Development and utilization of a macaque-based mammosphere culture technique for breast cancer research (2016)

Human breast cancers are thought to commonly arise from progressive neoplastic changes to the adult stem cells within the normal mammary gland. Research in this area of breast carcinogenesis currently relies heavily on the acquisition of mammary gland stem cells from the tissues of rodents and humans. While a great deal of information has been gained utilizing these models, there remain large gaps in our knowledge of breast cancer due to certain limitations with these species. The relevance of rodents as models for human breast cancer has been brought into question by notable differences between rodents and humans with regard to genetics, biology and mammary gland carcinogenesis. In contrast, the utility of human-derived samples is limited by ethical concerns and by the restricted availability of mammary tissues from women. Macaque monkeys are closely related to humans phylogenetically and these animals develop mammary gland tumors that are comparable to human breast cancers. Furthermore, mammary gland tissues can be easily collected from any demographic of animal. Despite their potential, only minimal breast cancer work has been undertaken in the macaques to date and research techniques common to both rodents and human are lacking for these species. This dissertation describes the optimization of a commonly-used mammary gland stem cell isolation technique, mammosphere culture, for the rhesus macaque (Macaca mulatta) and provides validation as to the reliability, relevance, and usefulness of this assay for human studies. Data obtained from this research demonstrated that the mammosphere culture technique is highly reproducible between homologous macaque tissue samples. This work also found that mammary gland biopsies collected from different anatomical locations on the same monkey share comparable mammosphere-forming ability and mammosphere-differentiation ability (collectively, the mammosphere potential). Finally, these initial studies identified macaque mammospheres to have proliferative and differentiating properties that are nearly identical to those described for human mammospheres. This dissertation also describes a series of macaque studies performed using the optimized mammosphere culture technique. In the first study, mammary gland tissues were obtained from female macaques in different reproductive demographics and the mammosphere potential of these animals was compared. The results suggested that the mammosphere potential of nulliparous mammary glands is significantly greater than that of multiparous mammary glands and that this difference is likely due to greater ratios of mammary gland stem cells within the nulliparous mammary gland. These data also suggested that there are differences in the mammosphere potential of mammary glands collected from animals at different stages of the reproductive cycle. An additional study comparing the mammosphere potential of young-multiparous and multiparous macaques collected during the menses stage of the menstrual cycle supported the parity-related findings of the first study. Data from the second study also identified significantly larger ratios of senescent cells in the mammosphere cultures of multiparous macaques as compared to young-nulliparous macaques. Finally, a study comparing the effects of ionizing radiation on mammospheres derived from young-nulliparous and multiparous macaques was performed. This last study found that stem cell-like cells of the young-nulliparous mammary gland were more resistant to the lethal effects of ionizing radiation than were those of the multiparous gland. The findings of these three studies are notable in that young-nulliparous girls are known to have a higher susceptibility to radiation-induced breast cancer than are multiparous women and these studies provide the first direct evidence as to the potential mechanistic reasons behind this observation. Specifically, as macaques appear to be relevant models for the study of the human breast, these data suggest that the increased susceptibility of young-nulliparous girls to radiation-induced carcinogenesis could arise from: 1) higher number of mammary stem cells within the breast; 2) a decreased predilection of these stem cells to undergo senescence; and 3) a decreased sensitivity of these stem cells to the lethal effects of ionizing radiation. In summary, macaque mammospheres appear to be relevant models for the study of the human breast. Use of this model allows for the study of mammary gland tissues from some demographics of interest (e.g., prepubescent individuals) that are impossible to investigate utilizing human tissues. The mammosphere culture techniques and data described in this dissertation serve as a foundation toward the use of macaques in future breast cancer research projects and other study data from this dissertation has provided novel insight as to the increased risk of radiation-induced breast cancers in young women.