Ductal carcinoma in situ: Molecular and cellular basis of malignancy.

e12582 Background: The goal of our study is to improve the diagnosis and treatment of DCIS through the identification of the factors driving its invasion and metastasis. Methods: We utilized the Mouse INtraDuctal (MIND) model to investigate the mechanism(s) by which some DCIS become invasive while others remain indolent. This model closely replicates the human condition by injecting non-invasive epithelial cells from patients into mouse mammary ducts to form in situ lesions. In some cases, these cells will display invasive behavior by overcoming the natural barriers of myoepithelium. To compare these two processes, we performed single-cell (sc) RNA-sequencing analysis of 60,000 cells from the pre-transplant time-point representing 17 DCIS (11 became invasive, 6 remained non-invasive). We also performed scATAC/RNA sequencing of a DCIS with associated IDC. Results: Our analysis revealed that the proportion of endothelial cells was higher in cases that became invasive, while the proportion of plasma and T cells was higher in those that remained non-invasive (t-test; P<0.05). This finding suggested that invasive cells reprogrammed the immune microenvironment to exclude immune cells as a means of evading immune surveillance. Additionally, we performed cell-cell interaction (CCI) analysis by CellPhoneDB. This analysis revealed highly ranked CCIs in invasive cases, including luminal cell interactions with pericytes via Wnt5A-SFRP1. On the other hand, highly ranked CCIs in non-invasive cases included HR-positive luminal cell interactions with T cells via CD226-Nectin-2 and CD96-Nectin1. Notably, CD226 and CD96 are T cell receptors that enhance CD8+ T cell activity. These results support the potential role of T-cell interactions with DCIS luminal cells in preventing their invasive transition. We conducted additional analysis of sc-RNA-sequencing data using a stem cell-based gene signature algorithm called CytoTRACE. Our analysis revealed that invasive cells were enriched in cell clusters with significantly higher stem cell scores than non-invasive cells. In another analysis of a patient DCIS with associated IDC using sc-ATAC and RNA sequencing, we discovered that the open chromatin regions in the stem cell cluster were enriched for FOXA1 binding motifs. This cluster also had the highest expression of two pioneer factors (FOXA1 and GRHL2), NEAT1 (paraspeckle protein), ERBB2, 3,4, and ANKRD30A (cell surface protein). Our findings suggest that the pioneer factors FOXA1 and GRHL2 play a crucial role as transcriptional drivers of stemness. Additionally, the ANKRD30A could be used to isolate stem cell clusters to validate their self-renewal potential. Conclusions: DCIS progression is facilitated by interactions between stromal, immune, and epithelial cells, which lead to epigenetic and transcriptional changes. This results in the formation of stem-like cells that are characterized by their plasticity and invasive capacity.