre- low density of AC133+ cells. These data provide a basis for further quent expression of AC133 on CSCs may reflect, in part, their preclinical and clinical use of the developed tracers for high-sensitivity common localization in a hypoxic environment (25). and high-resolution monitoring of AC133+ tumor stem cells. We previously reported the successful noninvasive detection of the AC133 epitope by antibody-based near-infrared fluorescence cancer stem cells | CSCs | glioblastoma molecular tomography (NIR FMT) in mice with s.c. xenografts of CD133-overexpressing tumor cells or traditional tumor cell ancer stem cells (CSCs) are highly undifferentiated tumor lines naturally displaying AC133 (26). However, we did not in- Ccells with characteristics similar to normal stem cells. These characteristics include long-term replication, self-renewal, and Significance aberrant differentiation (1, 2). Based on these characteristics, it has been hypothesized that only CSCs are able to propagate tumors for long periods of time and to initiate relapses or me- Cancer stem cells (CSCs) are thought to be responsible for tastases. Furthermore, CSCs are considered to be more resistant growth and dissemination of many malignant tumors and for to conventional radio- and chemotherapy than more differenti- relapse after therapy. Therefore methods for the noninvasive ated tumor cells (3–5). Hence, elimination of CSCs is challenging imaging of CSCs could have profound consequences for di- but necessary for successful tumor eradication. The stem cell agnosis and therapy monitoring in oncology. However, clini- hypothesis of cancer development and progression is conceptu- cally applicable methods for noninvasive CSC imaging are still ally attractive and is supported by many preclinical (1, 2, 5–7) lacking. The AC133 epitope of CD133 is one of the most in- and some clinical studies (4, 8). However, larger clinical trials tensely investigated CSC markers and is particularly important investigating the role of CSCs in patients have been hampered for aggressive brain tumors. Here we describe the development of clinically relevant tracers that permit high-sensitivity and by the lack of techniques to detect, localize, and quantify the + presence of CSCs noninvasively. Specifically, successful non- high-resolution monitoring of AC133 glioblastoma stem cells invasive imaging of unmanipulated CSCs with clinically relevant in both subcutaneous and intracerebral xenograft tumors using imaging probes (e.g., antibodies or other ligands binding CSC- positron emission tomography and near-infrared fluorescence specific cell-surface ) has not yet been reported (9–11). imaging, two clinically highly relevant imaging modalities. AC133 is an N-glycosylation–dependent epitope of the second Author contributions: F.B., S.P., M.M., M.B., H.M., and G.N. designed research; S.G., F.B., extracellular loop of CD133/prominin-1, a cholesterol-binding S.P., M.M., M.H., R.G., K.K., and M.B. performed research; M.H., J.S., C.C.H.-M., and A.-L.G. of unknown function that locates to plasma membrane contributed new reagents/analytic tools; S.G., F.B., S.P., M.M., E.F., R.G., X.Z., M.B., W.W., protrusions (12–14). Postnatally, the CD133 protein is expressed H.M., and G.N. analyzed data; and F.B., S.P., E.F., M.H., W.W., and G.N. wrote the paper. by certain epithelial and nonepithelial cells, by stem and pro- The authors declare no conflict of interest. genitor cells of various organs, and by CSCs of many different This article is a PNAS Direct Submission. types of malignant tumors (15). With a few exceptions, recog- Freely available online through the PNAS open access option. nition of the AC133 epitope by the AC133 mAb appears to be 1S.G., F.B. and S.P. contributed equally to this work. limited to cells harboring stem cell properties, and the AC133 2To whom correspondence should be addressed. E-mail: gabriele.niedermann@uniklinik- epitope—but not necessarily the CD133 protein—is down-reg- freiburg.de. ulated upon differentiation, presumably because of changes in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. glycosylation (12, 13, 15). 1073/pnas.1314189111/-/DCSupplemental.

