DOI: 10.6071/m3hm32
title: Single cell RNA sequencing (scRNAseq) of transplanted mT3 tumors

description: Background & Aims: Pancreatic ductal adenocarcinomas
 (PDAC) are characterized by fibrosis and an abundance of cancer-associated
 fibroblasts (CAFs). We investigated strategies to disrupt interactions
 among CAFs, the immune system, and cancer cells, focusing on adhesion
 molecule cadherin 11 (CDH11), which has been associated with other
 fibrotic disorders and is expressed by activated fibroblasts.
 Methods: We compared levels of CDH11mRNA in human
 pancreatitis and pancreatic cancer tissues and cells, compared with normal
 pancreas, and measured levels of CDH11 protein in human and mouse
 pancreatic lesions and normal tissues. We crossed
 p48-Cre;LSL-KrasG12D/+;LSL-Trp53R172H/+(KPC) mice with CDH11-knockout mice
 and measured survival times of offspring. Pancreata were collected and
 analyzed by histology, immunohistochemistry, and (single-cell) RNA
 sequencing; RNA and proteins were identified by imaging mass cytometry.
 Some mice were given injections of PD1 antibody or gemcitabine and
 survival was monitored. Pancreatic cancer cells from KPC mice were
 subcutaneously injected
 into Cdh11+/+ and Cdh11–/– mice and
 tumor growth was monitored. Pancreatic cancer cells (mT3) from KPC mice
 (C57BL/6), were subcutaneously injected
 into Cdh11+/+ (C57BL/6J) mice and mice were given
 injections of antibody against CDH11, gemcitabine, or small molecule
 inhibitor of CDH11 (SD133) and tumor growth was monitored.
 Results: Levels of CDH11mRNA and protein were
 significantly higher in CAFs than in pancreatic cancer epithelial cells,
 human or mouse pancreatic cancer cell lines, or immune cells.
 KPC/Cdh11+/– and KPC/Cdh11–/– mice survived
 significantly longer than KPC/Cdh11+/+ mice. Markers of stromal
 activation entirely surrounded pancreatic intraepithelial neoplasias in
 KPC/Cdh11+/+ mice and incompletely in KPC/Cdh11+/– and
 KPC/Cdh11–/– mice, whose lesions also contained fewer FOXP3+cells
 in the tumor center. Compared with pancreatic tumors
 inKPC/Cdh11+/+ mice, tumors of KPC/Cdh11+/– mice had
 increased markers of antigen processing and presentation; more lymphocytes
 and associated cytokines; decreased extracellular matrix components; and
 reductions in markers and cytokines associated with immunosuppression.
 Administration of the PD1 antibody did not prolong survival of KPC mice
 with 0, 1, or 2 alleles of Cdh11. Gemcitabine extended survival
 only of KPC/Cdh11+/– and KPC/Cdh11–/– mice or reduced
 subcutaneous tumor growth in mT3 engrafted Cdh11+/+ mice
 given in combination with the CDH11 antibody. A small molecule inhibitor
 of CDH11 reduced growth of pre-established mT3 subcutaneous tumors only if
 T and B cells were present in mice. Conclusions: Knockout or
 inhibition of CDH11, which is expressed by CAFs in the pancreatic tumor
 stroma, reduces growth of pancreatic tumors, increases their response to
 gemcitabine, and significantly extends survival of mice. CDH11 promotes
 immunosuppression and extracellular matrix deposition, and might be
 developed as a therapeutic target for pancreatic cancer
description: mT3 tumor was generated by injecting 25,000 mT3 cells (derived from a PDAC of a KPC C57BL/6 mouse) subcutaneously into the back flank of 10-week-old female C57BL/6 mice in a 1:1 suspension of Matrigel (Cat# 354234, Corning) and PBS. At 3 weeks post injection, the tumor was dissected and processed as described before to obtain single cell suspensions. Subsequently, immune cells and blood cells were removed by CD45+ magnetic bead-based depletion (Cat# 130-052-301, Miltenyi Biotech) and ACK lysis buffer (Cat# A1049201, Gibco), respectively, following manufacturer’s guidelines. Remaining cells were prepared for single cell sequencing using Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3 (Cat# 1000075, 10X Genomics) on a 10X Genomics Chromium Controller following manufacturers protocol and sequenced using Illumina NextSeq 500 sequencer. The Cell Ranger Single-Cell Software Suite (10X Genomics) was used to perform sample demultiplexing, barcode processing, and single-cell 3′ gene counting. Sequencing data was aligned to the mouse reference genome (mm10) using “cellranger mkfastq” with default parameters. Unique molecular identifier (UMI) counts were generated using “cellranger count”. Further analysis was performed in R using the Seurat package. Briefly, cells with fewer than 500 detected genes per cell and genes that were expressed by fewer than 5 cells were filtered out. Subsequently, cells with &gt;7800 genes were filtered out to remove noise from droplets containing more than one cell. Dead cells were excluded by retaining cells with &lt;5% mitochondrial reads. The data was subsequently normalized by employing a global-scaling normalization method “LogNormalize” followed by identification of 2,000 most variable genes in the dataset, data scaling and subsequently dimensionality reduction by principal component analysis (PCA) using the 2000 variable genes. Then, a graph based clustering was performed using Louvain algorithm implemented in Seurat and clusters of cells with distinct gene expression profiles were identified. A non-linear dimensional reduction was then performed via uniform manifold approximation and projection (UMAP) and the clusters expressing <i>Pdpn</i>, <i>Pdgfra</i>, <i>Thy1 </i>and <i>Dcn </i>markers were identified as CAFs. All animal experimental procedures were completed under an approved IACUC protocol at LLNL and conforming to the NIH Guide for the care and use of laboratory animals.
description: Raw gene expression counts for cells from immune-depleted mT3
 tumor (mT3.txt).