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Reductionist approach using primary and metastatic cell-derived extracellular vesicles prospect proteins associated with oral cancer prognosis

ABSTRACT: Oral squamous cell carcinoma (OSCC) shows high mortality rates that are largely associated with the presence of lymph node metastasis. However, the molecular mechanisms that drive OSCC metastasis are unknown. Here, we used a reductionist approach mapping the proteomic, miRNA, metabolomic and lipidomic profiles of extracellular vesicles (EVs) from human primary tumor (SCC-9 cells) and matched lymph node metastasis (LN1 cells) to explore the role of EV cargo in OSCC metastasis. Distinct omics profiles were associated with the metastatic phenotype, including 670 proteins, 217 miRNAs, 26 metabolites and 64 lipids differentially abundant between LN1 and SCC-9-derived EVs. A multi-omics integration indicated 11 ‘hub proteins’ significantly modulated in the metastatic site when compared to primary site-derived EVs, seven of them correlated with aggressiveness in cancer patients. The multi-omics approach allowed the prospection of proteins transported by EVs that potentially serves as prognostic markers in OSCC.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Adriana Franco Paes Leme

LAB HEAD: Adriana Franco Paes Leme

PROVIDER: PXD019216 | Pride | 2021-07-19

REPOSITORIES: Pride

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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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Reductionist approach using primary and metastatic cell-derived extracellular vesicles prospect proteins associated with oral cancer prognosis

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