Therapeutic effects of ablative radiation on local tumor require CD8+ T cells: changing strategies for cancer treatment

Abstract

Patients with locally advanced cancer or distant metastasis frequently receive prolonged treatment with chemotherapy and/or fractionated radiotherapy (RT). Despite the initial clinical response, treatment resistance frequently develops and cure in these patients is uncommon. Developments in RT technology allow for the use of high-dose (or ablative) RT to target local tumors, with limited damage to the surrounding normal tissue. We report that reduction of tumor burden after ablative RT depends largely on T-cell responses. Ablative RT dramatically increases T-cell priming in draining lymphoid tissues, leading to reduction/eradication of the primary tumor or distant metastasis in a CD8(+) T cell-dependent fashion. We further demonstrate that ablative RT-initiated immune responses and tumor reduction are abrogated by conventional fractionated RT or adjuvant chemotherapy but greatly amplified by local immunotherapy. Our study challenges the rationale for current RT/chemotherapy strategies and highlights the importance of immune activation in preventing tumor relapse. Our findings emphasize the need for new strategies that not only reduce tumor burden but also enhance the role of antitumor immunity.

Figure 1

Immunodeficiency abrogates the antitumor effect of RT. (A) WT C57BL/6 or nude mice (n = 10) were injected with 2 × 10⁶ B16 melanoma cells and treated 7 days later with 20 Gy. The radiation group in WT but not in nude mice showed significantly smaller tumor size (**P = .002 at day 10 after RT). (B) WT or nude mice (n = 8-12) were injected with 2 × 10⁵ B16-SIY and treated 10 days later with 25 Gy. The radiation group showed significantly smaller tumor size (***P < .001 on day 12 after RT). A similar trend of the inhibition was also detected with single 20 Gy. A total of 60% WT mice were cured, whereas 100% nude mice die with 20 Gy. (C) Tumor growth curve and (D) survival for WT mice injected with 10⁵ B16 and treated on day 14 with 15 Gy given on days 0, 1, and 2 after RT. A total of 200 μg/mouse anti-CD8 antibody was administered on days 0, 4, and 8 after RT (n = 5-9 per group). After RT plus depletion of CD8, the size of tumor increased significantly from RT alone (**P = .007 at day 14). Survival increased after RT (***P < .001), but with CD8 depletion survival was significantly reduced: *P < .05; **P < .01; ***P < .001. Similar experiments were repeated 3 times (A-D).

Figure 2

RT promotes priming of Ag-specific cells. (A) A total of 5 × 10⁵ B16-SIY tumor cells were subcutaneously injected into the lower back of C57BL/6 (n = 8-9 per group). Fourteen days after tumor challenge, mice received localized RT (20 Gy) on the tumors and were transferred intravenously with CFSE-labeled naive 2C cells. Four to 5 days after adoptive transfer, mice were killed for analysis of DLN and spleen. The degree of CFSE dilution via FACS was determined by gating on the 1B2⁺CD8⁺ lymphocyte population. The RT group has more proliferative T cells than the no RT group (***P < .001). (B) A total of 5 × 10⁵ B16-SIY tumor cells were subcutaneously injected into the lower back of C57BL/6 (n = 5 or 6 per group). Fourteen days after tumor challenge, mice received local RT (20 Gy) on the tumors and were killed 5 days later for tetramer⁺ cell analysis. DLN and spleen were harvested, collagenase digested, and then stained for FACS. Cells were gated on CD11c⁺ cells. Similar experiments were repeated twice. The RT group has more positive cells than the no-RT group (***P < .001). (C) A total of 2 × 10⁵ B16 tumor cells were subcutaneously injected into the lower back of C57BL/6 mice (n = 4-6 per group). Fourteen days after tumor challenge, mice received localized RT (20 Gy) on the tumors and were analyzed 48 hours later. DLN was isolated, collagenase-digested (1.5 mg/mL), and then stained for FACS. Cells were gated on CD11c⁺ cells. Mean ± SD for the no-RT group was 6.8 ± 4, and for the RT group 14.6 ± 2. Similar experiments were repeated at least twice.