E692–E701 | PNAS | Published online January 27, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1314189111 Downloaded by guest on October 1, 2021 vestigate patient-derived CSCs with the above-mentioned stem PNAS PLUS cell characteristics in that study, and NIR fluorescence, although penetrating tissues more deeply (2–4 cm) than visible- fluorescence, has limited importance for clinical whole-body imaging (27). We report here the successful noninvasive detection of tumor- associated AC133 by PET, using a radiolabeled AC133-specific mAb in mice xenografted with tumor cell lines overexpressing CD133 or with patient-derived AC133+ CSCs. PET is highly sensitive and is widely used for clinical whole-body diagnostic 64 imaging. As a PET nuclide, we used Cu (t1/2 = 12.7 h), which allows long-term tracking for at least 48 h, to follow the tumoral accumulation of relatively large molecules such as antibodies, that exhibit relatively slow tumor penetration (28). We chose S-2- (4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA, hereafter abbreviated as NOTA) as the 64Cu chelator, because high labeling efficiencies and high in vivo sta- bility have been reported for 64Cu-NOTA-antibody conjugates (29). In addition, we report the successful noninvasive detection of AC133+ CSCs with fluorescently labeled AC133 mAb and NIR imaging, a modality that is important for whole-body small- animal imaging and for intraoperative and endoscopic imaging and imaging of superficial tumors in humans (27, 30). In addition to imaging s.c. growing tumors, we report the successful anti- body-mediated imaging of orthotopic xenografts initiated from AC133+ glioblastoma stem cells in the brain of immunocom- promised mice, emphasizing the feasibility of noninvasive antibody- mediated imaging of brain tumors. Results Tracer Development and Characterization. After conjugation of the Fig. 1. Characterization of AC133+ cell lines and the modified AC133 chelator NOTA, we radiolabeled the AC133 mAb, which rec- mAbs. (A) Flow-cytometric detection of AC133 epitope expression on in vitro-cultured CD133-overexpressing U251 glioma cells and NCH421k glio- ognizes the AC133 epitope on CD133-overexpressing cells and − CSCs (Fig. 1A), and an isotype control antibody with 64Cu. The blastoma stem cells compared with CD133 U251 wild-type cells. Results NOTA-AC133 and NOTA-isotype control mAb conjugates were shown are representative of more than 10 independent experiments. (B) ± ± Flow-cytometric analysis of the binding specificity of the NOTA-AC133 and functionalized with an average of 5.1 0.8 and 2.3 0.3 che- Alexa 680-AC133 mAbs compared with unmodified AC133 mAb. The anal- lators per molecule of antibody, respectively, and in all radio- ysis was performed as described in Methods. Data are representative of labeling experiments the radiochemical purity was >95%, with three independent experiments. MFI, mean fluorescence intensity; OE, specific activities of 48.4 ± 9.5 MBq/nmol and 41.4 ± 5.6 MBq/nmol overexpressing. for 64Cu-NOTA-AC133 and the isotype control mAb, respec- tively. Conjugation of the AC133 mAb or the isotype control an- tibody to the NIR dye Alexa 680 resulted in 2.3 ± 0.2 and 1.5 ± 0.2 rated the PET imaging data (Fig. 2C). In contrast to the highly different uptake of the 64Cu-NOTA-AC133 mAb, both the AC133+ Alexa 680 molecules conjugated to the two mAbs, respectively. − Neither conjugation of NOTA nor that of Alexa 680 impaired and the AC133 tumors showed a similar uptake of 18F-fluoro- binding to the AC133 epitope, as revealed by flow cytometric deoxyglucose (FDG) (Fig. S1). titration of NOTA-AC133 and Alexa 680-AC133 against the unmodified AC133 antibody (Fig. 1B). FMT Imaging of Glioblastoma Stem Cell-Derived s.c. Xenografts. To develop animal models for imaging of AC133+ CSCs, which PET Imaging of s.c. CD133-Overexpressing Glioma Xenografts. We usually display much lower levels of AC133 than cell lines first established small-animal PET with the 64Cu-NOTA-AC133 overexpressing CD133, we chose the well-characterized glioma mAb in a robust nude mouse model with s.c. xenografts of U251 stem cell line NCH421k. This cell line has been derived from a glioma cells transduced with a CD133-encoding lentivirus result- primary glioblastoma [World Health Organization (WHO) grade ing in high expression of CD133 associated with high cell-surface IV] under stem cell culture conditions, and its CSC character- expression of the AC133 epitope (Fig. 1A). We s.c. injected these istics have been reported repeatedly (Fig. S2 and refs. 24, 31–33). AC133-high cells into the right flank and, as an internal control, NCH421k cells display 10- to 15-fold lower surface AC133 than − injected CD133 U251 wild-type cells into the left flank of each CD133-overexpressing U251 cells both in vitro (Fig. 1A) and in animal. The uptake of the 64Cu-NOTA-AC133 mAb in the CD133- vivo (Fig. S3A). We first established in vivo imaging of CSC- overexpressing xenografts was very high (Fig. 2A). As quantified containing tumors with NIR FMT, a sensitive and inexpensive from PET images, the uptake reached 37.9 ± 5.6% of the injected imaging technique permitting quantitative 3D detection of NIR − activity (IA)/g at 24 h and increased to values as high as 56.3 ± fluorophores in mice. Because CD133 derivatives of the NCH421k MEDICAL SCIENCES 16.2% 48 h after i.v. injection of the radiolabeled antibody. In line do not exist, we used an Alexa 680-labeled IgG1 isotype contrast, the uptake of antigen-negative tumors decreased antibody as specificity control. from 10.1 to 8.3% at 24 and 48 h, respectively, and was similar The Alexa 680-labeled AC133 mAb yielded a significantly to or slightly lower than that of liver and blood (Fig. 2B). At higher in vivo fluorescence signal in the tumor region than did − 48 h, the uptake ratios of AC133+ tumor to AC133 tumor, liver, the Alexa 680-labeled isotype control antibody (Fig. 3A). The ex blood, and background were 7.3 ± 1.9, 5.0 ± 0.6, 6.9 ± 1.2, and vivo biodistribution analysis confirmed that the fluorescently 32.0 ± 8.8, respectively. Ex vivo biodistribution studies at 48 h labeled AC133 mAb accumulated in the tumor (Fig. 3 B–D). In after administration of the 64Cu-NOTA-AC133 mAb corrobo- addition, flow-cytometric analyses of tumor single-cell suspensions

