Characterising cancer-associated fibroblast heterogeneity in non-smallcell lung cancer: relating molecular phenotype to function

Abstract

Despite recent therapeutic advances, non-small cell lung cancer (NSCLC) remains the leading cause of cancer death worldwide. To improve survival outcomes in this disease, novel therapeutic approaches are required. Relative to other cancers, NSCLC show low tumour purity with high proportions of immune and stromal cell populations. Cancer-associated fibroblasts (CAFs) are the most common stromal cell type in a range of solid tumours, where they have a number of tumour-promoting effects and are frequently associated with poor outcome. To date, therapeutic interventions targeting CAFs have shown largely disappointing results: this may be due in part to a lack of understanding of the variation within the CAF population. The aim of this work was to characterise the heterogeneity in the CAF population in NSCLC. First, we optimised our approach for the isolation of fibroblasts from primary lung tissues, determining that prolonged incubation with Collagenase is required. We next devised pipelines for the quality control of single-cell RNA sequencing data, identifying low-quality droplets and transcriptomic changes induced by prolonged enzymatic incubation. This approach was applied to 12 NSCLC and 6 patient-matched normal samples processed using the Drop-seq platform. Combining the resulting stromal cell data with those from a NSCLC dataset published during the course of this project identified 9 distinct populations, including 4 CAF groups. Two CAF populations showed overlap with the commonly-described “myofibroblastic” phenotype, and may have roles in the deposition and remodelling of extracellular matrix. Further functional characterisation requires in vitro work: as fibroblast culture is known to impact gene expression, we used the results of the above analyses to inform in vitro recapitulation of the identified ex vivo phenotypes. This approach allowed partial recreation of ex vivo gene expression profiles, although to an insufficient extent for preliminary functional analysis, and requires further refinement to allow accurate characterisation. Such studies should facilitate the development of more specific and successful stromal targeting strategies in NSCLC.

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Identifiers

ORCID for Christopher Hanley: orcid.org/0000-0003-3816-7220

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