【作者】 秦健；

【导师】 王书奎；

【作者基本信息】 东南大学 ， 临床检验诊断学， 2025， 博士

【摘要】 研究背景:结直肠癌(Colorectal cancer,CRC)在全球范围内发病率和死亡率均位于前列,其转移是导致患者预后不良的主要原因。肿瘤微环境(Tumor Microenvironment,TME)在CRC转移过程中发挥重要作用,其中肿瘤相关成纤维细胞(Cancer-Associated Fibroblasts,CAFs)是TME中的重要组成部分。CAFs具有高度异质性,不同CAFs亚群在肿瘤转移中的作用存在显著差异。单细胞测序(Single-cell sequencing,scRNA-seq)技术在揭示CAFs异质性中发挥了重要作用。胶原蛋白XI型α1链(Collagen Type XI Alpha 1Chain,COL11A1)作为在细胞外基质中发挥重要作用的纤维状胶原蛋白,在多种肿瘤中高表达并与肿瘤侵袭和转移相关,但其在CRC转移微环境中的特异性表达细胞亚群及调控机制尚不清楚。研究目的:本研究旨在通过scRNA-seq技术结合多组学分析及体内外功能实验,探究与CRC转移相关CAFs亚群COL11A1~+CAFs的分子特征,阐明其细胞起源及促转移的分子机制,为转移性CRC的精准治疗提供新靶点。研究方法:1.鉴定并验证CRC转移关键CAFs亚群(1)通过scRNA-seq分析临床收集的3例CRC未转移癌、2例CRC淋巴结转移原发灶、2例CRC肝转移原发灶和2例CRC肝转移灶组织样本,识别在CRC转移中发挥关键作用的CAFs亚群。(2)通过公共数据集分析和组织芯片实验检测COL11A1在CRC中的表达水平;通过组织多色免疫荧光实验,检测COL11A1在CRC原位癌和转移原发灶组织中表达水平和定位。(3)通过流式细胞术从新鲜CRC肿瘤组织样本中分选COL11A1~+CAFs;构建COL11A1~+CAFs与CRC细胞共培养体系,通过划痕实验和Transwell实验检测COL11A1~+CAFs对CRC迁移和侵袭能力的影响。(4)通过Western blot实验检测COL11A1~+CAFs对CRC细胞EMT关键信号分子表达的影响。(5)构建BALB/c裸鼠经脾肝转移模型,检测COL11A1~+CAFs在体内对CRC肝转移肿瘤形成的影响。2.探究COL11A1~+CAFs亚群的起源与形成机制(1)通过差异表达分析肠道特异性稳态成纤维细胞标志基因在各CAFs亚群中表达情况,识别肠道稳态成纤维细胞亚群;通过基于分区的图抽象算法PAGA分析和轨迹分析,探究各CAFs亚群分化轨迹。(2)通过配体-受体相互作用分析和肿瘤细胞分泌因子与CAFs亚群相关性分析,探究肿瘤细胞如何调控COL11A1~+CAFs形成。(3)通过SCENIC转录因子活性分析和ATAC-seq分析COL11A1~+CAFs中转录因子激活情况;Ch IP-q PCR实验探究促进COL11A1~+CAFs形成的关键转录因子。3.分析COL11A1~+CAFs调控CRC转移的分子机制(1)通过转录组测序及差异表达基因相关GSEA分析,探究COL11A1~+CAFs促进CRC转移的关键靶基因和下游调控通路。(2)通过Western blot实验和免疫组化实验检测LAMC2在CRC中的表达水平;采用CRISPR-Cas9技术敲除CRC细胞中LAMC2,划痕实验和Transwell实验检测COL11A1~+CAFs通过LAMC2调控CRC细胞转移的能力。(3)构建BALB/c裸鼠经脾肝转移模型,探究COL11A1~+CAFs通过调控LAMC2在体内对CRC肝转移肿瘤形成的影响。(4)通过配体-受体相互作用分析,探究COL11A1~+CAFs亚群分泌的COL11A1上调LAMC2的机制;通过GSEA分析和Western blot实验,探究COL11A1~+CAFs亚群通过LAMC2促进CRC细胞转移的下游关键调控通路。(5)通过小分子化合物库虚拟筛选、IC50实验和CRC类器官模型,筛选靶向LAMC2蛋白抑制剂。(6)通过划痕实验、Transwell实验检测靶向LAMC2蛋白的抑制剂F3385-1948对COL11A1~+CAFs调控CRC细胞侵袭和迁移能力的影响;构建BALB/c裸鼠经脾肝转移模型,评估小分子抑制剂F3385-1948通过靶向LAMC2对COL11A1~+CAFs调控CRC体内肝转移肿瘤形成的影响。研究结果:1.鉴定并验证CRC转移关键CAFs亚群scRNA-seq分析发现COL11A1~+CAFs亚群在CRC转移灶中呈现高比例分布;COL11A1在CRC转移灶中呈现更高比例分布中高表达,且与预后不良相关;COL11A1~+CAFs亚群条件培养基中COL11A1分泌水平升高;体内外实验结果显示COL11A1~+CAFs促进CRC细胞迁移、侵袭和EMT关键信号通路分子表达,并且在体内促进CRC肝转移肿瘤的形成。2.COL11A1~+CAFs亚群的起源与形成机制差异表达分析结果显示HSD17B2~+CAFs和PI16~+CAFs细胞亚群核心高表达肠道特异性稳态成纤维细胞标志基因;HSD17B2~+CAFs和PI16~+CAFs亚群朝向COL11A1~+CAFs亚群分化;肿瘤细胞通过分泌BMP2/4与CAFs细胞表面受体BMPR2作用,促进COL11A1~+CAFs细胞亚群形成;转录因子LEF1在COL11A1~+CAFs亚群中显著激活;体外实验加入外源性BMP2/4重组蛋白后,CAFs中COL11A1启动子上LEF1的富集水平显著升高。3.COL11A1~+CAFs通过靶向LAMC2调控CRC转移的分子机制转录组测序数据分析及组织芯片结果显示LAMC2在CRC肿瘤组织中高表达,且与患者不良预后相关;敲除LAMC2后,可显著抑制COL11A1~+CAFs亚群促进CRC细胞迁移能力、侵袭能力和EMT相关信号分子表达的水平;敲除LAMC2后可显著抑制COL11A1~+CAFs促进CRC体内肝转移肿瘤的形成;COL11A1~+CAFs分泌的COL11A1可与CRC细胞表面受体整合素α2β1(Integrinα2β1)结合,促进LAMC2表达;敲除LAMC2可显著抑制CRC细胞中Wnt/β-catenin信号通路激活;LAMC2小分子化合物库虚拟筛选出182类LAMC2蛋白小分子抑制剂;小分子抑制剂F3385-1948对多种CRC细胞系均表现出显著的剂量依赖性增殖抑制作用,诱导CRC类器官凋亡细胞比例显著增加;F3385-1948可显著抑制COL11A1~+CAFs亚群促进CRC细胞迁移能力、侵袭能力,并抑制COL11A1~+CAFs促进CRC细胞体内转移的能力。研究结论:本研究发现COL11A1~+CAFs亚群在CRC转移中起重要作用。HSD17B2~+CAFs和PI16~+CAFs亚群是COL11A1~+CAFs亚群的起源细胞亚群。CRC细胞分泌的BMP2/4可激活CAFs中转录因子LEF1,诱导COL11A1表达,从而促进COL11A1~+CAFs亚群的形成。COL11A1~+CAFs亚群可通过分泌COL11A1与CRC细胞表面Integrinα2β1结合,上调LAMC2表达,进而激活Wnt/β-catenin信号通路,最终驱动CRC侵袭与转移。通过小分子抑制剂F3385-1948靶向LAMC2蛋白,可抑制CRC细胞的转移能力,为转移性CRC治疗提供新的潜在靶点。更多还原