Gaedicke et al. PNAS | Published online January 27, 2014 | E693 Downloaded by guest on October 1, 2021 Fig. 2. PET/CT imaging and biodistribution of 64Cu-NOTA-AC133 mAb in mice bearing s.c. implanted U251 overexpressing CD133. Nude mice re- − ceived ∼8.0 ± 0.5 MBq 64Cu-NOTA-AC133 mAb via tail vein injection, and PET/CT images were acquired. The mice carried AC133 U251 wild-type and AC133/ CD133-overexpressing U251 gliomas in the left and right flanks, respectively. (A) Representative transverse tumor and coronal whole-body PET and fused PET/ CT sections at 24 and 48 h p.i. (B) Uptake of 64Cu-NOTA-AC133 mAb as measured by microPET in various organs and AC133− and AC133-overexpressing tumors at 24 and 48 h p.i. Values are the mean %IA/g of tissue. (C) Ex vivo biodistribution at 24 and 48 h p.i. Values are the mean %IA/g of tissue. n =7–8 mice per group. ***P < 0.001, t test; values represent means ± SD.

directly after the scans showed that the i.v.-injected Alexa 680- also could be detected directly postmortem on the excised tu- AC133 mAb had bound to almost all CSC marker-positive cells mor-bearing brains (Fig. 3F, Lower Left). in the tumor, verifying that the injected Alexa 680-AC133 mAb had penetrated the tumor tissue efficiently (Fig. 3E, Left). To PET Imaging of Tumor Stem Cell-Derived s.c. Xenografts. After the identify the CSCs ex vivo, the tumor single-cell suspensions were FMT studies had demonstrated that noninvasive mAb-mediated + costained with the AC141 antibody, which is specific for a second visualization of AC133 CSCs was possible in principle, we ex- + 64 stem cell-specific epitope of CD133. As expected, binding of the plored immuno-PET detection of AC133 CSCs. The Cu-NOTA- injected Alexa 680-labeled isotype control antibody to CSCs could AC133 mAb strongly marked s.c. growing NCH421k gliomas at 24 not be detected (Fig. 3E, Right). and 48 h postinjection (p.i.) (Fig. 4A, Left and Center), despite the considerably lower expression of AC133 on NCH421k cells as FMT Imaging of Intracerebral Xenograft Tumors. Antibodies have compared with CD133-overexpressing U251 cells (see Fig. 1A and only limited access to the brain because the undisturbed blood– Fig. S3A). Particularly remarkable was the much higher tracer up- brain barrier (BBB) is impermeable to macromolecules, and take liver [an organ exhibiting relatively high unspecific activity whether systemically administered antibodies can reach extra- because of high blood perfusion, antibody metabolism, and vascular targets in brain tumors with a disturbed BBB is of great potential transchelation of 64Cu (28, 36)]. The tumor-to-con- interest (34, 35). We therefore wanted to find out whether the tralateral background, tumor-to-blood pool, and tumor-to-liver AC133 mAb is suitable for imaging orthotopically growing contrasts were 21.4 ± 8.2, 2.7 ± 0.9, and 2.6 ± 0.8at24hand AC133+ glioma xenografts. We indeed could detect orthotopically 32.8 ± 19, 6.4 ± 2.5, and 5.4 ± 1.8 at 48 h p.i., respectively. The growing NCH421k gliomas noninvasively by NIR FMT imaging 64Cu-NOTA-isotype control antibody caused only a very weak upon i.v. injection of the Alexa 680-labeled AC133 mAb (Fig. 3F, tumor signal. As judged by visual inspection (Fig. 4A, Right) and Upper Left), and the signal caused by the Alexa 680-AC133 mAb according to in vivo and in vitro quantification (Fig. 4 B and C),

E694 | www.pnas.org/cgi/doi/10.1073/pnas.1314189111 Gaedicke et al. Downloaded by guest on October 1, 2021 PNAS PLUS

Fig. 3. NIR FMT imaging of xenografts containing AC133+ glioblastoma stem cells. Mice with s.c. growing NCH421k xenografts were injected i.v. with Alexa 680-AC133 mAb or Alexa 680-isotype control antibody. After 1, 2, 3, and 4 d, the mice were imaged using the FMT-1500 system. The pictures presented correspond to the last measurement acquired 96 h p.i. (A–D) 3D whole-body images (A), 3D images of the excised tumors (B) and organs (C), and quanti- fication of their fluorescence (D). (E) Flow-cytometric detection of i.v. injected Alexa 680-labeled AC133 mAb on AC133+ CSCs in single-cell suspensions of excised s.c. tumors. AC133+ CSCs were counterstained in single-cell suspensions with the AC141 mAb. Only cells falling in the CSC (FSC/SSC) gate are shown. Results shown are representative of four independent experiments. (F) 3D whole-body images and 3D images of excised brains of mice with intracerebral NCH421k xenografts 72 h after i.v. injection of Alexa 680-AC133 or Alexa 680-isotype control antibodies. For A–D and F, n = 5 mice per group. **P < 0.05; t test; values represent means ± SD.

