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Ex vivo isolation of adult stem cells from normal and tumour mouse mammary parenchyma

It is generally believed that tumours originate from adult tissues, in which most cells are quiescent, and that the proliferative advantage of tumour cells arises from their ability to bypass senescence. Once this is obtained, cells can accumulate further genetic alterations that drive progressive transformation into highly malignant derivatives. How normal cells evade the senescence program remains an unresolved issue.

Recent findings suggest the alternative possibility that stem/progenitor cells are the targets of transformation. Many tumours contain only a small subset of cells endowed with the property of uncontrolled growth (cancer stem cells). Although the program of senescence in stem cells is suppressed, their lifespan is restrained by signalling pathways (p19-p53; p16-Rb) that are activated by DNA damage (telomere dysfunction, environmental stresses). It is proposed that inactivation of these pathways in stem cells might contribute to tumour formation.

We set out to cultivate in vitro breast stem cells from the mouse mammary gland (BSC) based on their ability to survive in suspension as 'mammospheres' and to differentiate into myoepithelial and epithelial cells. Murine mammospheres do not result from passive cell aggregation and have clonal origin, as assessed by labelling cell membranes with different epifluorescent dyes. To prove the 'staminality' of these cellular populations we performed in vivo reconstitution experiments, by inoculating single cell suspensions of mammospheres in the mouse cleared fat pad.

Using the same experimental approach, we were also able to generate mammospheres from MMTV-ErbB2(cNeu) transgenic mice. These mice develop polyclonal mammary adenocarcinomas arising synchronously. The mammospheres arising from MMTV-ErbB2 mice show a dramatically prolonged lifespan upon serial passages and are bigger than their normal counterparts (more than 1000 cells/sphere vs 200–400 cells in the w.t. mice). To prove that cancer mammospheres are enriched in cancer BSC, we are performing transplantation experiments in which single cell suspensions of these mammospheres are inoculated in the normal gland of singenic mice.

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Bonizzi, G., Cicalese, A., Giulini, B. et al. Ex vivo isolation of adult stem cells from normal and tumour mouse mammary parenchyma. Breast Cancer Res 7 (Suppl 2), P3.05 (2005).

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  • Stem Cell
  • Cancer Stem Cell
  • Single Cell Suspension
  • Adult Stem Cell
  • Serial Passage