Abstract PS3-12-14: Targeting the Tumor Secretome to Enhance Immune Responses in Breast Cancer Liver Metastasis

Abstract Background: Nearly half of metastatic breast cancers spread to the liver, a metastatic site that responds poorly to immune checkpoint blockade. Although liver metastases are considered immunologically cold and this is thought to drive resistance, few studies investigate heterogeneity in the liver metastatic microenvironment. This gap in knowledge limits our understanding of the mechanisms driving therapy resistance. Objective: We aimed to characterize the heterogeneity of immune phenotypes in breast cancer liver metastases (BCLM) and postulated that comparison of immune hot and cold BCLM would reveal factors produced and secreted by the tumor to support an immune cold phenotype. Methods: We profiled a cohort of 20 human BCLM specimens using the NanoString nCounter system. Abundance of immune cells and secreted factors of interest were confirmed using multispectral fluorescence (mIF markers: PanCK+/tumor, CD4+ or CD8+/T cell, CD68+/macrophage, CD66b+/neutrophil) and immunohistochemistry (IHC). Similar approaches were conducted in a validation cohort of 19 BCLM. Single cell mRNA sequencing (scRNAseq) from 6 BCLM from GSE249361 were accessed and analyzed using CellChat to reveal differential communication between immune cells and AGR2-negative versus AGR2-expressing tumor cells. 4T1 or 66Cl-4 mouse mammary carcinoma spheroids were co-cultured with activated CD8+ splenocytes in the presence and absence of a secreted factor of interest, recombinant AGR2 (rAGR2). T cell distance from the center of spheroids was quantified using ImageJ. Three BCLM patient derived organoids (PDOs) were developed by the Hope for OTHERS rapid autopsy program at the University of Pittsburgh/UPMC Hillman Cancer Center. AGR2 levels were assessed in PDOs via IF. Results: nCounter analysis of 20 BCLM revealed that liver metastases lie across a spectrum of immune infiltration. Confirmatory multispectral fluorescence demonstrated that nearly half of the tumors (n=11) had abundant expression of macrophages and T cells, suggesting that they could be primed for response to checkpoint inhibitors. We compared differentially expressed genes between the most immune hot (n=5) and cold (n=5) metastases to reveal essential factors preventing immune infiltration in cold tumors. The most highly expressed gene in immune cold compared to immune hot BCLM was anterior gradient-2 (AGR2), a tumor secreted factor that correlated negatively with immune cell abundance in breast tumors. In a validation cohort of 19 BCLM, we confirmed that low AGR2 expression predicted a 2-fold increase in CD8+ T cell density. Datamining a scRNAseq dataset revealed that AGR2 is heterogeneously expressed in the tumor compartment. Using CellChat we showed that tumor cells lacking AGR2 have enhanced communication with CD8+ T cells through the MHC-I pathway, suggesting that AGR2 may dampen tumor antigen presentation. Alternatively, tumor cells expressing AGR2 have increased communication with several members of the tumor microenvironment through fibronectin signaling, revealing that tumor AGR2 may also enhance extracellular matrix dynamics. Preliminary mechanistic studies revealed that treatment with recombinant AGR2 decreases CD8+ splenocyte penetration into spheroids by 30%. Finally, AGR2 levels were heterogeneous in BCLM PDOs and they corresponded with the expression of AGR2 and the immune status in the original tumor. Conclusions: These findings suggest that BCLM produce and secrete AGR2 to limit T cell infiltration and activation. We are currently developing a platform with the BCLM PDOs to assess the impact of AGR2, and additional secreted factors, on immune cell responses using matching peripheral blood. We expect that these findings will support further development of AGR2 blocking antibodies as a companion or alternative immunotherapy approach in metastatic breast cancer. Citation Format: A. Phan, L. McCue, T. Hintelmann, M. M. Williams. Targeting the Tumor Secretome to Enhance Immune Responses in Breast Cancer Liver Metastasis [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-12-14.