Abstract 4269: Defining the impact of autophagy blockade on triple negative breast cancer and remodeling of the tumor-immune microenvironment in primary and lung metastatic settings

Abstract Background: Triple negative breast cancer (TNBC) cells have high basal rates of autophagy to meet metabolic demands. Our emerging in vitro data demonstrates that ULK3 is an important initiator of the autophagy program and is especially important in enhancing TNBC cell survival in response to stressors such as chemotherapy. Inhibiting autophagy can lead to TNBC cell death and potentially the subsequent induction of immunogenic cell death pathways. Here, we interrogate the effect of ULK3 ablation on TNBC migration, invasion and metastasis and whether there is an impact on the surrounding immune landscape. Methodology: We investigated the impact of autophagy impairment on TNBC cells viability and migration by using CRISPR-Cas9 ULK3 knockout in MDA-MB-231Luc and 4T1Luc cells, halting downstream autophagic machinery. Cell proliferation, migration, and ATP metabolism were monitored using advanced imaging tools such as Incucyte and Opera Phenix. The effects of ULK3 ablation on the metastatic process were examined in vivo by injecting parental 4T1Luc and ULK3 knockout (KO) cells into syngeneic BALB/c mice (500, 000 cells/mouse, n=10/group) and tracing metastasis to various organs (liver, bones) using bioluminescent imaging (BLI). Tissues were subsequently analyzed using 10x Genomics HD Visium Spatial Transcriptomics. Additionally, multiplex immunofluorescent analysis was performed on TNBC patient’s tissue microarrays. Results: Our in vitro data demonstrate that CRISPR-Cas9 ULK3 KO cells (MDA-MB-231Luc, 4T1Luc) exhibit reduced cell proliferation and migration, along with lower ATP metabolite levels, after 96 hours of incubation, compared to their parental counterparts. Our in vivo data confirmed reduced primary tumor growth and metastatic dissemination in BALB/c mice injected with 4T1Luc ULK3 KO cells compared to parental control. Ex vivo tissue analysis using spatial transcriptomics revealed distinct differences in the tumor microenvironment and immune infiltration between metastatic organs (liver vs bones), correlating higher tumor cell proliferation and higher T regulatory cell infiltration with poorer overall survival. Ongoing analysis of TMAs from Moffitt TNBC patients correlated similar immune e infiltration profiles with worse outcomes. Further analysis of autophagy markers in TNBC cells, along with their correlation to treatment regimens received, will help clarify the metabolic status of cells in patients. Conclusions: Our experimental approach highlights the potential of targeting the autophagy metabolic pathway to influence TNBC progression. These findings support the use of pharmacological autophagy inhibitors at specific disease stages and offer insights into how patients might respond to immunomodulatory agents based on the localization of their metastases. Citation Format: Tao Li, Hatem Soliman, Ernst Schonbrunn, Andrij Marusyk, Marilyn Bui, Conor C. Lynch, Marilena Tauro. Defining the impact of autophagy blockade on triple negative breast cancer and remodeling of the tumor-immune microenvironment in primary and lung metastatic settings [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4269.