Napolitano, Fabiana (2024) The tumor immune microenvironment modulates resistance to endocrine therapy in ER+ breast cancer. [Tesi di dottorato]

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Abstract

Background: Previous studies have reported the identification of a subgroup of HR+ breast tumors with enhanced immune infiltration. However, the relevance of its role in modulating the response to endocrine therapy (ET) is unclear. Patients and methods: Postmenopausal patients with stage I-III HR+ BC were treated with letrozole for 2-3 weeks until definitive surgery. Tumor tissue samples were collected at baseline and at the time of surgery. RNA was extracted, and RNA-seq was performed. The results were analyzed using Gene Sets Enrichment analysis, and enrichment in Hallmark pathways was evaluated. H&E slides, cyclic immunofluorescence, and spatial transcriptomic analyses were used to investigate the tumor immune microenvironment (TIME) in tumors sensitive vs. resistant to ET, as defined by ≤2.7% vs. ≥7.4% Ki67+ tumor cells, respectively. Results: Tumors resistant to estrogen deprivation exhibited higher stromal tumor-infiltrating lymphocytes and additional features of aggressive disease coupled with a worse survival outcome. In particular, high infiltration of CD8+ T and CD20+ B cells characterized the intra-tumoral microenvironment of resistant tumors, while sensitive tumors exhibited a TIME primarily including FOXP3+ cells. Utilizing a spatial transcriptomic approach, we observed upregulation of gene signatures related to antigen processing and presentation machinery (APM) and T cell immunity in endocrine-resistant tumors. Leveraging the CIBERSORT deconvolution method, we observed an increase in CD8+ T cell infiltration and a decrease in immunosuppressive Tregs in endocrine-resistant tumors upon treatment. Finally, RNA-sequencing results highlighted a potential role for the chemokines involved in CD8+ T cell recruitment (CXCL9, CXCL10, CXCL11) in endocrine-resistant tumors. When added to cell cultures' media, these chemokines can stimulate estrogen-independent proliferation of ER+ breast cancer cells, overcoming the therapeutic effect of estrogen suppression. In particular, CXCL11 seems to hold a prominent role in determining this effect, and the RNA gene expression levels at the time of the biopsy (prior to letrozole) were found to robustly differentiate between sensitive and resistant tumors (AUC ROC curve 0.77), thus predicting the response to endocrine therapy. Conclusions: In this study, we identified for the first time a subgroup of HR+ BC characterized by poor prognosis and resistance to endocrine therapy. Multiple approaches revealed features such as molecular aggressiveness and a TIME enriched with intra-tumoral CD8+ T cells. Digital spatial profiling showed increased T cell immunity and antigen processing and presentation pathways in ED-resistant tumors. Additionally, we found that these pathways were further enhanced by endocrine therapy. Finally, we explored the causal role of CD8+ T cells in determining resistance to endocrine therapy, highlighting how the chemokines involved in CD8+ T cell recruitment (CXCL11 in particular) were able to stimulate the proliferation of HR+ BC cell lines under conditions of estrogen deprivation. Taken together, our findings demonstrated that the presence of a CD8+ T cell-enriched TIME is associated with early resistance to endocrine therapies and poorer patient survival, and that CXCL11 expression could serve as a biomarker for patient selection and indicate potential treatment escalation strategies, including the use of immune checkpoint inhibitors.

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