Noninvasive positron emission tomography andfluorescence imaging of CD133+ tumor stem cellsSimone 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-69120Heidelberg, Germany; iCenter for Radiopharmaceutical Sciences, Swiss Federal Institute of Technology-Paul Scherrer Institute-University Hospital of Zurich, PaulScherrer 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 reviewAugust 9, 2013)

A technology that visualizes tumor stem cells with clinically relevanttracers could have a broad impact on cancer diagnosis andtreatment. The AC133 epitope of CD133 currently is one of thebest-characterized tumor stem cell markers for many intra- andextracranial tumor entities. Here we demonstrate the successfulnoninvasive detection of AC133+ tumor stem cells by PET and near-infrared fluorescence molecular tomography in subcutaneous andorthotopic glioma xenografts using antibody-based tracers. Partic-ularly, microPET with 64Cu-NOTA-AC133 mAb yielded high-qualityimages with outstanding tumor-to-background contrast, clearly de-lineating subcutaneous tumor stem cell-derived xenografts from sur-rounding tissues. Intracerebral tumors as small as 2–3 mm also wereclearly discernible, and the microPET images reflected the invasivegrowth pattern of orthotopic cancer stem cell-derived tumors withlow density of AC133+ cells. These data provide a basis for furtherpreclinical and clinical use of the developed tracers for high-sensitivityand high-resolution monitoring of AC133+ tumor stem cells.

cancer stem cells | CSCs | glioblastoma

Cancer stem cells (CSCs) are highly undifferentiated tumorcells with characteristics similar to normal stem cells. Thesecharacteristics include long-term replication, self-renewal, andaberrant differentiation (1, 2). Based on these characteristics, ithas been hypothesized that only CSCs are able to propagatetumors for long periods of time and to initiate relapses or me-tastases. Furthermore, CSCs are considered to be more resistantto conventional radio- and chemotherapy than more differenti-ated tumor cells (3–5). Hence, elimination of CSCs is challengingbut necessary for successful tumor eradication. The stem cellhypothesis of cancer development and progression is conceptu-ally attractive and is supported by many preclinical (1, 2, 5–7)and some clinical studies (4, 8). However, larger clinical trialsinvestigating the role of CSCs in patients have been hamperedby the lack of techniques to detect, localize, and quantify thepresence of CSCs noninvasively. Specifically, successful non-invasive imaging of unmanipulated CSCs with clinically relevantimaging probes (e.g., antibodies or other ligands binding CSC-specific cell-surface proteins) has not yet been reported (9–11).AC133 is an N-glycosylation–dependent epitope of the second

extracellular loop of CD133/prominin-1, a cholesterol-bindingprotein of unknown function that locates to plasma membraneprotrusions (12–14). Postnatally, the CD133 protein is expressedby certain epithelial and nonepithelial cells, by stem and pro-genitor cells of various organs, and by CSCs of many differenttypes of malignant tumors (15). With a few exceptions, recog-nition of the AC133 epitope by the AC133 mAb appears to belimited to cells harboring stem cell properties, and the AC133epitope—but not necessarily the CD133 protein—is down-reg-ulated upon differentiation, presumably because of changes inglycosylation (12, 13, 15).

AC133+ tumor stem cells have been described for glioblastomamultiforme (the most common and most aggressive primary braintumor in adults), various pediatric brain and central nervous sys-tem tumors (medulloblastoma, ependymoma, pineoblastoma,teratoid/rhabdoid tumors, and retinoblastoma), brain metastases,many different types of carcinomas including colon, pancreatic,lung, liver, and ovarian cancer, melanoma, sarcomas, and differenttypes of leukemia. Although AC133− tumor stem cells also exist(16–18), AC133+ cells found in these and other tumor types havebeen shown to be able to self-renew, to differentiate, and to rec-reate the original tumors when injected into immunocompromisedmice (8, 17, 19–22). Both, stemness and highly agressive malignanttumors often are associated with hypoxia (23), and hypoxia canpromote the expansion of CD133+ cells (24). Therefore the fre-quent expression of AC133 on CSCs may reflect, in part, theircommon localization in a hypoxic environment (25).We previously reported the successful noninvasive detection of

the AC133 epitope by antibody-based near-infrared fluorescencemolecular tomography (NIR FMT) in mice with s.c. xenograftsof CD133-overexpressing tumor cells or traditional tumor celllines naturally displaying AC133 (26). However, we did not in-

Significance

Cancer stem cells (CSCs) are thought to be responsible forgrowth and dissemination of many malignant tumors and forrelapse after therapy. Therefore methods for the noninvasiveimaging of CSCs could have profound consequences for di-agnosis and therapy monitoring in oncology. However, clini-cally applicable methods for noninvasive CSC imaging are stilllacking. The AC133 epitope of CD133 is one of the most in-tensely investigated CSC markers and is particularly importantfor aggressive brain tumors. Here we describe the developmentof clinically relevant tracers that permit high-sensitivity andhigh-resolution monitoring of AC133+ glioblastoma stem cellsin both subcutaneous and intracerebral xenograft tumors usingpositron emission tomography and near-infrared fluorescenceimaging, two clinically highly relevant imaging modalities.

