Metformin inhibits small intestinal neuroendocrine tumor growth in vivo

BACKGROUND: Small intestinal neuroendocrine tumors (SI-NETs) are slow-growing but highly metastatic, with most patients presenting metastases at diagnosis. Radical surgery remains the only potential curative option when feasible. Consequently, there is a critical need for novel therapeutic strategies that can limit tumor progression and enable more personalized treatment approaches in combination with current clinical practices. In this study, we evaluated the impact of metformin on SI-NET cell growth in vivo, characterized the associated microRNA expression profile, and identified potential driver genes modulated by metformin treatment. METHODS: A total of 22 SI-NET xenograft mouse models were established using CNDT2.5 and GOT1 cells. Mice were treated with metformin (2.56 mg/mL in drinking water) or water as control for 4 weeks. To explore the molecular impact of metformin, both small-RNA and total-RNA sequencing were performed on the dissected xenograft tumors. Proliferation and apoptosis were further evaluated by immunohistochemistry. RESULTS: In vivo treatment of SI-NET cells with metformin led to a reduction in tumor size in both CNDT2.5 and GOT1 xenograft models. Our sequencing analyses identified seven altered microRNAs and 1,776 differentially expressed genes in metformin-treated tumors compared to controls. To uncover potential driver genes in SI-NETs affected by metformin, we compared the differentially expressed genes from GOT1 xenograft model with those identified by comparing single-cell RNA profile of enterochromaffin cells to SI-NETs. This novel approach revealed a set of significantly regulated genes, including those involved in tumor proliferation, apoptosis, and metastasis, as well as genes related to voltage-gated calcium channels and signal transduction. CONCLUSIONS: Our novel findings support further investigation of metformin as a potential therapeutic agent in clinical trials for SI-NET patients, and suggest that identified miRNAs should be assessed as potential predictive biomarkers for metformin treatment. This study highlights novel candidate driver genes affected by metformin, which are associated with key cellular processes and enterochromaffin cell's function, offering insights into the underlying mechanisms in SI-NETs.