1 Abstract 2 Breast cancer (BC) encompasses multiple diseases made up of different molecular subtypes that are 3 characterized by distinct oncogenic drivers and unique treatment regimens. Despite these differences, across all 4 subtypes, individuals with advanced, recurrent, or metastatic disease still have limited treatment options and 5 poor overall survival outcomes. Immunotherapies offer an opportunity to treat patients regardless of molecular 6 subtypes. This proposal leverages intratumoral (IT) immunotherapy as an alternative to ‘license’ treated lesions 7 to yield productive ratios of T cells to suppressive immune subsets while amplifying immune checkpoint axes 8 and ultimately increasing sensitivity to immune checkpoint inhibitors (ICI). Using a model of TNBC and single 9 cell RNA sequencing, we demonstrate that IT plasmid IL-12 (pIL-12) can convert poorly immunogenic/low 10 TIL tumors into highly inflamed, immunologically active lesions through the coordinated upregulation of the 11 CXCR3 axis in infiltrating immune cells that impacts the migration, differentiation, and activation of both 12 innate and adaptive immune cells. This CXCR3 signature was also significantly enhanced in patients that had 13 an increase in CD8 T cell infiltration into treated tumors post IT pIL-12 therapy and prognostic of improved 14 overall survival. We hypothesize that targeting the CXCR3 axis IT will enhance TILs and convert patients into 15 ICI responders across all molecular subtypes of BC. The proposed work will leverage this preliminary data in 16 the following 3 aims: 1.) Demonstrate increased infiltration of tumor-specific T cells following IT pIL-12-EP 17 treatment and validate the induction of a CXCR3 or trafficking-associated gene signature in ICI responders 2.) 18 Evaluate the role of trafficking-associated proteins in enhancing responsiveness to anti-PDL1 in TNBC using a 19 CRISPR based screen; 3.) Assess IT injection of plasmid CXCL9 to determine if direct targeting of the CXCR3 20 axis is sufficient to enhance T cell infiltration. Dr. Crosby’s long-term goal is to build a research program that 21 contributes to an understanding of immune cell infiltration into tumors to better design, combine, and predict 22 responses to immunotherapies, with a specific focus on BC. A critical impediment to these types of studies is 23 the lack of oncogene-driven, spontaneous BC tumor models which hampers the translational applicability of 24 many pre-clinical findings. Key resources for Dr. Crosby’s independent research are the spontaneous HER2- 25 driven and p53/BRCA1/PTEN-driven TNBC models that she has created, published, and will uniquely possess 26 to perform these and many other studies. Addressing the basic question of how to enhance T cell infiltration 27 into tumors has significant implications for changing the paradigm of treatment for BC patients, particularly 28 using an intratumoral plasmid approach that is easily altered to follow up on ne...