Abstract 372: Macrophage contact-dependent stemness induction and progressive CSC enrichment during metastatic dissemination in breast cancer
Authors: Sharma Ved P. , Wang Yarong , Tang Binwu , Karagiannis George S. , Xue Emily A. , Entenberg David , Borriello Lucia , Coste Anouchka , Jones Joan G. , Surve Chinmay R. , Esposito Dominic , Oktay Maja H. , Wakefield Lalage M. , Condeelis John S. 
Published: September 2020

Abstract
Background: Cancer stem cells (CSCs) play an important role during metastatic progression of breast cancer. However, little is known, at the single cell level, about the process of stemness induction in non-stem cells or the dynamic behavior of CSCs during hematogenous dissemination.
Methods: Here, we employed high-resolution intravital multiphoton microscopy with a SOX2/OCT4 responsive fluorescent biosensor for stemness to directly observe the induction of stemness in single non-stem cells and their evolution through the metastatic cascade in living animals using orthotopic breast cancer xenograft model. We confirmed our findings in vitro using tumor cell-macrophage co-culture assays.
Results: We report that, both in vitro and in vivo, direct physical contact with macrophages induces stemness in non-stem cancer cells via juxtacrine Notch-Jagged1 signaling. In vivo, macrophage depletion with clodronate treatment showed a significant decrease in stem cells. In vitro, using either the fate mapping of non-stem cells with or without macrophage contact, or the origin-mapping of stem cells to find whether they originated from non-stem cells or pre-existing stem cells, we found that there was four-fold increase in new CSC induction after direct macrophage contact. In contrast, we did not see any role of macrophages in the expansion of pre-existing CSCs, both in vivo and in vitro, indicating that macrophage contact-dependent stem induction is the primary mechanism of CSC generation.
Using immunohistochemical staining in fixed tissue and live imaging of primary tumors and lungs in mice using optical windows, we found that during the course of dissemination of tumor cells from the primary site, CSCs become progressively enriched in the tumor cell population as they approach dissemination doorways (known as TMEM, Tumor MicroEnvironment of Metastasis), intravasate, circulate and arrive at the lung. Association with and passage through TMEM doorways is the step that generates the greatest enrichment in CSCs (~ 60-fold). On arrival in the lung, CSCs represent more than 75% of the disseminated tumor cell population, greatly enriched compared with their representation in the bulk primary tumor of ~ 1%.
Conclusion: Overall, these data indicate, for the first time, that macrophages associated with TMEM induce CSCs and promote TMEM-mediated CSC intravasation and early metastatic seeding. Our results are consistent with the dramatic enrichment of cancer stem cell markers in association with TMEM in breast cancer patients (Kim et al 2020 AACR abstract) and support a strategy for anti-metastatic therapy.
Citation Format: Ved P. Sharma, Yarong Wang, Binwu Tang, George S. Karagiannis, Emily A. Xue, David Entenberg, Lucia Borriello, Anouchka Coste, Joan G. Jones, Chinmay R. Surve, Dominic Esposito, Maja H. Oktay, Lalage M. Wakefield, John S. Condeelis. Macrophage contact-dependent stemness induction and progressive CSC enrichment during metastatic dissemination in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 372.