Abstract PS12-02: CXCL11 in the tumor immune microenvironment modulates resistance to endocrine therapy in hormone receptor-positive breast cancer

Abstract Despite advances in the treatment of hormone receptor-positive (HR+) breast cancer (BC), this disease subtype continues to be a major cause of mortality. The tumor immune microenvironment (TIME) plays a crucial yet not fully understood role in the natural history of HR+ BC. This study aims to clarify the impact of the TIME on HR+/HER2-negative BC response to antiestrogen treatment. We collected samples from postmenopausal patients with stage I-III HR+ BC, treated for 2-3 weeks with estrogen deprivation (ED) induced by letrozole. Treatment response was categorized based on the Ki67 scores at the time of the surgery (on treatment tumors, onTx): ED-sensitive (ED-S) if natural log (ln) of the Ki67 score ≤1.0 (i.e. ≤2.7% Ki67+ cells) vs. ED-resistant (ED-R) if ln ≥2.0 (i.e. ≥7.4% Ki67+ cells). Analysis of H&E tumor sections revealed that stromal tumor-infiltrating lymphocytes (sTILs) were significantly higher in ED-R compared to ED-S tumors (p=0.0001). Next, using both cyclic immunofluorescence (cycIF) and spatial transcriptomics, we found that the TIME of ED-R breast tumors was primarily infiltrated by CD8+ T cells, with their abundance being significantly higher in ED-R vs. ED-S tumors both prior to and after letrozole treatment (p<0.001 in preTx and p<0.001 in onTx tumors). In ED-R tumors specifically, CD8+ T cells were further enriched upon estrogen suppression (p<0.001), while Tregs decreased upon treatment (p<0.01). We next utilized a co-culture system to determine whether CD8+T cells can stimulate the proliferation of HR+BC cells. MCF7 and T47D cells were seeded in estrogen-free medium (EFM) in multi-well plates, with transwell inserts containing CD8+ T cells separated by a membrane with 3.0-µm pores. In this system, the cancer and T cells share the conditioned medium, thus allowing for paracrine cell-cell communication without direct contact between T cells and cancer cells. MCF7 and T47D exhibited 1.5- to 2.0-fold higher estrogen-independent proliferation when co-cultured with CD8+T cells (p=0.0115 and p=0.0003, respectively). Immune cell recruitment to cancer sites occurs through chemotactic cytokines. Thus, we investigated chemokine expression in RNA-seq data of ED-R vs. ED-S tumors. CXCL9, CXCL10, and CXCL11 chemokines were significantly enriched in ED-R compared to ED-S tumors (p<0.0001 for all genes; both preTx and onTx). Likewise, their receptor CXCR3 was similarly enriched in ED-R vs. ED-S tumors (preTx p=0.0106, onTx p<0.001). Publicly available datasets of patients with HR+BC treated with antiestrogen therapy showed that higher expression of CXCL9 (HR 1.36; p=0.016), CXCL10 (HR 1.71; p<0.0001), and CXCL11 (HR 1.5; p=0.0016) is associated with shorter relapse-free survival. We next investigated whether these chemokines play a causal role in resistance to ED using hormone-dependent MCF7 and T47D cells. Treatment with recombinant CXCL11 markedly upregulated phospho-AKT and phospho-ERK1/2 and stimulated estrogen-independent proliferation of both cell lines (p<0.0001). In addition, CXCL11 levels measured by ELISA increased 1.5- to 2.0-fold in media conditioned by breast cancer cells for 6 days when co-cultured with CD8+T cells compared to breast cancer cells alone. CXCR3 and CXCR7 are key receptors that interact with CXCL11. To assess their role in CXCL11-induced signaling, MCF7 and T47D cells were treated with a CXCR3 inhibitor (AMG487) and a CXCR7 antagonist, alone or in combination, in EFM with CXCL11. In particular, AMG487 strongly inhibited CXCL11-induced p-AKT/p-ERK activation in MCF7, while the effect was only partial in T47D cells. Taken together, our results suggest that CXCL11, a CD8+T cell-associated chemokine expressed in the breast TIME, promotes resistance to estrogen deprivation in early HR+BC, possibly through CXCR3. Further studies will investigate the use of inhibitors of CXCL11 receptors as a therapeutic strategy to reverse the TIME-mediated antiestrogen resistance. Citation Format: Fabiana Napolitano, Yunguan Wang, Dhivya R. Sudhan, Paula I. González-Ericsson, Luigi Formisano, Lei Guo, María Rosario Chica-Parrado, Chang-Ching Lin, Kyung-Min Lee, Hongli Ma, Nathaniel Evans, Alberto Servetto, Saurabh Mendiratta, Spencer Barnes, Yisheng V. Fang, Lin Xu, Gordon B. Mills, Marilyne Labrie, Ariella B. Hanker, Carlos L. Arteaga. CXCL11 in the tumor immune microenvironment modulates resistance to endocrine therapy in hormone receptor-positive breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr PS12-02.