Abstract 288: Middle-aged murine models of triple negative breast cancer (TNBC) show significant differences in tumor immune and stromal environments when compared to classic 6-8 week old models; Challenges and opportunities for preclinical efficacy studies
Authors: Falvo Paolo , Gruener Stephan , Orecchioni Stefania , Talarico Giovanna , Bravetti Giulia , Mitola Giulia , Pisati Federica , Barozzi Iros , Bertolini Francesco 
Published: March 2024

Abstract
The immune system orchestra suffer a deficit of function during aging, and the incidence of the large majority of cancer types is significantly increased in elderly humans. Although BC can occur in young women, it is way more common in middle-age and elderly women. There is evidence that a tonic immune system is pivotal for the success of therapies aimed at awakening a dormant or exhausted immunological response against cancer cells by mean of checkpoint inhibitors (CI). However, rodent models of cancers are currently using very young mice of a few weeks of age, which have a fully functional and tonic immune system and thymus and thus might be poorly representative of adult human cancer patients. We have recently reported that that a triple therapy (TT) involving antigen-presenting cell activation by vinca alkaloids and the generation of new TCF1+ stem cell-like T cell (scT) clones by an alkylating agent can significantly improve the efficacy of the CI anti-PD-1 in models of cancer known to be otherwise CI-resistant such as TNBC and lymphoma (Falvo et al, Cancer Res 2021; Orecchioni et al, JCM 2023). TT effect was due to T cells, as it was abrogated by their in vivo depletion. In the present study we investigated differences in TNBC growth kinetics, TT preclinical activity and tumor microenvironment (TME, including intratumoral immune and stroma cells) in young (6-8w, representative of a 6y old human) versus adult (12m, representative of 40y humans) mice. Models included 4T1 and EMT6 TNBC cells, the former generating a mostly lymphoid TME and the latter generating a mostly myeloid TME. In both models, TT efficacy was similar in young and in adult mice, as the treatment abrogated TNBC local and metastatic growth. CD8+ (but not CD4+) scTs, likely crucial players in TT efficacy, were only slightly reduced in adult mice in spite of age-related thymus involution. This notwithstanding, single cell RNA (10x), IHC and flow cytometry analyses of immune (CD45+EPCAM-) and stroma (CD45-EPCAM-) cell populations indicated major differences in the TME of young vs adult mice. Adults had significantly less CD4+ scTs, B naïve and NK cells and significantly increased memory B cells. TME matrix CAF were skewed in young, while pro-inflammatory stromal populations and myofibroblasts were skewed in adults. Matrix CAFs down regulated a signature involved in different ECM-remodeling abilities, and up regulated metabolic and hypoxic pathways. Expression of genes encoding for glycoproteins, basement membrane components, and collagens were also upregulated. Our data indicate profound differences between young and adult mice immune and stromal TMEs that should be taken into consideration when selecting the age of mice to be used in preclinical efficacy studies in orthotopic immune competent mice.
Citation Format: Paolo Falvo, Stephan Gruener, Stefania Orecchioni, Giovanna Talarico, Giulia Bravetti, Giulia Mitola, Federica Pisati, Iros Barozzi, Francesco Bertolini. Middle-aged murine models of triple negative breast cancer (TNBC) show significant differences in tumor immune and stromal environments when compared to classic 6-8 week old models; Challenges and opportunities for preclinical efficacy studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 288.