FAK regulates platelet extravasation and tumor growth after antiangiogenic therapy withdrawal

Abstract

Recent studies in patients with ovarian cancer suggest that tumor growth may be accelerated following cessation of antiangiogenesis therapy; however, the underlying mechanisms are not well understood. In this study, we aimed to compare the effects of therapy withdrawal to those of continuous treatment with various antiangiogenic agents. Cessation of therapy with pazopanib, bevacizumab, and the human and murine anti-VEGF antibody B20 was associated with substantial tumor growth in mouse models of ovarian cancer. Increased tumor growth was accompanied by tumor hypoxia, increased tumor angiogenesis, and vascular leakage. Moreover, we found hypoxia-induced ADP production and platelet infiltration into tumors after withdrawal of antiangiogenic therapy, and lowering platelet counts markedly inhibited tumor rebound after withdrawal of antiangiogenic therapy. Focal adhesion kinase (FAK) in platelets regulated their migration into the tumor microenvironment, and FAK-deficient platelets completely prevented the rebound tumor growth. Additionally, combined therapy with a FAK inhibitor and the antiangiogenic agents pazopanib and bevacizumab reduced tumor growth and inhibited negative effects following withdrawal of antiangiogenic therapy. In summary, these results suggest that FAK may be a unique target in situations in which antiangiogenic agents are withdrawn, and dual targeting of FAK and VEGF could have therapeutic implications for ovarian cancer management.

Figure 1. Tumor rebound after cessation of antiangiogenic therapy.

(A) Therapeutic schema for withdrawal and continuous treatment with antiangiogenic therapy. (B) Mean aggregate tumor weight and (C) number of nodules of tumors induced by i.p. injection of SKOV3ip1 human ovarian cancer cells into nude mice with various antiangiogenic treatment schedules. (D) Mean aggregate tumor weight of 2774 ovarian tumors after bevacizumab withdrawal or continuous treatment. (E) Visualization of tumor hypoxia and (F) vascular permeability after i.v. injection of hypoxyprobe and dextran, respectively. Scale bar: 100 μm. (G and H) The quantification of hypoxyprobe staining and the number of FITC⁺ dextran pixels after indicated treatments. (I–K) Vessel number and pericyte coverage after antiangiogenic therapy withdrawal and continuous treatment. (I) Representative immunofluorescence staining and (J and K) bar graphs. HPF, high-powered field. Scale bar: 100 μm. (B–D) n = 8–10 mice per group. (E, F, and I) Representative images from at least 5 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 (1-way ANOVA followed by a Tukey’s multiple comparison post-hoc test in B–D, G, H, J, and K). Averaged data are presented as the mean ± SEM. Paz, pazopanib; Bev, bevacizumab.

Figure 2. Antiangiogenic treatment, hypoxia, and platelet effect on cancer cells.

(A) The number of extravasated platelets in 2774 ovarian cancer cell–induced tumors in control mice and mice exposed to bevacizumab withdrawal or long-term treatment. (B) Representative immunofluorescence staining of tumors resected from control mice and mice exposed to bevacizumab withdrawal and long-term bevacizumab treatment. CD31 (red) is an endothelial marker, and GPIbβ (green) is a platelet marker. Scale bar: 100 μm. (C) ADP concentrations (μM) in supernatants of SKOV3ip1 cells grown in hypoxia (1% oxygen) or normoxia (21% oxygen) for 24, 48, and 72 hours (n = 3). (D) ADP levels (μM) in SKOV3ip1 tumors resected from mice exposed to long-term bevacizumab or withdrawal of bevacizumab. (E) Increase in proliferation of different ovarian cancer cell lines after exposure to platelets for 72 hours, as measured by EdU incorporation (n = 3). (F) Proliferation of HeyA8 ovarian cancer cells after coincubation with normal or 1% paraformaldehyde-fixed platelets (n = 3). (A and B) Quantification and representative images from tumors of at least 5 mice per group. (D) Quantification of ADP levels in tumors of 2 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 (1-way ANOVA followed by a Tukey’s multiple comparison post-hoc test in A, D, and F; 2-tailed Student’s t test in E). Averaged data are presented as the mean ± SEM.

Figure 3. Platelets are important mediators of tumor rebound growth after withdrawal of antiangiogenic therapy.

(A) Mean aggregate tumor weight of A2780 cell–induced tumors in mice receiving platelet transfusions or APA twice weekly. (B and C) Representative immunohistochemical staining for (B) Ki67 and (C) caspase-3 and their quantification in control tumors (black bars) and tumors transfused with platelets (gray bars) twice weekly. Scale bar: 50 μm. (D and E) Mean aggregate tumor weight in (D) A2780 cell– or (E) HeyA8 cell–induced i.p. tumors for each indicated treatment group. (A, D, and E) n = 8–10 mice per group. (B and C) Representative images from tumors of at least 5 mice per group. **P < 0.01, ***P < 0.001 (1-way ANOVA followed by a Tukey’s multiple comparison post-hoc test in A, D, and E; 2-tailed Student’s t test in B and C). Averaged data are presented as the mean ± SEM.

Figure 4. Role of platelet FAK on tumor growth.

(A) Mean aggregate tumor weight and (B) representative images of necropsy in WT or platelet-specific FAK-deficient mice that carried tumors induced by i.p. injection of ID8-VEGF murine ovarian cancer cells and were exposed to long-term treatment or withdrawal of anti-VEGF antibody (B20). (C) Quantification of extravasated platelets in ID8-VEGF tumors in WT or platelet-specific FAK-deficient mice. (D) Mean aggregate tumor weight and (E) number of tumor nodules of orthotopic SKOV3ip1 tumors after treatment with pazopanib, the FAK inhibitor GSK2256098 (FAKi), or a combination of the two. (F) Mean aggregate weight of orthotopic SKOV3ip1 tumors after withdrawal or long-term antiangiogenic therapy alone or in combination with FAK inhibitor GSK2256098. (A and D–F) n = 8–10 mice per group. (B and C) Quantification and representative images of tumors from at least 5 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 (1-way ANOVA followed by a Tukey’s multiple comparison post-hoc test in A and C–F). Averaged data are presented as the mean ± SEM.

Figure 5. Proposed model of the interactions among antiangiogenic therapy, platelets, and FAK and their effect on tumor growth.

Withdrawal of antiangiogenic therapy leads to tumor hypoxia, ADP production, increased angiogenesis, and platelet infiltration, thereby increasing tumor growth. On the other hand, continuous antiangiogenic therapy and/or FAK inhibitors might be a potential regimen to prevent tumor rebound after cessation of antiangiogenic therapy.