Primary breast cancer cell culture yields intra-tumor heterogeneous subpopulations expressing exclusive patterns of receptor tyrosine kinases

Background: It has become evident that intra-tumor heterogeneity of breast cancer impact on several biological processes such as proliferation, migration, cell death and also might contribute to chemotherapy resistance. The expression of Receptor Tyrosine Kinases (RTKs) has not been analyzed in the context of intra-tumor heterogeneity in a primary breast cancer cell culture. Several subpopulations were isolated from the MBCDF (M serial-breast cancer ductal F line) primary breast cancer cells and were successfully maintained in culture and divided in two groups according to their morphology and RTKs expression pattern, and correlated with biological processes like proliferation, migration, anchorage-independent cell growth, and resistance to cytotoxic chemotherapy drugs and tyrosine kinase inhibitors (TKIs).

Methods: Subpopulations were isolated from MBCDF primary breast cancer cell culture by limiting dilution. RTKs and hormone receptors were examined by Western blot. Proliferation was measure by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT assay). Cell viability was evaluated by Crystal Violet. Migration was assessed using Boyden chambers. Anchorage-independent cell growth was evaluated by colony formation in soft agar.

Results: Several subpopulations were isolated from the MBCDF breast cancer cells that were divided into two groups according to their morphology. Analysis of RTKs expression pattern showed that HER1, HER3, c-Met and VEGFR2 were expressed exclusively in cells from group 1, but not in cells from group 2. PDGFR was expressed only in cells from group 2, but not in cells from group 1. HER2, HER4, c-Kit, IGF1-R were expressed in all subpopulations. Biological processes correlated with the RTKs expression pattern. Group 2 subpopulations present the highest rate of cell proliferation, migration and anchorage-independent cell growth. Analysis of susceptibility to chemotherapy drugs and TKIs showed that only Paclitaxel and Imatinib behaved differently between groups. Group 1-cells were resistant to both Paclitaxel and Imatinib.

Conclusions: We demonstrated that subpopulations from MBCDF primary cell culture could be divided into two groups according to their morphology and a RTKs excluding-expression pattern. The differences observed in RTKs expression correlate with the biological characteristics and chemoresistance of each group. These results suggest that intra-tumor heterogeneity contributes to generate groups of subpopulations with a more aggressive phenotype within the tumor.