Schueddig, E.; Kochat, V.; Arslan, E.; Dallas, Y.; Yang, P.; Pedron, W.; Li, Z.; Henry, R.; Lin, J.; Mattohti, M.; Madan, R.; Fields, T.; Khan, S.; Golem, S.; Wagner, J. L.; Larson, K. E.; Balanoff, C.; Aripoli, A.; Huppe, A.; Winblad, O.; Peterson, J.; Hill, M.; Smith, C.; Jeffers, E. E.; Kilgore, L. J.; Navin, N.; Zang, C.; Wei, P.; Fabian, C.; Lewis, M. T.; Zhu, Q.; Thompson, A. M.; Godwin, A. K.; Koestler, D. C.; Rai, K.; Behbod, F.

Ductal carcinoma in situ (DCIS) exhibits substantial heterogeneity in its risk of progression to invasive breast cancer, yet the cellular and molecular determinants of high-risk lesions remain incompletely defined. Using spatially resolved single-cell transcriptomic and epigenomic profiling of 43 patient derived DCIS and DCIS/invasive ductal carcinoma (IDC) samples, we delineate cellular programs, spatial organization, and epigenetic regulatory mechanisms associated with invasive potential. We identify an epithelial population with stemness features within luminal hormone-responsive (LumHR) cells that progressively expands from benign tissue to DCIS and IDC, and is strongly associated with invasive progression and recurrence-linked transcriptional programs. Spatial mapping reveals discrete DCIS niches enriched for stem-like LumHR cells, characterized by elevated CEACAM6 expression and enhanced ligand receptor interactions, including CEACAM6-EGFR signaling between epithelial and stromal compartments, including cancer-associated fibroblasts, macrophages (APOC1 positive) and perivascular cells. These niches define a microenvironmental context that supports stemness and invasive potential. Epigenomic analyses implicate FOXA1 as a key regulator of these stem-like transcriptional states. Pharmacologic disruption of FOXA1 regulatory network using LSD1 inhibition suppresses stemness-associated transcriptional programs in vitro and significantly restrains tumor growth in vivo. Collectively, these findings define high risk DCIS as a stemness-driven disease embedded within specialized microenvironments, and identify associated regulatory networks as candidate biomarkers and therapeutic vulnerabilities.