Cadherin-11 deficiency-induced changes in pancreatic ductal adenocarcinoma
Data files
Oct 26, 2023 version files 189.20 MB
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KPC_Cdh11.Het_Stromal.zip
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KPC-Cdh11.Het_Immune.zip
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KPC-Cdh11.WT_Immune.zip
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KPC-Cdh11.WT_Stromal.zip
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README.md
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is one of the top five deadliest forms of cancer with very few treatment options. The 5-year survival rate for PDAC is 10% following diagnosis. Cadherin 11 (Cdh11), a cell-to-cell adhesion molecule, has been suggested to promote tumor growth and immunosuppression in PDAC, and Cdh11 inhibition significantly extended survival in mice with PDAC. However, the mechanisms by which Cdh11 deficiency influences PDAC progression and anti-tumor immune responses have yet to be fully elucidated. To investigate Cdh11-deficiency induced changes in PDAC tumor microenvironment (TME), we crossed p48-Cre; LSL-KrasG12D/+; LSL-Trp53R172H/+ (KPC) mice with Cdh11+/- mice and performed single-cell RNA sequencing (scRNA-seq) of the non-immune (CD45-) and immune (CD45+) compartment of KPC tumor-bearing Cdh11 proficient (KPC-Cdh11+/+) and Cdh11 deficient (KPC-Cdh11+/-) mice. Our analysis showed that Cdh11 is expressed primarily in cancer-associated fibroblasts (CAFs) and at low levels in epithelial cells undergoing epithelial-to-mesenchymal transition (EMT). Cdh11 deficiency altered the molecular profile of CAFs, leading to a decrease in the expression of myofibroblast markers such as Acta2 and Tagln and cytokines such as Il6, Il33 and Midkine (Mdk). We also observed a significant decrease in the presence of monocytes/macrophages and neutrophils in KPC-Cdh11+/- tumors while the proportion of T cells was increased. Additionally, myeloid lineage cells from Cdh11-deficient tumors had reduced expression of inflammatory cytokines that have previously been shown to play a role in immune suppression. In summary, our data suggests that Cdh11 deficiency significantly alters the fibroblast and immune microenvironments and contributes to the downregulation of inflammatory cytokines, leading to an increase in anti-tumor immunity and enhanced survival.
README
Both immune (CD45+) and stromal (CD45-) cell populations from KPC-Cdh11+/+ and KPC-Cdh11+/- mice were independently sequenced using a Chromium Single Cell 3' Reagent Kit and Chromium instrument (10X Genomics, Pleasanton, CA). Single-cell sequencing data was then analyzed using Cell Ranger (10x Genomics) and Seurat R package.
Datasets included:
KPC-Cdh11.WT_Immune Zipped folder with cell ranger files (barcodes.tsv, genes.tsv and matrix.mtx) containing immune data from KPC-Cdh11+/+ mice.
KPC-Cdh11.Het_Immune Zipped folder with cell ranger files (barcodes.tsv, genes.tsv and matrix.mtx) containing immune data from KPC-Cdh11+/- mice.
KPC-Cdh11.WT_Stromal Zipped folder with cell ranger files (barcodes.tsv, genes.tsv and matrix.mtx) containing stromal data from KPC-Cdh11+/+ mice.
KPC-Cdh11.Het_Stromal Zipped folder with cell ranger files (barcodes.tsv, genes.tsv and matrix.mtx) containing stromal data from KPC-Cdh11+/- mice.
A description of the file type and the variables present in each column is given here, https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/output/matrices
Methods
KPC-Cdh11+/+ and KPC-Cdh11+/- cohorts (n>=4) were euthanized at 4-5 months of age. Pancreases were excised and single cell suspensions were prepared using a collagenase digestion solution (3 mg/ml Collagenase I (ThermoFisher Scientific Catalog #17018029), 100 µg/mL DNase I (Roche Catalog #11284932001), Dispase II (Roche Catalog #4942078001) in RPMI1640 media supplemented with 10% FBS (ThermoFisher Scientific, Catalog #11875101) for 1 hour with shaking at 37°C. Digests were filtered through a 100 µm cell strainer and depleted of red blood cells using ACK lysis buffer, per manufacturer guidelines (Gibco, Catalog #A1049201). Remaining cells were analyzed for viability and CD45 expression. CD45-/7-AAD- (non-immune) and CD45+/7-AAD- (immune) cells populations were sorted via fluorescently activated cell sorting (FACS) and collected for scRNA-seq preparation. Both immune (CD45+) and non-immune (CD45-) cell populations were independently sequenced using a Chromium Single-cell libraries were prepared from sorted cell populations from KPC-Cdh11+/+, KPC-Cdh11+/- and Cdh11+/- mice using the Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3 (10x Genomics, Pleasanton, CA, USA; catalog no. 1000075) on a 10x Genomics Chromium Controller following manufacturers protocol and sequenced using an Illumina (San Diego, CA, USA) NextSeq 500 sequencer. The scRNA-seq data was demultiplexed and aligned against mouse reference genome mm10 using Cell Ranger Single-Cell Software Suite (10x Genomics, Pleasanton, CA, USA) to obtain Unique Molecular Identifier (UMI) counts.