Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages - PubMed (original) (raw)

. 2016 Apr 19;113(16):4470-5.

doi: 10.1073/pnas.1525349113. Epub 2016 Apr 4.

Nathaniel D Kirkpatrick 2, Yuhui Huang 2, Christian T Farrar 3, Koen A Marijt 2, Jonas Kloepper 2, Meenal Datta 4, Zohreh Amoozgar 2, Giorgio Seano 2, Keehoon Jung 2, Walid S Kamoun 2, Trupti Vardam 2, Matija Snuderl 2, Jermaine Goveia 2, Sampurna Chatterjee 2, Ana Batista 2, Alona Muzikansky 5, Ching Ching Leow 6, Lei Xu 2, Tracy T Batchelor 7, Dan G Duda 2, Dai Fukumura 8, Rakesh K Jain 8

Affiliations

Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages

Teresa E Peterson et al. Proc Natl Acad Sci U S A. 2016.

Abstract

Glioblastomas (GBMs) rapidly become refractory to anti-VEGF therapies. We previously demonstrated that ectopic overexpression of angiopoietin-2 (Ang-2) compromises the benefits of anti-VEGF receptor (VEGFR) treatment in murine GBM models and that circulating Ang-2 levels in GBM patients rebound after an initial decrease following cediranib (a pan-VEGFR tyrosine kinase inhibitor) administration. Here we tested whether dual inhibition of VEGFR/Ang-2 could improve survival in two orthotopic models of GBM, Gl261 and U87. Dual therapy using cediranib and MEDI3617 (an anti-Ang-2-neutralizing antibody) improved survival over each therapy alone by delaying Gl261 growth and increasing U87 necrosis, effectively reducing viable tumor burden. Consistent with their vascular-modulating function, the dual therapies enhanced morphological normalization of vessels. Dual therapy also led to changes in tumor-associated macrophages (TAMs). Inhibition of TAM recruitment using an anti-colony-stimulating factor-1 antibody compromised the survival benefit of dual therapy. Thus, dual inhibition of VEGFR/Ang-2 prolongs survival in preclinical GBM models by reducing tumor burden, improving normalization, and altering TAMs. This approach may represent a potential therapeutic strategy to overcome the limitations of anti-VEGFR monotherapy in GBM patients by integrating the complementary effects of anti-Ang2 treatment on vessels and immune cells.

Keywords: anti-angiogenic therapy; colony-stimulating factor 1; macrophage; tumor immunity; tumor microenvironment.

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Conflict of interest statement

Conflict of interest statement: R.K.J. received consultant fees from Ophthotech, SPARC, SynDevRx, and XTuit. R.K.J. owns equity in Enlight, Ophthotech, SynDevRx, and XTuit and serves on the Board of Directors of XTuit and on the Boards of Trustees of Tekla Healthcare Investors, Tekla Life Sciences Investors, Tekla Healthcare Opportunities Fund, and Tekla World Healthcare Fund. No reagents or funding from these companies was used in these studies. T.T.B. received consultant fees from Merck, Roche, Kirin Pharmaceuticals, Spectrum Pharmaceuticals, Novartis, and Champions Biotechnology. C.C.L. is an employee of MedImmune.

Figures

Fig. 1.

Fig. 1.

