Extracellular vesicles promote activation of pro-inflammatory cancer-associated fibroblasts in oral cancer

Abstract

Introduction: Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer and has a survival rate of ∼50% over 5 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.Methods: EVs were isolated from OSCC cell lines (Cal 27, SCC-9, SCC-25) using differential centrifugation. Nanoparticle tracking analysis was used for EV 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 h to assess activation. Flow cytometry was used to evaluate 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 showed several upregulated inflammatory pathways in those CAFs exposed to OSCC-derived EVs. Marker genes for inflammatory CAF subtypes were also upregulated, but not in CAFs activated by TGFβ. Finally, cytokine array analysis on secreted proteins revealed elevated levels of several pro-inflammatory cytokines from EV-activated CAFs, for instance IL-8 and CXCL5.Discussion: 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.

Extracellular vesicles as tools and targets in therapy for diseases

“…These HNSCC-derived EVs differ from TGF-activated CAF in that they activate the pro-inflammatory gene IL-6 and the hypoxia-related genes NF-B, HIF1, HK2, and PFKL. 123 There is growing evidence that miRNAs play a significant role in TGF-β signaling. TGF-1 has been shown to increase the expression of miR-21 in various cells.…”

Cancer-Associated Fibroblast Heterogeneity in Malignancy with Focus on Oral Squamous Cell Carcinoma

“…For instance, Hutton et al showed that CD105 + and CD105 − CAFs in PDAC were mutually exclusive cells that could not be converted into each other and that CD105 + CAFs induced increased tumor progression, while less abundant CD105 − CAFs supported favorable anti-tumor immunity [23]. CAF origin has been explored in other cancer types than PDAC, and, in addition to in vivo models, many in vitro studies clearly display how fibroblasts can become CAFs [24,25]. These studies have shown many origins of CAFs, including endothelial cells through endothelialmesenchymal transition (EndMT) [26], epithelial cells under specific circumstances through epithelial-mesenchymal transition (EMT) [27], bone marrow-derived mesenchymal stem cells (MSCs) [28], hematopoietic stem cells (HSCs) [29], cancer stem cells (CSCs) [30], adipocytes [31], pericytes [32], and stellate cells [33].…”

“…Physiological and genomic stress can also drive fibroblast activation info CAFs, with factors like reactive oxygen species [42] and double-stranded DNA breaks [43] serving as examples of this phenomenon. There is increasing evidence that cancer cell-derived extracellular vesicles (EVs) can also drive CAF activation [25,[44][45][46], though EV content differs and can include miRNAs, TGFβ, and cells surface proteins, as well as other proteins, mRNAs, and lncRNAs [44,47,48]. It should be noted, however, that many CAF subtypes and functions exist on a spectrum, rather than distinctive subtypes-and that these subtypes can have overlapping functions, a reality that amplifies the complexity inherent in defining CAFs subtypes.…”

“…This can be a significant challenge in the context of CAF research. Besides widely mentioned mesenchymal biomarkers such as VIM, αSMA, fibroblast activation protein (FAP), podoplanin (PDPN), integrin beta-1 (ITGB1, also known as CD29), FSP-1 (S100A4), and platelet-derived growth factor alfa (PDGFR-α), PDGFR-β [7,25], the role of matrix metalloproteinases (MMPs), through which CAFs can facilitate tumor growth, invasion, and metastasis has also been evaluated [49]. It is now commonplace for researchers to try to identify useful CAF markers through either flow sorting followed by RNA-seq and/or scRNA-seq to discover new potential CAF markers that are then validated in vivo.…”

FOXD2-AS1 promotes malignant cell behavior in oral squamous cell carcinoma via the miR-378 g/CRABP2 axis

“…As known, combined therapy of chemotherapy and radiotherapy exerts the most negative effect on the quality of life because of dry mouth side effects induced by these treatment, thereby influencing physical and mental state of patients [ 10 ]. The five-year survival rate for OSCC is only 50% and is not optimal [ 11 ]. With the rapid development of nanotechnology, nanodrug delivery systems has been widely utilized in targeted therapy for OSCC.…”