Malignant pleural mesothelioma (MPM) is an aggressive cancer, associated with poor prognosis. We assessed the feasibility of patient-derived cell cultures to serve as an ex vivo model of MPM. Patient-derived MPM cell cultures (n=16) exhibited stemness features and reflected intratumour and interpatient heterogeneity. A subset of the cells were subjected to high-throughput drug screening and coculture assays with cancer-specific cytotoxic T cells and showed diverse responses. Some of the biphasic MPM cells were capable of processing and presenting the neoantigen SSX-2 endogenously. In conclusion, patient-derived MPM cell cultures are a promising and faithful ex vivo model of MPM.
- pleural disease
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Malignant pleural mesothelioma (MPM) is an aggressive malignancy, with increasing incidence in many countries.1–3 Patients often present with advanced stage malignancy and malignant pleural effusion (MPE).2 Current treatment modalities offer limited life span benefit; thus, MPM is associated with poor prognosis and bears high mortality rate.3
Due to the complexity and heterogeneity of MPM, a biomarker-driven approach to therapy is likely to be required to control tumour progression, fluid formation and improve patients’ quality of life and survival. Preclinical in-vitro MPM models are an invaluable tool to study the disease and develop novel treatments.4–7 We designed a study to assess the feasibility of patient-derived MPM cell cultures, to serve as a faithful translational ex vivo platform suitable for the development of biomarker-guided therapies.
Patient-derived MPM cell cultures established from MPE specimens displayed morphological heterogeneity and features of cancer stemness
We generated a panel of 16 patient-derived MPM cell cultures originated from MPE specimens (online supplementary figures 1-2, online supplementary table 1). All cell cultures were in vitro morphologically stable, and displayed diverse growth rates and phenotypes, including cobblestone and spindle shapes (figure 1A–E). A blinded National Health Service Consultant Pathologist examined May-Grunwald Giemsa stained cytospin specimens of the MPM cell cultures and confirmed their malignant nature. They exhibited several cytological cancer hallmarks: atypical, prominent, pleomorphic, large and multiple nucleoli (figure 1A–E). MPM cell cultures were able to form tumour-spheroids exhibiting stemness potency and indicating the existence of a tumour stem cell subpopulation (figure 1F–M).
Genomic profiling of the patient-derived MPM cell cultures exhibited interpatient variation
We subjected to whole-genome sequencing three of the MPM cell cultures to profile their mutational landscape (online supplementary figures 3-5 and online supplementary table 2) and determine the mutational status of the known MPM-associated tumour suppressor genes: TRP53, NF2, BAP1, LATS2, SETD2 and CDKN2A.8 The analysis revealed a diverse spectrum of mutational profiles (non-synonymous, stop codon gain and frameshift insertion mutations) for these genes highlighting the importance of personalised and targeted therapies in MPM (figure 2A–B). These findings are in line with previously published data and indicate a resemblance at the molecular level between the patient-derived cell cultures and MPM tumours.8
High-throughput drug screening of patient-derived MPM cell cultures revealed heterogeneous response profiles
The three sequenced MPM cell cultures were subjected to a high-throughput drug screening assay (316 Food and Drug Administration (FDA) approved antitumour agents, online supplementary figure 6) and exhibited different response profiles, a finding that reflects the interpatient heterogeneity (figure 2C). We detected six drugs: bortezomib, carfilzomib, dactinomycin, dinaciclib, omacetaxine and idarubicin, which were consistently more cytotoxic compared with pemetrexed/cisplatin (positive control) the current clinical first-line chemotherapy regimen (figure 2D). Clusters of topoisomerase, mitotic (including microtubule stabilisers), histone deacetylase and mammalian target of rapamycin (mTOR) inhibitors were among the most effective drugs (online supplementary figure 7). We calculated the drug response curves to pemetrexed/cisplatin for the three MPM cell cultures. MESO-163 was more sensitive compared with MESO-031 and MESO-174 (figure 2E).
HLA-A2-restricted cancer-specific CD8+ T cells identified SSX2+ patient-derived MPM cell cultures
To evaluate the feasibility to use the MPM cell panel in T cell immunotherapy assays, we performed a series of coculture experiments with HLA-A2-restricted SSX2-specific cytotoxic CD8+ T cells (CTCs) and four HLA-A2+ MPM cell cultures (two epithelioid: MESO-044, MESO-278 and two biphasic: MESO-174, MESO-392; online supplementary figure 8).