Author contributions: F.B., S.P., M.M., M.B., H.M., and G.N. designed research; S.G., F.B.,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.contributed new reagents/analytic tools; S.G., F.B., S.P., M.M., E.F., R.G., X.Z., M.B., W.W.,H.M., and G.N. analyzed data; and F.B., S.P., E.F., M.H., W.W., and G.N. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1S.G., F.B. and S.P. contributed equally to this work.2To whom correspondence should be addressed. E-mail: gabriele.niedermann@uniklinik-freiburg.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1314189111/-/DCSupplemental.

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vestigate patient-derived CSCs with the above-mentioned stemcell characteristics in that study, and NIR fluorescence, althoughpenetrating tissues more deeply (2–4 cm) than visible-lightfluorescence, has limited importance for clinical whole-bodyimaging (27).We report here the successful noninvasive detection of tumor-

associated AC133 by PET, using a radiolabeled AC133-specificmAb in mice xenografted with tumor cell lines overexpressingCD133 or with patient-derived AC133+ CSCs. PET is highlysensitive and is widely used for clinical whole-body diagnosticimaging. As a PET nuclide, we used 64Cu (t1/2 = 12.7 h), whichallows long-term tracking for at least 48 h, to follow the tumoralaccumulation 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 64Cuchelator, 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 detectionof AC133+ CSCs with fluorescently labeled AC133 mAb andNIR imaging, a modality that is important for whole-body small-animal imaging and for intraoperative and endoscopic imagingand imaging of superficial tumors in humans (27, 30). In additionto imaging s.c. growing tumors, we report the successful anti-body-mediated imaging of orthotopic xenografts initiated fromAC133+ glioblastoma stem cells in the brain of immunocom-promised mice, emphasizing the feasibility of noninvasive antibody-mediated imaging of brain tumors.

ResultsTracer Development and Characterization. After conjugation of thechelator NOTA, we radiolabeled the AC133 mAb, which rec-ognizes the AC133 epitope on CD133-overexpressing cells andCSCs (Fig. 1A), and an isotype control antibody with 64Cu. TheNOTA-AC133 and NOTA-isotype control mAb conjugates werefunctionalized with an average of 5.1 ± 0.8 and 2.3 ± 0.3 che-lators per molecule of antibody, respectively, and in all radio-labeling experiments the radiochemical purity was >95%, withspecific activities of 48.4 ± 9.5 MBq/nmol and 41.4 ± 5.6 MBq/nmolfor 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 ± 0.2Alexa 680 molecules conjugated to the two mAbs, respectively.Neither conjugation of NOTA nor that of Alexa 680 impairedbinding to the AC133 epitope, as revealed by flow cytometrictitration of NOTA-AC133 and Alexa 680-AC133 against theunmodified AC133 antibody (Fig. 1B).

PET Imaging of s.c. CD133-Overexpressing Glioma Xenografts. Wefirst established small-animal PET with the 64Cu-NOTA-AC133mAb in a robust nude mouse model with s.c. xenografts of U251glioma cells transduced with a CD133-encoding lentivirus result-ing in high expression of CD133 associated with high cell-surfaceexpression of the AC133 epitope (Fig. 1A). We s.c. injected theseAC133-high cells into the right flank and, as an internal control,injected CD133− U251 wild-type cells into the left flank of eachanimal. The uptake of the 64Cu-NOTA-AC133 mAb in the CD133-overexpressing xenografts was very high (Fig. 2A). As quantifiedfrom PET images, the uptake reached 37.9 ± 5.6% of the injectedactivity (IA)/g at 24 h and increased to values as high as 56.3 ±16.2% 48 h after i.v. injection of the radiolabeled antibody. Incontrast, the uptake of antigen-negative tumors decreasedfrom 10.1 to 8.3% at 24 and 48 h, respectively, and was similarto or slightly lower than that of liver and blood (Fig. 2B). At48 h, the uptake ratios of AC133+ tumor to AC133− tumor, liver,blood, and background were 7.3 ± 1.9, 5.0 ± 0.6, 6.9 ± 1.2, and32.0 ± 8.8, respectively. Ex vivo biodistribution studies at 48 hafter administration of the 64Cu-NOTA-AC133 mAb corrobo-

rated the PET imaging data (Fig. 2C). In contrast to the highlydifferent uptake of the 64Cu-NOTA-AC133 mAb, both the AC133+

and the AC133− tumors showed a similar uptake of 18F-fluoro-deoxyglucose (FDG) (Fig. S1).

