ScRNA-seq of Small Intestine Neuroendocrine Tumors

Here we profiled ten low-grade small intestine NET (SiNET) tumor samples as well as one mixed lung tumor by single-cell or single-nuclei RNA-seq. We find that SiNETs are largely separated into two distinct subtypes, in which the neuroendocrine cells upregulate epithelial or neuronal markers, respectively. Surprisingly, in both subtypes the neuroendocrine cells are largely non-proliferative while higher proliferation is observed in multiple non-malignant cell types. Specifically, B and plasma cells are highly proliferative in the epithelial-like SiNET subtype, potentially reflecting the outcome of high Migration Inhibitory Factor (MIF) expression in those tumors, which may constitute a relevant target. Finally, our analysis of a mixed lung neuroendocrine tumor identifies a population of putative progenitor cells that may give rise to both neuroendocrine and non-neuroendocrine (squamous) cells, potentially explaining the origin of the mixed histology. Taken together, our results provide important insights and hypotheses regarding the biology of neuroendocrine neoplasms. Fresh tumor samples were collected at the time of surgery and processed immediately. Tumors were minced and enzymatically dissociated using a tumor dissociation kit (Miltenyi Biotec) in a GentleMACS Octo-dissociator at low speed. A single-cell suspension was obtained by filtering through a 60-100 µm cell strainer, followed by RBC removal (Roche) and dead cell removal (Miltenyi). Cell viability and density were assessed using Trypan blue staining and a Countess II automated hemocytometer. 10x Genomics Single-Cell Chromium Controller was used to load 8,000-10,000 cells per channel. For frozen tumors, dissociation was performed with nuclei isolation using ST-based buffers (Slyper et al.) or the EZ-lysis method (Sigma). Tissue was homogenized in ice-cold buffer using a Dounce tissue grinder (Sigma), followed by centrifugation (500g, 4°C), washing, and resuspension in a PBS-based buffer with BSA and RNase inhibitor. Nuclei suspensions were filtered through a 40 µm strainer and counted, with a final concentration of 1,000 nuclei/µl for 10x Chromium loading. For library preparation, the 10x Genomics Chromium Next GEM Single Cell 3′ GEM, Library & Gel Bead Kit v3.1 was used for fresh tumor/single-cell samples, while the Chromium Next GEM Single 5′ GEM was used for frozen tumor/single-nuclei samples. Libraries were generated following the standard 10x Genomics single-cell RNA-seq protocol, including reverse transcription, cDNA amplification, fragmentation, and adapter/sample index attachment. Sequencing was performed on an Illumina NovaSeq-6000 using either: SP-100 kit (2 pooled libraries per lane) S1-100 kit (4 pooled libraries per two lanes) Read lengths: Read 1: 26 nt, Read 2: 55 nt, Index 1: 8 nt, Index 2: 0 nt. *************************************************************** Raw files for human/patient samples were not submitted to GEO due to concerns about submitting personally identifiable sequence data for open access. ***************************************************************