UBC Theses and Dissertations
Clonal heterogeneity of normal and transformed mammary stem cells Nguyen, Long Viet
The normal mammary gland contains “stem cells” with extensive in vivo growth and bi-lineage differentiation potential and a surface phenotype of basal cells (BCs). BCs also contain cells with more limited growth and differentiation activity in vitro. An analogous luminal-restricted progenitor (LPs) subset has surface characteristics of both basal and luminal cells. I hypothesized that the growth and differentiation activity displayed by individual mammary epithelial cells from both subsets would be highly diverse, and that the properties of tumours produced from these cells would be affected by their cell of origin. To address this hypothesis, I first developed a lentiviral-mediated barcoding strategy that involves transducing each cell with a unique 27-base pair non-coding DNA sequence so that the number of its clonal progeny can be inferred from high-throughput sequencing data obtained on the progeny of bulk-transduced populations. The use of “spiked-in” control cells carrying a known barcode provided an internal calibration for clone size calculations and allowed clones of ≥100 cells to be reliably detected. Application of this strategy to normal mouse and human mammary cells identified expected bi-lineage clones but an unanticipated predominance of lineage-restricted clones produced in primary transplants. These experiments also revealed that many clones apparent in secondary hosts were not detected in the primary hosts, indicating their origin from cells with very delayed growth activity. Application of the barcoding strategy to normal human BCs and LPs transduced with lentiviruses encoding KRASG¹²D ± PI3KCAH¹⁰⁴⁷R ± TP53R²⁷³C showed tumour formation in subsequently transplanted immunodeficient mice was rapid (within 8 weeks) and efficient from both cell types (8-12/18 donors, 1/200-1/4,000 transduced cells). However, tumours generated from LPs contained larger clones than tumours generated from BCs. Surprisingly, none of the LP-derived tumours were ERα⁺ (typical of luminal-like breast cancers) whereas 60% of the BC-derived tumours were. Earlier analysis of xenografts of similarly transduced cells revealed changes in both the number and phenotype of the cells present. Taken together, these findings underscore the diverse regenerative activity of normal mammary cells and provide definitive evidence that the cell of origin can affect the properties of human breast tumours generated using identical oncogenes.
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