FMT Imaging of Glioblastoma Stem Cell-Derived s.c. Xenografts. Todevelop animal models for imaging of AC133+ CSCs, whichusually display much lower levels of AC133 than cell linesoverexpressing CD133, we chose the well-characterized gliomastem cell line NCH421k. This cell line has been derived from aprimary glioblastoma [World Health Organization (WHO) gradeIV] under stem cell culture conditions, and its CSC character-istics have been reported repeatedly (Fig. S2 and refs. 24, 31–33).NCH421k cells display 10- to 15-fold lower surface AC133 thanCD133-overexpressing U251 cells both in vitro (Fig. 1A) and invivo (Fig. S3A). We first established in vivo imaging of CSC-containing tumors with NIR FMT, a sensitive and inexpensiveimaging technique permitting quantitative 3D detection of NIRfluorophores in mice. Because CD133− derivatives of the NCH421kline do not exist, we used an Alexa 680-labeled IgG1 isotypeantibody as specificity control.The Alexa 680-labeled AC133 mAb yielded a significantly

higher in vivo fluorescence signal in the tumor region than didthe Alexa 680-labeled isotype control antibody (Fig. 3A). The exvivo biodistribution analysis confirmed that the fluorescentlylabeled AC133 mAb accumulated in the tumor (Fig. 3 B–D). Inaddition, flow-cytometric analyses of tumor single-cell suspensions

Fig. 1. Characterization of AC133+ cell lines and the modified AC133mAbs. (A) Flow-cytometric detection of AC133 epitope expression on invitro-cultured CD133-overexpressing U251 glioma cells and NCH421k glio-blastoma stem cells compared with CD133− U251 wild-type cells. Resultsshown are representative of more than 10 independent experiments. (B)Flow-cytometric analysis of the binding specificity of the NOTA-AC133 andAlexa 680-AC133 mAbs compared with unmodified AC133 mAb. The anal-ysis was performed as described in Methods. Data are representative ofthree independent experiments. MFI, mean fluorescence intensity; OE,overexpressing.

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directly after the scans showed that the i.v.-injected Alexa 680-AC133 mAb had bound to almost all CSC marker-positive cellsin the tumor, verifying that the injected Alexa 680-AC133 mAbhad penetrated the tumor tissue efficiently (Fig. 3E, Left). Toidentify the CSCs ex vivo, the tumor single-cell suspensions werecostained with the AC141 antibody, which is specific for a secondstem cell-specific epitope of CD133. As expected, binding of theinjected Alexa 680-labeled isotype control antibody to CSCs couldnot be detected (Fig. 3E, Right).

FMT Imaging of Intracerebral Xenograft Tumors. Antibodies haveonly limited access to the brain because the undisturbed blood–brain barrier (BBB) is impermeable to macromolecules, andwhether systemically administered antibodies can reach extra-vascular targets in brain tumors with a disturbed BBB is of greatinterest (34, 35). We therefore wanted to find out whether theAC133 mAb is suitable for imaging orthotopically growingAC133+ glioma xenografts. We indeed could detect orthotopicallygrowing NCH421k gliomas noninvasively by NIR FMT imagingupon i.v. injection of the Alexa 680-labeled AC133 mAb (Fig. 3F,Upper Left), and the signal caused by the Alexa 680-AC133 mAb

also could be detected directly postmortem on the excised tu-mor-bearing brains (Fig. 3F, Lower Left).

PET Imaging of Tumor Stem Cell-Derived s.c. Xenografts. After theFMT studies had demonstrated that noninvasive mAb-mediatedvisualization of AC133+ CSCs was possible in principle, we ex-plored immuno-PET detection of AC133+ CSCs. The 64Cu-NOTA-AC133 mAb strongly marked s.c. growing NCH421k gliomas at 24and 48 h postinjection (p.i.) (Fig. 4A, Left and Center), despite theconsiderably lower expression of AC133 on NCH421k cells ascompared with CD133-overexpressing U251 cells (see Fig. 1A andFig. S3A). Particularly remarkable was the much higher tracer up-take liver [an organ exhibiting relatively high unspecific activitybecause of high blood perfusion, antibody metabolism, andpotential transchelation of 64Cu (28, 36)]. The tumor-to-con-tralateral background, tumor-to-blood pool, and tumor-to-livercontrasts were 21.4 ± 8.2, 2.7 ± 0.9, and 2.6 ± 0.8 at 24 h and32.8 ± 19, 6.4 ± 2.5, and 5.4 ± 1.8 at 48 h p.i., respectively. The64Cu-NOTA-isotype control antibody caused only a very weaktumor signal. As judged by visual inspection (Fig. 4A, Right) andaccording to in vivo and in vitro quantification (Fig. 4 B and C),

Fig. 2. PET/CT imaging and biodistribution of 64Cu-NOTA-AC133 mAb in mice bearing s.c. implanted U251 gliomas 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 tumorsat 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 pergroup. ***P < 0.001, t test; values represent means ± SD.