this signal was in the range of or slightly lower than that of the signal caused by the 64Cu-NOTA-isotype control antibody was blood pool (heart and blood vessels), presumably reflecting not significantly above background (Fig. 6A, Right, and Fig. 6B). a high vascularization of the aggressively growing tumors and unspecific accumulation of a proportion of the antibody in the Correlation of MicroPET Images with Histopathologic Tumor Appearance. interstitial space via the enhanced permeation and retention The PET signal of orthotopic CD133-overexpressing U251 gliomas effect (37). was homogeneous and sharply delineated from the surrounding brain tissue, whereas that of the NCH421k tumors was more het- PET Imaging of Intracerebral Xenograft Tumors. We first performed erogeneous. Particularly striking was the reduced and diffuse signal microPET of intracerebrally growing tumor lesions in mice in the periphery of these CSC-derived tumors (compare Fig. 5A, bearing CD133-overexpressing gliomas. MicroPET after i.v. in- Left and Center and Fig. 6A, Left). These differences in microPET jection of the 64Cu-NOTA-AC133 mAb permitted the detection signal intensity correlated with histopathological differences. On not only of relatively large intracerebral U251 gliomas over- H&E-stained brain sections, the U251 tumors appeared as very expressing CD133 but also of very small ones (Fig. 5A, Left and compact, homogeneous, and well-delineated tumor masses (Fig. 7 A and B), reflecting noninfiltrative growth behavior. This appearance Center). These small lesions were imaged as soon as 12 d after is typical of orthotopic xenografts of conventional glioma cell lines, tumor cell implantation, when they had reached a size of only which generally do not recapitulate the invasive growth pattern 2–3 mm in diameter, as determined by contrast-enhanced CT. In that is a major feature (17, 31) of the highly malignant tumor contrast, the 64Cu-NOTA-AC133 mAb caused only a very weak − entity human glioblastoma multiforme (38) and certainly con- signal in AC133 U251 wild-type tumors (Fig. 5A, Right, and Fig. tributes to the failure of current therapies. In contrast, NCH421k 5B). We then asked whether intracerebral tumors initiated from + xenografts exhibited a very different cyto-architecture. The H&E MEDICAL SCIENCES AC133 glioma stem cells could be detected also. To this end, staining appeared more heterogeneous, with a lower intensity in the NCH421k glioblastoma stem cells were injected into the fore- 1- to 2-mm-thick periphery of the tumors than in the center (Fig. brains of immunodeficient mice. MicroPET after i.v. injection of 7D). Higher magnifications revealed tumor cells migrating along 64 the Cu-NOTA-AC133 mAb clearly allowed the detection of blood vessels in the tumor periphery, forming irregular tumor intracerebral xenografts initiated from AC133+ glioblastoma margins (Fig. 7E). Together, these histopathological features reflect stem cells (Fig. 6A, Left, and Fig. 6B). At 48 h, the uptake ratio the highly infiltrative growth behavior characteristic of human of AC133+ NCH421k stem cell-containing tumors to back- glioblastoma multiforme. AC133 staining of brain tissue sections ground in the brain was 14 ± 3.4. In contrast, the brain tumor also revealed a high density (72 ± 20%) of intensely stained

Gaedicke et al. PNAS | Published online January 27, 2014 | E695 Downloaded by guest on October 1, 2021 Fig. 4. PET/CT imaging and biodistribution of 64Cu-NOTA-AC133 and isotype control mAbs in mice bearing s.c. implanted xenografts containing AC133+ glioblastoma stem cells. NOD/SCID mice bearing NCH421k xenografts in the right flank were given ∼6.4 ± 1.7 MBq of either 64Cu-NOTA-AC133 or 64Cu-NOTA- isotype control mAb via tail vein injection, and PET/CT images were acquired. (A) Representative transverse tumor and coronal whole-body PET and fused PET/ CT sections at 24 and 48 h p.i. The yellow arrows indicate the liver; “A” indicates aorta branching into the two common iliac arteries. (B) Uptake of both 64Cu- NOTA-AC133 and 64Cu-NOTA-isotype control mAb as determined by microPET in various organs and in the tumor. Values are the mean %IA/g of tissue. (C)Ex vivo biodistribution. Values are the mean %IA/g of tissue. n = 5 mice per group. ***P < 0.001, t test; values represent means ± SD.

AC133-expressing cells in CD133-overexpressing U251 gliomas 17, 19, 31), and we demonstrate here the successful noninvasive (Fig. 7C and Fig. S3 B and C). In contrast, the AC133+ cell density imaging of intracerebral xenografts with a low density of (20.0 ± 11%) and cellular AC133 expression levels were much AC133+ glioblastoma stem cells by both the Alexa 680-labeled lower in the NCH421k tumors, and the AC133+ tumor cells tended AC133 mAb (using FMT) and the 64Cu-NOTA-AC133 mAb to occur in clusters (Fig. 7F and Fig. S3 B and C). The reduction in (using microPET). the percentage of AC133+ cells compared with the initial 90–100% Current imaging techniques used for standard treatment in the NCH421k implant suspension may be the result mainly of monitoring in patients with solid tumors assess the shrinkage of differentiation and the invasive growth behavior. Nonetheless, the tumor as a response criterion. However, when non-CSCs microPET using the 64Cu-NOTA-AC133 mAb clearly enabled the outnumber the CSCs, tumor shrinkage may reflect largely the detection of the highly invasive, CSC-derived NCH421k tumors. elimination of treatment-sensitive bulk tumor cells, and whether long-term self-renewing CSCs also are being eliminated can be Discussion answered only with CSC-specific tracers and sensitive imaging The AC133 epitope of CD133/prominin is perhaps the most modalities. Because CSCs also may be responsible for the initi- intensely investigated of the known CSC markers (8, 13, 17, 19, ation of local recurrences and distant metastases, specific and 20, 22). Although of great interest and importance, noninvasive sensitive CSC imaging techniques could enable the early detection imaging of AC133+ CSCs or other types of CSCs with clinically of these events as well. relevant tracers and imaging modalities has not yet been repor- PET and NIR fluorescence imaging are sensitive imaging mo- ted (9–11). Here, we demonstrate the successful noninvasive dalities that are becoming increasingly important in clinical practice antibody-mediated imaging of AC133+ glioblastoma stem cells (27, 30, 39–41). We show here that particularly 64Cu-NOTA- by PET and NIR fluorescence imaging in xenograft tumor AC133 mAb-mediated microPET yields high-quality, high- models in mice. Despite the relatively low AC133 expression on resolution images with outstanding tumor-to-background con- CSCs, we obtained high-quality PET images with the 64Cu-loaded trast. 64Cu-NOTA-AC133 mAb-mediated microPET permitted NOTA-conjugated AC133-specific antibody. The AC133 epitope the detection of very small brain tumor lesions (2–3mminsize) is particularly important as a CSC marker of brain tumors (3, 8, and also reflected differences in invasive behavior between

