IDDF2024-ABS-0121 Single-cell transcriptomic analysis of small intestinal neuroendocrine tumors revealed potential mechanisms of mesenteric fibrosis

<h3>Background</h3> Nearly half of patients with small intestinal neuroendocrine tumor (SI-NET) experienced local mesenteric fibrosis (MF), significantly impacting their prognosis. However, the mechanism by which SI-NET causes MF remains unclear. We aim to elucidate the molecular mechanisms underlying SI-NET fibrosis and explore potential therapeutic targets. <h3>Methods</h3> We conducted single-cell RNA sequencing (scRNA-seq) on 5 primary tumor specimens and their corresponding adjacent non-tumor tissues, integrating five public SI-NET scRNA-seq data. The results of the analyses were validated through spatial transcriptomics, bulk RNA-sequencing, immunohistochemistry staining, enzyme-linked immunosorbent assays and in vitro experiments (IDDF2024-ABS-0121 Figure 1 (A)). <h3>Results</h3> All samples were categorized into eight major cell lineages and further subdivided into 50 cell subclusters (IDDF2024-ABS-0121 Figure 1 (B-D)). Compared with SI-NET without MF (SINET_NF), fibrosis-related genes and pathways were significantly upregulated in tumor cells of SI-NET with MF (SINET_MF), among which Insulin-like Growth Factor Binding Protein 3 (IGFBP3) was especially overexpressed and abundantly secreted, indicating the potential of IGFBP3 as a biomarker to distinguish SI-NET patients with MF (IDDF2024-ABS-0121 2 (A-F)). Overexpression of IGFBP3 was positively correlated with stromal cells, which implied its involvement in stromal remodeling and angiogenesis (IDDF2024-ABS-0121 Figure 2 (G)). LUM+ fibroblasts (FIB_LUM), responsible for extracellular matrix synthesis, were significantly enriched in SINET_MF (IDDF2024-ABS-0121 Figure 3 (A-C)). IGFBP3 was demonstrated to bind to the TGFβ receptors on FIB_LUM, subsequently activating the TGFβ signaling pathway and promoting intestinal fibrosis, further validated through bulk RNA-sequencing and in vitro experiments (IDDF2024-ABS-0121 Figure 3 (D-F)). Additionally, EC_ESM1, which facilitates vascular budding, showed increased abundance in SINET_MF, with IGFBP3 likely enhancing their proliferation via the PI3K-AKT signaling pathway (IDDF2024-ABS-0121 Figure 4). Moreover, immature PVL cells may induce vascular leakage through the ANGPT2-TIE2 axis within the ECs of SINET_MF, which partially explains the increased invasiveness of tumor cells of SINET_MF (IDDF2024-ABS-0121 Figure 5 (A-E)). <h3>Conclusions</h3> We elucidated the molecular mechanisms underlying the specific secretion of IGFBP3 by tumor cells of SINET_MF, which participate in the regulation of extracellular matrix remodeling and angiogenesis (IDDF2024-ABS-0121 Figure 5 (F)). IGFBP3 might be a potential drug target and biomarker for SI-NET patients with MF.