Transdifferentiation of glioblastoma cells into vascular endothelial cells - PubMed (original) (raw)

Transdifferentiation of glioblastoma cells into vascular endothelial cells

Yasushi Soda et al. Proc Natl Acad Sci U S A. 2011.

Abstract

Glioblastoma (GBM) is the most malignant brain tumor and is highly resistant to intensive combination therapies and anti-VEGF therapies. To assess the resistance mechanism to anti-VEGF therapy, we examined the vessels of GBMs in tumors that were induced by the transduction of p53(+/-) heterozygous mice with lentiviral vectors containing oncogenes and the marker GFP in the hippocampus of GFAP-Cre recombinase (Cre) mice. We were surprised to observe GFP(+) vascular endothelial cells (ECs). Transplantation of mouse GBM cells revealed that the tumor-derived endothelial cells (TDECs) originated from tumor-initiating cells and did not result from cell fusion of ECs and tumor cells. An in vitro differentiation assay suggested that hypoxia is an important factor in the differentiation of tumor cells to ECs and is independent of VEGF. TDEC formation was not only resistant to an anti-VEGF receptor inhibitor in mouse GBMs but it led to an increase in their frequency. A xenograft model of human GBM spheres from clinical specimens and direct clinical samples from patients with GBM also showed the presence of TDECs. We suggest that the TDEC is an important player in the resistance to anti-VEGF therapy, and hence a potential target for GBM therapy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1.

Fig 1.

TDECs. GFAP-Cre/p53+/− mouse brain was transduced by Tomo H-RasV12 LVs and Tomo Akt LVs as described (13). A representative image of ECs observed by confocal microscopy is shown. (A) Regular ECs lined the vessel lumen and expressed EC marker vWF (ii) but not the tumor marker GFP (i). DAPI was used as the nuclear marker, and the image was incorporated in the merge panel (iii). (B) In contrast, TDECs expressed both the GFP marker (i, iv, vii, and x) and EC markers vWF (ii), CD31 (v), CD34 (viii), and CD144 (xi). Some GFP+ ECs formed vessels with GFP− regular ECs (vi, arrowheads). DAPI was used as the nuclear marker, and the image was incorporated in the merge panels (iii, vi, ix, and xii). (C) TDECs expressed Flag-tagged H-RasV12 in addition to GFP and CD31 (i_–_v, arrows). They also expressed nestin in addition to GFP and vWF (vi_–_x, R and T indicate regular ECs and TDECs, respectively). (D) Representative result of flow cytometry for dissociated brain tumors. In the CD45− population (Top), ECs were CD31+CD34+ and constituted 7.1% of the whole tumor (Middle) and GFP+ ECs (TDECs) represented 24.4% of total ECs (Bottom). The asterisk represents the percentage of cells in each quadrant. All confocal pictures are single-slice images at an Airy factor of 1.0. [Magnification: all confocal images were taken at 63× with 3× (A and B) or 2× (C) electrical zoom (total magnification: 189× or 126×).]

Fig. 2.

Fig. 2.

Hypoxia and expression of HIF-1α and receptors of angiogenic growth factors. (A) Hypoxyprobe assay of the tumor. The hypoxic area (p02 < 10 mmHg) was detected by the anti-Hypoxyprobe antibody. The dotted line shows the approximate border of the hypoxic area. (Scale bar: 1 mm.) (B) Regular tumor ECs expressed VEGF-R2 (i–v), but GFP+ TDECs did not express VEGF-R2 (vi–x). (C) GFP+ TDECs (T) expressed FGF-R1, as did regular ECs (R) and surrounding tumor cells. All confocal pictures are single-slice images at an Airy factor of 1.0. [Magnification: 63× with 3× electrical zoom (total magnification: 189×) except A, which was 1.25×.]

Fig. 3.

Fig. 3.

Blood flow in TDEC-forming vessels. Biotinylated lectin injection assay showed that both regular vessels (A) and TDEC-forming vessels (B) are functional and allowed blood flow. (C) Nonfunctional TDEC-forming vessels were also observed. All confocal pictures are single-slice images at an Airy factor of 1.0. [Magnification: 63× with 3× electrical zoom (total magnification: 189×).]

Fig. 4.

Fig. 4.

