# Single-Cell Comparison of Small Intestinal Neuroendocrine Tumors and Enterochromaffin Cells from Two Patients

## TL;DR

This study compares cancerous gut cells with normal hormone-producing cells at the single-cell level to uncover how these tumors develop and change functionally.

## Contribution

The study uses single-cell RNA sequencing to identify tumor-specific gene activity changes and functional reprogramming in small intestinal neuroendocrine tumors.

## Key findings

- Tumor cells lose traits of hormone-producing intestinal cells and gain traits of nerve cells.
- SI-NET cells are transcriptionally similar to serotonergic enterochromaffin cells.
- Functional reprogramming involves loss of epithelial differentiation and gain of neuronal signaling.

## Abstract

Small intestinal neuroendocrine tumors develop from hormone-producing cells in the gut, yet the changes that cause these cells to become cancerous are still not well understood. Most earlier studies analyzed whole tissue samples, which makes it difficult to separate signals coming from normal cells from those of hormone-producing normal cells. In this study, we examined tumors at the level of individual cells and compared them directly with their corresponding normal hormone-producing counterparts. This allowed us to identify gene activity changes that are specific to the tumor cells. We discovered that the tumor cells lose traits that are typical of endocrine intestinal cells and gain traits that are more commonly associated with nerve cells. These shifts suggest that the tumor cells undergo functional reprogramming as they develop. Our findings offer new insight into the tumor biology of our patients and may help us guide future research into targeted treatment strategies.

Background: Several studies have attempted to identify the initiating drivers of small intestinal neuroendocrine tumor (SI-NET) development and the molecular mechanisms underlying their progression and metastatic spread. Previous gene expression studies have used bulk microarrays or RNA sequencing to compare tumor tissue with normal intestinal mucosa. However, the intestine comprises multiple distinct cell types, and bulk analyses are limited by this cellular heterogeneity, which can confound tumor-specific signals. Methods: We performed single-cell RNA sequencing on primary SI-NETs and paired normal mucosa from two patients to directly compare tumor cells with their cells of origin, the enterochromaffin (EC) cells. To minimize type I errors, we applied a two-step validation strategy by overlapping differentially expressed genes with an external single-cell dataset and cross-referencing candidate genes for enteroendocrine expression in the Human Protein Atlas. Results: For further distinction and characterization, ECs were subdivided into serotonergic and non-serotonergic clusters. This analysis revealed that the SI-NET cells are transcriptionally more similar to serotonergic ECs, consistent with serum metabolite profiles derived from clinical parameters. Our analyses uncovered a loss-of-expression program characterized by regulators of epithelial differentiation and in parallel, a gain-of-expression program displayed neuronal signaling gene induction, implicating functional reprogramming toward neuronal-like properties. Together, these specific losses and gains suggest that our patient-derived SI-NETs undergo adaptation through both loss of enteroendocrine functions and acquisition of neurobiological-promoting signaling pathways. Conclusions: These findings nominate candidate drivers for further functional validation and highlight potential therapeutic strategies in our patient cohort, including restoring suppressed Notch signaling and targeting aberrant neuronal signaling networks. However, even with a two-step validation procedure, the modest cohort size limits statistical power and generalizability, particularly for the proposed association to a serotonergic phenotype. Larger, multi-patient single-cell studies are required to confirm these mechanisms and establish their clinical relevance.