E696 | www.pnas.org/cgi/doi/10.1073/pnas.1314189111 Gaedicke et al. Downloaded by guest on October 1, 2021 PNAS PLUS

Fig. 5. PET/CT imaging and biodistribution of 64Cu-NOTA-AC133 mAb in mice bearing orthotopic U251 glioma xenografts. Nude mice bearing orthotopic xenografts of U251 glioma cells overexpressing CD133 or orthotopic xenografts of CD133− U251 wild-type cells received 7.5 ± 0.8 MBq 64Cu-NOTA-AC133 mAb via tail vein injection, and PET/CT images were acquired 24 and 48 h p.i. (A) Representative contrast-enhanced microCT and fused microPET/CT sections from mice bearing CD133-overexpressing U251 gliomas and from mice bearing U251 wild-type tumors. For the PET/CT images, an upper threshold correspondingto the maximum tracer uptake of the CD133-overexpressing U251 tumors (43%IA/g) was chosen. (B) Uptake of 64Cu-NOTA-AC133 mAb as determined by microPET in brain tumor, normal brain tissue in the contralateral hemisphere, liver, and in the heart/blood pool. Values are the mean %IA/g of tissue. n =5–6 mice. ***P < 0.001, t test; values represent means ± SD.

orthotopically growing U251 (noninvasive) and NCH421k (in- whether intraabdominal tumors with AC133+ CSCs within or vasive) gliomas. Whereas sharply delineated PET signals reflected close to the liver also can be detected readily using this tracer. − 64

the compact and spherical microscopic appearance of U251 tumors, AC133 tumors or the Cu-labeled isotype control antibody MEDICAL SCIENCES more diffuse PET signals seemed to reflect the lower density of caused only signals as low as background, not exceeding that of AC133+ cells and the chaotic and infiltrative growth pattern de- the blood pool. Because CSCs show rather low AC133 expres- tected microscopically in the orthotopic NCH421k tumors. In ad- sion, these data considered together suggest that the 64Cu-NOTA- dition, the 64Cu-NOTA-AC133 mAb could image s.c. xenografts AC133 mAb is a very specific and highly sensitive tool for the containing AC133+ CSCs clearly. The tracer uptake was signif- noninvasive detection of AC133+ CSCs. icantly higher in these flank tumors than in liver and blood, A proportion of the WHO grade IV and of which cause the highest background signals in immuno-PET with other tumor entities contains high frequencies of AC133+ cells. intact antibodies. However, future studies are needed to determine The AC133+ fraction among the highly aggressive glioblastomas

Gaedicke et al. PNAS | Published online January 27, 2014 | E697 Downloaded by guest on October 1, 2021 Fig. 6. PET/CT imaging and biodistribution of 64Cu-NOTA-AC133 and isotype control mAbs in mice bearing orthotopic xenografts initiated from AC133+ glioblastoma stem cells. Mice bearing orthotopic xenografts containing patient-derived NCH421k glioblastoma stem cells were given 7.3 ± 1.9 MBq of either 64Cu-NOTA-AC133 or 64Cu-NOTA-isotype control mAb via tail vein injection, and PET/CT images were acquired. (A) Representative contrast-enhanced microCT and fused microPET/CT sections are shown. For the PET/CT images, an upper threshold corresponding to the maximum 64Cu-NOTA-AC133 mAb uptake (34.6% IA/g) in the NCH421 tumor was chosen. (B) Uptake of both 64Cu-NOTA-AC133 and 64Cu-NOTA-isotype control mAbs as determined by microPET in brain tumor, normal brain tissue in the contralateral hemisphere, liver, and in the heart/blood pool. Values are the mean %IA/g of tissue; n = 5 mice per group. ***P < 0.001, **P < 0.05, t test; values represent means ± SD.

