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Immunomodulation of T helper cells by tumour microenvironment in oral cancer is associated with rapid membrane-associated Vitamin D signalling pathway

ABSTRACT: The immune system plays a key role in the protective response against oral cancer, however the tumour microenvironment (TME) is able to impair this anti-cancer response by modulating T helper (Th) responses and promoting an anti-inflammatory environment. Regulatory T cells (Tregs) and Th2 effector cells (Teff) have been associated with bad prognosis in oral squamous cell carcinoma (OSCC), however it is unknown the main chemoattractant or immunomodulatory mechanisms associated with the enrichment of these subsets in OSCC. We characterised T helper-like lineages in Teff and Tregs and evaluated the main immunomodulatory changes induced by the TME in OSCC. Our phenotypic data revealed a higher distribution of tumour-infiltrating CCR8+ and Th2-like Treg in OSCC in comparison with non-malignant samples, whereas the percentages of Th1 cells were reduced in cancer. We then analysed the presence of CCR8 ligands and the chemoattractant effect of tumour secretomes, however despite observing higher presence of CCR8 ligand CCL18, we only observed reduced migration of Teff to tumour secretomes. The direct effect of the TME was then analysed by exposing T cell subsets to cancer secretomes and we observed that the TME induced higher expression of CCR8 and immunomodulatory molecules on both subsets. Finally, the proteomic analysis of the TME suggests that these changes were associated with the rapid membrane-associated vitamin D signalling pathway. In summary, the TME from OSCC promotes immunomodulatory changes by regulating CCR8 expression and disbalancing the Th1/Th2 repertoire.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

DISEASE(S): Oral Squamous Cell Carcinoma

SUBMITTER: Estefania Nova-Lamperti

LAB HEAD: Estefania Nova-Lamperti

PROVIDER: PXD023049 | Pride | 2021-04-29

REPOSITORIES: Pride

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Project description:The immune system plays a key role in the protective response against oral cancer; however, the tumour microenvironment (TME) is able to impair this anti-cancer response by modulating T helper (Th) responses and promoting an anti-inflammatory environment. Regulatory T cells (Tregs) and Th2 effector cells (Teff) have been associated with bad prognosis in oral squamous cell carcinoma (OSCC), however, it is unknown the main immunomodulatory mechanisms associated with the enrichment of these subsets in OSCC. We characterized Th-like lineages in Tregs and Teff and evaluated the main immunomodulatory changes induced by the TME in OSCC. Our phenotypic data revealed a higher distribution of tumour-infiltrating CCR8+ and Th2-like Treg in OSCC in comparison with non-malignant samples, whereas the percentages of Th1 cells were reduced in cancer. We then analyzed the direct effect of the TME by exposing T cell subsets to cancer secretomes and we observed that the OSCC secretome induced CCR8 expression and reduced cytokine production on both subsets. Transcriptomic analysis showed that the co-culture with OSCC secretome induced several gene changes associated with the vitamin D (VitD) signaling pathway in T cells. In addition, a proteomic analysis identified the presence of several proteins associated with prostaglandin E2 (PGE2) production by rapid membrane VitD signaling and an reduce presence of the VitD binding protein. Thus, we analyzed the effect of VitD and PGE2 and we observed that VitD promote a regulatory Th2-like responses and CCR8 expression, whereas PGE2 also modulate CCR8 but inhibited cytokine production in combination with VitD. Finally, we evaluated the presence of CCR8 ligand in OSCC and we observed higher presence of chemokine CCL18, which was also able to up regulate CCR8 in activated Th cells. Overall, our data showed that the immunomodulatory changes induced by the TME regarding CCR8 expression and regulatory Th2 phenotypes are associated with PGE2 mediated VitD signaling pathway and CCL18 expression in OSCC.

Project description:Malignant germ-cell-tumours (GCTs) are characterised by microRNA (miRNA/miR-) dysregulation, with universal over-expression of miR-371~373 and miR-302/367 clusters regardless of patient age, tumour site, or subtype (seminoma/yolk-sac-tumour/embryonal carcinoma). These miRNAs are released into the bloodstream, presumed within extracellular-vesicles (EVs) and represent promising biomarkers. Here, we comprehensively examined the role of EVs, and their miRNA cargo, on (fibroblast/endothelial/macrophage) cells representative of the testicular GCT (TGCT) tumour microenvironment (TME). Small RNA next-generation-sequencing was performed on 34 samples, comprising representative malignant GCT cell lines/EVs and controls (testis fibroblast [Hs1.Tes] cell-line/EVs and testis/ovary samples). TME cells received TGCT co-culture, TGCT-derived EVs, and a miRNA overexpression system (miR-371a-OE) to assess functional relevance. TGCT cells secreted EVs into culture media. MiR-371~373 and miR-302/367 cluster miRNAs were overexpressed in all TGCT cells/subtypes compared with control cells and were highly abundant in TGCT-derived EVs, with miR-371a-3p/miR-371a-5p the most abundant. TGCT co-culture resulted in increased levels of miRNAs from the miR-371~373 and miR-302/367 clusters in TME (fibroblast) cells. Next, fluorescent labelling demonstrated TGCT-derived EVs were internalised by all TME (fibroblast/endothelial/macrophage) cells. TME (fibroblast/endothelial) cell treatment with EVs derived from different TGCT subtypes resulted in increased miR-371~373 and miR-302/367 miRNA levels, and other generic (eg, miR-205-5p/miR-148-3p) and subtype-specific (seminoma, eg, miR-203a-3p; yolk-sac-tumour, eg, miR-375-3p) miRNAs. MiR-371a-OE in TME cells resulted in increased collagen contraction (fibroblasts) and angiogenesis (endothelial cells), via direct mRNA downregulation and alteration of relevant pathways. TGCT cells communicate with nontumour stromal TME cells through release of EVs enriched in oncogenic miRNAs, potentially contributing to tumour progression.

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Immunomodulation of T helper cells by tumour microenvironment in oral cancer is associated with rapid membrane-associated Vitamin D signalling pathway

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