Figure 3

Chemotherapy diminishes the effect of radiation-mediated eradication of metastases and T-cell priming. (A) A total of 2 × 10⁵ B16-CCR7 cells were subcutaneously injected; and on days 14, 15, and 16, mice received 15 Gy. On days 7 and 14 after RT, 200 mg/kg dacarbazine (also for human melanoma) was administered intraperitoneally. The radiation group showed a significantly smaller tumor size (***P < .001 at day 13 after RT). Additional dacarbazine after RT led to significant regrowth (**P < .007 at day 26 after RT, *P = .015 day 32 after RT; n = 3-5). (B) Tumor growth curve: 10⁵ 4T1 tumor cells were injected; and on days 15, 16, and 17, mice received 15 Gy. On days 7 and 14 after RT, 20 mg/kg paclitaxel was administered intraperitoneally. The radiation group showed significantly smaller tumor size (**P = .008 at day 23; n = 4-9 per group). (C) Metastasis assay: 10⁵ 4T1 tumor cells were subcutaneously injected; and on days 12, 13, and 14, Balb/c mice received local RT of 15 Gy. The tumors were removed on day 21. On days 7 and 12 after RT, 20 mg/kg paclitaxel was administered intraperitoneally No colonies were detected after radiation, whereas addition of chemotherapy completely eliminated the effect of radiation (n = 4 or 5 per group). (D) A total of 5 × 10⁵ B16-SIY melanoma cells were injected subcutaneously. On day 17, mice were transferred with 2 × 10⁶ CFSE-labeled 2C cells and locally RT with 20 Gy. A total of 200 mg/kg dacarbazine intraperitoneally was given 2 days after adoptive transfer. DLN and spleen were harvested on day 21 for analysis. (E) A total of 5 × 10⁵ B16-SIY melanoma cells were injected subcutaneously. Mice received local tumor RT of 20 Gy once or 5 Gy × 4. Single-treatment 200 μg/mouse of anti-CD8 antibody was administered on days 0, 4, 8, and 12 after RT. Repeated treatment of radiation showed significant regrowth of tumor mass (*P = .03 at day 25; n = 4-6). (F) A total of 8 × 10⁶ human lung tumor A549 cells were subcutaneously injected into B6/Rag^−/− mice; and 4 weeks later, the mice were adoptively transferred with 2 × 10⁶ LN cells from OT-I transgenic mice. Three days later, mice received 20 Gy of local RT. RT (P = .48) or T cells (P = .3) alone showed no significant differences from the no treatment group, whereas the radiation + T-cell group showed significantly smaller tumor size (*P = .018 at day 60). Similar experiments were repeated at least twice (A-F).

Figure 4

The synergy of RT plus ad-LIGHT immunotherapy eradicates distant metastases. 4T1 tumor: Balb/c mice were subcutaneously injected with 10⁵ cells on the lower back. Mice received local RT (12 Gy) on days 14 and 15 and intratumoral injection with ad-control (2 × 10¹⁰ virus particles [vp]) or ad-LIGHT (2 × 10¹⁰ vp) on days 15 and 16 (n = 24-41 pooled from 5 experiments). B16-CCR7 tumor: C57BL/6 mice were subcutaneously injected with 10⁵ cells on the lower back. Mice received local RT (12 Gy) on days 14 and 15 and intratumoral injection with ad-control (2 × 10¹⁰ vp) or ad-LIGHT (2 × 10¹⁰ vp) on days 15, 16, and 17 (n = 6-9 pooled from 2 experiments). On day 25 after tumor injection, tumors were surgically removed. Mice were killed on day 35 for tumor colonogenic assay (n = 4 or 5 per group). No colonies were detected in combination group in both types of tumor. Similar experiments were repeated 3 times.