Abstract B10: A fluorescent cancer stem cell sensor reveals dynamic induction of a stem phenotype in non-stem tumor cells on contact with macrophages in vitro and in vivo
Authors: Tang Bingwu , Sharma Ved , Wang Yarong , Raviv Yuval , Entenberg David , Oktay Maja , Condeelis John , Wakefield Lalage 
Published: June 2020

Many tumors are hierarchically organized, with a minor population of cancer stem cells (CSCs)/tumor-initiating cells at the apex of the hierarchy. These CSCs are highly enriched for the ability to drive tumor initiation, metastasis, and disease recurrence after therapy. Thus, a better understanding of the CSC population will be critical to the design of more effective anticancer therapeutics. To address this challenge, we have generated a lentiviral-based cancer stem cell sensor that reports with high temporal resolution on the cancer stem cell phenotype as a dynamic state. Expression of a destabilized fluorescent protein is driven by activation of an artificial enhancer element (SORE6) that responds to the master stem cell transcription factors Oct4 and Sox2 or their paralogs. We have extensively validated this reporter as marking cells with the expected characteristics of cancer stem cells, including the ability to self-renew, initiate, and sustain tumorigenesis and metastasis and exhibit chemoresistance (Tang et al., Stem Cell Reports 4:155-159). Intravital videomicroscopy and ex vivo imaging of orthotopically-implanted MDAMB231 breast tumors carrying the SORE6 stem cell sensor revealed that 60% of the CSCs in the primary tumor are found in association with macrophages. Macrophage depletion with clodronate liposomes in vivo reduced the number of CSCs in the tumor, indicating a role for macrophages in supporting or expanding the CSC population. Importantly, intravital imaging showed instances in which contact with intratumoral macrophages appeared to cause an induction of the stem phenotype in non-stem tumor cells and that this induction is concentrated at TMEM intravasation doorways where macrophages are enriched. Modeling this phenomenon in heterotypic cocultures in vitro, we showed that coculture of tumor cells with M2-like macrophages but not endothelial cells could increase the proportion of CSCs. This increase was contact dependent and involved Notch signaling. Single cell fate-mapping experiments in the coculture model revealed that the macrophages caused a &gt;4x increase in the rate of direct conversion of non-stem to stem cells, without affecting CSC proliferation. The induced CSCs were capable of initiating tumorsphere formation in vitro, thus confirming their functionality. In summary, this novel imaging approach allowed us to make the important finding that macrophage contact can induce phenotypic plasticity in more differentiated tumor cells, thereby allowing them to acquire a stem phenotype during intravasation and dissemination. These results suggest that targeting the interaction between macrophages and tumor cells may be a new strategy to reduce the cancer stem cell population both locally and systemically.

Citation Format: Bingwu Tang, Ved Sharma, Yarong Wang, Yuval Raviv, David Entenberg, Maja Oktay, John Condeelis, Lalage Wakefield. A fluorescent cancer stem cell sensor reveals dynamic induction of a stem phenotype in non-stem tumor cells on contact with macrophages in vitro and in vivo [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B10.