Abstract 2560: Age-dependent differences in breast tumor microenvironment: Challenges and opportunities for efficacy studies in preclinical models

Abstract Immunity declines with age, leading to functional deficits and an increased incidence of cancer in the elderly. Despite this, most cancer models utilize young (6-8 week old) mice, which are not representative of adult cancer patients. Triple negative breast cancer (TNBC) typically occurs around 30-50 years of age, which can be approximated to 12 months in mice. We previously demonstrated that a Triple-Therapy (TT) including APC activation via vinorelbine and the generation of TCF1+ stem-cell-like T cells (scTs) by cyclophosphamide (CTX), significantly enhances anti-PD-1 efficacy in TNBC and lymphoma models resistant to anti-PD1 treatment by promoting T-cell-mediated tumor abrogation. In this study, we examine TT effects on TNBC growth and the tumor microenvironment (TME) in young (6-8 weeks) vs adult (12 months) mice. We investigated two models, one (4T1) with predominantly lymphoid TME and the other (EMT6) with a predominant myeloid TME. Regular-dose-TT efficacy was similar across age groups, as - notably - CD8+ scTs were only slightly reduced in adults. At variance, low-dose TT and/or metronomic CTX were significantly less effective in adult mice. This might explain why encouraging preclinical results of metronomic CTX where sometimes difficult to replicate with adult cancer patients. Single-cell analyses revealed substantial differences in the TME: adults exhibited fewer CD4+ scTs, naïve B cells, and NK cells, but more memory B cells. Cancer-associated fibroblasts (CAFs) with an extracellular matrix (ECM) deposition signature (Matrix-CAFs) were more prevalent in young mice, whereas pro-inflammatory stromal populations and myofibroblasts were more frequent in adults. In adult mice, Matrix-CAFs displayed reduced ECM-remodeling capacity, decreased collagen deposition, and distinct interaction patterns with other cells in the TME. To gain further insight, non-epithelial cells from the mammary gland and scRNA data of Tabula Muris Senis were contextualised with the TME age-related signature. Age-related transcriptional differences observed in the TME already show an age-related trend in the stroma of the normal mammary gland, including a reduction of T cells, and an increase in the ECM remodeling ability of fibroblasts. Nevertheless, the extent of these differences is larger in the TME as compared to normal ageing breast, suggesting the tumor cells can leverage and exacerbate these differences, potentially to the tumor’s advantage. These findings underscore the importance of considering age-dependent TME variations when designing preclinical studies. Citation Format: Paolo Falvo, Stephan Gruener, Stefania Orecchioni, Giovanna Talarico, Giulia Mitola, Giulia Bravetti, Federica Pisati, Juliane Winkler, Iros Barozzi, Francesco Bertolini. Age-dependent differences in breast tumor microenvironment: Challenges and opportunities for efficacy studies in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2560.