Age-dependent differences in the tumor microenvironment of preclinical models of triple-negative breast cancer: challenges and opportunities for efficacy studies.

The immune system orchestra suffer a deficit of function during aging, and the incidence of most cancer types is significantly increased in elderly humans. Although breast cancer (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, current cancer models in rodents use 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 (PMIDs: 33268528, 37048617). 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 (PMID: 33268528). 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 despite age-related thymus involution. This notwithstanding, single-cell transcriptomics, 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. Cancer-associated fibroblasts with a matrix deposition signature (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 TMEs that should be taken into consideration when selecting the age of orthotopic immune competent mice to be used in preclinical efficacy studies.