PO.TB10.07 · 肿瘤生物学
多组学揭示ESCC淋巴结转移中CAF的多样性
Multi-omics reveals the diversity of CAF in ESCC lymph node metastasis
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
1. 引言 食管鳞状细胞癌（ESCC）是中国最常见且致死性最高的恶性肿瘤之一，占癌症相关死亡率的第五位。癌症相关成纤维细胞（CAF）在肿瘤微环境（TME）中发挥重要作用。淋巴结转移是导致ESCC患者预后较差的关键风险因素。然而，淋巴结转移的起始和机制仍不明确。我们此前的研究已揭示CAF可通过分泌MFGE8激活AKT/STAT3信号来促进ESCC淋巴结转移。因此，我们决定进一步探索CAF在ESCC淋巴结转移中的作用。
2. 材料与方法 我们收集了ESCC肿瘤样本进行空间转录组测序。结合我们的单细胞RNA测序数据和已发表的单细胞测序队列，我们进行了多组学时空分析，以揭示ESCC中CAF的多样性。我们还进行了多重免疫组织化学（mIHC）以在ESCC样本中验证该亚型。我们从患者和小鼠模型中收集新鲜ESCC样本，从肿瘤中分离CAF用于体外和体内实验。进行天狼星红染色以分析胶原成分。培养KYSE30、KYSE180、KYSE410、KYSE520和小鼠mEC25细胞系用于体内和体外实验。
3. 结果 我们整合了自己的单细胞RNA测序数据（GSE203067）和另一个已发表队列（GSE160269），共包含64例ESCC患者。经过无监督聚类，我们得到6个成纤维细胞亚簇（包括周细胞和平滑肌细胞）。组织分布显示MMP11+ Fib富集于原发肿瘤和转移淋巴结，而PI16+ Fib富集于正常食管。随后我们通过去卷积算法分析空间转录组测序数据，揭示了ESCC肿瘤中不同成纤维细胞亚型的空间分布。MMP11+ Fib在伴淋巴结转移患者的原发肿瘤中占比更高。基因集富集分析表明，涉及细胞外基质和上皮-间质转化的通路在MMP11+ Fib中上调。Western blot和免疫荧光染色验证了MMP11在来自ESCC患者和小鼠ESCC模型的CAF中的表达。基于空间定位的细胞-细胞相互作用分析表明，MMP11+ Fib与肿瘤细胞之间的POSTN-(ITGAV/ITGB5)配体-受体信号被显著激活。
4. 结论 ESCC中的CAF显示出巨大的多样性，而MMP11+ Fib是一种促肿瘤和促转移的CAF亚型，是ESCC治疗的潜在靶点。
查看英文原文 English abstract
1. Introduction Esophageal squamous cell carcinoma (ESCC) is among the most common and lethal malignancies in China, accounting for the fifth-highest rate of cancer-associated mortality. Cancer-associated fibroblast (CAF) plays an important role in tumor microenvironment (TME). Lymph node metastasis is a key risk factor that leads to worse prognosis of ESCC patients. However, the initiation and mechanism of lymph node metastasis is still uncertain. Our previous study has revealed that CAF can promote ESCC lymph node metastasis through secreting MFGE8 to activate AKT/STAT3 signaling. Thus, we decided to further explore the role of CAF in ESCC lymph node metastasis.
2. Materials and methods We collected ESCC tumor samples for spatial transcriptome sequencing. Combined with our single-cell RNA sequencing data and published single-cell sequencing cohort, we performed multi-omics spatiotemporal analysis to reveal the diversity of CAF in ESCC. We also performed multi-plex immunohistochemistry (mIHC) to verify the subtype in ESCC samples. We collected fresh ESCC samples from patients and murine models to isolate CAF from tumor for in vitro and in vivo experiment. Sirius red staining was performed for collagen composition analysis. KYSE30, KYSE180, KYSE410, KYSE520, and mouse mEC25 cell lines were cultured for in vivo and in vitro experiments.
3. Results We integrated our single-cell RNA sequencing data (GSE203067) and another published cohort (GSE160269), which contained 64 ESCC patients. After unsupervised clustering, we got 6 subclusters of fibroblast (including pericyte and smooth muscle cell). Tissue distribution showed that MMP11+ Fib was enriched in primary tumor and metastatic lymph node, while PI16+ Fib was enriched in normal esophagus. Then we analyzed spatial transcriptome sequencing data via deconvolution algorithm, and revealed the spatial distribution of different fibroblast subtypes in ESCC tumor. MMP11+ Fib showed a higher proportion in primary tumor of patients with lymph node metastasis. Gene Set Enrichment Analysis indicated pathways with extracellular matrix and epithelial-mesenchymal transition were upregulated in MMP11+ Fib. Western blot and immunofluorescent staining verified the expression of MMP11 in CAF from ESCC patients and murine ESCC model. Cell-cell interaction analysis, based on spatial localization, indicated the POSTN-(ITGAV/ITGB5) ligand-receptor signaling was significantly activated between MMP11+ Fib and tumor cell.
4. Conclusion CAF in ESCC showed a huge diversity, and the MMP11+ Fib is a pro-tumor and pro-metastasis subtype of CAF, which is a potential target for ESCC treatment.
利益披露 Disclosure
L. Tan, None.