Abstract P4-06-11: Cryoablation of murine mammary tumors induce robust immune response
Authors: Klein JD , Aukers Z , Kennedy J , Ciocca RM , Sabol JL , Carp NZ , Wallon UM 
Published: May 2019

Abstract
Background: Breast cancer is traditionally not considered as a highly immunogenic disease.
Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with reported high genomic instability and high mutation rate, indicating the possible presence of neoantigens. Cryoablation, the destruction of cells by ultra-low temperatures, can release these neoantigens and induce a tumor specific immune response.
We hypothesized these neoantigens might be sufficient to trigger a robust immune response to prevent and/or reduce spread and relapse of TNBC.
In this pilot study we cryoablated orthotopical 4T1 tumors in immune competent Balb/c mice and compared the results to surgery to evaluate 1) possible induction of immune responses and 2) effects on metastases formation.
Methods: We used 4T1 mammary carcinoma cells to initiate tumor growth in the mammary fatpad. Tumors were treated by cryoablation, cryoablation followed by surgery (cryo-surgery), or surgery alone. Tumor growth was followed and allowed to reach 3-4mm in largest dimension. Animals were euthanized 7 days post-treatment and tissues were collected to assess cytokine levels and presence of dissociated 4T1 cells. Single-cell suspensions of tumor, tumor-draining lymph node [TDLN], and spleen were tested for secretion of mouse Th1/Th2 cytokines using a bead array and measured by flow cytometery. Possible metastatic spread was assessed by a clonogenic assay using cells from venous blood, lung, and brain. Cell suspensions were seeded in growth medium supplemented with the selection agent 6-thioguanine, allowing only resistant 4T1 cells to form colonies.
Results: Cryoablation transformed tumors into a gelatinous mass surrounded by a fibrotic capsule, as typically seen in the clinic. Frozen sections of tumors revealed a necrotic core and infiltrating lymphocytes in the microenvironment. These animals displayed robust increases of Th1 and Th2 cytokines in both spleen and TDLN compared to animals with cryo-surgery treatment. TDLN of animals with surgically excised tumors secreted only IL-2. Circulating tumor cells were found in animals prior to treatment, while no 4T1 colonies formed from cell suspensions of lung and brain tissue [N=8]. At end-point, the surgery alone group had more 4T1 foci formed from lung and brain [mean foci /animal = 6.25 and 0.75, respectively; N=6] than the other two groups. Two animals in this group progressed and were euthanized early due to numerous lung metastases. The cryoablated group had the lowest number of foci formed in the lung and brain [2.25 and 0 respectively; N=8], and all animals were healthy at the predetermined end-point. Mean foci formation in the cryo-surgery group [N=7] was in-between the two other groups and one animal was euthanized early due to metastatic burden 5 days after surgery.
Conclusion: Cryoablation of TNBC can induce stimulatory immune responses in vivo. These immune responses might explain why animals treated with cryoablation, though having circulating tumor cells at the time of treatment, exhibited fewer micro metastatic growths compared to surgery alone and the cryo-surgery combination. On-going experiments aim to identify long-term effects of cryoablation on the formation of metastatic foci and growth.
Citation Format: Klein JD, Aukers Z, Kennedy J, Ciocca RM, Sabol JL, Carp NZ, Wallon UM. Cryoablation of murine mammary tumors induce robust immune response [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-11.