Project description:Introduction: Malignant pleural mesothelioma (MPM) is an aggressive malignancy with poor prognosis. Unlike many other cancers, MPM is mostly characterized by inactivation of tumor suppressor genes. Its highly malignant nature in absence of tumor driving oncogene mutations indicates an extrinsic supply of stimulating signals by cells of the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are an abundant cell type of the TME and have been shown to drive the progression of several cancer types. The aim of the current study was to isolate and characterize patient-derived mesothelioma-associated fibroblasts (Meso-CAFs), and evaluate their impact on MPM cells. Methods: Meso-CAFs were isolated from surgical specimens of MPM patients and analyzed by comparative genomic hybridization, transcriptomics and proteomics. Human MPM cell lines were retrovirally transduced with GFP. The impact of Meso-CAFs on tumor cell growth, migration, and response to pathway inhibitors and cisplatin was investigated in 2D and 3D co-culture models by videomicroscopy and automated image analysis. Results: Meso-CAFs possess normal genomes without gene copy number aberrations typical for MPM cells. They express CAF markers and lack MPM marker expression. Their proteome and secretome profiles clearly differ from normal lung fibroblasts with particularly strong differences in the fraction of actively secreted proteins. The presence of Meso-CAFs in co-culture resulted in increased proliferation and migration of MPM cells. A similar effect on cell growth was induced by conditioned medium. Met/PI3K or WNT signaling inhibition abolished the Meso-CAF-mediated growth stimulation. While Meso-CAFs reduced the efficacy of EGFR inhibition in co-cultures, they did not provide protection of tumor cells against cisplatin. Conclusion: Our study provides the first characterization of human patient-derived Meso-CAFs and demonstrates a strong impact of Meso-CAFs on tumor cell growth and migration, two key aspects of MPM aggressiveness, indicating a substantial role of Meso-CAFs in driving MPM progression. Moreover, we show that Meso-CAFs significantly influence drug response and identify signaling pathways required for Meso-CAF-mediated growth stimulation. These data could be relevant for improving therapeutic strategies in MPM.

Project description:Introduction: Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer and has a survival rate of ~50% over five years. New treatment strategies are sorely needed to improve survival rates – and a better understanding of the mechanisms underlying tumorigenesis is needed to develop these strategies. The role of the tumor microenvironment (TME) has increasingly been identified as crucial in tumor progression and metastasis. One of the main constituents of the TME, cancer-associated fibroblasts (CAFs), plays a key role in influencing the biological behavior of tumors. Multiple mechanisms contribute to CAF activation, such as TGFβ signaling, but the role of extracellular vesicles (EVs) in CAF activation in OSCC is poorly understood. Assessing the impact of oral cancer-derived EVs on CAF activation will help to better illuminate OSCC pathophysiology and may drive development of novel treatments options. Materials and Methods: EVs were isolated from OSCC cell lines (Cal 27, SCC-9, SCC-25) using differential centrifugation. Nanoparticle tracking analysis was used for EV quantification and size characterization, and Western blot to confirm the presence of EV protein markers. Oral fibroblasts were co-cultured with enriched EVs, TGFβ, or PBS over 72 hours to assess activation. Flow cytometry was used to evaluate the difference in CAF markers. RNA collected from fibroblasts was extracted and the transcriptome was sequenced. Conditioned media from the co-cultures was evaluated with cytokine array profiling. Results: OSCC-derived EVs can activate oral fibroblasts into CAFs that are different from those activated by TGFβ, suggesting different mechanisms of activation and different functional properties. Gene set enrichment analysis using expression data from activated CAFs showed several upregulated inflammatory pathways in those CAFs exposed to OSCC-derived EVs. Marker genes for inflammatory CAF subtypes were also upregulated. This was not seen in CAFs activated by TGFβ. Finally, cytokine array analysis on secreted proteins from CAFs revealed elevated levels of several pro-inflammatory cytokines from CAFs activated by EVs, for instance IL-8 and CXCL5. Conclusions: Taken together, our results reveal the ability of OSCC-derived EVs to activate fibroblasts into CAFs. These CAFs seem to have unique properties, differing from TGFβ-activated CAFs. Gaining an understanding of the interplay between EVs and stromal cells such as CAFs could lead to further insights into OSCC tumorigenesis and potential novel therapeutics.

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Pan-cancer single-cell analysis reveals the heterogeneity and plasticity of cancer-associated fibroblasts in the tumor microenvironment

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