Imaging and Selective Elimination of Glioblastoma Stem Cells with Theranostic Near-Infrared-Labeled CD133-Specific Antibodies

Abstract

Near-infrared photoimmunotherapy (NIR-PIT), which employs monoclonal antibody (mAb)-phototoxic phthalocyanine dye IR700 conjugates, permits the specific, image-guided and spatiotemporally controlled elimination of tumor cells. Here, we report the highly efficient NIR-PIT of human tumor xenografts initiated from patient-derived cancer stem cells (CSCs). Using glioblastoma stem cells (GBM-SCs) expressing the prototypic CSC marker AC133/CD133, we also demonstrate here for the first time that NIR-PIT is highly effective against brain tumors. The intravenously injected theranostic AC133 mAb conjugate enabled the non-invasive detection of orthotopic gliomas by NIR fluorescence imaging, and reached AC133+ GBM-SCs at the invasive tumor front. AC133-targeted NIR-PIT induced the rapid cell death of AC133+ GBM-SCs and thereby strong shrinkage of both subcutaneous and invasively growing brain tumors. A single round of NIR-PIT extended the overall survival of mice with established orthotopic gliomas by more than a factor of two, even though the harmless NIR light was applied through the intact skull. Humanised versions of this theranostic agent may facilitate intraoperative imaging and histopathological evaluation of tumor borders and enable the highly specific and efficient eradication of CSCs.

Keywords: CD133; cancer stem cells; glioblastoma; near-infrared photoimmunotherapy.

Figure 1

Validation of the immunoreactivity of the AC133-IR700 conjugate. (A) Side-by-side flow-cytometric analysis of the binding specificity of the AC133-IR700 conjugate compared to the unmodified AC133 mAb. Mixtures of HCT116 WT cells (AC133 positive) and p53-deficient HCT116 cells (AC133 negative) were incubated with serial dilutions of unmodified or modified antibody and analyzed by flow cytometry using an anti-mouse PE-conjugated F(ab')₂ fragment. (B) Flow-cytometric analysis demonstrating binding of AC133-IR700 to CD133-OE U251 glioma cells and NCH421k GBM-SCs. (C) Immunofluorescence staining demonstrating specific binding of the AC133-IR700 conjugate (red) to CD133-OE U251 cells but not to WT U251 cells. The cells were incubated with AC133-IR700 alone (left) or - to block its binding - together with the competitive unlabeled AC133 mAb (right) for 6 h. To visualize the CSC marker CD133, the cells were stained with AC141-PE (green). Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (D) NIR-FMT imaging of mice bearing WT U251 (AC133-) and CD133-OE U251 (AC133+) tumors 24 h after i.v. injection of the AC133-IR700 conjugate.

Figure 2

Target-specific cell death induced by AC133-IR700-mediated PIT in vitro. (A) NIR light irradiation-induced death of CD133-OE U251 cells pre-incubated for 6 h with AC133-IR700, as determined by Trypan blue exclusion. No cell death was detected after incubation with IR700-conjugated isotype control mAb or in control cultures without antibody. Data are means ± SE (n ≥ 5, ****p < 0.0001 for cultures treated with AC133-IR700 compared to the control cultures using an unpaired t-test). (B) Microscopic analysis of CD133-OE U251 cells treated by AC133-IR700-mediated PIT in comparison to cultures treated with an IR700-labeled isotype control antibody or without antibody. Note the cell shrinkage and membrane blebbing in the AC133-IR700-containing cultures. Scale bar, 50 μm. (C) AC133-IR700-mediated PIT did not have phototoxic effects on WT U251 cells (n ≥ 5). (D) NIR light irradiation efficiently induced cell death of NCH421k GBM-SCs as determined by Trypan blue exclusion (n ≥ 5, ****p < 0.0001 for cultures treated with AC133-IR700 compared to the control cultures using an unpaired t-test). (E) PIT abolished sphere formation in cultures of NCH421k GBM-SCs treated as described in Materials and Methods. Scale bar, 200 μm. Except the cultures termed “untreated”, all cultures were exposed to NIR light as described in Materials and Methods.

Figure 3

AC133-IR700-mediated PIT delays tumor initiation by NCH421k GBM-SCs in an s.c. mouse model. (A) NIR-FMT and BLI of mice bearing s.c. inoculated NCH421k GBM-CSs before and after PIT. NCH421k cells were pre-incubated with the AC133-IR700 conjugate overnight before cell implantation. NIR light (100 J/cm²) was applied to the implantation site in the left, but not in the right, flank immediately after implantation. Representative images are shown. (B) Inhibition of tumor initiation assessed by tumor volume measurements (n = 4; ****p < 0.0001; two-way repeated measures ANOVA). (C) Representative image of a mouse bearing two NCH421k tumors 50 days after inoculation. (D) Tumor growth assessed by BLI correlates with the volumetric measurements (n = 4). (E) Excised tumors 60 days after cell implantation.