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this signal was in the range of or slightly lower than that of theblood pool (heart and blood vessels), presumably reflectinga high vascularization of the aggressively growing tumors andunspecific accumulation of a proportion of the antibody in theinterstitial space via the enhanced permeation and retentioneffect (37).

PET Imaging of Intracerebral Xenograft Tumors. We first performedmicroPET of intracerebrally growing tumor lesions in micebearing CD133-overexpressing gliomas. MicroPET after i.v. in-jection of the 64Cu-NOTA-AC133 mAb permitted the detectionnot only of relatively large intracerebral U251 gliomas over-expressing CD133 but also of very small ones (Fig. 5A, Left andCenter). These small lesions were imaged as soon as 12 d aftertumor cell implantation, when they had reached a size of only2–3 mm in diameter, as determined by contrast-enhanced CT. Incontrast, the 64Cu-NOTA-AC133 mAb caused only a very weaksignal in AC133− U251 wild-type tumors (Fig. 5A, Right, and Fig.5B). We then asked whether intracerebral tumors initiated fromAC133+ glioma stem cells could be detected also. To this end,NCH421k glioblastoma stem cells were injected into the fore-brains of immunodeficient mice. MicroPET after i.v. injection ofthe 64Cu-NOTA-AC133 mAb clearly allowed the detection ofintracerebral xenografts initiated from AC133+ glioblastomastem cells (Fig. 6A, Left, and Fig. 6B). At 48 h, the uptake ratioof AC133+ NCH421k stem cell-containing tumors to back-ground in the brain was 14 ± 3.4. In contrast, the brain tumor

signal caused by the 64Cu-NOTA-isotype control antibody wasnot significantly above background (Fig. 6A, Right, and Fig. 6B).

Correlation of MicroPET Images with Histopathologic Tumor Appearance.The PET signal of orthotopic CD133-overexpressing U251 gliomaswas homogeneous and sharply delineated from the surroundingbrain tissue, whereas that of the NCH421k tumors was more het-erogeneous. Particularly striking was the reduced and diffuse signalin the periphery of these CSC-derived tumors (compare Fig. 5A,Left and Center and Fig. 6A, Left). These differences in microPETsignal intensity correlated with histopathological differences. OnH&E-stained brain sections, the U251 tumors appeared as verycompact, homogeneous, and well-delineated tumor masses (Fig. 7 Aand B), reflecting noninfiltrative growth behavior. This appearanceis typical of orthotopic xenografts of conventional glioma cell lines,which generally do not recapitulate the invasive growth patternthat is a major feature (17, 31) of the highly malignant tumorentity human glioblastoma multiforme (38) and certainly con-tributes to the failure of current therapies. In contrast, NCH421kxenografts exhibited a very different cyto-architecture. The H&Estaining appeared more heterogeneous, with a lower intensity in the1- to 2-mm-thick periphery of the tumors than in the center (Fig.7D). Higher magnifications revealed tumor cells migrating alongblood vessels in the tumor periphery, forming irregular tumormargins (Fig. 7E). Together, these histopathological features reflectthe highly infiltrative growth behavior characteristic of humanglioblastoma multiforme. AC133 staining of brain tissue sectionsalso revealed a high density (72 ± 20%) of intensely stained

Fig. 3. NIR FMT imaging of xenografts containing AC133+ glioblastoma stem cells. Mice with s.c. growing NCH421k xenografts were injected i.v. with Alexa680-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 presentedcorrespond 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 ofexcised 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 intracerebralNCH421k 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.

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AC133-expressing cells in CD133-overexpressing U251 gliomas(Fig. 7C and Fig. S3 B and C). In contrast, the AC133+ cell density(20.0 ± 11%) and cellular AC133 expression levels were muchlower in the NCH421k tumors, and the AC133+ tumor cells tendedto occur in clusters (Fig. 7F and Fig. S3 B and C). The reduction inthe percentage of AC133+ cells compared with the initial 90–100%in the NCH421k implant suspension may be the result mainly ofdifferentiation and the invasive growth behavior. Nonetheless,microPET using the 64Cu-NOTA-AC133 mAb clearly enabled thedetection of the highly invasive, CSC-derived NCH421k tumors.

