Human models of Merkel cell carcinoma oncogenesis

默克尔细胞癌(MCC)是一种侵袭性很强的皮肤肿瘤，由于病因不同，可分为两种亚型。而在约80%的病例中，MCC是由Merkel细胞多瘤病毒(MCPyV)(病毒阳性；VP-MCC)引起的，病毒阴性(VN)-MCC是由紫外线诱导的突变引起的，因此具有高突变负荷的特点。皮肤MCC是从哪些细胞产生的，这两种亚型是否具有相同的来源仍然存在争议。一些细胞，如默克尔细胞、表皮干细胞、成纤维细胞或B细胞系细胞被讨论为可能的候选细胞。在第一个资助期间，我们研究了由鳞状细胞癌(SCC)和VN-MCC部分组成的联合肿瘤。利用遗传分析，我们证明了MCC是从SCCs转分化而来的。因此，这些MCC代表上皮性肿瘤，就像两个VP-MCC一样，我们能够根据遗传相似性证明它们起源于毛母细胞瘤。由于毛母细胞瘤起源于毛囊中的KRT17+细胞，具有分化潜能，我们的假设是毛囊中的KRT17+MERKEL祖细胞是VP-MCC的起源细胞。毛囊起源的这一假说也得到了最近发表的MCC小鼠模型的支持，由于这些细胞的位置，可以进一步解释VP-MCC通常较低的突变负荷。相反，VN-MCC的起源可能是毛囊间表皮暴露于阳光下的前体细胞或SCC细胞，因为VN-MCC在高达25%的病例中被发现与SCC有关。在拟议的项目中，通过在体外进行适当的基因操作，将在第一个资助期产生所需的载体，从而追溯从鳞状细胞癌到微囊癌的转分化。此外，MCPyV癌蛋白(小T和大T抗原)将在人皮肤器官中表达，以产生复杂的人Merkel细胞癌模型。在第一个资助期，在维尔茨堡成功地建立了由多能干细胞生成毛发皮肤器官的项目。此外，不同的载体系统的产生和测试，允许单独或组合表达T抗原，无论是结构性的，Dox诱导的或细胞类型特异性的(例如，在KRT17+细胞中)在皮肤器官中。因此，所有的工具都可用于研究复杂人体模型系统中VP-MCC起源的问题。

Merkel cell carcinoma (MCC) is a very aggressive skin tumor in which - due to divergent etiology - two subtypes can be distinguished. While in about 80% of cases MCC is caused by Merkel cell polyomavirus (MCPyV) (virus-positive; VP-MCC), the virus-negative (VN)-MCC result from UV-induced mutations and are thus characterized by high mutational load. From which cells of the skin MCCs arise and whether the two subtypes have the same origin is still controversial. A number of cells such as Merkel cells, epidermal stem cells, fibroblasts or B cell lineage cells are discussed as possible candidates. During the first funding period, we investigated combined tumors consisting of a squamous cell carcinoma (SCC) and a VN-MCC part. Using genetic analyses, we demonstrated that the MCCs developed by transdifferentiation from the SCCs. Thus, these MCCs represent epithelial tumors, as do two VP-MCCs, for which we were able to demonstrate origination from trichoblastomas based on genetic similarities. Because trichoblastomas derive from KRT17+ cells of the hair follicle and show Merkel cell differentiation potential, our hypothesis is that KRT17+ Merkel cell progenitor cells localized in the hair follicle are cells of origin for VP-MCCs. This hypothesis of a hair follicle origin is also supported by a recently published MCC mouse model and, because of the location of these cells, may furthermore explain the generally low mutation load of VP-MCCs. In contrast, the origin of VN-MCC would be sun-exposed precursor cells of the interfollicular epidermis or, not infrequently, SCC cells, since VN-MCC is found associated with SCC in up to 25% of cases. In the proposed project, the transdifferentiation from SCC to MCC will be retraced by appropriate genetic manipulation in vitro with the required vectors already been generated in the first funding period. In addition, the MCPyV oncoproteins (small T and large T antigen) will be expressed in human skin organoids to generate a complex human Merkel cell carcinoma model. In the first funding period, the generation of hair-bearing skin organoids from pluripotent stem cells was successfully established in Würzburg. In addition, different vector systems were generated and tested, allowing individual or combinational expression of T antigens either constitutively, Dox-inducible or cell type-specific (e.g. in KRT17+ cells) in skin organoids. Thus, all the tools are available to investigate the question of VP-MCC origin in a complex human model system.