| INTRODUCTIONHuman breast cancers are heterogeneous both within tumor and between tumors. The presence of five molecular subtypes of breast cancer has been demonstrated. Among these subtypes, luminal A and basal-like are two distinct subtypes in pathology and in molecular profiles. We speculated that luminal A and basal-like tumor has its own cancer stem cells respectively. In this study, we have isolated breast cancer stem/progenitor cells from basal-like breast cancer and luminal A breast cancer and explored their biological behaviors.Part I Isolation, Propagation and identification of Breast Cancer Cells with Stem/Progenitor Cell Properties Object:To isolate, propagate and identify human breast cancer with stem cell-like properties in vitro and identify their stem cell-like properties through phenotypic and functional assay. To explore the method and influential factors on the formation of mammospheres in serum free culture system.Methods:Single cell suspensions derived from human breast cancer tissues were seeded in ultra low attachment plates in serum free media supplemented with bFGF, EGF and B27 for enriching human breast stem/progenitor cells as nonadherent mammospheres. Serial passaging in serum free media supplemented with bFGF, EGF and B27 was performed to determine self-renewal ability of mammosphere-derived cells. Differentiation was induced by culturing mammospheres in DMEM-F12 supplemented with serum without growth factors. Expression of CD34, nestin and epithelial specific antigen (ESA) on mammospheres was detected by indirect immunofluorescence under confocal laser scanning microscopy. The mammosphere-derived cells were implanted into nude mice to assess their tumorigenesis ability.Result:We got nonadherent mammospheres from 30 samples which had not endured chemotherapy. Cells from seven samples formed nonadherent mammospheres out of 12 cases which had received chemotherapy. The mammosphere-derived cells in our culture display stem cell-like properties for they can be serially passaged and form mammospheres in serum-free medias and differentiate along different mammary epithelial lineages in serum-supply medias. The mammosphere-derived cells expressed the putative epithelial cell marker ESA, but not CD34 and nestin, as indicated that our cultures were epithelial stem cell origin but not hematopoietic stem cells or neural stem cells origin. New tumors identical with the primary tumors formed when cells were injected into the mammary fat pad of nude mice. This suggested that cells in our culture were tumorigenic breast cancer cells.Conclusion:The mammosphere-derived cells from human breast cancer fresh tissues in our culture display stem cell-like properties and can be used for cancer stem cell research.PartⅡA comparison of Stem/Progenitor Cells Properties in cells derived from Basic-Like and Luminal-A Breast CancerObject:To isolate breast cancer stem/progenitor cells from basal-like breast cancer and luminal A breast cancer and explored their biological behaviors.Methods:Human breast cancer stem/progenitor cells were enriched in suspension cultures as nonadherent mammospheres. Proliferative capacity of mammosphere-derived cells in serum free media was assessed though determinations of the growth curve. Serial sphere formation assay was performed to determine self-renewal ability of breast cancer stem cells. Differentiation was induced by culturing mammosphere-forming cells in DMEM-F12 supplemented with serum without growth factors. Growth potential in vivo was evaluated by implanting mammosphere-derived cells into nude mice to constitute animal model and measuring the size of xenograft tumor.Results:Cells from all samples including basal-like subtype and luminal A subtype grew in serum-free conditions as mammosphere. Basic-like tumors have a high stem cell frequency (5.1%±1.08%) than that of luminal A tumors (2.02%±0.30%) and yielded mammospheres with a larger diameter which contained more cells. Mammospheres from both basic-like tumors and luminal A tumors could be serially passaged. Basic-like tumors showed a progressive decrease in sphere-forming efficiency, increased differentiation tendency and failed to passage beyond 15 generations with most cells adhering and terminally differentiating, while mammospheres derive from luminal A tumors could be serially passaged over 20 generations in vitro though more and more single cells lost their sphere-forming potentials. All mice of basal-like subtype group gave rise to new tumors when 105 cells per animal were injected while three of five animals could form tumors in luminal A subtype group. The tumor size of basal-like subtype group increases more rapidly than that of luminal A subtype group. Conclusion:mammosphere-forming cells from basal-like breast cancers showed different capabilities for self-renewal, clonogenicity, and tumorigenicity from that of mammospheres-derived cells from luminal A breast cancers.PartⅢPhenotypic Characterization of cancer Stem/Progenitor Cells derived from Basic-Like and Luminal-A Breast CancerObject:To isolate breast cancer stem/progenitor cells from basal-like breast cancer and luminal A breast cancer and explore their phenotypic variation.Methods:The expression of CK14, CK18 and CK19 on mammospheres and differentiated cells was detected by indirect immunofluorescence under confocal laser scanning microscopy. The expression of CD44, CD24 and the ratio of CD44+/CD24-/low cell population was evaluated by flow cytometry.Result:mammospheres derived from basal-like tumors showing positivity for CK14 and CK19, but not for CK18. differentiated cells revealed positivity for all these three markers. Mammospheres derived from luminal carcinomas expressed CK18 and CK19, but not CK14. Staining for these cytokeratins, revealed positivity for both CK18 and CK19, but not CK14.CD44+/CD24- tumor cells were detected in all basal-like subtype cases with a proportion 27.7%±1.4%,while absent or low in luminal A breast cancer.Conclusion:This paper shows, for the first time, that basal-like breast cancers and luminal A breast cancer exhibited different phenotypic and functional characteristics. This implied that they could originate from different mammary epithelial cells in different stage. |
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