TDECs in tumor-initiating cells transplanted into immunocompromised mice. Representative images of TDECs in brain tumors developed in NOD-SCID mice transplanted with tumor-initiating cell line. Cell line 005 (A), 005 subclone cells (B), and another tumor-initiating cell line 006 (C). TDECs expressed GFP and EC markers vWF and CD34. (D) Representative image of regular ECs and TDECs in brain tumors developed in DsRed nude mice transplanted with 005 cells by confocal microscopy. Regular tumor vessel ECs expressed vWF and host marker DsRed but not tumor marker GFP (i_–_iv), whereas GFP+ vWF+ TDECs did not express DsRed (v_–_viii). TDECs are indicated by arrows, and regular ECs are indicated by arrowheads (v_–_viii). All confocal pictures are single-slice images at an Airy factor of 1.0. [Magnification: 63× with 3× electrical zoom (total magnification: 189×).] (E) Representative results of flow cytometry of dissociated tumors developed in DsRed nude mice transplanted with 005 subclone cells. In the CD45−CD31+ EC fraction (Left), DsRed-positive cells did not express GFP (Right). (F) Representative results of flow cytometry for dissociated tumors developed in NOD-SCID mice. In the CD45−CD31+ EC fraction, cells expressing NOD-SCID mouse-specific H-2Kd did not express GFP. The asterisk represents the percentage of cells in each quadrant.

Fig. 5.

Fig. 5.

Endothelial differentiation of tumor-initiating cells in vitro. (A) Morphological changes of 005 cells cultured in N2 medium, DFS medium, and EGM with or without DFO. (B) Expression of HIF-1α in 005 cells cultured in the indicated conditions. Nuclear protein was extracted from the cells and analyzed by Western blotting using anti-HIF-1α antibody (Upper) or anti-lamin B1 antibody (Lower). (C) Representative confocal microscopy images of 005 cells cultured in N2 medium without DFO (Upper) or in EGM with DFO (Lower). The confocal microscopy images are maximum projection images of consecutive single-slice images at an Airy factor of 1.0. (Magnification: 40×.) (D) Flow cytometry of CD31 and VEGF-R2 expression in 005 cells cultured in various conditions. (E) Tube formation assay of 005 cells cultured in the indicated conditions and seeded on Matrigel. The 005 cells were cultured in DFS medium with DFO and in EGM with or without DFO under normoxia-formed tube structure, and all these cells were GFP+. The hypoxic condition (low O2) in DFS medium and EGM without DFO also induced tube formation.

Fig. 6.

Fig. 6.

Effect of inhibition of VEGF on TDEC formation. (A) Concentration of mVEGF in culture supernatant of 005 cells in various conditions. VEGF was released from 005 cells constitutively, and DFO treatment enhanced the production of VEGF significantly in DFS medium and EGM. Data represent mean ± SD of triplicate assays. (B) Effect of anti-VEGF NAb on tube formation. The 005 cells cultured in the indicated conditions were seeded on Matrigel, and tube formation was observed after 20 h. We omitted human VEGF from the EGM in this assay and used 1 μg/mL anti-VEGF NAb. (C) Effect of anti-VEGF receptor small molecule inhibitor AG28262 on tube formation of 005 cells. We cultured cells and observed tube formation under the same condition indicated in B, except for the addition of NAb. (D) Survival curve of the GBM mice treated with AG28262. GFAP-Cre transgenic mice received stereotaxic injection of LVs in the hippocampus of the brain. Mice were administrated 100 mg·kg−1·d−1 AG28262 orally for 6 wk from the sixth week after lentiviral injection. Control mice were administrated vehicle (0.5% carboxyl methyl cellulose). The survival curve was obtained by the Kaplan–Meier method, and the statistical difference was examined by the log-rank test. (E) Frequency of TDEC-forming vessels in the mouse GBM. Tumors were obtained from the mice that developed tumors and examined by immunofluorescence assay using a confocal microscope. Data represent mean ± SD from six (control) or five (AG28262) mice. *<0.5% by the Mann–Whitney U test; **<0.5% by the Wilcoxon signed-rank test; NS, not significant by the Wilcoxon signed-rank test.

Fig. 7.

Fig. 7.

TDEC formation in a xenograft model using human GBM spheres and patient samples. Representative images of regular ECs (A and C) and TDECs (B and D) in brain tumors developed in NOD-SCID mice transplanted with human GBM spheres. (A_–_D) (i) DAPI nuclear staining; (ii) GFP; (iii) and (iv) expression of indicated antigens; (v) merging image. (A and B) Tumors were stained with anti-vWF antibody, which reacts with both mouse and human vWF, and with an antibody specific for human nestin. (A) Regular ECs expressed vWF (iii) but not GFP (ii) or human nestin (iv). (B) TDECs expressed vWF (iii), GFP (ii), and human Nestin (iv; v, showing the merge with an arrow). A regular EC is indicated by the arrowhead (v). (C and D) Tumors were also stained with antibodies specific for mouse CD31 or human CD31. (C) Regular ECs expressed mouse CD31 (iii) but not human CD31 (iv). (D) TDECs expressed human CD31 (iv) but not mouse CD31 (iii). (E and F) Representative images of blood vessels of clinical samples of patients with GBM. (i) vWF; (ii) EGFR; (iii) merging image; (iv) merging image with Hoechst 33258 nuclear staining. (E) Vessels of normal brain expressed vWF (i) but not EGFR (ii). (F) vWF+ ECs (i) strongly expressed EGFR (ii), and surrounding tumor cells expressed EGFR (ii).

Comment in

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