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

Abstract

A technology that visualizes tumor stem cells with clinically relevant tracers could have a broad impact on cancer diagnosis and treatment. The AC133 epitope of CD133 currently is one of the best-characterized tumor stem cell markers for many intra- and extracranial tumor entities. Here we demonstrate the successful noninvasive detection of AC133(+) tumor stem cells by PET and near-infrared fluorescence molecular tomography in subcutaneous and orthotopic glioma xenografts using antibody-based tracers. Particularly, microPET with (64)Cu-NOTA-AC133 mAb yielded high-quality images with outstanding tumor-to-background contrast, clearly delineating subcutaneous tumor stem cell-derived xenografts from surrounding tissues. Intracerebral tumors as small as 2-3 mm also were clearly discernible, and the microPET images reflected the invasive growth pattern of orthotopic cancer stem cell-derived tumors with low density of AC133(+) cells. These data provide a basis for further preclinical and clinical use of the developed tracers for high-sensitivity and high-resolution monitoring of AC133(+) tumor stem cells.

Keywords: CSCs; cancer stem cells; glioblastoma.

Fig. 1.

Characterization of AC133⁺ cell lines and the modified AC133 mAbs. (A) Flow-cytometric detection of AC133 epitope expression on in vitro-cultured CD133-overexpressing U251 glioma cells and NCH421k glioblastoma stem cells compared with CD133⁻ U251 wild-type cells. Results shown are representative of more than 10 independent experiments. (B) Flow-cytometric analysis of the binding specificity of the NOTA-AC133 and Alexa 680-AC133 mAbs compared with unmodified AC133 mAb. The analysis was performed as described in Methods. Data are representative of three independent experiments. MFI, mean fluorescence intensity; OE, overexpressing.

Fig. 2.

PET/CT imaging and biodistribution of ⁶⁴Cu-NOTA-AC133 mAb in mice bearing s.c. implanted U251 gliomas overexpressing CD133. Nude mice received ∼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.

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 quantification 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.

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.

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 corresponding to 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.

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.

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.