Glioblastoma resistance to anti-VEGF therapy is associated with myeloid cell infiltration, stem cell accumulation, and a mesenchymal phenotype

Abstract

Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis. Inhibiting the VEGF-VEGF receptor (R) signal transduction pathway in glioblastoma has recently been shown to delay progression, but the relative benefit and mechanisms of response and failure of anti-VEGF therapy and VEGFR inhibitors are not well understood. The purpose of our study was to evaluate the relative effectiveness of VEGF sequestration and/or VEGFR inhibition on orthotopic tumor growth and the mechanism(s) of treatment resistance. We evaluated, not only, the effects of anti-VEGF therapy (bevacizumab), anti-VEGFR therapy (sunitinib), and the combination on the survival of mice bearing orthotopic gliomas, but also the differential effects of the treatments on tumor vascularity, cellular proliferation, mesenchymal and stem cell markers, and myeloid cell infiltration using flow cytometry and immunohistochemistry. Bevacizumab significantly prolonged survival compared with the control or sunitinib alone. Both antiangiogenic agents initially reduced infiltration of macrophages and tumor vascularity. However, multitargeted VEGFR inhibition, but not VEGF sequestration, rapidly created a vascular gradient and more rapidly induced tumor hypoxia. Re-infiltration of macrophages was associated with the induction of hypoxia. Combination treatment with bevacizumab and sunitinib improved animal survival compared with bevacizumab therapy alone. However, at the time of tumor progression, a significant increase in CD11b(+)/Gr1(+) granulocyte infiltration was observed, and tumors developed aggressive mesenchymal features and increased stem cell marker expression. Collectively, our results demonstrate a more prolonged decrease in tumor vascularity with bevacizumab than with sunitinib, associated with a delay in the development of hypoxia and sustained reduction of infiltrated myeloid cells.

Fig. 1.

Anti-VEGF but not VEGF receptor inhibitor therapy prolongs survival in an orthotopic glioma xenograft model. (A) Kaplan–Meier graph showing improved survival in nude mice with U87 tumors treated with bevacizumab (Bev) or sunitinib compared with untreated controls. (B) Kaplan–Meier graph showing improved survival in nude mice with U87 tumors treated with bevacizumab (Bev) + sunititnib compared with bevacizumab alone.

Fig. 2.

VEGFR inhibition produces greater reduction in central tumor vascularity than does anti-VEGF therapy. (A) Representative light microscopy images showing immunohistochemical detection of factor VIII (brown staining) in U87 tumors from control, bevacizumab (bev), sunitinib, and bev + sunitinib treated animals (200×) at 2, 4, and 6 weeks. (B) Bar graph demonstrating the average percentage of change in fractional area of factor VIII staining in central (c), middle (m), and peripheral (p) tumor locations in all treatment groups. *P < .05 compared with untreated controls; #P < .05 compared with sunitinib; ♦ P < .05 compared with the same treatment group at either 2 or 4 weeks. (C) Percent change in vascular density comparing the central vs peripheral tumor regions at each time point for each treatment group. *P < .05 compared with untreated controls; #P < .05 compared with sunitinib; +P < .05 compared with bev treatment. (D) Glioma cell proliferation using Ki-67 analysis at each time point. *P < .05 compared with untreated controls; #P < .05 compared with sunitinib; + P < .05 compared with the same treatment group at any of the 4.

Fig. 3.

VEGFR inhibition induces greater tumor hypoxia than anti-VEGF therapy (A) Representative light microscopy image at 200× magnification showing immunohistochemical detection of carbonic anhydrase 9 (CA9; brown nuclear staining) demonstrating regions of tumor hypoxia in the 4 treatment groups at 4 and 6 weeks. (B) Bar graph depicting the quantification of fractional staining of CA9. *P < .05 compared with controls. (C) An inverse correlation between factor VIII staining and fractional area of CA9 staining was seen for all tumor treatment groups.

Fig. 4.

