Heterogeneity of primary tumors (PTs) is reflected by genetic and epigenetic diversity, as well as diverse PT microenvironments. Whether PT microenvironments might influence the fate of disseminating tumor cells (DTC) has never been explored in situ. We previously defined, in breast cancer, a dormancy signature (DS) associated with longer metastasis-free periods. 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, while clinical evidence links hypoxic tumors to increased therapy resistance and a worse outcome. 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 H2B-Dendra2 (green-to-red fluorescence) 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 vivo in primary tumors we implanted induction NANo IntraVItal Devices (iNANIVIDs) carrying a hypoxia-mimetic agent (desferrioxamine - DFOM) in T-HEp3 tumors or exposed cultured MDA-MB-231 or ZR-75-1 cells in vitro to 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 upregulation of dormancy markers. We found a significant increase in quiescent lung DTCs of hypoxia induced T-HEp3 or MDA-MB-231 cells, traceable >2 weeks after extravasation by fluorescent label retention. Significantly more single, non-proliferating HEp3 DTCs originating from the iNANIVID induced hypoxic regions showed a dormant profile compared to DTCs originating from a normoxic milieu. Only the hypoxic pre-treated group was able to form micrometastases at 10 days after injection, suggesting the presence of a more aggressive sub clone in this group. Analysis in 3D culture models revealed that ZR-75-1 cells (ER+) were more prone to enter a prolonged quiescent state after exposure to hypoxia (1% O2) while this response was not observed in MDA-MB-231 (TN). The induction of quiescence in ZR-75-1 was NR2F1 dependent. Lastly, using a spontaneously metastatic PyMT driven Dendra2-tagged breast cancer model in immunocompetent mice, we found that ∼75% of dormant DTCs upregulate 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 PT microenvironments increase phenotypic heterogeneity of DTCs and lead to the expression of the DS. 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 V. Verkhusha, David Entenberg, Kevin W. Eliceiri, James Castracane, Patricia J. Keely, John S. Condeelis, Julio A. Aguirre-Ghiso. Phenotypic heterogeneity of disseminated tumor cells is predetermined by primary tumor hypoxic microenvironments. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4394.