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| Status |
Public on Nov 14, 2024 |
| Title |
Single-cell RNA-sequencing of human spleens reveals an IDO-1+ Tolerogenic Dendritic Cell subset in pancreatic cancer patients that is absent in normal individuals. |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Local and systemic immunosuppression are prominent features of pancreatic cancer, rendering anti-tumor effector cells inactive and immunotherapeutic approaches ineffective. The spleen, an understudied point of antigen-presentation and T cell priming in humans, holds particular importance in pancreatic cancer due to its proximity to the developing tumor. As main effectors of antigen presentation, dendritic cells display antigens to lymphocytes, thereby bridging the innate and adaptive immune response. While tumor-infiltrating anti-inflammatory dendritic cells have been described, splenic dendritic cells have historically just been considered to stimulate the anti-tumor immune response. Here, we describe, for the first time, the presence of an immunosuppressive, tolerogenic IDO1+ dendritic cell subset in the spleens of pancreatic cancer patients that likely contributes to systemic immunosuppression that is associated with pancreatic ductal adenocarcinoma. Network analysis of scRNA seq data reveals extensive communication networks between the identified tolerogenic DC cluster and numerous immune cell populations in the spleen. Interactions with innate and adaptive immune cells suggest a broad influence on leukocyte trafficking and immune regulation within the spleen microenvironment. The identification of signaling pathways involving AHR and IDO-1, CCL19, NECTIN2, CLEC2D, and others elucidates potential mechanisms underlying the immunosuppressive functions of this cell type.
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| Overall design |
We performed single-cell RNA-sequencing (scRNA-seq.) of primary human spleens and compared the immune cell signatures of spleens from 4 PDAC patients with the spleens of 3 normal individuals. PDAC patient spleen samples were collected on-site during distal pancreatectomy with splenectomy and processed within 2 h. Normal sample spleen processing began approximately three to 4 h post-mortem. Spleens were diced and stomached in the Stomacher 80 Biomaster (Seward TM) according to the manufacturer’s protocol. After red blood cell lysis, dead cells were removed using Dead Cell Removal MicroBeads (Miltenyi; Catalog-No.: 130-090-101). Splenocytes were given to the UNMC Genomics Core Facility to generate the scRNA-eq libraries (10x Genomics' Chromium Single Cell Platform 3′ Version 3), following standard protocol. Transcript reads were aligned to the GRCh38 Human Genome reference using the Cell Ranger 3.0.0 pipeline (10x Genomics, Inc.) to generate the matrix files containing cell barcodes, UMIs (unique molecular identifiers), and transcript counts. UMI count matrices were generated for each sample and imported into Seurat R toolkit (Version 4.September 9, 9049). Only those genes expressed in more than three3 cells and cells that expressed more than 250 genes were kept; low-quality cells (≤250 genes/cell, ≤500 UMIs/cells, and ≥20 % mitochondrial genes) were excluded. A total of 30,864 cells in all samples were analyzed. The expression data was normalized and scaled using default settings. Principal component analysis (PCA) was performed based on 2000 highly variable genes for dimensionality reduction. Data integration of PDAC and normal spleen samples was performed using the anchor-based Canonical Correlation Analysis (CCA) integration method using Seurat v5. Cell type identification and annotation in the generated dataset were performed by reference-based mapping using the Azimuth v0.5.0 package within R. Briefly, we utilized the Tabula Sapiens normal human spleen scRNAseq dataset as reference dataset for this purpose.
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| Contributor(s) |
Hollingsworth MA, Mundry CS, Triplett AA, Grandgenett PM, Shah OS, Chaitankar V, McAndrews KL, Mehla K, Ly QP, Cox JL, Eberle KC, Swanson BJ, Lazenby A, Klute KA |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
| BioProject |
PRJNA1033837 |
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| Submission date |
Nov 13, 2023 |
| Last update date |
Nov 15, 2024 |
| Contact name |
Michael A Hollingsworth |
| Organization name |
University of Nebraska Med Center
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| Department |
Eppley Cancer Institute
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| Street address |
BCC 8.12.384, 601 S Saddle Creek Road
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| City |
Omaha |
| State/province |
NE |
| ZIP/Postal code |
68106-6805 |
| Country |
USA |
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| Platforms (1) |
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| Samples (7)
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| Supplementary file |
Size |
Download |
File type/resource |
| GSE247635_RAW.tar |
207.4 Mb |
(http)(custom) |
TAR (of MTX, TSV) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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