Molecular interplay between dormant bone marrow-resident cells (BMRCs) and CTCs in breast cancer.. Molecular interplay between dormant bone marrow-resident cells (BMRCs) and CTCs in breast cancer.

Despite widespread knowledge that bone marrow-resident breast cancer cells (BMRCs) affect tumor progression, signaling mechanisms of BMRCs implicated in maintaining long-term dormancy have not been characterized. To overcome these hurdles, we developed a novel experimental model of tumor dormancy employing circulating tumor cells (CTCs) derived from metastatic breast cancer patients (de novo CTCs), transplanted them in immunocompromised mice, and re-isolated these cells from xenografted mice bone marrow (ex vivo BMRCs) and blood (ex vivo CTCs) to perform downstream transcriptomic analyses. Here we report that the balance between mTORC1 vs mTORC2 signaling regulates BMRC mitotic and/or dormancy characteristics. Overall design: Blood samples were collected from 6 (2 each of ER/PR+, HER2+ and triple negative) metastatic breast cancer patients in accordance to a protocol approved by Institutional Ethical Review Board at Houston Methodist Research Institute. After red blood cell lysis, the remaining buffy coat layer was subjected to FACS for enrichment of CTC populations by (i) doublet discrimination and dead cell elimination (DAPI-), followed by (ii) depletion of cells normally present in the peripheral blood using lineage-specific (CD45-/CD34-/CD105-/CD90-/CD73-) antibodies. Next, we implanted the residual CTC-enriched/lineage-negative cells into NOD scid-gamma (NSG) mice by intra-cardiac injection. Mice were euthanized after 4-8 months of in vivo selection, and bone marrow and blood were subjected to FACS to isolate ex vivo BMRCs and CTCs. The ex vivo BMRC/CTCs were defined as live (DAPI-), HLA-ABC+ (confirming human origin), PanCK+ (epithelial phenotype) or CD44+/CD24- (stem-like phenotype). Sorted cells were collected directly into a pre-chilled tube maintained at 4oC containing RNA lysis buffer and total RNA was collected, followed by RNA and cDNA amplifications, quality controls and gene expression arrays using the Affymetrix HTA 2.0 array. Only mice that were confirmed to be free of overt metastasis, were used for this study. Ex vivo BMRC/CTC gene expression was compared to whole gene expression data from CTCs isolated from 10 metastatic breast cancer patients (de novo CTCs) publicly available at GSE99394. Comparison between ex vivo BMRC/CTCs and de novo CTCs highlighted the transcriptomic landscape of dormant breast cancer BMRC/CTCs and identified mTORC2 signaling as a key regulator of metastatic dormancy.