Dual cediranib+MEDI3617 therapy enhances survival and reduces tumor burden in Gl261 and U87 tumors compared with cediranib therapy alone. Mice bearing Gl261 (A_–_D) or U87 (E_–_H) tumors were treated with control (green traces), MEDI3617 (orange traces), cediranib (red traces/bars), or dual therapy (blue traces/bars). (A) In Gl261 tumors, both cediranib and MEDI3617 monotherapies led to significantly higher overall median survival (24 d) than control treatment (20 d) (cediranib *P = 0.017; MEDI3617 *P = 0.011; n = 10). Dual therapy (n = 11) led to a significantly higher median survival (38 d) than control (***P < 0.0001) or cediranib treatment (†P = 0.002). (B) There was a significant difference in the growth rate of tumors treated with dual therapy compared with both control-treated (***P < 0.0001) and cediranib-treated (†P = 0.0076) tumors as measured by OFDI. (C) Dual therapy-treated tumors were significantly smaller than cediranib-treated tumors at day 20 as measured by MRI (†P = 0.0089). (D) There was no change in the extent of necrosis at day 20 (P = 0.11). (E) In the U87 model, both cediranib and dual therapy-treated mice had a significantly higher overall median survival (26 d and 13 d, respectively; n = 13) than control-treated mice (5 d; n = 12; ***P < 0.0001). (F and G) There was no difference in tumor growth (F) or volume (G) between dual therapy-treated tumors and cediranib-treated tumors in the U87 model. (H) There was a significant increase in ischemic hypoxic changes (early necrosis) in dual therapy-treated tumors compared with cediranib-treated tumors at day 6 (†P = 0.030). cedi, cediranib; cedi+M3671, cediranib+MEDI3617. Error bars represent the SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control unless otherwise indicated.

Fig. S1.

Fig. S1.

Representative images of necrosis in Gl261 and U87 tumors. (A) Representative image of H&E staining to highlight necrosis found in Gl261 tumors at day 20. (B) Representative image of early necrosis exhibiting diffuse hypoxic ischemic changes in U87 tumors at day 6.

Fig. S2.

Fig. S2.

Dual cediranib+MEDI3617 therapy increases the development of early necrosis in U87 tumors. Mice bearing U87 tumors were treated with control (green bar), MEDI3617 (orange bar), cediranib (red bar), or dual therapy (blue bar). H&E-stained tissues were analyzed for the area of diffuse hypoxic changes (early necrosis). *Dual therapy significantly increased early necrosis in U87 tumors compared with cediranib- (P = 0.0039), MEDI3617- (P = 0.043) and control-treated (P = 0.0026) tumors. Error bars represent the SEM.

Fig. S3.

Fig. S3.

In vitro cell viability in Gl261 and U87 tumors treated with cediranib, MEDI3617, or dual cediranib+MEDI3617 therapy. Gl261 or U87 cells were treated with increasing concentrations of cediranib or MEDI3617. Cell viability was measured using an MTT assay after 72 h of incubation with therapies. (A and D) Cediranib concentrations ranged from 1–106 nM. Cediranib monotherapy was cytotoxic to cells at concentrations above 103 nM. (B and E) MEDI3617 concentrations ranged from 0.00113–1.13 nM. MEDI3617 had no effect on cell viability. (C and F) Cells were treated with dual-therapy regimens of cediranib (102 nM, 103 nM, or 106 nM) + MEDI3617 (0.113 nM or 0.00113 nM). As in cediranib monotherapy, dual therapy reduced cell viability at higher concentrations of cediranib. These effects were the same with different concentrations of MEDI3617, suggesting that the main cytotoxic effects are caused solely by cediranib.

Fig. S4.

Fig. S4.

Dual cediranib+MEDI3617 therapy does not affect tumor apoptosis or proliferation. Gl261 (A and B) and U87 (C and D) tissues were collected from mice treated with control (green bars), MEDI3617 (orange bars), cediranib (red bars), or dual therapy (blue bars) at various time points after beginning treatment. Sections were stained for cleaved caspase 3 (apoptosis) or Ki67 (proliferation) and DAPI (nuclei). (A and B) In Gl261 tissue, neither apoptosis (A) nor proliferation (B) was altered by dual therapy at days 14 or 21. (C and D) Similarly, in U87 tissue dual therapy had no effect on apoptosis (C) or proliferation (D). Error bars represent the SEM.

Fig. 2.

Fig. 2.

