Cryoablation Plus Immune Checkpoint Inhibitors Enhanced Dendritic Cell and T Cell Activation in TNBC Murine Model

Flavia Sardela de Miranda,1– 3 Rachel L Babcock,1– 4 Maria F Mahecha,1,2 Prrishti J Gukhool,1,2 Geetha P Boligala,3,4 Amanda K Garcia,5 Dalia Martinez-Marin,4 Ava G Oliver,5 Thomas Hintelmann,5 Kathryn L Furr,1,2 Chhanda Bose,6 Sharilyn Almodovar,5 Sharda P Singh,1,2,7 Kevin Pruitt,4 Michael W Melkus,1,2 Rakhshanda Layeequr Rahman1,2 1Department of Surgery, Texas Tech University Health Sciences Center, Lubbock, TX, 79430, USA; 2Breast Center of Excellence, Texas Tech University Health Sciences Center, Lubbock, TX, 79430, USA; 3Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX, 79430, USA; 4Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA; 5Department of Immunology and Molecular Microbiology, Texas Tech University Health Sciences Center, Lubbock, TX, 79430, USA; 6Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, Lubbock, TX, 79430, USA; 7Department of Internal Medicine, Tech University Health Sciences Center, Lubbock, TX, 79430, USACorrespondence: Michael W Melkus, Department of Surgery, TTUHSC, 3601 4th Street, Lubbock, TX, 79430-8312, USA, Email [email protected] Rakhshanda Layeequr Rahman, Breast Health Center, Creighton University-CHI Health Lakeside, 16901 Lakeside Hills Ct, Omaha, NE, 68130, USA, Email [email protected]: Cryoablation eradicates tumors through repeated freeze–thaw cycles and preserves tumor-associated antigens, triggering inflammatory signals capable of priming anti-tumor immunity, yet its therapeutic potential in triple-negative breast cancer (TNBC) remains largely unexplored. Immune checkpoint inhibitors (ICIs) have shown clinical benefit in TNBC but come with significant immune-related toxicities. Combining cryoablation with ICIs in TNBC may amplify the efficacy of cryoablation, which is significantly less toxic than ICIs, thereby providing opportunities for lowering the doses of ICIs in clinical practice. Here, we investigated the therapeutic impact of cryoablation with ICIs in an orthotopic bilateral murine TNBC model.Methods: Two weeks after tumor induction, primary tumors were cryoablated while the abscopal tumors were not manipulated and represented distant tumors. Twenty-four hours pre- and post-cryoablation, mice received an intra-peritoneal injection of PBS or ICIs (anti-CTLA-4, PD-1, or PD-L1). Tumors, tumor-draining lymph nodes (TdLNs), spleen, and peripheral blood were assessed for immune profiling a week later.Results: Preliminary analyses demonstrated that combining cryoablation with anti-CTLA-4 enhanced T cell activation systemically compared to cryoablation alone or in combination with PD-1/PD-L1 blockade. Notably, relative to cryoablation monotherapy, combination with anti-CTLA-4 increased the frequencies of activated CD4⁺ and CD8⁺ T cells in the abscopal tumors, while also inducing a higher frequency and activation of conventional dendritic cells in the abscopal TdLNs.Conclusion: These results suggest combination of cryoablation with anti-CTLA-4 therapy enhances systemic antitumor immunity by boosting antigen presentation. Our results support further investigation into this combination strategy to prevent tumor recurrence and metastasis while minimizing toxicity of treatment.Keywords: triple-negative breast cancer, cryoablation, immune checkpoint inhibitors, anti-tumor immune response, abscopal effect