Antiangiogenic agents modulate CD11b⁺/F4/80⁺/Gr1⁻ myeloid cell recruitment to tumors. (A) Representative flow cytometry analyses from tumor at 4 and 6 weeks in each group. (B) Upper panel, bar graph demonstrating the average percentage of CD11b+/F4/80+/Gr1- myeloid cells at each time point for the 4 treatment groups. Lower panel, bar graph showing average percentage of CD11b+/Gr1- cells expressing VEGFR1+. (C) Top, representative photomicrographs of CD68 macrophage staining at the corresponding time points. Bottom, bar graph of fractional area of CD68 staining. *P < .05 compared with controls; #P < .05 compared with sunitinib treated animals. (D) Positive correlation between number of CD11b+/F4/80+/Gr1- macrophages using flow cytometry and fractional area of CA9 staining.

Fig. 4.

Antiangiogenic agents modulate CD11b⁺/F4/80⁺/Gr1⁻ myeloid cell recruitment to tumors. (A) Representative flow cytometry analyses from tumor at 4 and 6 weeks in each group. (B) Upper panel, bar graph demonstrating the average percentage of CD11b+/F4/80+/Gr1- myeloid cells at each time point for the 4 treatment groups. Lower panel, bar graph showing average percentage of CD11b+/Gr1- cells expressing VEGFR1+. (C) Top, representative photomicrographs of CD68 macrophage staining at the corresponding time points. Bottom, bar graph of fractional area of CD68 staining. *P < .05 compared with controls; #P < .05 compared with sunitinib treated animals. (D) Positive correlation between number of CD11b+/F4/80+/Gr1- macrophages using flow cytometry and fractional area of CA9 staining.

Fig. 5.

Antiangiogenic agents modulate CD11b⁺/F4/80^-/Gr1⁺ myeloid cell recruitment to tumors. (A) Representative flow cytometry analyses from tumor at 4 and 6 weeks in each group. (B) Bar graph demonstrating the average percentage of CD11b+/F4/80-/Gr1+ myeloid cells at each time point for the 4 treatment groups; *, P < 0.05.

Fig. 6.

Antiangiogenic therapy induces mesenchymal changes to glioma tumor tissue in vivo. (A) Tumors treated with antiangiogenic therapy induced aggressive (invasive) behavior and histologic characteristics characteristic of mesenchymal tumors (see white arrows). Expression of mesenchymal markers vimentin and smooth muscle actin (SMA) are increased following antiangiogenic therapy, whereas e-cadherin expression decreases. (B) Tumors treated with bevacizumab and sunitinib show a significant increase in the expression of the glioma stem cell markers nestin and SOX2. (C) Expression levels of TGF beta and F4/80 in U87 glioblastoma xenograft tumors in response to antiangiogenic therapy. Tissue slides from xenografts were stained with anti-TGF beta (green) and anti-F4/80 (red) antibodies as described in “Materials and Methods.” The bar graph represents the percentage of TGF beta-positive cells in each condition under ×200. (D) The expression of ZEB2 and nestin in U87 glioblastoma xenograft tumors in response to antiangiogenic therapy. Xenografts tissue was stained with anti-ZEB2 (green) and anti-nestin (red) antibodies as described in “Materials and Methods.” The bar graph represents the percentage of ZEB2-positive cells for each treatment condition under ×200.

Fig. 6.

Antiangiogenic therapy induces mesenchymal changes to glioma tumor tissue in vivo. (A) Tumors treated with antiangiogenic therapy induced aggressive (invasive) behavior and histologic characteristics characteristic of mesenchymal tumors (see white arrows). Expression of mesenchymal markers vimentin and smooth muscle actin (SMA) are increased following antiangiogenic therapy, whereas e-cadherin expression decreases. (B) Tumors treated with bevacizumab and sunitinib show a significant increase in the expression of the glioma stem cell markers nestin and SOX2. (C) Expression levels of TGF beta and F4/80 in U87 glioblastoma xenograft tumors in response to antiangiogenic therapy. Tissue slides from xenografts were stained with anti-TGF beta (green) and anti-F4/80 (red) antibodies as described in “Materials and Methods.” The bar graph represents the percentage of TGF beta-positive cells in each condition under ×200. (D) The expression of ZEB2 and nestin in U87 glioblastoma xenograft tumors in response to antiangiogenic therapy. Xenografts tissue was stained with anti-ZEB2 (green) and anti-nestin (red) antibodies as described in “Materials and Methods.” The bar graph represents the percentage of ZEB2-positive cells for each treatment condition under ×200.