Dual cediranib+MEDI3617 therapy extends vascular normalization in viable tissue compared with cediranib therapy alone. Gl261 tumors were collected from mice treated with cediranib (red bars) or dual therapy (blue bars) at days 14 or 21 after beginning treatment. Sections were stained for CD31 (for vessels), desmin (for perivascular cells), or collagen IV (for BM) and DAPI (for nuclei). (A, B, D) Both MVD (A) and perivascular cell coverage (the percentage of the desmin/CD31 double-positive area in the CD31+ area) (B) were higher in the dual therapy-treated tumors than in cediranib-treated tumors at days 14 and 21, but BM coverage (D) was higher only at day 21. (C) There was no significant difference in BM thickness among groups. (E) Representative images of CD31 (green)/desmin (red) staining in the normal brain (nl brain) and in control (cont)-, cediranib (cedi)-, and dual therapy (cedi+M3617)-treated tumors on day 21. (F) Representative images of CD31 (green)/collagen IV (red) staining. Error bars represent the SEM. *P < 0.05 compared with control unless otherwise indicated. (Scale bars, 50 μm.)

Fig. 3.

Fig. 3.

Dual cediranib+MEDI3617 therapy promotes edema control in Gl261 tumors. Gl261-bearing mice (n = 5–7) were treated with control (green bars), cediranib (red bars), or dual therapy (blue bars). Water content (edema) was measured on day 10 after treatment start by wet/dry weight ratio in the contralateral hemisphere (Contra) (A), ipsilateral hemisphere (Ipsi) (B), and tumor (C). Cediranib monotherapy significantly decreased water content in both the contralateral (P = 0.029) and ipsilateral (P = 0.024) hemispheres. Dual therapy also decreased water content compared with control-treated tumors, although the decrease did not reach statistical significance (contralateral P = 0.095; ipsilateral P = 0.052). There was no significant difference in edema in any of the tissues treated with dual therapy as compared with cediranib treatment. cedi, cediranib; cedi+M3617, cediranib+MEDI3617; Cont, control. Error bars represent the SEM. *P < 0.05 compared with control unless otherwise indicated.

Fig. 4.

Fig. 4.

Dual cediranib+MEDI3617 therapy improves vessel normalization in U87 tumors as compared with cediranib monotherapy. U87 tissues were collected from mice treated with control (green bars), MEDI3617 (orange bars), cediranib (red bars), or dual cediranib+MEDI3617 (blue bars) therapy at days 3 or 8 after beginning treatment. Sections were stained for CD31, either desmin or collagen IV (BM), and DAPI. (A and C) Both MVD (A) and BM thickness (C) were significantly decreased at day 3 by both cediranib and dual therapy as compared with control treatment and remained low at day 8. (B) In the dual therapy-treated tumors, perivascular cell coverage (the percentage of the desmin/CD31 double-positive area in the CD31+ area) also was significantly higher on day 3 as compared with control tumors. (D) There was no significant difference in BM coverage among groups. (E) Representative images of CD31 (green)/desmin (red) staining in the normal brain (nl brain) and in control (cont)-, cediranib (cedi)-, and dual therapy (cedi+M3617)-treated tumors. (F) Representative images of CD31 (green)/collagen IV (red) staining in the normal brain and in control-, cediranib-, and dual therapy-treated tumors. Error bars represent the SEM. *P < 0.05 compared with control unless otherwise indicated. (Scale bars, 50 μm.)

Fig. S5.

Fig. S5.

Dual cediranib+MEDI3617 therapy promotes edema control in U87 tumors. U87 tumor-bearing mice (n = 5–8) were treated with control (green bars), MEDI3617 (orange bars), cediranib (red bars), or dual therapy (blue bars). Water content (edema) was measured on days 3, 5, and 10 after treatment start by the wet/dry weight ratio in the contralateral hemisphere (Contra) (A), ipsilateral hemisphere (Ipsi) (B), and tumor (C). Cediranib monotherapy significantly decreased water content compared with control in all tissues measured at day 3 (contralateral, *P = 0.0061; ipsilateral, **P = 0.0004; tumor, **P = 0.0002). Cediranib monotherapy also decreased edema on day 5 compared with control in both the contralateral (**P = 0.0002) and ipsilateral (**P = 0.0007) hemispheres. Dual therapy also decreased edema on day 3 (contralateral, *P = 0.0370; ipsilateral, **P = 0.0004; tumor, ***P = 0.0001) and day 5 (contralateral and ipsilateral, ***P < 0.0001) compared with control. There was no significant difference in edema in any of the tissues treated with dual therapy compared with those treated with cediranib except for an unexpected increase in the water content of the contralateral hemisphere at day 10 (*P = 0.013). Error bars represent the SEM. *P < 0.05 compared with control unless otherwise indicated.

