COL10A1 expression distinguishes a subset of cancer-associated fibroblasts present in the stroma of high-risk basal cell carcinoma

Basal cell carcinoma (BCC) is the most frequently diagnosed skin cancer and the most common malignancy in humans. Different morphological subtypes of BCC are associated with low- or high-risk of recurrence and aggressiveness, but the underlying biology of how the individual subtypes arise remains largely unknown. Because the majority of BCCs appear to arise from mutations in the same pathway, we hypothesized that BCC development, growth and invasive potential is also influenced by the tumor microenvironment and in particular by cancer-associated fibroblasts (CAFs) and their secreted factors. In this work, we aimed to characterize the stroma of the different BCC subtypes with a focus on CAF populations. To investigate the stromal features of the different BCC subtypes, we applied laser-capture microdissection (LCM) followed by RNA sequencing. A cohort of 15 BCC samples from 5 different “pure” subtypes (superficial, nodular, micronodular, sclerosing and basosquamous; n=3 each) were selected and included in the analysis. Healthy skin was used as a control (n=6). We show that the stroma of the different BCC subtypes have distinct gene expression signatures. Nodular and micronodular seem to have the most similar signatures, while superficial and sclerosing the most different. By comparing low- and high-risk BCC subtypes, we observed that Collagen 10A1 (COL10A1) is overexpressed in the stroma of sclerosing/infiltrative and basosquamous but not micronodular high-risk subtypes. Those findings were confirmed by immunohistochemistry in a cohort of 89 different BCC and 13 healthy skin samples. Moreover, scRNAseq analysis of BCCs of two independent datasets showed that the COL10A1-expressing population of cells is associated with the stroma adjacent to invasive BCC and shows extracellular matrix remodeling features. In order to investigate the stromal features characterizing the different BCC morphological subtypes, we selected FFPE specimens of superficial, nodular, micronodular, sclerosing/infiltrating and basosquamous BCC subtypes. Healthy skin samples were included as controls. To specifically analyze stromal cell populations and extracellular matrix components directly adjacent to tumor cells or healthy epidermis, we applied Laser-Capture Microdissection (LCM) followed by RNA sequencing.

基底细胞癌（Basal cell carcinoma, BCC）是临床最常被确诊的皮肤恶性肿瘤，亦是人类最常见的恶性肿瘤。不同形态学亚型的基底细胞癌对应不同的复发风险与侵袭性高低，但目前学界对各亚型的具体起源生物学机制仍知之甚少。由于绝大多数基底细胞癌似乎均起源于同一信号通路的突变，我们提出假说：基底细胞癌的发生、生长及侵袭潜能同样受肿瘤微环境调控，尤其与肿瘤相关成纤维细胞（cancer-associated fibroblasts, CAFs）及其分泌的因子密切相关。本研究旨在对不同基底细胞癌亚型的间质进行特征分析，重点聚焦于肿瘤相关成纤维细胞群。为探究不同基底细胞癌亚型的间质特征，我们采用激光捕获显微切割（laser-capture microdissection, LCM）技术联合RNA测序开展研究。本研究纳入了来自5种不同"pure"亚型（浅表型、结节型、微结节型、硬化型及基底鳞状型，每种亚型各3例，共计15例基底细胞癌样本）的队列进行分析，并以6例健康皮肤组织作为对照。研究结果显示，不同基底细胞癌亚型的间质具有独特的基因表达特征：结节型与微结节型的表达谱最为相似，而浅表型与硬化型则差异最大。通过对比低风险与高风险基底细胞癌亚型，我们发现胶原10A1（Collagen 10A1, COL10A1）在硬化型/浸润型及基底鳞状型这类高风险亚型的间质中呈高表达，但在微结节型高风险亚型中未观察到此现象。该发现通过对89例基底细胞癌及13例健康皮肤样本队列进行免疫组化验证得以确认。此外，对两项独立数据集的基底细胞癌进行单细胞RNA测序（single-cell RNA sequencing, scRNAseq）分析显示，表达COL10A1的细胞群与侵袭性基底细胞癌邻近的间质相关，且具备细胞外基质重塑的功能特征。为进一步探究不同基底细胞癌形态学亚型的间质特征，我们选取了浅表型、结节型、微结节型、硬化型/浸润型及基底鳞状型基底细胞癌的福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded, FFPE）样本，并纳入健康皮肤样本作为对照。为特异性分析紧邻肿瘤细胞或健康表皮的间质细胞群及细胞外基质成分，我们再次采用激光捕获显微切割技术联合RNA测序进行检测。