554 Differential immunotherapy responses in novel murine breast cancer cell lines

<h3>Background</h3> Breast Cancer (BC) accounts for over 2 million new cancer diagnoses per year and is the leading cause of cancer mortality among females.¹ BC has historically been considered poorly immunogenic with low immune checkpoint blockade (ICB) therapy responsiveness. Encouragingly, recent studies have demonstrated efficacy of ICB treatment in improving treatment outcomes when given in combination with chemotherapy.^{2 3} To further improve patient survival, it is critically important to develop novel murine models that reflect physiologically relevant genetic events leading to BC. Here, we demonstrate generation of two independent novel mammary cancer cell lines, and their aPD-1, aCTLA4 ICB responsiveness as mono- or combination therapy <i>in vivo</i>. <h3>Methods</h3> Two novel mammary BC cell lines MM001 and MM008 were isolated from spontaneously arising tumors in C57BL/6 MMTV-Cre⁺;p53^fl/+ mice. Cells were serially passaged for multiple rounds by orthotopic injections in mammary fat pads of C57BL/6 (WT) mice, leading to development of conditioned MM001i and MM008i cell lines. We next investigated aPD-1 and/or aCTLA4 therapy responsiveness in WT and severely immunocompromised (NSG) mice injected with MM001i or MM008i tumor cells. Control groups were treated with isotype control antibodies. Mice that demonstrated complete tumor regression on ICB treatment were rechallenged with the same cell line to evaluate the immune memory responses. <h3>Results</h3> Reproducibly growing tumors were obtained in WT and NSG mice. 50% of MM001i and MM008i tumors reached the endpoint (1000mm³) around days 18 and 21 respectively, in WT mice. Interestingly, despite originating from the same genetic background, MM001i and MM008i demonstrated distinct responses to treatment. MM001i tumors responded poorly to aPD-1, while CTLA-4 and combination treatment showed a significant reduction in tumor burden. Contrastingly, MM008i tumors demonstrated significant responsiveness and complete remission in all three treatment groups compared to isotype control. Memory rechallenge experiment in treated mice under remission demonstrated complete MM008i tumor regression, strongly suggesting memory responses against MM008i. Conversely, severely immunocompromised NSG mice injected with either MM001i or MM008i demonstrated no therapy efficacy, thus confirming the critical role of immune system in targeting MM001i and MM008i. <h3>Conclusions</h3> Two novel and distinct murine mammary cell lines have been established to model breast cancer <i>in vivo</i>. Given the unique response profile of each cell line to immunotherapy despite the common background, further investigation of the differences between the two could yield insights into the mechanisms and potential biomarkers underpinning treatment susceptibility and resistance to checkpoint blockade. <h3>References</h3> Wilkinson L, Gathani T. Understanding breast cancer as a global health concern. <i>Br. J. Radiol</i> 2022;<b>95</b>:20211033. Schmid P, Cortes J, Pusztai L, McArthur H, Kümmel S, Bergh J, <i>et al</i>. Pembrolizumab for early triple-negative breast cancer. <i>N Engl J Med</i> 2020;<b>382</b>:810–821. Cortes J, Rugo H, Cescon D, Seock-Ah I, Yusof M, Gallardo C, <i>et al</i>. Pembrolizumab plus chemotherapy in advanced triple-negative breast cancer. <i>N Engl J Med</i> 2022;<b>387</b>:217–226. <h3>Ethics Approval</h3> All animal studies were approved by the University of Texas Health San Antonio IUCUC, protocol #20220056AR.