Dissecting immune-related intercellular crosstalk along mammary gland development and breast cancer

The mammary gland is a dynamic tissue that changes drastically during postnatal developmental stages, along its “temporary” final differentiation in lactation, and during tissue reorganization resulting from estrous cycle and involution. In this active ecosystem, cell-cell communication between immune and non-immune compartments is crucial during both tissue homeostasis and carcinogenesis, but these networks remain largely unknown. Here, we used a breast cancer mouse model carrying the polyomavirus middle T-antigen (PyMT) oncogene and non-carrier littermates, to collect samples at various time points from healthy and cancerous tissues. By calibration of unique tissue dissociation protocol followed by single-cell RNA-sequencing analysis, we identified a wide range of immune and non-immune cell types and states from both conditions. Interestingly, we identified populations that were markedly expanded during early postnatal development, such as Schwann cells and innate lymphoid cells (ILCs). In addition, we classified cell states that were expanded only in the oncogene carriers, including distinct macrophage and fibroblast populations. By applying physically interacting cell sequencing (PIC-seq), we identified PICs comprised of immune and epithelial cells in both normal and cancer breast tissues. Importantly, PIC-seq analysis highlighted a higher incidence of immune/epithelial cell (CD45+/EPCAM+) physical crosstalk in tumor samples as soon as week 6 post-natal, during neoplasia but prior to tumor appearance. Together, our data shows that exploring the transient cell state composition and the intercellular communications along a time course can reveal important cell phenotypes and novel dynamics in the immune-related signaling that shape the pre-neoplastic microenvironment and could predispose the development of cancer.