Dll1-Mediated Notch Signaling Drives Tumor Cell Cross-talk with Cancer-Associated Fibroblasts to Promote Radioresistance in Breast Cancer

Resistance to radiotherapy is a major obstacle for effective cancer treatment. Both cancer-associated fibroblasts (CAF) within the tumor microenvironment (TME) and Notch signaling are impli-cated in radioresistance, but their potential interrelationship is unclear. Here, we report that irradiated samples obtained from luminal breast cancer patient tumors express higher levels of the Notch ligand Dll1 and have a greater number of aSMA-and FAP-expressing activated CAFs. Single cell transcriptomic profiles fur-ther revealed enrichment of an alpha SMA(+) myofibroblastic subpopu-lation of CAF in Dll1(+) tumors. In murine and human patient-derived xenograft models, Dll1(+) tumor cells were more radio-resistant than Dll1(-) tumor cells, and genetic and pharmacologic blocking of Dll1-mediated Notch signaling decreased the number of Dll1(+) cancer stem cells (CSC) and CAFs and increased Dll1(+)tumor cell radiosensitivity. Dll1(+) cells recruited CAFs in an IL6-dependent fashion and promoted Wnt ligand secretion by Notch2/3-expres-sing CAFs, thereby driving Wnt/beta-catenin-dependent increases in Dll1(+) CSC function to promote metastasis. Treatment with the porcupine inhibitor LGK-974 that inhibits Wnt ligand secretion or pharmacologic blockade of IL6 or Dll1 suppressed CAF-dependent enhancement of Dll1(+) CSC function and metastasis in radioresis-tant tumors. Together, these findings reveal an essential cross-talk between Dll1(+) cancer cells and CAFs, which promotes metastasis and radioresistance, which could be therapeutically exploited to improve the outcome of patients with breast cancer. Significance: Dll1(+) breast cancer cells activate Notch signaling in cancer-associated fibroblasts that increases Wnt ligand secretion and leads to beta-catenin-driven radioresistance and metastasis, opening new therapeutic avenues for breast cancer.