Flávia Sardela De Miranda ¹ Dalia Martinez-Marin ² Rachel L Babcock ^1,2 Maribel Castro ¹ Geetha P Boligala ^2* Sonia Y Khan ^1,3* Kathryn L Furr ^1* Isabel Castro-Piedras ¹ Nicholas Wagner ¹ Dakota E Robison ^1* Karla Daniele ^1* Sharda P Singh ¹ Kevin Pruitt ^2* Michael W. Melkus ^1* Rakhshanda Layeequr Rahman ^1,4*

• ¹ Texas Tech University Health Sciences Center, Lubbock, United States
• ² University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
• ³ The University of Texas Rio Grande Valley, Edinburg, Texas, United States
• ⁴ MetroHealth Medical Center, Cleveland, Ohio, United States

Despite recent advances, triple-negative breast cancer (TNBC) patients remain at high risk for recurrence and metastasis, which creates the need for innovative therapeutic approaches to improve patient outcomes. Cryoablation is a promising, less invasive alternative to surgical resection, capable of inducing tumor necrosis via freeze/thaw cycles. Necrotic cell death results in increased inflammatory signals and release of preserved tumor antigens, which have the potential to boost the local and systemic anti-tumor immune response. Thus, compared to surgery, cryoablation enhances the activation of T cells leading to an improved abscopal effect, defined as the occurrence of a systemic response after local treatment. We previously showed with a bilateraltumor mouse model of TNBC that cryoablation of the primary tumor leads to increased infiltration of distant tumors by tumor infiltrating lymphocytes (TILs) and decreased rates of recurrence and metastasis. However, the early drivers of the cryoablation-generated abscopal effect are still unknown and knowledge of the mechanism could provide insight into improving the anti-tumor immune response through pharmacologic immune modulation in addition to cryoablation. Here we show that cryoablation of mouse mammary carcinomas results in smaller distant tumors that harbor increased frequencies of anti-tumor cells [such as natural killer (NK) cells], accompanied by a systemic increase in the frequency of migratory conventional type 1 dendritic cells (cDC1; CD103 + XCR1 + ), compared to resection. The changes in cell frequencies are mirrored by the immune gene signature of the distant tumors, with cryoablation inducing genes involved with NK cell activation and leukocyte-mediated toxicity, including IL11ra1 and Pfr1. These results better define the early mechanisms through which cryoablation improves tumor elimination, which is mediated by enhanced frequencies of anti-tumoral cells such as NK and cDC1s at the distant tumor and in the spleen of mice treated with cryoablation, respectively.