Abstract PS3-13-13: Cryoablation Combined with Immune Checkpoint Inhibitors Enhances Conventional Dendritic Cell Activation in Abscopal Tumors and Tumor-Draining Lymph Nodes

Abstract Background: Immune checkpoint inhibitors (ICIs), such as anti-CTLA-4 which acts primarily during T cell priming and anti-PD-1 which targets exhausted T cells, have shown clinical efficacy in triple-negative breast cancer (TNBC) but are associated with immune-related adverse events. These limitations underscore the need for combination strategies that enhance therapeutic outcomes and reduce ICI dosing requirements. Cryoablation, a non-surgical procedure, “kills” the tumor through freeze-thaw cycles while preserving tumor-associated antigens (TAAs). This procedure induces tumor necrosis and release of damage-associated molecular patterns that trigger antitumor immune responses and may enhance the abscopal effect - systemic immune response against distant tumors. The immune activation is mediated by immune cells recruited to the ablation site and tumor-draining lymph nodes (TdLNs). Conventional dendritic cells (cDC) play a central role in antitumor immunity by capturing TAAs and presenting them to T cells. Among cDC subsets, cDC1 - including lymphoid resident (CD8α+) and migratory (CD103+) populations - specialize in cross-presenting antigens to CD8+ T cells, while cDC2 primarily present antigens to CD4+ T cells. We hypothesized that combining cryoablation with ICIs enhances antitumor immunity by promoting antigen presentation and T cell activation. Methods: In a murine TNBC cryoablation model, 4T1-12B cells were bilaterally injected into BALB/c mice. The left (primary) tumors were cryoablated at 2 weeks, while the right tumors (abscopal tumors) were left intact to represent distant metastatic tumors to examine the immune abscopal effect. PBS or 100 µg ICI (anti-CTLA-4, PD-1, or PD-L1) were administered by I.P. injection 24h pre- and post-cryoablation. Mice were sacrificed a week later and evaluated for early immune activation. The primary and abscopal tumors and TdLNs, peripheral blood, and spleen were collected, processed and analyzed for immune system activation by flow cytometry. Results: Preliminary analyses demonstrated that combining cryoablation with anti-CTLA-4 therapy enhanced CD4+ and CD8+ T cell activation in both the spleen and peripheral blood compared to cryoablation alone or in combination with PD-1/PD-L1 blockade. Notably, relative to cryoablation monotherapy, the addition of anti-CTLA-4 increased the frequency and activation of both cDC1 and cDC2 subsets in the abscopal TdLNs. Among cDC1 populations, the lymphoid resident subset was significantly elevated in the cryoablation plus anti-CTLA-4 group. Furthermore, abscopal tumors from this group showed a higher frequency of activated CD4+ and CD8+ T cells, as well as NKT cells. Conclusions: These results suggest combination of cryoablation with anti-CTLA-4 therapy enhances systemic antitumor immunity more effectively than cryoablation alone or with PD-1/PD-L1 inhibition. One possible explanation is that cryoablation destroys the entire tumor immune microenvironment, resulting in fewer effector T cells for PD-1/PD-L1 inhibitors to act upon, which limits efficacy in this context. In contrast, anti-CTLA-4 may act earlier in the immune response by promoting de novo T cell priming. We observed increased frequencies and activation of cDC1 and cDC2 in the TdLNs of abscopal tumors, which likely contributed to the elevated numbers of activated CD4+ and CD8+ T cells in the abscopal tumor microenvironment. Altogether, these findings highlight the potential of cryoablation combined with anti-CTLA-4 to enhance antitumor immunity. Our results support further investigation into this combination strategy to assess its long-term potential to prevent tumor recurrence and metastasis. Citation Format: F. Sardela de Miranda, R. L. Babcock, G. P. Boligala, M. F. Mahecha, P. J. Gukhool, A. K. Garcia, A. G. Oliver, C. Bose, S. Almodovar, K. Pruitt, S. P. Singh, M. W. Melkus, R. Layeequr Rahman. Cryoablation Combined with Immune Checkpoint Inhibitors Enhances Conventional Dendritic Cell Activation in Abscopal Tumors and Tumor-Draining Lymph Nodes abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-13-13.