【Abstract】 Background:Colorectal cancer(CRC)ranks among the top three malignancies globally in terms of both incidence and mortality,with metastasis being the primary cause of poor patient prognosis.The tumor microenvironment(TME)plays a critical role in CRC metastasis,and cancer-associated fibroblasts(CAFs)are a key component of the TME.CAFs exhibit high heterogeneity,and distinct CAFs subpopulations contribute differentially to tumor progression.Single-cell RNA sequencing(sc RNA-seq)has been instrumental in revealing CAFs heterogeneity.Collagen Type XI Alpha 1 Chain(COL11A1),a fibrillar collagen critical for extracellular matrix(ECM)function,is highly expressed in multiple cancers and associated with tumor invasion and metastasis.However,the specific cellular subpopulations expressing COL11A1 in the CRC metastatic microenvironment and the molecular mechanisms regulating CRC metastasis remain unclear.Objectives:This study aims to investigate the molecular characteristics and cellular origin of the COL11A1~+CAFs subpopulation associated with CRC metastasis through sc RNA-seq,multi-omics analyses,and functional experiments,and to elucidate its pro-metastatic mechanisms,thereby providing novel therapeutic targets for CRC metastasis.Methods:1.Identification and Validation of of Key CAFs Subpopulations in CRC Metastasis(1)sc RNA-seq analysis of clinical samples,including 3 primary CRC tumors,2 primary tumors with lymph node metastasis,2 primary tumors with liver metastasis,and 2 liver metastatic lesions,to identify CAFs subpopulations critical for CRC metastasis.(2)Validation of COL11A1 expression levels in CRC using public datasets and tissue microarrays;localization of COL11A1 in primary and metastatic CRC tissues via multiplex immunofluorescence.(3)Isolation of COL11A1~+CAFs from fresh CRC tissues by flow cytometry;establishment of co-culture systems with CRC cells to assess their impact on migration and invasion using scratch and Transwell assays.(4)Western blot analysis of EMT-related signaling molecules in CRC cells influenced by COL11A1~+CAFs.(5)Construction of a BALB/c nude mouse splenic-liver metastasis model to evaluate the regulatory role of COL11A1~+CAFs in CRC metastasis in vivo.2.Investigation of the Origin and Formation Mechanisms of COL11A1~+CAFs(1)Differential expression analysis of intestinal homeostatic fibroblast marker genes across CAFs subpopulations to identify homeostatic subpopulations;PAGA and trajectory analysis to map CAFs differentiation pathways.(2)Ligand-receptor interaction analysis and correlation studies between tumor-secreted factors and CAFs subpopulations to explore tumor cell regulation of COL11A1~+CAFs formation.(3)Transcription factor(TF)enrichment analysis and ATAC-seq to assess TF activation in COL11A1~+CAFs.Ch IP-q PCR to validate key TFs driving COL11A1~+CAFs formation.3.COL11A1~+CAFs Regulate CRC Metastasis via LAMC2-Targeted Mechanisms(1)Transcriptomic sequencing and GSEA to identify key target genes(and downstream pathways regulated by COL11A1~+CAFs.(2)Western blot and immunohistochemistry to detect LAMC2 expression in CRC.CRISPR-Cas9-mediated LAMC2 knockout to assess its role in COL11A1~+CAFs-driven CRC cell migration,invasion,and EMT signaling.