DOI: 10.5061/dryad.gxd2547s0
title: Cadherin-11 deficiency-induced changes in pancreatic ductal adenocarcinoma

description: 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.
description: KPC-Cdh11+/+ and 
 KPC-Cdh11+/- cohorts (n&gt;=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
 <em>KPC-Cdh11<sup>+/+</sup></em>,
 <em>KPC-Cdh11<sup>+/-</sup></em> and
 <em>Cdh11<sup>+/-</sup> </em>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.
description: Both immune (CD45<sup>+</sup>) and stromal
 (CD45<sup>-</sup>) cell populations from
 KPC-Cdh11<sup>+/+</sup> and
 KPC-Cdh11<sup>+/-</sup> 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<sup>+/+</sup> mice. KPC-Cdh11.Het_Immune Zipped
 folder with cell ranger files (barcodes.tsv, genes.tsv and matrix.mtx)
 containing immune data from KPC-Cdh11<sup>+/-</sup> mice.
 KPC-Cdh11.WT_Stromal Zipped folder with cell ranger files (barcodes.tsv,
 genes.tsv and matrix.mtx) containing stromal data from
 KPC-Cdh11<sup>+/+</sup> mice. KPC-Cdh11.Het_Stromal Zipped
 folder with cell ranger files (barcodes.tsv, genes.tsv and matrix.mtx)
 containing stromal data from KPC-Cdh11<sup>+/- </sup>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](https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/output/matrices)