T1 Exploring the tumour stroma in pleural mesothelioma using single-cell and single-nucleus transcriptomics

Introduction

Pleural mesothelioma (PM) is a devastating malignancy primarily related to asbestos exposure. It is considered a stromal rich malignancy with cancer-associated fibroblasts (CAFs) the dominant cell type within the tumour stroma, however little is known about CAF biology in PM. Single cell RNA-sequencing (scRNA-seq) allows for detailed characterisation of CAFs but has yet to be applied to PM. We have generated a single cell transcriptomic dataset from both fresh tissue for scRNA-seq, and isolated nuclei from frozen samples for single nuclei RNA-sequencing (snRNA-seq). We then explore CAF heterogeneity in PM to potentially modulate this population for therapeutic benefit.

Methods

Ethical approval was gained under Mesobank Research Ethics Committee reference 18/EE/0161. Fresh parietal pleura collected via VATS were dissociated into a single cell suspension and the library constructed using the 10X Genomics 3' v3.1 pipeline for scRNA-seq (n=7). Frozen tumour blocks from Mesobank were processed for single nuclei isolation and submitted using the same pipeline for snRNA-seq (n=15). Libraries were combined and sequenced on Illumina NovaSeq 6000. Healthy pleura scRNA-seq data previously published was included (GSE243446). Raw reads were processed using CellRanger 7.1.0 and aligned to GRCh38 reference. Further quality control and data processing was carried out using scanpy and integrated using scANVI.

Results

282,674 cells/nuclei from 32 samples (healthy pleura n=7, asbestos-exposed fibrinous pleuritis n=5, epithelioid n=9, biphasic n=7 and sarcomatoid n=4) are present in this dataset (figure 1). We identify a cluster of mesenchymal cells found only in sarcomatoid/biphasic PM with HMGA2 marker gene expression and enrichment in neuronal related gene sets. Fibroblast heterogeneity is observed with populations consistent with lipofibroblast (APOE⁺ PI16^- COL15A1^-), adventitial (PI16⁺) and myofibroblasts (ACTA2⁺). The lipofibroblast subtype is absent in malignant samples and instead replaced by myofibroblasts.

Conclusions

We have generated a single cell atlas of asbestos exposed pleura and demonstrate fibroblast heterogeneity in PM. We have identified a novel cluster of mesenchymal cells that appear only in sarcomatoid/biphasic PM. Future analysis will focus on CAF-mesothelioma cell interactions and whether these can be harnessed for therapeutic benefit.