LBPO.TB01 · 肿瘤生物学 · Late-Breaking
结直肠癌中癌相关成纤维细胞与正常成纤维细胞的空间程序随错配修复状态而分化
Spatial programs of cancer-associated and normal fibroblasts diverges with mismatch repair status in colorectal cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：结直肠癌（CRC）中成纤维细胞来源的转录特征与不良预后相关，且在错配修复正常（MMRp）疾病中较错配修复缺陷（MMRd）疾病中升高。然而，成纤维细胞亚型按MMR状态的空间组织尚未明确，限制了对调控这些不同微环境的基质邻域的认识。我们推测CRC中存在按MMR分层的基质生态位，具有由多种成纤维细胞亚型构成的不同组成。
方法：来自41例CRC供者（MMRp n=34，MMRd n=7）的新鲜原发肿瘤接受单细胞RNA测序（scRNA-seq）以定义成纤维细胞亚型，随后通过多重全切片成像将其映射到匹配的福尔马林固定石蜡包埋（FFPE）切片以及一个独立的存档FFPE队列（MMRp n=50，MMRd n=30）。肿瘤区域按肿瘤细胞与基质细胞密度定义（肿瘤核心：高肿瘤/低基质；浸润边缘：低肿瘤/高基质）。
结果：scRNA-seq解析出5种在各MMR背景下均保守的成纤维细胞亚型。其中两种为癌相关成纤维细胞（CAF）：富含纤维状ECM基因的肌成纤维细胞样CAF（myCAF_ECM）、炎症性CAF（iCAF）。三种为正常成纤维细胞（NF）：上皮下肌成纤维细胞（SEMF；收缩性基因）、固有层成纤维细胞（LPF；生长因子活性基因）和隐窝成纤维细胞（CF；纤维状与非纤维状ECM基因）。
无论MMR状态如何，肿瘤核心外周600 µm以内的浸润边缘相对于其他区域显示出高4倍的CAF密度（p<0.01）。该区域构成一个CAF富集生态位，CAF在其中形成灶状聚集体。聚集体组成按MMR状态分化：在MMRp肿瘤中，myCAF_ECM的丰度比iCAF高16倍（p<0.01），而在MMRd肿瘤中，iCAF的丰度比myCAF_ECM高3倍（p<0.03）。
NF在肿瘤基质中总体上与CAF一样丰富，且呈MMR依赖性分布。在MMRp肿瘤中，LPF在CAF生态位内的丰度比更深的浸润边缘高5倍（p<0.01），而CF峰值密度在更深浸润边缘高2倍（p<0.02）。在MMRd肿瘤中，SEMF在生态位内的丰度高2倍（p<0.05）。在全部5种亚型中，SEMF在两种MMR背景下均定位于最接近肿瘤细胞的位置，且对SEMF-肿瘤相互作用的配体-受体分析（LIANA）显示，相对于正常结直肠组织，肿瘤中ECM重塑通路富集。
结论：CRC含有以灶状CAF聚集体和不同NF定位为特征的MMR依赖性成纤维细胞生态位。我们的数据独特地凸显了CRC中NF的丰度与模式分布，并促使对TME中NF-肿瘤串扰的进一步研究。
查看英文原文 English abstract
Background: Fibroblast-derived transcriptional signatures in colorectal cancer (CRC) associate with poor prognosis and are elevated in mismatch repair-proficient (MMRp) versus mismatch repair-deficient (MMRd) disease. Yet the spatial organization of fibroblast subtypes by MMR status is undefined, limiting insight into stromal neighborhoods that modulate these distinct microenvironments. We hypothesized that CRC harbors MMR-stratified stromal niches with distinct compositions of various fibroblast subtypes.
Methods: Fresh primary tumors from 41 CRC donors (MMRp n=34, MMRd n=7) underwent single-cell RNA sequencing (scRNA-seq) to define fibroblast subtypes, which were subsequently mapped on matched formalin-fixed, paraffin-embedded (FFPE) sections and an independent archival FFPE cohort (MMRp n=50, MMRd n=30) via multiplex whole-slide imaging. Tumor regions were defined by tumor vs. stromal cell densities (tumor core: high tumor/low stromal; invasive margin: low tumor/high stromal).
Results: scRNA-seq resolved 5 fibroblast subtypes conserved across MMR settings. Two were cancer-associated fibroblasts (CAFs): myofibroblastic CAFs enriched for fibrillar ECM genes (myCAF_ECM), inflammatory CAFs (iCAFs). Three were normal fibroblasts (NFs): subepithelial myofibroblasts (SEMFs; contractility genes), lamina propria fibroblasts (LPFs; growth factor activity genes), and crypt fibroblasts (CFs; fibrillar and non‑fibrillar ECM genes).
The invasive margin within 600 µm of the tumor core periphery showed 4-fold higher CAF density (p<0.01) relative to other areas, regardless of MMR status. This region constituted a CAF-enriched niche, within which CAFs formed focal aggregates. Aggregate composition diverged by MMR status: in MMRp tumors, myCAF_ECM were 16‑fold more prevalent than iCAFs (p<0.01), whereas in MMRd tumors, iCAFs were 3‑fold more prevalent than myCAF_ECM (p<0.03).
NFs were collectively as abundant as CAFs in the tumor stroma, with MMR‑dependent distributions. In MMRp tumors, LPFs were 5‑fold more prevalent within the CAF-niche versus the deeper invasive margin (p<0.01), whereas CF peak densities were 2-fold higher in deeper invasive margin (p<0.02). In MMRd tumors, SEMFs were 2‑fold more prevalent in the niche (p<0.05). Among all 5 subtypes, SEMFs localized closest to tumor cells in both MMR settings, and ligand-receptor analysis (LIANA) of SEMF-tumor interactions showed enrichment of ECM‑remodeling pathways in tumors relative to normal colorectal tissues.
Conclusions: CRC contains MMR‑dependent fibroblast niches characterized by focal CAF aggregates and distinct NF localization. Our data uniquely highlight NF prevalence and patterning in CRC and prompt further investigation into NF-tumor crosstalk in the TME.
利益披露 Disclosure
D. Barua,
Merck Employment.
T. Tan,
Merck Sharp & Dohme Employment.
S. Ho,
Merck Sharp & Dohme Employment.
M. T. Wong,
Merck Sharp & Dohme Employment.
I. B. H. Tan,
Merch Sharpe & Dohme ).
J. H. Yearley,
Merck Employment.
S. Liu,
Merck Employment.