Figure 4

Strong therapeutic effect of a single round of AC133-IR700-mediated PIT on well-established s.c. xenograft tumors initiated by NCH421k GBM-SCs. (A) When the tumors had reached a size of 100 mm³ (approx. 40 days after implantation), 100 μg of AC133-IR700 was injected i.v. and FMT and BLI were performed 24 h later. Thereafter, the tumor in the left flank, but not the one in the right flank, was irradiated with NIR light at a dose of 100 J/cm². Representative images are shown. (B) Reduced expression of the neural stem and progenitor cell markers Sox2, Nestin, and Musashi, and increased expression of the glial differentiation marker GFAP after AC133-IR700-mediated PIT, as assessed by Western blotting of tumor cell lysates. (C) Confocal microscopic images of tumor sections showing loss of cells expressing the stem and progenitor cell marker Nestin after NIR-PIT. The sections were stained with DAPI to visualize nuclei and with Alexa Fluor^® 488-labeled Nestin mAb. Scale bar, 25 μm. In B and C, representative results are shown (n = 3). (D) Substantial tumor regression and strong growth delay after AC133-IR700-mediated PIT. BLI correlated with the volumetric measurements obtained with caliper readings (n = 5; ****p < 0.0001, **p < 0.01; two-way repeated measures ANOVA). (E) Representative photograph of a mouse 4.5 weeks after PIT. (F) T₂-weighted axial MR images of a mouse bearing established s.c. NCH421k tumors before and 2 weeks after PIT. The left panels show the tumors on the left and right flanks before NIR light irradiation; the right panels show the non-irradiated (upper) and irradiated (lower) tumors 15 days after i.v. injection of AC133-IR700. (G) Tumors were harvested approx. 5 weeks after PIT. (H) Differences in final tumor weight between irradiated and non-irradiated tumors (n = 4).

Figure 5

Biodistribution of the i.v. injected AC133 mAb conjugate in mice bearing orthotopic AC133+ brain tumors. (A) Mice with CD133-OE U251 brain tumors were injected i.v. with AC133-IR700; 24 h later, organs were excised and imaged using the FMT-1500 system. (B) Confocal microscopic images of brain sections encompassing the NCH421k tumor periphery. The sections were stained with DAPI to visualize nuclei and with Alexa Fluor^® 488-labeled Nestin mAb. Note the infiltrative growth pattern of the NCH421k cells. Scale bar, 50 μm. (C) H&E staining of NCH421 brain tumor sections also showing irregular tumor margins (T: tumor). Scale bar, 50 μm. (D) Immunohistological detection of the i.v. injected AC133-IR700 conjugate at the margins of orthotopically growing NCH421k brain tumors. The brain tumor sections were stained with DAPI to visualize nuclei and PE-labeled AC141 mAb to visualize CD133 expression. Scale bar, 50 μm. Pictures shown are representative of three independent experiments.

Figure 6

Theranostic imaging and efficient PIT-mediated eradication of NCH421k GBM-SCs in 2-week established orthotopic brain tumors. (A) Non-invasive detection of AC133-IR700 by NIR-FMT before and after NIR light irradiation. 100 μg of AC133-IR700 was injected i.v. 24 h earlier. Representative images are shown. (B) NIR-FMT imaging of brains harbouring NCH421k GBM xenografts 24 h after injection of AC133-IR700 before (upper) and after (lower) ex vivo excision of the brain tumor. (C) Histological evaluation of NCH421 brain tumors revealed necrosis and a lower tumor cell density after NIR-PIT. Note that there was no significant necrosis in mice exposed to either AC133-IR700 injected i.v. or NIR light alone. All sections were stained with H&E. Scale bar, 100 μm. Representative images are shown. (D) BLI assessing the growth of the NCH421k brain tumors treated (red) or not (black) by PIT. All mice received AC133-IR700 i.v.; whereas the control mice were not irradiated, the mice in the PIT group were irradiated with 50 and 100 J/cm² of NIR light 24 and 72 h after tracer injection (n = 7 mice per group). (E) Representative BLI images of a control and an irradiated mouse at different time points after treatment. (F) Survival of the mice according to Kaplan-Meier (n = 7 mice in each group, **p < 0.01 for mice receiving NIR irradiation compared to the control group using a log-rank test).