was reported to range from 19 to 29% (19) or from ≤1–50% (8). could be detectable by 64Cu-NOTA-AC133 PET. Nevertheless, In the latter study, a considerable proportion of tumors contained future studies are needed to define more precisely the lower limit >10% or even >25% AC133+ cells, and the AC133+ cells tended of detection in terms of the percentage of tumor cells that can be to organize in clusters. For medulloblastomas, 6–21% AC133+ noninvasively detected using AC133 mAb-based tracers. cells have been reported (19), and for teratoid/rhabdoid brain High-density areas of normal stem cells also may pose a chal- tumors 1–36% have been reported at diagnosis and 30–70% at lenge in CSC imaging, because the currently known CSC mark- relapse (42). For colon cancer, 2–19% AC133+ cells were repor- ers are shared by subsets of normal tissue stem cells. However, ted, compared with 0.4–2% in the surrounding tissue (20), and for human hematopoietic stem cells, a subset of which is AC133+, ovarian cancer the reported range is 0.3–35% (43). Other tumor constitute only a very minor fraction (<0.2%) of blood cells (45) entities (e.g., lower-grade gliomas) usually contain only smaller and express considerably less surface AC133 than NCH421k populations of AC133+ tumor cells (8). For pancreatic cancers the glioblastoma stem cells. Prostate, liver, kidney, skin, and retina mean is 1.8% (21); and for lung cancers the mean is 5% and that also contain AC133+ stem and progenitor cells (15). The AC133 of surrounding tissue is <1% (44). We were able to image diffusely epitope also is a known marker of fetal human neural stem cells. growing glioblastoma xenografts with a low density (mean: 20%) However, postnatally, the typical stem cell regions in the brain of AC133+ CSCs with high tumor-to-background contrast ratio (the subventricular zone and the hippocampus) seem not to (mean: 14 at 48 h p.i.). Because specific tumor detection would be contain CD133+ stem cells (46). This absence of stem cells is a possible at a tumor-to-background contrast ratio of 2 or even less, good prerequisite for AC133 mAb-based brain tumor imaging. it is likely that much lower frequencies of AC133+ tumor cells The AC133 epitope can be detected on differentiated human

E698 | www.pnas.org/cgi/doi/10.1073/pnas.1314189111 Gaedicke et al. Downloaded by guest on October 1, 2021 PNAS PLUS

Fig. 7. Histopathological examination of orthotopically growing brain tumors. Brain sections of mice bearing U251 gliomas overexpressing CD133 or gliomas derived from NCH421k glioblastoma stem cells. (A and D) Brain sections were stained with H&E and scanned by a whole-slide scanner. (Scale bars: 1 mm.) (B and E) Higher-magnification views of the areas boxed in the tumor periphery in A and D including the tumor margins. (C and F) Confocal microscopic images of brain sections encompassing the tumor periphery. The sections were stained with DAPI to visualize nuclei and with PE-labeled AC133 mAb. Note the plasma membrane staining for AC133. Pictures shown are representative of three independent experiments.

luminal epithelia by immunohistochemistry of tissue fixed in Methods paraformaldehyde (16). However, because fixation strongly Cell Lines. The U251 cell line was obtained from American Type Culture affects the recognition of the epitope (13), its accessibility in Collection (ATCC). NCH421k is a glioblastoma stem cell line that was derived these epithelia to the AC133 mAb in noninvasive imaging is directly from a primary glioblastoma tumor sample. The establishment of the difficult to predict. CD133-overexpressing U251 glioma cell line and of the NCH421k glioblas- Current imaging techniques used in patients with primary or toma stem cell line have been described previously (26, 31). NCH421k cells metastatic brain tumors quantify the tumor burden indirectly were cultured under normoxic conditions (21% O2) in Neurobasal-A medium (Invitrogen) supplemented with 20 ng/mL EGF and 20 ng/mL FGF-2 (Prospec), through edema, vascular integrity (i.e., contrast enhancement 1× penicillin/streptomycin (PAA Laboratories), 0.5× minimum essential me- using low-molecular-weight contrast agents), or metabolic ac- dium (MEM) nonessential amino acids, 1× Glutamax-I, and B27 supplement tivity (mainly using amino acid PET). Although it is known that, (all from Invitrogen) on normal plastic, where they grew as spheres. CD133- in highly malignant brain tumors, the BBB is disturbed and that overexpressing U251 cells and wild-type U251 cells were cultured in medium its permeability is increased for macromolecules (34), reports on containing 10% (vol/vol) FBS. All three cell lines were transduced with a lentivirus antibody-mediated PET of brain tumor lesions are scarce (40). coding for a fusion protein consisting of the latest generation of firefly luciferase Our microPET imaging and biodistribution analyses show that and a neomycin resistance cassette (-mCherry-IRES-FFneo or L1-FF-IRESneo) the 64Cu-NOTA-AC133 mAb is exquisitely suited for imaging constructed in the G.N. laboratory according to standard procedures. orthotopically growing glioblastoma xenografts, indicating that Antibodies. AC133.1 hybridoma cells (14) (ATCC HB-12346) were cultured in this antibody tracer penetrates these tumors quite efficiently. DMEM with Ultra Low IgG FBS (Invitrogen), and the AC133 mAb was purified Antibody-mediated brain tumor CSC imaging also may have from the hybridoma supernatant according to standard methods. The IgG1 advantages over other currently used brain tumor imaging isotype control antibody was purchased from BioXcell. techniques in distinguishing true tumor relapse from pseudo- progression, because it is based on a molecular antigen/antibody Antibody Conjugation with Alexa 680. Conjugation of Alexa Fluor 680 (Alexa interaction that is more tumor-specific than imaging based on 680) to the AC133 and IgG1 isotype control antibodies was conducted with vascular integrity or metabolic activity. In addition, our data the Alexa Fluor-680 SAIVI Rapid Antibody Labeling Kit from Invitrogen ’ suggest that noninvasive antibody-mediated imaging could be according to the manufacturer s instructions. Protein concentrations of the very useful in general for assessing receptor expression and Alexa Fluor 680-conjugated mAbs and the degree of labeling were determined with a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific). evaluating antibody therapeutics in neurooncology. In conclusion, we have developed antibody-based PET and Antibody Conjugation with NOTA. To AC133 or the isotype control antibody (in NIR fluorescence tracers that enable the specific and highly metal-free PBS), p-SCN-Bn-NOTA (Macrocyclics) dissolved in DMSO was + sensitive detection of AC133 CSCs. FMT and PET detection of added at a ratio of 1:32 (final volume 500 μL, 0.1 M bicarbonate buffer, + AC133 CSCs is important for preclinical studies in human tu- pH 9.5). Conjugation was allowed to proceed at room temperature for 5 h.

