Abstract 3445: Immune cells infiltration and activation are higher in breast cancers resistant to antiestrogen therapy
Authors: Napolitano Fabiana , Sudhan Dhivya R. , Wang Yunguan , González-Ericsson Paula I. , Formisano Luigi , Lee Kyung-Min , Lin Chang-Ching , Chica-Parrado María Rosario , Ma Hongli , Evans Nathaniel , Servetto Alberto , Mendiratta Saurabh , Barnes Spencer , Fang Yisheng V. , Balko Justin M. , Mills Gordon B. , Labrie Marilyne , Hanker Ariella B. , Arteaga Carlos L. 
Published: April 2023

Abstract
We investigated the tumor microenvironment (TME) in estrogen receptor positive (ER+) primary breast cancers from stage I-III patients treated with estrogen deprivation (ED, induced with letrozole) for 10-21 days. We used primary breast cancer biopsies, collected from 198 patients, to assess tumor-infiltrating lymphocytes (TILs), transcriptomic profiles, and TME composition. Diagnostic biopsy and surgical on-treatment tumor specimens were collected; Ki67, ERα, PR, and HER2 expression were measured using automated quantitative analysis (AQUA). Tumors were categorized as estrogen deprivation-sensitive (ED-S) or -resistant (ED-R) based on the natural log (ln) of the Ki67 score (ln ≤1.0 or ≤2.7% Ki67+ cells vs. ln ≥2.0 or ≥7.4%, respectively) in the on-treatment (surgical) biopsy. Scoring of stromal (s)TILs, using hematoxylin and eosin staining of on-treatment specimens, revealed that sTILs were elevated in ED-R compared to ED-S tumors (p&lt;0.0001). We next constructed tissue microarrays (TMAs) of on-treatment tumor sections and performed cyclic immunofluorescence (CyCIF) with 38 antibodies, including markers of hormone receptors, immune cells, signaling pathways, proliferation, DAPI (nuclei), and others. We then segmented single cells from each tumor core and designated them as immune, cancer, or stromal cells based on expression of a set of markers. Briefly, cells that stained strongly for CD45 (leukocyte marker), and/or CD4 (T lymphocyte marker), or CD68 (macrophage marker) were considered immune cells. CD45/CD4/CD68-negative cells were categorized as tumor, if E-cadherin and/or cytokeratin-positive, or stromal cells, if -negative. To investigate the TME composition, we next assessed cell specific spatial enrichment by quantifying the expression of immune markers in the area immediately adjacent to each tumor cell. Relative to ED-S, ED-R samples exhibited higher CD45+ cells (p&lt;0.0001) and higher CD8+ T cells (p=0.0329), while CD4+ T cells showed no difference. Conversely, immune-suppressive T-reg (CD4+FOXP3+) cells were enriched in the ED-S samples (p=0.0004). In addition, PD1, a marker for T cell exhaustion, was higher in the ED-S group (p=0.0004), as were CD68+ cells (p&lt;0.0001). To confirm and expand upon these findings, RNA-sequencing was performed on the on-treatment tumor sections. Gene set enrichment analysis of hallmark gene signatures revealed that immune-related gene sets, such as “IFNα response”, “IFNɣ response”, “allograft rejection”, and “inflammatory response” were enriched in ED-R vs. ED-S tumors. In summary, our study shows that activated anti-tumor immune cells are enriched in the TME of ER+ breast tumors resistant to estrogen deprivation, whereas ED-sensitive tumors show a more immunosuppressed or immune cold milieu. These data suggest a potential causal link between antiestrogen treatment and modulation of antitumor immunity in ER+ breast cancers.
Citation Format: Fabiana Napolitano, Dhivya R. Sudhan, Yunguan Wang, Paula I. González-Ericsson, Luigi Formisano, Kyung-Min Lee, Chang-Ching Lin, María Rosario Chica-Parrado, Hongli Ma, Nathaniel Evans, Alberto Servetto, Saurabh Mendiratta, Spencer Barnes, Yisheng V. Fang, Justin M. Balko, Gordon B. Mills, Marilyne Labrie, Ariella B. Hanker, Carlos L. Arteaga. Immune cells infiltration and activation are higher in breast cancers resistant to antiestrogen therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3445.