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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 >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.

Cancer stem cells (CSCs) are a subpopulation of tumor cells that are uniquely capable of initiating and sustaining tumorigenesis, and they have also been implicated in driving disease recurrence after cancer therapy. We have previously developed a novel lentiviral-based reporter system for direct visualization, quantitation and isolation of cells with CSC properties. The construct consists of a tandemly-repeated composite SOX2-OCT4 response element (“SORE6”) driving expression of a destabilized fluorescent protein reporter (Tang et al. Stem Cell Reports, 2015). TGF-βs are pleiotropic regulatory factors with complex roles in tumorigenesis. We and others have hypothesized that TGF-β can regulate the CSC population in breast cancer with stimulatory or inhibitory effects depending the model and stage of cancer development. Using the MCF10Ca1h and MCF7 breast cancer models, which retain tumor suppressor responses to TGF-β, here we show that TGF-β treatment significantly reduces the size of the CSC population in vitro. Although TGF-β had relatively little effect on invasion and migration of the bulk population in these models, it clearly inhibited migration and invasion of the CSCs, suggesting that biological responses to TGF-β can vary depending on the position of the cell in the differentiation hierarchy. Combining our stem cell reporter with a TGF-β pathway reporter, we further show that CSCs in the MCF10Ca1h model have higher endogenous activation of the TGF-β pathway than does the bulk tumor cell population, and by time-lapse video microscopy we find that CSCs with active TGF-β signaling are relatively quiescent. Furthermore, neutralization of TGF-β in vivo leads to an increased representation of CSCs in MCF10Ca1h and MCF7 tumors. Thus our preliminary results suggest that in breast cancer models where TGF-β acts as a tumor suppressor, TGF-β signaling is preferentially activated in the CSC compartment and keeps a subpopulation of CSCs in a quiescent and stationary state. These findings have important implications for the clinical use of TGF-β pathway antagonists. In contrast, in the metastatic breast cancer MDA-MB231 model, in which TGF-β acts as a pro-progression factor, we show that TGF-β can increase the size of the CSC population and enhance invasion. In this model, the CSCs are enriched for the ability to form macroscopic lung metastases when compared with non-CSCs, and we show that this effect is due in part to a selective survival advantage of the CSCs in the first 24 hours after arrival at the metastatic site. The role of TGF-β in this process is currently under investigation. Citation Format: Binwu Tang, George Baker, Joshua Zent, Yuval Raviv, Lalage Wakefield. Transforming growth factor-beta regulation of cancer stem cell dynamics at different stages of breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4782. doi:10.1158/1538-7445.AM2017-4782