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PURPOSE: Small intestinal neuroendocrine tumors (SI-NET) frequently present as multifocal lesions, but the molecular mechanisms underlying their development and heterogeneity remain unclear. This study aimed to characterize the phenotypes of tumor cell populations across anatomic sites in patients with multifocal SI-NET and identify local microenvironmental factors influencing tumor development.EXPERIMENTAL DESIGN: Spatial transcriptomics was performed on 72 tissue microarray cores derived from 4 patients with multifocal SI-NETs that included tumoral and nontumoral tissues from various anatomic layers of the small intestine and regional metastatic sites. Unsupervised clustering, overrepresentation analysis, and ligand-receptor (L-R) pair analysis were used to define the tumor cell subtypes and associated signaling networks. External datasets were used for validation. Protein expression of selected genes was evaluated by immunohistochemistry and immunofluorescence.RESULTS: Unsupervised clustering revealed four major tumor cell subtypes: "mucosal," "mesenteric," "lymphatic," and "deep," based on their anatomic location and transcriptomic profiles. Each subtype exhibited distinct gene expression patterns and L-R interactions. The "mesenteric" and "lymphatic" subtypes exhibited distinct L-R pairs, such as NRG1-ERBB3 (HER3) and CXCL12-CXCR4, respectively. 5HT-HTR1D was found in all subtypes except "mucosal." Across the four subtypes, SST-SSTR1/2, PTN-NCL, MDK-NCL, and GJD2-GJD2 were consistently detected, suggesting fundamental roles in SI-NET biology.CONCLUSIONS: Although further validation is needed, our findings indicate that multifocal SI-NETs consist of spatially distinct tumor cell subtypes affected by local cellular interactions, providing insight into SI-NET intratumoral heterogeneity, possible microenvironment-triggered tumorigenesis, and potential subtype-targeted therapeutic strategies.