Decoding intra-tumor heterogeneity in breast tumors.

Introduction: Cellular intra-tumor heterogeneity in triple negative breast tumors (TNBC) leads to phenotypic variation between tumor subpopulations and poses a critical challenge on choosing biomarkers and therapies. We wish to decipher the complete sets of tumor cellular subclones within each tumor and the altered protein pathways that characterize those subclones in order to suggest the optimal, patient-specific, therapy.

Materials and Methods: We analyzed breast cancer protein markers in thousands of individual breast tumor cells. We used 2 syngeneic mouse models of TNBC: AT3 (on C57BL6 mice), and 4T1 (on BALB/c mice), along with the AT3 and 4T1 parental cells grown in vitro. Tumors were digested into single cell suspensions and labeled with fluorescently-tagged antibodies against 11 breast cancer biomarkers including HER2, EGFR, EpCAM, CD44, CD24, PD-L1, c-Kit, CD133, E-Cadherin, cMET and MUC1. 4T1 and AT3 cells were irradiated and grown under normal conditions. Cells were examined for differential expression of these markers using multicolor flow cytometry. Information-theoretic analysis was used to discern tumor subpopulations.

Results: We resolved an unbalanced network structure that occurred in AT3 and 4T1 models in response to irradiation, comprising of 10 distinct unbalanced protein networks (processes). The same processes were existed before irradiation however the percentage of the cells harboring these processes was different. For example, in 4T1 model a process, in which HER2 and EGFR were upregulated, appeared in a small group of cells (0.3%) before irradiation. However, this group expanded gradually 48hrs post-irradiation up to 5% and reached 21% of the population after 6d post-irradiation. Similarly the group of cells harboring process with upregulated cMet and Muc1 along with downregulated Ecadherin has been expanded significantly during the irradiation treatment.

Conclusions: These results demonstrate that irradiation resulted in the expansion of the previously existing very small cellular groups within the tumors. We hypothesize that this expansion may lead to the resistance of TNBC to irradiation. “Moreover, these results suggest that in order to sensitize certain triple negative cells to irradiation, the central proteins from the expanded subpopulations, such as HER2 and cMet in 4T1 should be inhibited.” We currently study the in vivo effect of anti-Her2 and anti-cMet drugs on the response of TNBC tumors to the irradiation therapy.

Acknowledgments: STEP Foundation and Abisch-Frenkel Foundation.