Endothelial membrane remodeling is obligate for anti-angiogenic radiosensitization during tumor radiosurgery

Abstract

Background: While there is significant interest in combining anti-angiogenesis therapy with conventional anti-cancer treatment, clinical trials have as of yet yielded limited therapeutic gain, mainly because mechanisms of anti-angiogenic therapy remain to a large extent unknown. Currently, anti-angiogenic tumor therapy is conceptualized to either "normalize" dysfunctional tumor vasculature, or to prevent recruitment of circulating endothelial precursors into the tumor. An alternative biology, restricted to delivery of anti-angiogenics immediately prior to single dose radiotherapy (radiosurgery), is provided in the present study.

Methodology/principal findings: Genetic data indicate an acute wave of ceramide-mediated endothelial apoptosis, initiated by acid sphingomyelinase (ASMase), regulates tumor stem cell response to single dose radiotherapy, obligatory for tumor cure. Here we show VEGF prevented radiation-induced ASMase activation in cultured endothelium, occurring within minutes after radiation exposure, consequently repressing apoptosis, an event reversible with exogenous C(16)-ceramide. Anti-VEGFR2 acts conversely, enhancing ceramide generation and apoptosis. In vivo, MCA/129 fibrosarcoma tumors were implanted in asmase(+/+) mice or asmase(-/-) littermates and irradiated in the presence or absence of anti-VEGFR2 DC101 or anti-VEGF G6-31 antibodies. These anti-angiogenic agents, only if delivered immediately prior to single dose radiotherapy, de-repressed radiation-induced ASMase activation, synergistically increasing the endothelial apoptotic component of tumor response and tumor cure. Anti-angiogenic radiosensitization was abrogated in tumors implanted in asmase(-/-) mice that provide apoptosis-resistant vasculature, or in wild-type littermates pre-treated with anti-ceramide antibody, indicating that ceramide is necessary for this effect.

Conclusions/significance: These studies show that angiogenic factors fail to suppress apoptosis if ceramide remains elevated while anti-angiogenic therapies fail without ceramide elevation, defining a ceramide rheostat that determines outcome of single dose radiotherapy. Understanding the temporal sequencing of anti-angiogenic drugs and radiation enables optimized radiosensitization and design of innovative radiosurgery clinical trials.

Figure 1. VEGF inhibits radiation-induced apoptosis via repression of ASMase activation.

(A) ASMase activity following 10 Gy is inhibited by VEGF pre-treatment. Irradiated BAEC samples were collected at the indicated times and ASMase activity measured by quantifying conversion of [¹⁴C]sphingomyelin to the product [¹⁴C]phosphocholine. Data (mean±s.d.) represent duplicate determinations from 2 experiments. (B) Radiation-induced ceramide generation is inhibited by VEGF pre-incubation. VEGF (1 ng/ml) was added 15 min before irradiation. Ceramide was quantified at the indicated times by the diacylglycerol kinase assay. Data (mean±s.d.) represent triplicate determinations from 2 experiments. (C, D) VEGF pre-treatment inhibits radiation-induced apoptosis. C₁₆-ceramide (C₁₆, 1 µM) was added 30 min prior to irradiation. Samples were fixed in 10% paraformaldehyde prior to bis-benzimide staining. For apoptosis quantification, data (mean±s.d) represent duplicate determinations of at least 400 bis-benzimide stained nuclei counted from 2 experiments. Caspase 3 activity was measured by quantitation of the luminescence of cleaved DEVD-AMC substrate. Data (mean±s.d.) represent duplicate points from 3 experiments.

Figure 2. VEGF regulates a pro-apoptotic ceramide rheostat via ASMase repression.

(A) ASMase activation in response to DC101. A19 BAEC were incubated with 5 µg/ml DC101 and ASMase activity was measured every 2 h for 24 h by quantifying conversion of [¹⁴C]sphingomyelin to the product [¹⁴C]phosphocholine as in Fig. 1A . Data (mean±s.d.) represent triplicate determinations from 2 experiments. (B) DC101 (5 µg/ml) induces ceramide elevation. Ceramide was quantified by the diacylglycerol kinase assay as in Fig. 1B at the times indicated. Data (mean±s.d.) represent triplicate determinations from 2 experiments. (C) DC101 induces apoptosis. DC101(5 µg/ml) was added and cells collected every 2 h, fixed and apoptosis quantified using bis-benzimide method as in Fig. 1C . (D) Disruption of GEMs inhibits DC101-induced apoptosis. Nystatin (30 µg/ml) was added 30 min prior to the addition of escalating doses of DC101 and apoptosis quantified after 24 h as in Fig. 1C . (E) Anti-ceramide antibody MAS0020 inhibits apoptosis induction by DC101. MAS0020 was added at 250 ng/ml 30 min prior to the addition of increasing concentrations of DC101. After 24 h, apoptosis was quantified as in Fig. 1C . (F) bFGF inhibits DC101-induced apoptosis by inhibition of ceramide generation. bFGF (1 ng/ml) and C₁₆-ceramide (1 µM) were added as detailed in Fig. 1C , and apoptosis was quantified after 24 h.

