Joshi Priyanka, Ghadi Dnyanada S, Waghmare Sanjeev K
Stem Cell Biology Group, Waghmare Lab, Cancer Research Institute, Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, Maharashtra, India; Homi Bhabha National Institute, Mumbai, India.
Stem Cell Biology Group, Waghmare Lab, Cancer Research Institute, Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, Maharashtra, India; Homi Bhabha National Institute, Mumbai, India.
Methods Cell Biol. 2022;171:63-80. doi: 10.1016/bs.mcb.2022.06.002. Epub 2022 Jul 21.
Skin squamous cell carcinoma (skin SCC) is the most frequently occurring cancer. Skin is the first line of defense that provides protection from the external environment. Skin consists of epidermis, dermis, and hypodermis. The epidermis comprises of inter-follicular epidermis, hair follicles, sebaceous glands, and sweat glands. Stem cells within these epidermal compartments play crucial role in epidermal regeneration and repair. Various factors such as higher exposure to ultraviolet light (UV) of sun, genetic predisposition, exposure to carcinogens, etc. that give rise to skin cancer. Within the skin SCC, there exists a pool of cancer stem cells (CSCs) that are highly quiescent with self-renewal capacity. Further, isolation and molecular characterization of CSCs would enable to unravel mechanism involved in tumor progression, metastasis, relapse, and resistance to chemotherapeutic agents. To understand the sequential events of carcinogenesis, the two-stage skin carcinogenesis murine model is proposed, which employs the topical application of a chemical carcinogen, DMBA that causes several activating mutations occurring in the genes responsible for cell proliferation and growth. Further, initiation is followed by tumor promotion, which is induced by repeated application of tumor-promoting agent, TPA, which fixes the activating mutations resulting in the formation of a benign papilloma. Subsequently, papilloma further progresses to highly malignant SCC. Here, using the two-stage skin carcinogenesis murine model, we provide a detailed protocol for the isolation of CSCs from murine skin SCC. FACS sorting of CSCs is followed by assays such as invitro-spheroid assay, in vivo-tumorigenesis-limiting dilution and in vivo-tumorigenesis-serial transplantation assay and expression profiling.
皮肤鳞状细胞癌（皮肤SCC）是最常见的癌症。皮肤是抵御外部环境的第一道防线。皮肤由表皮、真皮和皮下组织组成。表皮包括毛囊间表皮、毛囊、皮脂腺和汗腺。这些表皮区域内的干细胞在表皮再生和修复中起关键作用。多种因素，如过度暴露于阳光中的紫外线（UV）、遗传易感性、接触致癌物等，会引发皮肤癌。在皮肤SCC中，存在一群具有自我更新能力且高度静止的癌症干细胞（CSCs）。此外，对CSCs的分离和分子特征分析将有助于揭示肿瘤进展、转移、复发以及对化疗药物耐药所涉及的机制。为了解致癌作用的连续事件，提出了两阶段皮肤致癌小鼠模型，该模型采用局部应用化学致癌物二甲基苯并蒽（DMBA），其会导致负责细胞增殖和生长的基因发生多种激活突变。此外，启动阶段之后是肿瘤促进阶段，通过重复应用肿瘤促进剂佛波酯（TPA）诱导，TPA会固定激活突变，导致良性乳头瘤形成。随后，乳头瘤进一步发展为高度恶性的SCC。在此，我们使用两阶段皮肤致癌小鼠模型，提供了从小鼠皮肤SCC中分离CSCs的详细方案。对CSCs进行荧光激活细胞分选（FACS）后，进行体外球体形成试验、体内肿瘤发生极限稀释试验和体内肿瘤发生连续移植试验以及表达谱分析等检测。
