Defining the cell type responsible for parity-induced protection against breast cancer

Parity (childbearing) significantly decreases a woman’s risk of breast cancer (BCa). Two proposed mechanisms of parity-induced protection investigated herein are a reduction in mammary stem cells (MaSCs) or an increase in differentiation in the parous mammary gland. MaSCs are postulated to mediate BCa risk as they are long-lived allowing a greater chance of accumulating genetic mutations likely to facilitate a carcinogenic transformation. Thus, a reduction in MaSCs by pregnancy may equate to a reduced risk of BCa. In order to investigate this, I first set out to ensure the isolation method used was highly selective for MaSCs. Cells expressing the proposed stem cell marker Stem Cell Antigen 1 (Sca-1) were characterized, as preliminary data indicated its superior ability to enrich for MaSCs beyond that of classical markers (defined by heat-stable antigen (CD24) positivity and high expression of integrin alpha 6 (CD49f)). Then, analyses of DNA damage markers and in vitro repair responses were used to assess the carcinogen sensitivity of MaSCs following treatment with a chemical carcinogen. Finally, primary and serial mammary fat pad stem cell assays were used to assess MaSC numbers and activity following pregnancy. Sca-1 was found not to further enrich for MaSC activity with a higher selectivity than the classical markers of MaSCs, thus CD24+CD49fhi cells were assessed for carcinogen sensitivity and stem cell activity following pregnancy. CD24+CD49fhi MaSC-enriched cells were found to be susceptible to carcinogens but not reduced by parity. Rather, parity appears to reduce a rare subset of cells that display stem cell activity outside the classical CD24+CD49fhi MaSC-enriched population. The terminal differentiation of the mammary gland that occurs at pregnancy is believed to permanently alter the gene expression profile. Preliminary data indicated the cell-specific target of these gene expression changes by parity was the stromal fibroblasts. The gene expression changes suggested a reduction in proliferative ability. To confirm this, the in vitro proliferative rate of parous fibroblasts was assessed in comparison to age-matched nulliparous fibroblasts. To then see if this proliferative rate was capable of affecting the proliferation of adjacent BCa cells, fibroblasts were co-cultured with low and high grade BCa cells in 3D matrices. Finally the ability of the parous fibroblasts to regulate in vivo tumour formation was assessed by co-injecting fibroblasts and BCa cells into mice. Parity was observed to reduce the proliferative rate in primary cultures of fibroblasts in 50% of the batches isolated over the course of the study. When tested using in vitro 3D proliferation assays or in vivo tumour growth assays, the effect of the parous fibroblasts was equally variable. In contrast, nulliparous fibroblasts were shown to be capable of tumour-stimulating behaviour on several occasions, an effect not observed in their parous counterparts. Altogether this work shows that a non-classical stem cell within the CD24+CD49lo/med population is decreased with parity and parous mammary fibroblasts have a less tumour stimulatory phenotype. This work thus identifies two cell types to further explore with the long-term goal to develop preventative therapies mimicking parity protection against BCa