Abstract A43: Phenotypic heterogeneity of disseminated tumor cells is predetermined by primary tumor hypoxic microenvironments
Authors: Fluegen Georg , Avivar-Valderas Alvaro , Wang Yarong , Padgen Michael R. , Williams James K. , Verkhusha Vladislav , Cheung Julie F. , Entenberg David , Castracane James , Keely Patricia J. , Condeelis John , Aguirre-Ghiso Julio A. 
Published: May 2019

Abstract
Heterogeneity within and between primary tumors (PTs) is reflected by genetic and epigenetic diversity and varying PT microenvironments. Further, whether PT microenvironments might influence the fate of disseminating tumor cells (DTC) has never been explored. 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. Clinical evidence links hypoxic tumors to increased therapy resistance and a worse outcome. However, a main response of tumor cells to hypoxia is growth arrest, but how this response is linked to the clinical outcome is unknown. We hypothesized that hypoxic PT microenvironments may spawn a subpopulation of DTCs that, by virtue of becoming dormant, might escape therapies and eventually fuel incurable metastasis. We used H2B-GFP 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 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 significant upregulation of p27, NR2F1 and DEC2 (dormancy genes), as well as induction of hypoxia markers (GLUT1, HIF1α). Human HNSCC PT samples showed the same link between spontaneous hypoxic regions and up regulation of dormancy markers. We further found a significant increase in quiescent lung DTCs of hypoxia induced H2B-GFP T-HEp3 or H2B-Dendra2 MDA-MB-231 cells, traceable &gt; 2 weeks after extravasation using H2B-GFP and H2B-Dendra2-RED label retention. Using human Vimentin to screen for HEp3 tumor cells in lungs, we found that single, unproliferating DTCs originating from the iNANIVID induced hypoxic regions showed a dormant profile (upregulation of p27, NR2F1, DEC2 and TGFβ2) compared to DTCs originating from a normoxic milieu. Simultaneously, only the hypoxic pre-treated group was able to form micro-metastasis at 10 days after injection, suggesting the presence of a more aggressive sub clone in this group. 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) in an NR2F1-dependent manner. This response is not observed in triple negative/DS- breast cancer cells. Lastly, using a spontaneously metastatic PyMT driven Dendra2-tagged breast cancer model, we found that ~75% of dormant DTCs up regulate the dormancy marker NR2F1 at or soon after reaching the lung, suggesting a rapid induction of dormancy upon reaching target organs. We propose that hypoxic primary tumor stress microenvironments increase phenotypic heterogeneity of DTCs and leads to the expression of 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 R. Padgen, James K. Williams, Vladislav Verkhusha, Julie F. Cheung, David Entenberg, James Castracane, Patricia J. Keely, John Condeelis, Julio A. Aguirre-Ghiso. Phenotypic heterogeneity of disseminated tumor cells is predetermined by primary tumor hypoxic microenvironments. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A43.