DOI: 10.5061/dryad.0cfxpnw54
title: Colorectal cancer interleukin-10 blockade scRNA-seq

description: Objective: PD-1 checkpoint inhibition and adoptive cellular therapy have
 limited success in patients with microsatellite stable colorectal cancer
 liver metastases (CRLM). We demonstrate that interleukin-10 (IL-10)
 blockade enhances endogenous T cell and chimeric antigen receptor T
 (CAR-T) cell anti-tumor function in CRLM slice cultures.Design: We created
 organotypic slice cultures from human CRLM (n = 38) and tested the
 anti-tumor effects of a neutralizing antibody against IL-10 (αIL-10). We
 evaluated slice cultures with single and multiplex immunohistochemistry,
 in situ hybridization, single cell RNA sequencing, and time-lapse
 fluorescent microscopy. In addition, we studied the effects of αIL-10 on
 carcinoembryonic antigen (CEA)-specific CAR-T cells exogenously
 administered to both human CRLM slice cultures and a CRLM murine model.
 Results: There was little effect of PD-1 blockade in CRLM slice cultures.
 In contrast, αIL-10 generated 1.8-fold increase in T cell-mediated
 carcinoma cell death, and increased proportion of CD8+ T cells and
 inflammatory polarization of macrophages. In addition to effects on
 endogenous immune cells in human CRLM, αIL-10 also rescued murine CAR-T
 cell proliferation and cytotoxicity from myeloid cell-mediated
 immunosuppression. In human CRLM slices, αIL-10 dramatically improved
 CEA-specific CAR-T cell cytotoxicity, generating nearly 70% carcinoma
 apoptosis across multiple human tumors. We saw a less dramatic, but
 similar effect of pretreatment of CAR-T cells with an IL-10 receptor
 blocking antibody, demonstrating that IL-10 inhibits CAR-T function in the
 CRLM tumor microenvironment. Conclusion: Neutralizing the effects of IL-10
 in human CRLM has therapeutic potential as a stand-alone treatment and to
 augment the function of adoptively transferred CAR-T cells.
description: Following surgical resection of CRLM specimens
 greater than 2 cm in diameter, sterile 6 mm tumor tissue cores were punch
 biopsied and immediately placed in BELZER-UW solution on ice. Within
 hours, cores were cut into 250 μm thick slices by vibratome and placed
 with media onto Millicell Cell Culture Inserts in a 24-well cell culture
 plate. Tumor slices were treated with anti-IL-10 antibody (20 µg/mL,
 #501407, Biolegend), with FOLFOX (1 µg/ml 5-Fluorouracil in combination
 with 1 µg/ml oxaliplatin) or or with FOLFIRI (1 µg/ml 5-Fluorouracil in
 combination with 2 µg/ml irinotecan). Control group consisted of slices
 treated with 0.2% DMSO in medium.<br>The dose of anti-IL-10 was
 chosen based upon a dose titration experiment using 5, 10, and 20 µg/ml
 concentrations of antibody, showing a maximal effect at 20
 µg/ml. For single-cell analysis, tumor slices were
 dissociated using the MACS Tumor Dissociation Kit according to the
 Miltenyi Biotec “dissociation of soft tumors” protocol. Cells were
 processed by 10xGenomics Chromium using the single-cell 3' RNA
 version 2 protocol. RNAseq libraries were sequenced on the NextSeq500
 instrument for 150 cycles (26 bp for Read 1 and 124 bp for Read 2). Reads
 were aligned to the human genome (GRCh38) and quantified using the Cell
 Ranger version 2.0 with default settings. The data from two tumors treated
 with anti-IL-10R was combined with the data of Jabbari et al. (2020) from
 eight tumors treated with FOLFOX/FOLFIRI available at
 &lt;https://doi.org/10.5061/dryad.pvmcvdngt&gt;. Gene expression
 data was aggregated and saved as a single AnnData object in HDF5 format
 (H5AD).  For bulk tumor analysis, RNA was extracted from slices using Qiagen RNEasy kit per the manufacturer’s instructions. Samples were submitted to the Northwest Genomics Center for sequencing on a NovaSeq6000 instrument. Demultiplexed, unaligned BAM files were produced by Picard ExtractIlluminaBarcodes and IlluminaBasecallsToSam were converted to FASTQ format using SamTools bam2fq (v1.4). Sequence read and base quality was checked using the FASTX-toolkit (v0.0.13). Sequences were aligned to GRCh38 with reference transcriptome GENCODE release 29 using STAR. Gene level expression quantification was generated with RNA-SeQC (v2.3.3) and RSEM (v1.3.1). RPKM and TPM for transcript isoforms were quantified on the merged lane-level data with RSEM (v1.3.1). Gene-level RPKMs were quantified with RNA-SeQC (v2.3.3) on a standard collapsed reference annotation.
description: Bulk RNAseq alignments and gene expression tables are contained in gzipped archives (sample.tar.gz). Single-cell RNAseq data is contained in an H5AD file that can be read, analyzed, and converted using ScanPy (https://scanpy.readthedocs.io) with its "read_h5ad" function. We also provide a CSV files (sample_identification_singlecell.csv and sample_identification_bulk.csv) with sample and treatment information.