(3)BALB/c nude mouse splenic-liver metastasis model to evaluate LAMC2-dependent regulation of CRC metastasis by COL11A1~+CAFs.(4)Ligand-receptor interaction analysis to elucidate COL11A1-driven LAMC2upregulation.(5)Virtual screening of small molecule compound libraries,IC50 assays,and CRC organoid models were employed to screen for LAMC2-targeting inhibitors.(6)Wound healing and Transwell assays were performed to evaluate the effects of LAMC2-targeting inhibitor F3385-1948 on COL11A1~+CAFs-regulated CRC cell migration and invasion.BALB/c nude mouse splenic-liver metastasis model was established to assess the impact of F3385-1948 on COL11A1~+CAFs-mediated CRC liver metastasis formation via LAMC2targeting.Results:1.Identification and Validation of of Key CAFs Subpopulations in CRC MetastasisThe sc RNA-seq analysis identified COL11A1~+CAFs as a key subpopulation enriched in CRC metastatic lesions.COL11A1 expression was significantly higher in metastatic tissues and correlated with poor prognosis.The conditioned medium from COL11A1~+CAFs increased COL11A1 secretion levels.In vitro and in vivo experiments demonstrated that COL11A1~+CAFs promoted CRC cell migration,invasion,and expression of EMT-related signaling molecules,while enhancing the formation of liver metastasis in nude mice.2.Investigation of the Origin and Formation Mechanisms of COL11A1~+CAFsHSD17B2~+CAFs and PI16~+CAFs express homeostatic fibroblast markers.These subpopulations differentiate into COL11A1~+CAFs.Tumor-secreted BMP2/4 interacts with BMPR2 on CAFs to promote COL11A1~+CAFs formation.Transcription factor LEF1 is activated in COL11A1~+CAFs.Exogenous BMP2/4 increases LEF1 enrichment at the COL11A1 promoter.3.COL11A1~+CAFs Regulate CRC Metastasis via LAMC2-Targeted MechanismsTranscriptomic sequencing and tissue microarrays confirmed high LAMC2 expression in CRC tissues,which was associated with poor prognosis.CRISPR-Cas9-mediated LAMC2knockout suppressed COL11A1~+CAFs-induced CRC cell migration,invasion,and EMT signaling.In vivo,LAMC2 knockout inhibited COL11A1~+CAFs-driven liver metastasis.COL11A1 secreted by COL11A1~+CAFs bound to Integrinα2β1 on CRC cells,upregulating LAMC2 expression and activating the Wnt/β-catenin pathway.Virtual screening identified 182LAMC2 inhibitors,with F3385-1948 showing dose-dependent inhibition of CRC cell proliferation,induction of organoid apoptosis,and suppression of metastasis in vitro and in vivo.Conclusion:This study demonstrates that the COL11A1~+CAFs subpopulation plays a critical role in CRC metastasis.The HSD17B2~+CAFs and PI16~+CAFs subpopulations serve as the cellular origins of COL11A1~+CAFs.CRC cell-secreted BMP2/4 activates the transcription factor LEF1in CAFs,thereby inducing COL11A1 expression and promoting the formation of the COL11A1~+CAFs subpopulation.The COL11A1~+CAFs subpopulation secretes COL11A1 to bind Integrinα2β1 on CRC cells,upregulating LAMC2 expression and subsequently activating the Wnt/β-catenin signaling pathways,thereby driving CRC invasion and metastasis.Targeting LAMC2 with the small-molecule inhibitor F3385-1948 effectively suppresses CRC metastasis,providing a novel therapeutic strategy for metastatic CRC.更多还原