Potent induction of tumor immunity by combining tumor cryoablation with anti-CTLA-4 therapy

Abstract

Thermal ablation to destroy tumor tissue may help activate tumor-specific T cells by elevating the presentation of tumor antigens to the immune system. However, the antitumor activity of these T cells may be restrained by their expression of the inhibitory T-cell coreceptor CTLA-4, the target of the recently U.S. Food and Drug Administration-approved antibody drug ipilumimab. By relieving this restraint, CTLA-4-blocking antibodies such as ipilumimab can promote tumor rejection, but the full scope of their most suitable applications has yet to be fully determined. In this study, we offer a preclinical proof-of-concept in the TRAMP C2 mouse model of prostate cancer that CTLA-4 blockade cooperates with cryoablation of a primary tumor to prevent the outgrowth of secondary tumors seeded by challenge at a distant site. Although growth of secondary tumors was unaffected by cryoablation alone, the combination treatment was sufficient to slow growth or trigger rejection. In addition, secondary tumors were highly infiltrated by CD4(+) T cells and CD8(+) T cells, and there was a significant increase in the ratio of intratumoral T effector cells to CD4(+)FoxP3(+) T regulatory cells, compared with monotherapy. These findings documented for the first time an effect of this immunotherapeutic intervention on the intratumoral accumulation and systemic expansion of CD8(+) T cells specific for the TRAMP C2-specific antigen SPAS-1. Although cryoablation is currently used to treat a targeted tumor nodule, our results suggest that combination therapy with CTLA-4 blockade will augment antitumor immunity and rejection of tumor metastases in this setting.

Figure 1. Cryoablation and anti-CTLA-4 combination therapy synergize to mediate rejection of a second TRAMP C2 tumor challenge

A. Scheme to study the immune response to cryoablation and anti-CTLA-4 combination therapy. C57BL/6 male mice were challenged with TRAMP C2 prostate tumor cells on the left flank. Cryoablation was administered when the tumors reached about 5-8 mm in diameter. Mice were challenged the following day after cryoablation with a second injection of TRAMP C2 on the right flank and some of the mice were also treated with CTLA-4 blocking antibodies (clone 9H10) or hamster IgG isotype control antibodies. Calipers were used to monitor the growth of the second tumor. T cell infiltrates into the second tumor were analyzed on day 15. B. Mean and individual tumor growth. Error bars represent standard error of the mean. C. Tumor free survival. Data shown is representative of at least 3 independent experiments. Tumor free survival was plotted as a Kaplan-Meier curve and the log-rank test was used for statistical analysis. Number of mice that rejected the right flank tumor challenge: No Treatment: 0/5; Anti-CTLA-4 only 0/10; Cryoablation+IgG: 0/5; Cryo+Anti-CTLA-4: 4/9. One mouse from the Cryo+Anti-CTLA-4 group was omitted from the analysis due to partial ablation of the primary tumor.

Figure 2. CD4+ T cells and CD8+ T cells infiltrate the secondary tumors of combination treated mice

A. Immunofluorescence, B. Representative plots of flow cytometric analysis and C. Quantification of T cells in secondary tumors from untreated, anti-CTLA-4, cryoablation+IgG and cryoablation+anti-CTLA-4 combination treated mice. Right flank tumors were pooled from 6 mice for each experimental group. C. Shows pooled data from 3 independent experiments. Statistical significance was calculated by one-way ANOVA using the Bonferroni post test with the following notations for P value: * P = 0.01-0.05, and *** P < 0.001. Error bars represent standard error of the mean.

Figure 3. Depletion of CD8+ T cells diminishes the therapeutic effect

Kaplan-Meier curve showing tumor free survival of mice with no treatment and cryoablation/anti-CTLA-4 combination treated mice with and without depletion of CD8+ T cells. Data were pooled from 3 independent experiments. The log-rank test was used for statistical analysis.

Figure 4. Secondary tumors from combination treated mice are infiltrated by TRAMP-C2 specific CD8+ T cells

A. Flow cytometry of tumors after staining with SPAS-1 peptide-loaded MHC class I tetramers. Plots shown are gated on CD3+CD8+ cells. B. Quantification of tetramer positive cells in tumors. Tumors were harvested 15 days after injection of the right flank tumor. Right flank tumors were pooled from 6 mice for each experimental group and data were pooled from 3 independent experiments. Experimental data were analyzed by one-way ANOVA using the Bonferroni post test with the following notations for P value: * P = 0.01-0.05, ** P = 0.001-0.01, and *** P < 0.001. Error bars represent standard error of the mean.

Figure 5. Cryoablation+Anti-CTLA-4 combination treatment induces SPAS-1 specific CD8+ T cells to accumulate in spleens

A. Percentage of CD8+ T cells that are tetramer+ in spleens. Representative plots (gated on CD8+ cells) are shown. B. Percentage of CD8+tetramer+ T cells in the spleen. Data were pooled from 3 independent experiments. C. Detection of IFNγ secretion by splenocytes in response to SPAS-1 peptide (SNC9_H8) by ELISPOT. Data were pooled from 3 independent experiments. Experimental data were analyzed by one-way ANOVA using the Bonferroni post test with the following notations for P value: * P = 0.01-0.05 and *** P < 0.001. Error bars represent standard error of the mean.

Figure 6. Combination treated tumors have an increased Teff to Treg ratio compared to controls

A. Representative flow cytometry plots showing percentage (gated on CD4+ cells) of CD4+FoxP3+ T cells in pooled tumors. B. Number of FoxP3+ Cells per mg of tumor. C. Effector to Treg ratio in pooled tumors. Right flank tumors were pooled from 6 mice for each experimental group and data were pooled from 3 independent experiments. Experimental data were analyzed by one-way ANOVA using the Bonferroni post test with the following notations for P value: * P = 0.01-0.05, ** P = 0.001-0.01, and *** P < 0.001. Error bars represent standard error of the mean.