Effect of Radiofrequency Ablation and Checkpoint Inhibition on Immune Response and Tumor-Specific Cytotoxicity in a Pre-Clinical Mouse Melanoma Model

Objective: In our previous in vivo work, mice treated with radiofrequency ablation (RFA) in combination with anti-CTLA-4 and PD1 checkpoint inhibitors (CPI) for treatment of B16-ovalbumin (OVA) melanoma tumors showed a decrease in primary tumor volume (p<0.001). Comparison of RFA+CPI to RFA only-treated mice suggested improved survival rates [93% (14/15) and 67% (10/15), respectively], but the difference was not statistically significant. The purpose of this study was to evaluate, in vitro, the effect of CPI and RFA on the systemic immune response.

Methods: All animal procedures were approved by the Animal Care and Use Committee. The following female c57BL/6J mice cohorts were evaluated: a non-tumor placebo group (n=5); primary tumor, T1 (n=5); secondary tumor, T2 (n=5); T1+T2 (n=15); RFA alone T1+RFA+T2 (n=15); CPI alone T1+CPI+T2 (n=15); T1+CPI+RFA+CPI+T2 (n=15). T1 was inoculated with B16-OVA cells, 14 days before RFA. On day 0, RFA was performed on T1. To assess therapeutic outcomes on a distant tumor, T2 was inoculated with B16-OVA cells on day 2. CPI groups received three doses of anti-CTLA-4 and PD-1 either 4 days pre-RFA (CPI alone), or 4 days pre-RFA and the day after RFA (RFA+CPI). Serum samples were collected on days 0, 2, 4, and 14 to measure cytokine concentration by electrochemiluminescence. On day 14, mice were euthanized and splenocytes were collected and cryopreserved. IFNg production was detected by ELISpot from splenocytes co-cultured with B16-OVA cells. Cell mediated cytotoxicity and viability of B16-OVA and B16-F10 cells co-cultured with splenocytes were evaluated by lactate dehydrogenase (LDH) release and Trypan Blue exclusion assays, respectively. LDH release was calculated using the formula: % cytotoxicity=(Experimental release/Target maximum release) x 100.

Results: Based on the cytokine profile in serum, mice treated with RFA (RFA+CPI or RFA alone) showed a strong inflammatory response 6h post-RFA. While RFA-treated mice showed increased production of IL-6 and IL-1b, only mice treated with CPI+RFA generated an early regulatory response to control inflammation (increased IL-4, IL-5 and IL-10) on day 2. This was the only group that showed sustained and balanced Th1 (IFNg and IL-2) and Th2 (IL-4, IL-5 and IL-10) responses with a low inflammatory profile (IL-6, IL-1b and TNFα) on day 14. ELISpot assays revealed that splenocytes collected from the RFA+CPI cohort co-cultured with B16-OVA cells produced more IFNg compared to RFA alone (2-fold, p<0.0005). Co-culture of B16-OVA and B16-F10 tumor cells with splenocytes collected from the RFA+CPI cohort showed higher cytotoxicity (46%, 68%, respectively, p<0.0001) and trended towards lower viability (1%, 12%, respectively) compared to RFA, which expressed <2% cytotoxicity in both cohorts and >50% viability in both cohorts.

Conclusions: These results suggest that RFA in combination with CPI presented a more sustained and balanced production of pro and anti-inflammatory cytokines that are involved in T cell activation and regulation of inflammatory responses. Cytotoxicity assays suggest that RFA in combination with CPI may induce enhanced T cell responses capable of conferring tumor-specific antigen recognition and cross protection against non-OVA B16 melanoma cells in vivo. This mechanistic characterization of immunomodulation after the combination of RFA and CPI may explain the more specific and effective anti-tumoral immune responses reported in clinical trials of CPI with ablation for hepatocellular carcinoma.