Table_13_Transcriptomic Profiling of In Vitro Tumor-Stromal Cell Paracrine Crosstalk Identifies Involvement of the Integrin Signaling Pathway in the Pathogenesis of Mesenteric Fibrosis in Human Small Intestinal Neuroendocrine Neoplasms.docx

AimAnalysis of the pathophysiology of mesenteric fibrosis (MF) in small intestinal neuroendocrine tumors (SI-NETs) in an in vitro paracrine model and in human SI-NET tissue samples.
MethodsAn indirect co-culture model of SI-NET cells KRJ-I and P-STS with stromal cells HEK293 was designed to evaluate the paracrine effects on cell metabolic activity, gene expression by RT2 PCR Profilers to analyse cancer and fibrosis related genes, and RNA sequencing. The integrin signaling pathway, a specific Ingenuity enriched pathway, was further explored in a cohort of human SI-NET tissues by performing protein analysis and immunohistochemistry.
ResultsRT Profiler array analysis demonstrated several genes to be significantly up- or down-regulated in a cell specific manner as a result of the paracrine effect. This was further confirmed by employing RNA sequencing revealing multiple signaling pathways involved in carcinogenesis and fibrogenesis that were significantly affected in these cell lines. A significant upregulation in the expression of various integrin pathway – related genes was identified in the mesenteric mass of fibrotic SI-NET as confirmed by RT-qPCR and immunohistochemistry. Protein analysis demonstrated downstream activation of the MAPK and mTOR pathways in some patients with fibrotic SI-NETs.
ConclusionThis study has provided the first comprehensive analysis of the crosstalk of SI-NET cells with stromal cells. A novel pathway – the integrin pathway – was identified and further validated and confirmed in a cohort of human SI-NET tissue featured by a dual role in fibrogenesis/carcinogenesis within the neoplastic fibrotic microenvironment.

研究目的：本研究旨在通过体外旁分泌模型与人体小肠神经内分泌肿瘤（small intestinal neuroendocrine tumors, SI-NETs）组织样本，探讨小肠神经内分泌肿瘤相关肠系膜纤维化（mesenteric fibrosis, MF）的病理生理学机制。
研究方法：本研究构建小肠神经内分泌肿瘤细胞KRJ-I、P-STS与基质细胞HEK293的间接共培养模型，以评估旁分泌效应对细胞代谢活性的影响；通过RT2 PCR Profilers分析癌症及纤维化相关基因的表达，并开展RNA测序（RNA sequencing）。针对经Ingenuity富集得到的特异性整合素信号通路（integrin signaling pathway），本研究通过蛋白质分析与免疫组化（immunohistochemistry），在一组人体小肠神经内分泌肿瘤组织队列中展开进一步探究。
研究结果：RT2 Profiler芯片分析显示，旁分泌效应可使部分基因以细胞特异性方式出现显著上调或下调。RNA测序进一步验证了这一结果，揭示出这些细胞系中多个参与癌变及纤维化发生的信号通路受到显著影响。经实时定量PCR（RT-qPCR）与免疫组化验证，纤维化型小肠神经内分泌肿瘤的肠系膜病灶中，多种整合素通路相关基因的表达显著上调。蛋白质分析显示，部分纤维化型小肠神经内分泌肿瘤患者体内存在丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）与哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）通路的下游激活现象。
研究结论：本研究首次全面分析了小肠神经内分泌肿瘤细胞与基质细胞之间的交互对话。本研究鉴定出一条全新通路——整合素通路（integrin pathway），并在一组人体小肠神经内分泌肿瘤组织队列中对该通路进行验证与确认，该通路在肿瘤性纤维化微环境中同时参与纤维化发生与癌变过程，发挥双重作用。