Blockade of tumor necrosis factor alpha signaling in tumor-associated macrophages as a radiosensitizing strategy

Abstract

Most cancer patients receive radiotherapy during the course of their disease. Improvements in the therapeutic index have been based mainly on physical improvements in delivery, as radiosensitizer development to target tumor cells has yet to yield effective agents. Recent investigations have focused on the tumor stroma as a target for radiosensitization. Here, we report that depletion of tumor-associated macrophages (TAMvarphi) by systemic or local injection of the macrophage-depleting liposomal clodronate before radiotherapy can increase the antitumor effects of ionizing radiation (IR), either as a large single dose (20 Gy) or as a fractionated dose (2 Gy x 10). Coimplantation of tumor cells with bone marrow-derived macrophages (BMDMvarphi) increased tumor radioresistance. Studies using mice with germline deletions in tumor necrosis factor receptors 1 and 2 (TNFR1,2(-/-)) or TNFalpha (TNF(-/-)), or treatment of wild-type mice with a soluble TNF receptor fusion protein (Enbrel), revealed that radioresistance mediated by BMDMvarphi required intact TNFalpha signaling. Radiation exposure upregulated vascular endothelial growth factor (VEGF) in macrophages and VEGF-neutralizing antibodies enhanced the antitumor response to IR. Thus, the radioprotective effect of TNFalpha was mediated by TAM-produced VEGF. Our findings offer a mechanistic basis to target macrophage populations generally or TNFalpha-induced macrophage VEGF specifically as tractable strategies to improve the efficacy of radiotherapy.

Figure 1.. Depletion of macrophages enhances radiation.

(a). Systemic macrophage depletion with Lip-Clod (200μl i.p.) prior to inoculation of B16.SIY cells significantly (p=0.002) enhanced IR (20 Gy) compared with IR alone in WT (n=11–16/group). (b). Systemic depletion of macrophages after tumors were established enhanced IR (p=0.071) compared to IR alone (n=11–16/group). (c). Intratumoral macrophage depletion significantly (p=0.037) delayed tumor regrowth compared to IR alone (n=10–12/group). ♦ Lip-PBS; ◊ 20 Gy + Lip-PBS; ■ Lip-Clod; □ 20 Gy + Lip-Clod

Figure 2.. TNFα signaling in BMDMφ mediates tumor radioresistance.

(a). Base line radiation response to 20 Gy of B16.SIY tumors growing in WT (n=12/group). ♦B16; ◊ IR in B16 (b). Co-injection of WT BMDMφ with B16.SIY tumor cells significantly accelerates tumor regrowth following 20 Gy compared with baseline (a), (p=0.03, n=12/group). ♦WT-Mac + B16; ◊ IR in WT-Mac + B16 (c). Co-injection of TNF−/− BMDMφ with B16.SIYcells significantly decreased tumor regrowth following 20 Gy compared to WT (b), (p=0.03, n=12/group). ♦ TNF−/− Mac + B16; ◊ IR in TNF−/− Mac + B16

Figure 3.. TNFα signaling in BMDMφ mediates tumor radioresistance through autocrine/paracrine signaling.

(a). Growth of B16.SIY tumors in TNFR1,2−/− mice (n=15/group). ♦ B16; ◊ IR in B16 (b). Co-injection of WT BMDMφ with B16.SIY cells significantly accelerates tumor regrowth following 20 Gy (p=0.003, n=12/group). ♦ WT-Mac + B16; ◊ IR in WT-Mac + B16 (c). Co-injection of WT BMDMφ with B16.SIY cells significantly accelerates tumor regrowth following 20 Gy compared with co-injection of TNFR1,2−/− BMDMφ and B16.SIY cells (p=0.041). ♦ TNFR−/− Mac + B16; ◊ IR in TNFR−/− Mac + B16

Figure 4.. Radiation induction of VEGF in BMDMφ.

(a). A significant increase in VEGF was detected in WT BMDMφ compared with TNFR1,2−/− BMDMφ with 5 Gy (b). Luminex assay confirmation of VEGF induction by IR in WT BMDMφ. The mean of triplicates from one representative experiment is shown.

Figure 5.. Radiation induction of VEGF in TAMφ.

(a). Western blot analysis of VEGF expression of CD11b⁺F4/80⁺ TAMφ isolated from tumors grown in WT and TNFR1,2−/−. (b). VEGF levels were significantly elevated in CD11b⁺F4/80⁺ TAMφ from WT compared with TNFR1,2−/− (p=0.015, Luminex). (c) Increase neovasculature/angiogenesis in tumors grown in WT compared to TNFR1,2−/− post IR. Arrows indicate functional vessels containing red blood cells. (d). VGFR2⁺ staining shows a significant decrease in perfused vessels post IR in tumors grown in TNFR1,2−/− compared to WT control (p=0.0001) and WT treated with 20 Gy (p=0.002).

Figure 6.. Enbrel® enhances radiosensitivity by blocking the VEGF induction in TAMφ.

(a). Enbrel® + 20 Gy produced a significant decrease in B16.SIY regrowth compared to 20 Gy alone (p=0.022). ♦ PBS; ◊ 20 Gy + PBS; ■ Enbrel; □ 20 Gy + Enbrel (b). Western blot of VEGF expression by CD11b⁺F4/80⁺ TAMφ from WT treated with Enbrel® + 20 Gy. (c). Luminex showing inhibition of VEGF induction in CD11b⁺F4/80⁺ TAMφ isolated from tumors treated with Enbrel® + 20 Gy in WT. The mean of triplicates from one representative experiment is shown. (d). Representative tumor tissue sections showing a high density of intact neovasculature/angiogenesis in WT mice post IR and was reduced with IR + Enbrel® (upper panel); Blue VEGFR2⁺ microvessel density and red NG2⁺ pericyte coverage were compared between groups (lower panel). Enbrel® + 20 Gy inhibited VEGFR2⁺ microvessel repair/neovasculature with less pericyte coverage noted by NG2⁺ staining.