mor xenograft models. The present study also sets the founda- Excess chelator was removed by G25-Sephadex size-exclusion chromatogra- MEDICAL SCIENCES tion for the application of fluorescent and positron emitter- phy on PD-10 columns. The columns were washed with PBS supplied with labeled AC133 mAbs, or humanized versions thereof, in human 1 μM EDTA to remove metal ions, followed by washing with only PBS. studies with clinical NIR fluorescence and PET scanners. In Fractions containing the immunoconjugate were collected in PBS and con- centrated to 1 mg/mL with Amicon Ultra 4 centrifugation tubes (Millipore), addition, our study confirms that the AC133 mAb also may be followed by buffer exchange to ammonium acetate buffer, pH 5.2. useful for therapeutic targeting, including the treatment of brain 64 64 tumors such as the highly aggressive and presently incurable Labeling of NOTA-AC133 mAb with Cu. CuCl2 was obtained from the De- glioblastoma multiforme (32) as well as for the development of partment of Preclinical Imaging and Radiopharmacy, Eberhard Karls Uni- theranostic probes (47). versity, Tübingen Germany. Labeling was performed in 250-μL ammonium

Gaedicke et al. PNAS | Published online January 27, 2014 | E699 Downloaded by guest on October 1, 2021 acetate buffer (0.1 M, pH 8.2) with 240 μg NOTA-AC133 or 240 μg isotype by TrueQuant software (PerkinElmer), using calibrated standards of the la- 64 control mAb. About 100 MBq CuCl2 was added and incubated for 40 min at beled mAbs. 37 °C. The labeling reaction was stopped by adding 100 μL 0.1 M EDTA so- 64 64 lution to chelate-free CuCl2. Quality control was performed by an isocratic PET and CT Imaging. Mice were injected i.v. with either 30 μg Cu-NOTA- HPLC run (Ramona Star HPLC system; Raytest GmbH) on a BioSilect SEC 250–5 AC133 or isotype control mAb. PET imaging was performed with a microPET size-exclusion column (Bio-Rad Laboratories) with PBS as eluent at a flow Focus 120 (Concorde). To account for the physical decay of 64Cu, the ac- rate of 1 mL/min. The retention times for the labeled compounds were quisition time was 20 min at 24 h p.i. and 45 min at 48 h p.i. PET acquisition (mean ± SD) 6:48 ± 0:11 min and 9:44 ± 0:08 min for the labeled mAb and was followed immediately by CT imaging (CT Imaging microCT scanner). For − − free 64Cu(EDTA)2 , respectively. Free 64Cu(EDTA)2 was separated by fil- studies of intracranial tumors, mice were injected i.v. with 100 μL Imeron 350 trating through an Amicon 10-kDa cutoff filter. Together with this purifi- as contrast agent immediately before the start of the CT scan. The head cation step, the labeled compound was buffer exchanged into 0.9% NaCl region of each mouse was scanned in one bed position for 90 s using a 360° solution for injection and adjusted to the final volume. rotation step, a tube voltage of 40 keV, and a tube current of 1 mA.

Determination of NOTA Chelators per Antibody. The number of NOTA chela- PET and CT Image Analysis. PET images were reconstructed by the routine 2D tors per antibody molecule was determined by isotope dilution and con- ordered subset expectation maximization (OSEM2D) algorithm provided by firmed by LC-MS using an Agilent 1260 nano/capillary LC system coupled to an the scanner software, as previously described (48). The resolution of the Agilent 6530 QTOF equipped with a Chip-Cube. reconstructed images ranged between 1.5 and 1.7 mm. Quantitative analysis of radiotracer uptake was performed with AMIDE software, and the repor- Titration of Labeled Antibodies. Preservation of binding affinity upon labeling ted values represent the mean activity concentration expressed as percent of with either Alexa 680 or NOTA was ascertained through side-by-side flow injected activity per gram of tissue (%IA/g), assuming a tissue density of 1 g/mL. cytometric titration analysis of labeled and unlabeled antibody, respectively. Image contrast was assessed by calculating the ratio of radiotracer uptake to Serially diluted antibody was incubated with 5 × 105 HCT116 colon carcinoma contralateral brain, left ventricular cavity, and liver. Images of the CT scans were cells (which are AC133+) and 5 × 105 p53-deficient HCT116 cells (which are reconstructed with a voxel size of 0.12 × 0.12 × 0.12 mm and a T30 kernel, using − AC133 ) suspended in 100 μL FACS buffer (0.5% BSA/2 mM EDTA in PBS) for the software provided by the manufacturer. Fusion of the PET and CT images 15 min. After washing, the cells were incubated with 1.5 μg anti-mouse IgG was performed by ROVER software (ABX).

