基底乳腺干细胞不同亚群的识别

The mammary stem cells (MaSC) have been perceived to be the cells of origin for different subtypes of breast cancer, yet a clear understanding of cell hierarchy in the mammary gland is lacking. In particular, few studies examined different stem/progenitor cells within the basal lineage, where new studies showed that these cells could be the cells of origin for hormonal receptor positive breast cancers. Multiple lines of evidences from published studies speculate that there are different subpopulations of basal MaSCs, yet isolation and characterization of these potential different MaSCs has not been possible. Most recently, we accidently discovered two cell populations with distinct GFP intensity (low vs. high) in the basal lineage from a CAG-GFP-FVB mouse model. Our preliminary findings indicate that these two cell populations harbor their own MaSCs, which are distinct from each other in terms of in vitro colony formation, gene expression, and neoplastic transformation potential in a xenoestrogen exposed mouse model. Specifically, GFPhi cells are mainly devoid of ductal structures but confined to the tertiary alveolar structures while GFPlo cells are the opposite. GFPhi cells are also enriched for alveolar gene signature, increase significantly upon pregnancy, and are more prone to form hyperplastic lesions in regenerated glands upon in vivo transplantation. We thus hypothesize that there are two distinct MaSC populations reside in the basal lineage with one unipotent MaSCs (GFPlo) give rise to ductal basal cells and the other bipotent MaSCs (GFPhi) give rise to alveolar basal and luminal cells. We will take advantage of our GFP-FVB mouse model to separate the GFPlo and GFPhi basal cells and use a novel in vitro sphere formation and differentiation (SFD) assay developed in our laboratory to qualify and quantify these MaSCs. Our novel hypothesis will be tested with three specific aims. First, we will determine phenotypic differences of MaSCs from GFPlo and GFPhi basal cells with our in vitro SFD assay and the in vitro colony formation assay. Second, we will determine functional differences of MaSCs from GFPlo and GFPhi basal cells with the in vivo limited dilution transplantation assay. Third, we will determine gene expression differences of MaSCs from GFPlo and GFPhi basal cells by doing the RNAseq on MaSC-enriched spheres as well as single-cell gene analysis on single MaSC with a panel of genes selected from the RNAseq data. Validation of our proposed hypothesis will completely re-shape the paradigm of current cell hierarchy model in mammary gland. Achievement of our specific aims will not only provide potential candidate stem cell specific markers for future isolation of distinct MaSC populations reside within the basal lineage, but will also help pinpoint which stem/progenitor cells is indeed the most susceptible cell population for tumorigenic transformation, which will have significant implications in disease prevention for human breast cancer.

乳腺干细胞因与乳腺癌的成因息息相关而备受关注，最新研究预示基底干细胞极有可能是雌激素受体阳性乳癌的原发细胞，但许多研究证据表明基底干细胞可能含有不同的亚群。对这些干细胞亚群的剖析将有助于我们了解乳腺癌的成因。但目前对这一领域的研究纯属空白，本课题组基于对GFP-FVB小鼠乳腺的导管和腺泡呈现出荧光表达不同这一偶然发现，提出基底干细胞含有单能导管干细胞和双能腺泡干细胞的科学假说。我们将根据荧光表达量的差异分选出导管和腺泡基底细胞亚群，然后采用干细胞定性定量分析技术、基因测序和单细胞基因表达技术来：1）探索不同干细胞亚群在不同生长发育阶段的形态数量变化；2）阐述不同干细胞亚群的自我更新和细胞分化能力的异同；3）甄别不同干细胞亚群之间的基因表达差异，以期找到用以区分不同干细胞亚群的特定的细胞表面分子标记物。研究结果将为今后探索基底干细胞在乳腺癌形成和发展过程中所起的作用做出铺垫。

乳腺干细胞因与乳腺癌的成因息息相关而备受关注，最新研究预示基底干细胞极有可能是雌激素受体阳性乳癌的原发细胞，但许多研究证据表明基底干细胞可能含有不同的亚群。对这些干细胞亚群的剖析将有助于我们了解乳腺癌的成因。但目前对这一领域的研究纯属空白，本课题组基于对GFP-FVB小鼠乳腺的导管和腺泡呈现出荧光表达不同这一偶然发现，提出基底干细胞含有单能导管干细胞和双能腺泡干细胞的科学假说。 为了验证这一假说，我们根据GFP荧光表达量的差异通过流式分选出导管和腺泡基底细胞亚群，然后采用体内和体外干细胞定性定量分析技术、干细胞体外传代培养以及单细胞基因表达技术来系统比对这两群基底干细胞形态功能的差异性。研究发现：荧光强度高的基底干细胞的数量在孕鼠和老龄小鼠乳腺中的含量相比较于未怀孕的年轻的小鼠乳腺来说显著增加；荧光强度高和荧光强度低的基底干细胞在体外培养中所形成的三维结构呈现出显著的形态差异，其中由荧光强度高的基底干细胞所形成的三维结构高表达K14和ERα，低表达Stat5，而由荧光强度低的基底干细胞所形成的三维结构刚好相反；荧光强度高和荧光强度低的基底干细胞的再生能力在体内移植实验中没有显著的区别，但其在体外传代培养实验中的自我更新和分化能力存在显著差异，其中荧光强度高的基底干细胞自我更新和分化能力更强；单细胞基因表达分析显示荧光强度高的基底干细胞在表达基底细胞特征基因的同时也表达管腔细胞的一些特征基因，显示其具有双能性。综上所述，本项目的研究表明基底干细胞包含着不同的亚群，其中荧光强度高的基底干细胞亚群具有与雌激素受体阳性乳癌的一些相似特征。我们由此推断这一基底干细胞亚群极有可能是雌激素受体阳性乳癌的原发细胞。故而，本研究结果将为今后探索基底干细胞在乳腺癌形成和发展过程中所起的作用做出铺垫。