Fig. 5.

Fig. 5.

Cediranib and MEDI3617 have differential effects on TAM recruitment and polarization in Gl261 tumors. Immune cell profiles in Gl261 tumors were analyzed by flow cytometry on day 10. (A) The percentage of F4/80+ TAMs was significantly different among treatment groups (ANOVA P = 0.046; n = 7 or 8). Cediranib monotherapy tended to reduce the percentage of TAMs compared with MEDI3617 monotherapy and dual therapy (P = 0.054 and P = 0.096, respectively). (B) Representative images of the gating strategy to define TAM phenotypes for each treatment group. TAMs were defined as one of four phenotypes: M1-like TAM (CD11c+MRC1−), M2-like (CD11c−MRC1+), M1–M2 intermediate (CD11c+MRC1+), and double-negative (CD11c−MRC1−) TAMs. (C) There was no significant difference among treatment groups in the percentage of M1-like TAMs. (D) There was no significant difference among any of the groups in the percentage of M2-like TAMs. (E) There was a significant difference among treatment groups in the percentage of M1–M2 intermediate TAMs that did not display clear M1- or M2-like activation (ANOVA P = 0.0031). Furthermore, both cediranib monotherapy and dual therapy significantly reduced the percentage of these TAMs compared with MEDI3617 monotherapy (*P = 0.0019 and *P = 0.029). (F) There was a trend toward a difference among treatment groups in the M1/M2 ratio (Kruskal–Wallis test, P = 0.098). Dual therapy tended to increase M1/M2 ratio compared with cediranib monotherapy (P = 0.053). The dashed line indicates an M1/M2 ratio of 1. Total TAMs were normalized to CD45+ cells for comparisons among treatment groups. All TAM phenotypes described were normalized to total F4/80+ cells for comparisons among treatment groups. Error bars represent the SEM.

Fig. S6.

Fig. S6.

The gating strategy used to determine TAM recruitment and phenotype in Gl261 tumors. Single-cell suspensions were stained for the following markers: 7AAD (cell viability), CD45, F4/80, CD11c, and MRC1. Cells were first gated according to side-scattered area (SSC-A) and forward-scattered area (FSC-A). Further gating according to SSC-A and side-scattered width (SSC-W) and FSC-A and forward-scattered width (FSC-W) was used to identify single cells. 7AAD was used to identify dead cells. Viable cells then were gated on CD45 to identify CD45+dsRed− tumor-infiltrating immune cells. CD45+ cells were further gated on F4/80 to identify CD45+F4/80+ TAMs. Finally, CD11c and MRC1 were used to identify specific TAM phenotypes: M1-like TAMs (CD11c+MRC1−), M2-like TAMs (CD11c−MRC1+), M1–M2 intermediate TAMs (CD11c+MRC1+), and double-negative TAMs (CD11c−MRC1−).

Fig. S7.

Fig. S7.

Dual cediranib+MEDI3617 therapy has no effect on granulocyte or T-cell recruitment to Gl261 tumors. Gl261 tumors were collected from mice treated with control (n = 7), MEDI3617 (n = 7), cediranib (n = 7), or dual cediranib+MEDI3617 therapy (n = 8) at day 10 after the beginning of treatment. After disassociation, the resulting cell suspensions were stained with CD45, Gr1, CD4, and CD8 antibodies for flow cytometric analysis of myeloid and lymphoid populations within the tumors. There was no difference among the treatment groups in the percentages of CD45+ cells (A), CD45+Gr1+ granulocytes (B), CD4+ T lymphocytes (C), or CD8+ T lymphocytes (D). Error bars represent the SEM.

Fig. S8.

Fig. S8.