As expected, the exposure of cancer-specific CTCs, to SSX2-peptide loaded MPM cells at varying peptide concentrations triggered the induction of cytotoxic immune responses (online supplementary figures 9-13). Interestingly, the two biphasic MPM cell cultures were able to induce a CTC immune response even without the SSX2-peptide pulse. In specific, we detected increased: CD107a expression, killing capacity and production of two antitumour cytokines: interferon γ and tumour necrosis factor α (figure 2A–C) by the CTCs. The biphasic cell cultures MESO-174 and MESO-392 exhibited statistically significantly higher levels of SSX2 gene expression compared with the epithelioid MESO-044 and MESO-278 (figure 3D). These findings suggest that the two biphasic MPM cell cultures were able to endogenously process and present the SSX-2 epitope peptide.
We report the establishment and validation of a panel of 16 patient-derived MPM cell cultures as an ex-vivo model of the disease. The cells were polyclonal, displayed tumour stemness features and demonstrated diverse morphologies, growth rates and mutational landscapes. Three and four MPM cell cultures were subjected to high-throughput drug screening and CTCs coculture assays and exhibited diverse responses. We detected a correlation between the response to pemetrexed/cisplatin and the observed clinical phenotype and discovered two biphasic cell cultures that harboured an SSX2+ subpopulation. Cell cultures from the same patient were established at different clinical course timepoints, which may provide the potential to study molecular alterations due to MPM progression.
Strengths and limitations
The patient-derived ex vivo MPM model offers the potential to serve as a faithful and practical tool to study MPM and accelerate research.4–7 The diverse profiles of MPM cell cultures reflect the interpatient variability. Single sequencing in combination with high-throughput drug screening of the MPM cell panel offer the potential to study treatment response mechanisms, identify neoantigens and importantly investigate the molecular pathways which are involved in tumour stem cell resistance to treatments and tumour relapse.9 The discovery of neoantigens, such as SSX2, is important as they are an attractive drug target due to specificity.10 The development and validation of tumour classification molecular signatures with regards to treatment response could improve patient clinical management. Coculture of tumour and cancer-specific CTCs has the potential to unveil their multifaceted crosstalk and develop novel immunotherapies.
The model described herein has limitations. The establishment of a cell culture is a relative lengthy procedure (1–2 months), the success rate (45%) of cell culture establishment requires improvement and the reasons for which some samples fail to generate a cell line require investigation. The drug screening hits need to be validated with further experiments that directly measure cell death.
In conclusion, patient-derived MPM cell cultures exhibit a remarkable resemblance to the clinical phenotype and features of MPM, which are suggestive of a faithful ex-vivo human model.
The authors would like to thank the Oxford Respiratory Trials Unit, the Target Discovery Institute and the Nuffield Department of Medicine, University of Oxford for the support to conduct the research project.
NIK and RA are joint first authors.
NMR and IP are joint senior authors.
Contributors NIK, NMR and IP conceived and designed the study. NIK, RA, MAH, XY and YP did laboratory assays and analysed the data. MM did cytological and histological analyses. NIK and MAH did gene expression assays and analysed the data. NIK, RA and SM analysed the whole-genome sequencing data. EB, RMM, RJH and VG collected clinical data. NIK and RA analysed clinical data. NIK, RA, SH and DE did the high-throughput drug screening and analysed the data. NIK, SJ, CV and MD provided resources and prepared the ethics protocols. GTS and TD provided materials and knowledge. NMR and IP supervised the study. All authors reviewed the manuscript.
Funding Oxford Centre for Histopathology Research and the Oxford Radcliffe Biobank are supported by the NIHR Oxford Biomedical Research Centre (Molecular Diagnostics theme), the Oxford Cancer Centre (CRUK), the NIHR Thames Valley and South Midlands Clinical Research Network, individual stakeholders, and the University of Oxford’s Medical Sciences Division. NIK and NMR are supported by a National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC) grant. TD and YP are supported by Medical Research Council, UK. This work was supported by an Oxfordshire Health Services Research Committee (OHSRC) Research Grant (ID: 1336) to NIK and a Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS, ID: 2018-I2M-2–002) to TD.
Competing interests IP works for AstraZeneca in a non-related field.
Patient consent for publication Not required.
Ethics approval Oxford Radcliffe Biobank Tissue Access Committee approval reference number: 19/A107.
Provenance and peer review Not commissioned; externally peer reviewed.