DiscussionThe AC133 epitope of CD133/prominin is perhaps the mostintensely investigated of the known CSC markers (8, 13, 17, 19,20, 22). Although of great interest and importance, noninvasiveimaging of AC133+ CSCs or other types of CSCs with clinicallyrelevant tracers and imaging modalities has not yet been repor-ted (9–11). Here, we demonstrate the successful noninvasiveantibody-mediated imaging of AC133+ glioblastoma stem cellsby PET and NIR fluorescence imaging in xenograft tumormodels in mice. Despite the relatively low AC133 expression onCSCs, we obtained high-quality PET images with the 64Cu-loadedNOTA-conjugated AC133-specific antibody. The AC133 epitopeis particularly important as a CSC marker of brain tumors (3, 8,

17, 19, 31), and we demonstrate here the successful noninvasiveimaging of intracerebral xenografts with a low density ofAC133+ glioblastoma stem cells by both the Alexa 680-labeledAC133 mAb (using FMT) and the 64Cu-NOTA-AC133 mAb(using microPET).Current imaging techniques used for standard treatment

monitoring in patients with solid tumors assess the shrinkage ofthe tumor as a response criterion. However, when non-CSCsoutnumber the CSCs, tumor shrinkage may reflect largely theelimination of treatment-sensitive bulk tumor cells, and whetherlong-term self-renewing CSCs also are being eliminated can beanswered only with CSC-specific tracers and sensitive imagingmodalities. Because CSCs also may be responsible for the initi-ation of local recurrences and distant metastases, specific andsensitive CSC imaging techniques could enable the early detectionof these events as well.PET and NIR fluorescence imaging are sensitive imaging mo-

dalities that are becoming increasingly important in clinical practice(27, 30, 39–41). We show here that particularly 64Cu-NOTA-AC133 mAb-mediated microPET yields high-quality, high-resolution images with outstanding tumor-to-background con-trast. 64Cu-NOTA-AC133 mAb-mediated microPET permittedthe detection of very small brain tumor lesions (2–3 mm in size)and also reflected differences in invasive behavior between

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) Exvivo biodistribution. Values are the mean %IA/g of tissue. n = 5 mice per group. ***P < 0.001, t test; values represent means ± SD.

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orthotopically growing U251 (noninvasive) and NCH421k (in-vasive) gliomas. Whereas sharply delineated PET signals reflectedthe compact and spherical microscopic appearance of U251 tumors,more diffuse PET signals seemed to reflect the lower density ofAC133+ cells and the chaotic and infiltrative growth pattern de-tected microscopically in the orthotopic NCH421k tumors. In ad-dition, the 64Cu-NOTA-AC133 mAb could image s.c. xenograftscontaining AC133+ CSCs clearly. The tracer uptake was signif-icantly higher in these flank tumors than in liver and blood,which cause the highest background signals in immuno-PET withintact antibodies. However, future studies are needed to determine

whether intraabdominal tumors with AC133+ CSCs within orclose to the liver also can be detected readily using this tracer.AC133− tumors or the 64Cu-labeled isotype control antibodycaused only signals as low as background, not exceeding that ofthe blood pool. Because CSCs show rather low AC133 expres-sion, these data considered together suggest that the 64Cu-NOTA-AC133 mAb is a very specific and highly sensitive tool for thenoninvasive detection of AC133+ CSCs.A proportion of the WHO grade IV glioblastomas and of

other tumor entities contains high frequencies of AC133+ cells.The AC133+ fraction among the highly aggressive glioblastomas

Fig. 5. PET/CT imaging and biodistribution of 64Cu-NOTA-AC133 mAb in mice bearing orthotopic U251 glioma xenografts. Nude mice bearing orthotopicxenografts of U251 glioma cells overexpressing CD133 or orthotopic xenografts of CD133− U251 wild-type cells received 7.5 ± 0.8 MBq 64Cu-NOTA-AC133 mAbvia 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 frommice bearing CD133-overexpressing U251 gliomas and frommice bearing U251 wild-type tumors. For the PET/CT images, an upper threshold corresponding tothe maximum tracer uptake of the CD133-overexpressing U251 tumors (43%IA/g) was chosen. (B) Uptake of 64Cu-NOTA-AC133 mAb as determined bymicroPET 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 = 5–6mice. ***P < 0.001, t test; values represent means ± SD.