Peritoneal macrophage gene expression is altered by dual therapy. Peritoneal macrophages isolated from C57BL/6 mice were untreated (control; green bars) or were treated with MEDI3617 (100 nM) (orange bars), cediranib (0.5 μM) (red bars), or dual therapy (blue bars) for 24 h. (A) Gene-expression analysis showed that the M1-associated genes Cxcl9 (P = 0.011), Cxcl11 (P = 0.033), and Tnfα (P < 0.0001) were more highly expressed in dual therapy-treated macrophages than in untreated macrophages. Furthermore, dual therapy also increased the expression of _Cxcl9_ (_P_ = 0.011), _Cxcl11_ (_P_ = 0.020), and _Tnfα_ (_P_ = 0.0002) compared with MEDI3617 treatment. Finally, _Cxcl9_ (_P_ = 0.011), _Cxcl11_ (_P_ = 0.034), and _Tnfα_ (_P_ = 004) also were more highly expressed in dual therapy-treated macrophages than in cediranib-treated macrophages. (_B_) Gene expression analysis showed that the M2-associated genes did not change significantly in any of the treatments. Samples were measured in duplicate and experiments were repeated twice (_n_ = 2 per treatment group). *_P_ < 0.05, **_P_ > 0.01, ***P > 0.001.

Fig. 6.

Fig. 6.

Inhibition of macrophage recruitment by CSF-1 blockade compromises the survival benefit of dual cediranib+MEDI3617 therapy. Mice bearing Gl261 tumors were treated with control (n = 16; solid green trace), anti–CSF-1 (αCSF-1; n = 18; dotted green trace), dual cediranib+MEDI3617 therapy (n = 28; solid blue trace), or anti–CSF-1 + dual therapy (n = 26; dotted blue trace). Anti–CSF-1 by itself had no effect on overall median survival compared with control treatment (7 d vs. 6 d). When combined with dual therapy, however, anti–CSF-1 significantly reduced overall median survival compared with dual therapy alone (10 d vs. 17 d; *P = 0.003). Dual therapy with or without combined anti–CSF-1 treatment significantly increased overall median survival compared with control treatment (P = 0.0035 and P < 0.0001, respectively). These data confirm the role of macrophages as essential mediators of the survival benefits observed with dual therapy.

Fig. S9.

Fig. S9.

CSF-1 blockade decreases TAMs. Mice bearing Gl261 tumors were treated with control (n = 5, green bar), dual cediranib+MEDI3617 (n = 5, blue bar), or dual therapy + anti–CSF-1 (n = 5, blue checkered bar) for 10 d before analysis with flow cytometry. The addition of anti–CSF-1 treatment led to a significant decrease in macrophage recruitment compared with control-treatment (*P = 0.0135). Error bars represent the SEM.

Fig. S10.

Fig. S10.

Animal survival after treatment with cediranib and/or MEDI3617 with or without the inhibition of macrophage recruitment by CSF1-blockade. Mice bearing Gl261 tumors were treated with control (n = 16, solid green trace), anti–CSF-1 (n = 18, dotted green trace), MEDI3617 (n = 6, solid orange trace), anti–CSF-1+MEDI3617 (n = 7, dotted orange trace), cediranib (n = 12, solid red trace), anti–CSF-1+cediranib (n = 12, dotted red trace), dual therapy (n = 28, solid blue trace), or anti–CSF-1 + dual therapy (n = 26, dotted blue trace). Cediranib improved survival both as a monotherapy (P = 0.0032) and in combination with anti–CSF-1 (P = 0.0071). Tumor-bearing animals treated with dual therapy had significantly longer survival than the tumor-bearing animals treated with cediranib alone or with anti–CSF-1 (P = 0.0006 and P = 0.0013, respectively). The addition of anti–CSF-1 had no effect on overall median survival when combined with control, MEDI3617, or cediranib treatment as compared with each therapy alone. When combined with dual therapy, however, anti–CSF-1 significantly reduced overall median survival as compared with dual therapy alone (10 d vs. 17 d; *P = 0.003) and impaired the survival benefit of dual therapy over cediranib monotherapy (P = 0.4174). Dual therapy with and without combined anti–CSF-1 treatment led to significant increases in overall median survival compared with control treatment (P = 0.0035 and P < 0.0001, respectively). These data confirm the role of macrophages as essential mediators of the survival benefits observed with dual therapy.

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