Live imaging of breast tumors shows macrophage-dependent induction and TMEM-mediated enrichment of cancer stem cells during metastatic dissemination

Cancer stem cells (CSCs) play an important role during metastasis, but the dynamic behavior and induction mechanisms of CSCs are not well understood. We employed high-resolution intravital microscopy using a CSC biosensor to directly observe CSCs in live mice with mammary tumors. CSCs display the slow-migratory, invadopod-rich phenotype that is the hallmark of disseminating tumor cells. CSCs are enriched near macrophages, particularly near macrophage-containing intravasation sites called Tumor Microenvironment of Metastasis (TMEM) doorways. A dramatic enrichment of CSCs occurs on association with TMEM doorways, contributing to the finding that CSCs represent ∼>60% of circulating tumor cells. Mechanistically, stemness is induced in non-stem cancer cells upon their direct contact with macrophages via Notch signaling. In breast cancers from patients, the density of TMEM doorways correlates strongly with the proportion of cancer cells expressing stem cell markers, indicating that in human breast cancer TMEM doorways are not only cancer cell intravasation portals but also CSC programming sites. One Sentence Summary Intravital imaging reveals macrophage-mediated induction of cancer stem cells in vivo and their dramatic enrichment on dissemination through TMEM doorways. ### Competing Interest Statement The authors have declared no competing interest. * SORE6>GFP : cancer stem cell reporter construct ([Fig S1A][1]) minCMV>GFP : minCMV control construct SORE6-GFP : fluorescent protein product of SORE6>GFP construct SORE6+ : same as SORE6-GFP SORE6- : tumor cells containing the SORE6>GFP construct but not expressing fluorescent protein product (SORE-GFP), due to lack of stem transcription factors activity CSC : Cancer stem-like cell [1]: #F8