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was reported to range from 19 to 29% (19) or from ≤1–50% (8).In the latter study, a considerable proportion of tumors contained>10% or even >25% AC133+ cells, and the AC133+ cells tendedto organize in clusters. For medulloblastomas, 6–21% AC133+

cells have been reported (19), and for teratoid/rhabdoid braintumors 1–36% have been reported at diagnosis and 30–70% atrelapse (42). For colon cancer, 2–19% AC133+ cells were repor-ted, compared with 0.4–2% in the surrounding tissue (20), and forovarian cancer the reported range is 0.3–35% (43). Other tumorentities (e.g., lower-grade gliomas) usually contain only smallerpopulations of AC133+ tumor cells (8). For pancreatic cancers themean is 1.8% (21); and for lung cancers the mean is 5% and thatof surrounding tissue is

luminal epithelia by immunohistochemistry of tissue fixed inparaformaldehyde (16). However, because fixation stronglyaffects the recognition of the epitope (13), its accessibility inthese epithelia to the AC133 mAb in noninvasive imaging isdifficult to predict.Current imaging techniques used in patients with primary or

metastatic brain tumors quantify the tumor burden indirectlythrough edema, vascular integrity (i.e., contrast enhancementusing low-molecular-weight contrast agents), or metabolic ac-tivity (mainly using amino acid PET). Although it is known that,in highly malignant brain tumors, the BBB is disturbed and thatits permeability is increased for macromolecules (34), reports onantibody-mediated PET of brain tumor lesions are scarce (40).Our microPET imaging and biodistribution analyses show thatthe 64Cu-NOTA-AC133 mAb is exquisitely suited for imagingorthotopically growing glioblastoma xenografts, indicating thatthis antibody tracer penetrates these tumors quite efficiently.Antibody-mediated brain tumor CSC imaging also may haveadvantages over other currently used brain tumor imagingtechniques in distinguishing true tumor relapse from pseudo-progression, because it is based on a molecular antigen/antibodyinteraction that is more tumor-specific than imaging based onvascular integrity or metabolic activity. In addition, our datasuggest that noninvasive antibody-mediated imaging could bevery useful in general for assessing receptor expression andevaluating antibody therapeutics in neurooncology.In conclusion, we have developed antibody-based PET and

NIR fluorescence tracers that enable the specific and highlysensitive detection of AC133+ CSCs. FMT and PET detection ofAC133+ CSCs is important for preclinical studies in human tu-mor xenograft models. The present study also sets the founda-tion for the application of fluorescent and positron emitter-labeled AC133 mAbs, or humanized versions thereof, in humanstudies with clinical NIR fluorescence and PET scanners. Inaddition, our study confirms that the AC133 mAb also may beuseful for therapeutic targeting, including the treatment of braintumors such as the highly aggressive and presently incurableglioblastoma multiforme (32) as well as for the development oftheranostic probes (47).

MethodsCell Lines. The U251 cell line was obtained from American Type CultureCollection (ATCC). NCH421k is a glioblastoma stem cell line that was deriveddirectly from a primary glioblastoma tumor sample. The establishment of theCD133-overexpressing U251 glioma cell line and of the NCH421k glioblas-toma stem cell line have been described previously (26, 31). NCH421k cellswere cultured under normoxic conditions (21% O2) in Neurobasal-A medium(Invitrogen) supplemented with 20 ng/mL EGF and 20 ng/mL FGF-2 (Prospec),1× penicillin/streptomycin (PAA Laboratories), 0.5× minimum essential me-dium (MEM) nonessential amino acids, 1× Glutamax-I, and B27 supplement(all from Invitrogen) on normal plastic, where they grew as spheres. CD133-overexpressing U251 cells and wild-type U251 cells were cultured in mediumcontaining 10% (vol/vol) FBS. All three cell lines were transduced with a lentiviruscoding for a fusion protein consisting of the latest generation of firefly luciferaseand a neomycin resistance cassette (L1-mCherry-IRES-FFneo or L1-FF-IRESneo)constructed in the G.N. laboratory according to standard procedures.

Antibodies. AC133.1 hybridoma cells (14) (ATCC HB-12346) were cultured inDMEMwith Ultra Low IgG FBS (Invitrogen), and the AC133 mAb was purifiedfrom the hybridoma supernatant according to standard methods. The IgG1isotype control antibody was purchased from BioXcell.

Antibody Conjugation with Alexa 680. Conjugation of Alexa Fluor 680 (Alexa680) to the AC133 and IgG1 isotype control antibodies was conducted withthe Alexa Fluor-680 SAIVI Rapid Antibody Labeling Kit from Invitrogenaccording to the manufacturer’s instructions. Protein concentrations of theAlexa Fluor 680-conjugated mAbs and the degree of labeling were determinedwith a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific).

Antibody Conjugation with NOTA. To AC133 or the isotype control antibody (inmetal-free PBS), p-SCN-Bn-NOTA (Macrocyclics) dissolved in DMSO wasadded at a ratio of 1:32 (final volume 500 μL, 0.1 M bicarbonate buffer,pH 9.5). Conjugation was allowed to proceed at room temperature for 5 h.Excess chelator was removed by G25-Sephadex size-exclusion chromatogra-phy on PD-10 columns. The columns were washed with PBS supplied with1 μM EDTA to remove metal ions, followed by washing with only PBS.Fractions containing the immunoconjugate were collected in PBS and con-centrated to 1 mg/mL with Amicon Ultra 4 centrifugation tubes (Millipore),followed by buffer exchange to ammonium acetate buffer, pH 5.2.