Figure 3. Anti-angiogenic treatment (DC101) radiosensitizes via ASMase activation.

(A) DC101-pre-treatment increases radiation-induced ceramide elevation. Cells were pre-treated for 16 h with 0.2 µg/ml DC101, irradiated at 8 Gy, and ceramide was measured by diacylglycerol kinase assay as in Fig. 1B . Data (mean±s.d.) represent triplicate determinations from 2 experiments. (B) DC101 increases radiation-induced apoptosis. Cells were pre-incubated for 16 h with 0.2 µg/ml DC101. At 8 h post-irradiation, apoptosis was quantified using bis-benzimide staining. (C) Anti-ceramide antibody inhibits DC101-enhanced radiation-induced apoptosis. BAEC were treated with 350 ng/ml MAS0020 15 min prior to 0.2 µg/ml DC101, and after 16 h irradiated at 5 Gy. Apoptosis was quantified 8 h post-irradiation by bis-benzimide staining. Data in (2C–F, 3A, and 3C) represent means±s.d. of duplicate determinations from at least 400 scored nuclei from 2 separate experiments.

Figure 4. ASMase is required for anti-VEGF (G6–31) mAb radiosensitization in vivo.

(A and B) Impact of 1 h pre-treatment with G6–31 (5 mg/kg) on tumor response to 14.5 Gy. N equals number of animals per group. (C and D) Impact of a 1 h pre-treatment with G6–31 (5 mg/kg) on tumor response to 13.5 Gy using tumor xenografts grown in asmase^+/+ (left panel) or asmase^−/− littermates (right panel). No tumor growth delay was observed in tumors growing in ASMase deficient mice after 13.5 Gy pre-treated with G6–31 as compared to tumors growing in the asmase^+/+ littermates. Data (mean±s.e.m.) are collated from 5 animals per group. Arrows indicate the day of treatment. (E) Impact of anti-ceramide treatment on G6–31-enhanced radiation-response. G6–31 (5 mg/kg) was injected i.v. 1 h before 14.5 Gy irradiation. Anti-ceramide Ab MAS0020 (25 µg) was injected i.v. immediately before irradiation. Anti-ceramide treated mice displayed a significantly reduced tumor growth delay. Data (means ± s.e.m.) were collated from 4 mice per group.

Figure 5. Role of ASMase in anti-angiogenic therapy.

(A) Impact of a 1 h pre-treatment with DC101 (1600 µg/animal) on the tumor response to 13.5 Gy using MCA/129 fibrosarcomas grown in asmase^+/+ or (B) in asmase^−/− littermates. Data (mean±s.e.m.) are collated from asmase^+/+ control mice (n = 7), DC101-treated asmase^+/+ mice (n = 9), and asmase^−/− mice (n = 10 each). (C) Quantification of the G6–31 effect on radiation-induced endothelial cell apoptosis. Endothelial cells (mean±s.e.m.) from cross sections of 14.5 Gy-irradiated MCA/129 fibrosarcomas were stained with both an endothelial cell specific Ab (MECA-32) and TUNEL. Data was collated from 20 fields from 1 of 2 similar experiments employing 2 animals per group at the times shown. (D) Quantification of the effect of DC101 on 13.5 Gy radiation-induced endothelial cell apoptosis. Data (mean±s.e.m.) represent TUNEL-positive endothelial cells collated from 20 fields from 1 of 2 similar experiments employing 2 animals per group.

Figure 6. Impact of timing of DC101 addition on the radiation response of MCA/129-fibrosarcomas.

DC101 (1600 µg/kg) was injected i.v. at the indicated times before (A) and after (B) irradiation. Data (means ±s.e.m.) were collated from 5 mice per group. Arrows indicate time of irradiation.