Clonal analysis of human mammary cell transformation and X-ray sensitivity

Intra-tumoural biological, transcriptional and genomic heterogeneity are hallmarks of human breast cancers. However, tumour propagating activity appears confined to subsets of cells within each tumour. This finding is thought to indicate a persistence of mechanisms that maintain a hierarchical growth and differentiation structure in the normal mammary gland. Because little is known about the responses of either primary normal or malignant human mammary cells to existing therapies, this thesis sought to examine the intrinsic sensitivity of different purified human mammary colony-forming cell (CFC) types and tumours derived from them to ionizing radiation. Luminal progenitor (LP) CFCs were found to be ~1.5-fold more radioresistant than basal cell (BC) CFCs and LPs also showed evidence of checkpoint adaptation, slower repair activity and greater predisposition of γH2AX foci accumulation. Two human breast cancer cell lines (MDA-MB231 and SUM149) and a non-tumorigenic human mammary cell line (MCF-10A) were all found to be more radioresistant than the normal LP-CFCs. CFCs isolated from 8-week tumours generated in mice transplanted with normal human BCs or LPs transduced with KRASG¹²D showed even greater radioresistance and this was further increased in serially passaged derivative lines with more aggressive growth properties. To examine the responsiveness of malignant cells with tumor-initiating cell (TIC) activity in vivo, a dose-response analysis was first undertaken of their frequencies in the MDA-MB231 and SUM149 cells. Limiting dilution analysis (LDA) and single-cell transplants showed the frequency of TICs in both to be very high (