Abstract 3000: Hypoxic primary tumor stress microenvironments prime DTCs in lungs for dormancy
Authors: Fluegen Georg , Avivar-Valderas Alvaro , Wang Yarong , Padgen Michael , Estrada Yeriel , Williams James K. , Entenberg David , Eliceiri Kevin , Keely Patricia J. , Castracane James , Verkhusha Vladislav V. , Condeelis John , Aguirre-Ghiso Julio A. 
Published: August 2015

Abstract
The mechanisms behind dormancy of disseminated tumor cells (DTCs) are unclear. Further, whether primary tumor microenvironments might influence DTC fate has never been explored in situ. We found that breast tumors enriched for a specific dormancy signature (DS+) displayed longer metastasis-free periods than those poor (DS-) for the signature. Key genes in the DS induce quiescence and are also regulated by hypoxia. Interestingly, a main response of tumor cells to hypoxia is growth arrest. We hypothesized that hypoxic primary tumor microenvironments may spawn a subpopulation of DTCs that, by virtue of becoming dormant, might escape therapies and eventually fuel incurable metastasis. We used H2B-EGFP inducible HEp3 HNSCC and photo-switchable (green-to-red fluorescence) H2B-Dendra2 expressing MDA-MB-231 and ZR-75-1 human breast cancer cell lines to identify cells from hypoxic microenvironments. To initiate spatially defined hypoxic microenvironments in primary tumors we implanted induction NANo IntraVItal Devices (iNANIVIDs) carrying a hypoxia-mimetic agent (desferrioxamine - DFOM) in T-HEp3 tumors in vivo (chicken chorioallantoic membrane (CAM) model) or exposed cultured MDA-MB-231 or ZR-75-1 cells in vitro to either 21% or 1% O2. The regions influenced by the DFOM-iNANIVID displayed upregulation of p27, NR2F1 and DEC2 (dormancy genes), as well as induction of hypoxia markers (GLUT1, HIF1α). We found that lung DTCs derived by mouse tail vein injection of hypoxia induced H2B-EGFP T-HEp3 or H2B-Dendra2 MDA-MB-231 cells were primed to enter dormancy in lungs &gt; 2 weeks after extravasation, as measured using H2B-EGFP and H2B-Dendra2-RED label retention. Using human Vimentin to screen for HEp3 tumor cells in lungs, we found that DTCs originating from the iNANIVID induced hypoxic regions showed a dormant profile as evidenced by significant upregulation of p27, NR2F1 and DEC2 compared to DTCs originating from a normoxic milieu. Further, analysis in 3D culture models revealed that ER+/DS+ breast cancer cells (ZR-75-1) are more prone to enter a prolonged quiescent state after a brief exposure to hypoxia (1% O2) while this response is not observed in triple negative/DS- breast cancer cells (MDA-MB-231). We propose that hypoxic primary tumor stress microenvironments induce a subpopulation of tumor cells to express the DS. Upon spreading, these DTCs may be more prone to enter dormancy, evade anti-proliferative therapies and eventually fuel metastasis.
Citation Format: Georg Fluegen, Alvaro Avivar-Valderas, Yarong Wang, Michael Padgen, Yeriel Estrada, James K. Williams, David Entenberg, Kevin Eliceiri, Patricia J. Keely, James Castracane, Vladislav V. Verkhusha, John Condeelis, Julio A. Aguirre-Ghiso. Hypoxic primary tumor stress microenvironments prime DTCs in lungs for dormancy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3000. doi:10.1158/1538-7445.AM2015-3000