Labeling of NOTA-AC133 mAb with 64Cu. 64CuCl2 was obtained from the De-partment of Preclinical Imaging and Radiopharmacy, Eberhard Karls Uni-versity, Tübingen Germany. Labeling was performed in 250-μL ammonium

Fig. 7. Histopathological examination of orthotopically growing brain tumors. Brain sections of mice bearing U251 gliomas overexpressing CD133 or gliomasderived 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 microscopicimages of brain sections encompassing the tumor periphery. The sections were stained with DAPI to visualize nuclei and with PE-labeled AC133 mAb. Notethe plasma membrane staining for AC133. Pictures shown are representative of three independent experiments.

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acetate buffer (0.1 M, pH 8.2) with 240 μg NOTA-AC133 or 240 μg isotypecontrol mAb. About 100 MBq 64CuCl2 was added and incubated for 40 min at37 °C. The labeling reaction was stopped by adding 100 μL 0.1 M EDTA so-lution to chelate-free 64CuCl2. Quality control was performed by an isocraticHPLC run (Ramona Star HPLC system; Raytest GmbH) on a BioSilect SEC 250–5size-exclusion column (Bio-Rad Laboratories) with PBS as eluent at a flowrate of 1 mL/min. The retention times for the labeled compounds were(mean ± SD) 6:48 ± 0:11 min and 9:44 ± 0:08 min for the labeled mAb andfree 64Cu(EDTA)2−, respectively. Free 64Cu(EDTA)2− was separated by fil-trating through an Amicon 10-kDa cutoff filter. Together with this purifi-cation step, the labeled compound was buffer exchanged into 0.9% NaClsolution for injection and adjusted to the final volume.

Determination of NOTA Chelators per Antibody. The number of NOTA chela-tors per antibody molecule was determined by isotope dilution and con-firmed by LC-MS using anAgilent 1260 nano/capillary LC system coupled to anAgilent 6530 QTOF equipped with a Chip-Cube.

Titration of Labeled Antibodies. Preservation of binding affinity upon labelingwith either Alexa 680 or NOTA was ascertained through side-by-side flowcytometric titration analysis of labeled and unlabeled antibody, respectively.Serially diluted antibody was incubated with 5 × 105 HCT116 colon carcinomacells (which are AC133+) and 5 × 105 p53-deficient HCT116 cells (which areAC133−) suspended in 100 μL FACS buffer (0.5% BSA/2 mM EDTA in PBS) for15 min. After washing, the cells were incubated with 1.5 μg anti-mouse IgGphycoerythrin (PE)-conjugated F(ab’)2 fragment (Dianova) in 100 μL FACSbuffer for 20 min. Samples then were washed twice and analyzed ona FACSVerse (BD Biosciences).

Animal Experiments. All animal experiments were performed in accordancewith the German Animal License Regulations and were approved by theanimal care committee of the Regierungspräsidium Freiburg (registrationnumber: G-10/64).

Tumor cell Implantation and Tumor Growth Monitoring. For s.c. tumor models,5 × 106 U251 wild-type, CD133-overexpressing U251 or NCH421k cells wereimplanted into the flanks of 6- to 8-wk-old immunodeficient mice. BALB/cnude and the more immunodeficient NOD/SCID mice (Charles River) wereused for U251 and NCH421k cells, respectively. The growth of the xenograftswas monitored by caliper measurement. When the s.c. growing tumorsreached a size of 500–1,000 mm3, they were used for in vivo imagingexperiments. For orthotopic brain tumor models, 2.5 × 105 wild-type orCD133-overexpressing U251 or NCH421k cells stably transduced with lucif-erase were implanted manually 3 mm anterior and 3 mm to the right of thebregma in the brains of 6- to 8-wk-old nude mice. The cells, which weresuspended in 4 μL PBS, were injected at a depth of 3 mm with a Hamiltonsyringe, which was held in position for 5 min. After the injection, the surfacewas cleaned with a sterile cotton swab, and the burr hole was filled withbone wax. Thereafter, tumor growth was monitored noninvasively using invivo bioluminescence imaging on an IVIS spectrum imaging system (Perki-nElmer) three times per week (Fig. S2D). Mice with orthotopic brain tumorswere imaged with FMT or PET when the bioluminescence signal reached anintensity between 5 × 107 and 1 × 108 photons/s. When the NCH421k tumor-bearing mice met the above criteria, they were randomized into groups andwere injected with either AC133 or isotype control antibodies.