phycoerythrin (PE)-conjugated F(ab’)2 fragment (Dianova) in 100 μLFACS buffer for 20 min. Samples then were washed twice and analyzed on Ex Vivo Biodistribution. After the imaging studies, the animals were killed by a FACSVerse (BD Biosciences). cervical dislocation, and tumor and other organs were sampled. In animals injected with fluorescent antibody, either ex vivo FMT imaging or FACS Animal Experiments. All animal experiments were performed in accordance analysis of tumor single-cell suspensions (as described below) was performed. with the German Animal License Regulations and were approved by the After PET/CT imaging, radioactivity within the tumor and the normal organs animal care committee of the Regierungspräsidium Freiburg (registration was measured using a gamma counter Packard Cobra II (PerkinElmer). All number: G-10/64). values were decay-corrected and expressed as %IA/g ± SD, by measuring a standard of known activity from the respective injected probe. Tumor cell Implantation and Tumor Growth Monitoring. For s.c. tumor models, 5 × 106 U251 wild-type, CD133-overexpressing U251 or NCH421k cells were Histopathology. Paraformaldehyde (4%)-fixed brains were cut in a Vibratome implanted into the flanks of 6- to 8-wk-old immunodeficient mice. BALB/c (Leica VT-1000S; Leica) in horizontal sections 60 μm apart. Brain sections were nude and the more immunodeficient NOD/SCID mice (Charles River) were mounted and stained with H&E. For analysis of AC133 expression in tumor used for U251 and NCH421k cells, respectively. The growth of the xenografts tissue, 60-μm sections were stained using a PE-labeled antibody against was monitored by caliper measurement. When the s.c. growing tumors human AC133.1 (Miltenyi Biotec). For negative control, slices were stained reached a size of 500–1,000 mm3, they were used for in vivo imaging with PE-labeled IgG1 isotype control antibody. After washings and nuclear experiments. For orthotopic brain tumor models, 2.5 × 105 wild-type or staining with DAPI, sections were mounted and analyzed with a krypton-argon CD133-overexpressing U251 or NCH421k cells stably transduced with lucif- laser scanning confocal imaging system (TCSNT; Leica Microsystems AG). erase were implanted manually 3 mm anterior and 3 mm to the right of the bregma in the brains of 6- to 8-wk-old nude mice. The cells, which were FACS Analysis of Tumor Single-Cell Suspensions. At 96 h after injection of Alexa suspended in 4 μL PBS, were injected at a depth of 3 mm with a Hamilton 680-labeled mAbs, pieces of s.c. grown tumors were digested with 0.7 U/mL syringe, which was held in position for 5 min. After the injection, the surface Liberase-Blendzyme (Roche)/Accutase (eBioscience)/100 U DNase I (Invi- was cleaned with a sterile cotton swab, and the burr hole was filled with trogen)/10 mM MgCl2 for 30 min at 37 °C. Nondigested pieces were digested bone wax. Thereafter, tumor growth was monitored noninvasively using in further with Accutase for 30 min and then were pressed through a cell vivo bioluminescence imaging on an IVIS spectrum imaging system (Perki- strainer. Red blood cells were removed using ice-cold RBC Lysis Buffer nElmer) three times per week (Fig. S2D). Mice with orthotopic brain tumors (eBioscience). Cells then were washed with PBS, resuspended in FACS buffer, were imaged with FMT or PET when the bioluminescence signal reached an and transferred through a preseparation filter. The cells were incubated intensity between 5 × 107 and 1 × 108 photons/s. When the NCH421k tumor- with FcR blocking reagent (Miltenyi) for 10 min, followed by incubation with bearing mice met the above criteria, they were randomized into groups and 5 μg/mL anti-AC141-PE (Miltenyi), and then were analyzed by flow cytometry. were injected with either AC133 or isotype control antibodies. FACS Analysis of AC133 Expression on Cultured Tumor Cells. Tumor cells FMT in Vivo Imaging. Alexa 680-labeled AC133 or isotype control mAb (70 μg) (CD133-overexpressing U251 and wild-type U251 as well as NCH421k cells) was injected i.v. The fluorescence signals were visualized using an FMT1500 were collected, incubated with FcR blocking reagent, stained with anti- system (PerkinElmer) at several time points p.i. The mice were anesthetized AC133.1-PE antibody from Miltenyi Biotec, and then subjected to FACS analysis. by gas anesthesia (isoflurane/oxygen mixture) and then were placed into an imaging cassette. After the cassette was positioned in the FMT1500 imaging Statistical Analysis. Results are presented as means ± SD. Data were com- system, reflectance images were captured in white light and fluorescence pared using the unpaired two-tailed Student t test. A P value <0.05 was (2D planar). For 3D imaging, a field enclosing the tumor was selected, and considered significant. Analyses were performed using GraphPad Prism the tomographic scan was carried out. The scan data were analyzed using software version 6.0 (GraphPad Software Inc.). reconstruction software provided by the manufacturer (PerkinElmer). For tomographic data analysis, 3D regions of interest were drawn around the ACKNOWLEDGMENTS. We thank Dr. Felix Heinemann and Dr. Ursula Nestle for tumor, and the total amount of fluorochrome (in picomoles) was calculated discussions. This work was supported by a grant from the Clotten Foundation.

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