FMT in Vivo Imaging. Alexa 680-labeled AC133 or isotype control mAb (70 μg)was injected i.v. The fluorescence signals were visualized using an FMT1500system (PerkinElmer) at several time points p.i. The mice were anesthetizedby gas anesthesia (isoflurane/oxygen mixture) and then were placed into animaging cassette. After the cassette was positioned in the FMT1500 imagingsystem, reflectance images were captured in white light and fluorescence(2D planar). For 3D imaging, a field enclosing the tumor was selected, andthe tomographic scan was carried out. The scan data were analyzed usingreconstruction software provided by the manufacturer (PerkinElmer). Fortomographic data analysis, 3D regions of interest were drawn around thetumor, and the total amount of fluorochrome (in picomoles) was calculated

by TrueQuant software (PerkinElmer), using calibrated standards of the la-beled mAbs.

PET and CT Imaging. Mice were injected i.v. with either 30 μg 64Cu-NOTA-AC133 or isotype control mAb. PET imaging was performed with a microPETFocus 120 (Concorde). To account for the physical decay of 64Cu, the ac-quisition time was 20 min at 24 h p.i. and 45 min at 48 h p.i. PET acquisitionwas followed immediately by CT imaging (CT Imaging microCT scanner). Forstudies of intracranial tumors, mice were injected i.v. with 100 μL Imeron 350as contrast agent immediately before the start of the CT scan. The headregion of each mouse was scanned in one bed position for 90 s using a 360°rotation step, a tube voltage of 40 keV, and a tube current of 1 mA.

PET and CT Image Analysis. PET images were reconstructed by the routine 2Dordered subset expectation maximization (OSEM2D) algorithm provided bythe scanner software, as previously described (48). The resolution of thereconstructed images ranged between 1.5 and 1.7 mm. Quantitative analysisof radiotracer uptake was performed with AMIDE software, and the repor-ted values represent the mean activity concentration expressed as percent ofinjected activity per gram of tissue (%IA/g), assuming a tissue density of 1 g/mL.Image contrast was assessed by calculating the ratio of radiotracer uptake tocontralateral brain, left ventricular cavity, and liver. Images of the CT scans werereconstructed with a voxel size of 0.12 × 0.12 × 0.12mm and a T30 kernel, usingthe software provided by the manufacturer. Fusion of the PET and CT imageswas performed by ROVER software (ABX).

Ex Vivo Biodistribution. After the imaging studies, the animals were killed bycervical dislocation, and tumor and other organs were sampled. In animalsinjected with fluorescent antibody, either ex vivo FMT imaging or FACSanalysis of tumor single-cell suspensions (as described below) was performed.After PET/CT imaging, radioactivity within the tumor and the normal organswas measured using a gamma counter Packard Cobra II (PerkinElmer). Allvalues were decay-corrected and expressed as %IA/g ± SD, by measuringa standard of known activity from the respective injected probe.

Histopathology. Paraformaldehyde (4%)-fixed brains were cut in a Vibratome(Leica VT-1000S; Leica) in horizontal sections 60 μm apart. Brain sections weremounted and stained with H&E. For analysis of AC133 expression in tumortissue, 60-μm sections were stained using a PE-labeled antibody againsthuman AC133.1 (Miltenyi Biotec). For negative control, slices were stainedwith PE-labeled IgG1 isotype control antibody. After washings and nuclearstaining with DAPI, sections were mounted and analyzed with a krypton-argonlaser scanning confocal imaging system (TCSNT; Leica Microsystems AG).

FACS Analysis of Tumor Single-Cell Suspensions.At 96 h after injection of Alexa680-labeled mAbs, pieces of s.c. grown tumors were digested with 0.7 U/mLLiberase-Blendzyme (Roche)/Accutase (eBioscience)/100 U DNase I (Invi-trogen)/10 mM MgCl2 for 30 min at 37 °C. Nondigested pieces were digestedfurther with Accutase for 30 min and then were pressed through a cellstrainer. Red blood cells were removed using ice-cold RBC Lysis Buffer(eBioscience). Cells then were washed with PBS, resuspended in FACS buffer,and transferred through a preseparation filter. The cells were incubatedwith FcR blocking reagent (Miltenyi) for 10 min, followed by incubation with5 μg/mL anti-AC141-PE (Miltenyi), and then were analyzed by flow cytometry.

FACS Analysis of AC133 Expression on Cultured Tumor Cells. Tumor cells(CD133-overexpressing U251 and wild-type U251 as well as NCH421k cells)were collected, incubated with FcR blocking reagent, stained with anti-AC133.1-PE antibody fromMiltenyi Biotec, and then subjected to FACS analysis.

Statistical Analysis. Results are presented as means ± SD. Data were com-pared using the unpaired two-tailed Student t test. A P value

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