Arebro Julia, Towle Rebecca, Lee Che-Min, Bennewith Kevin L, Garnis Cathie
Department of Integrative Oncology, British Columbia Cancer Research Center, Vancouver, BC, Canada.
Division of ENT Diseases, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden.
Front Cell Dev Biol. 2023 Sep 7;11:1240159. doi: 10.3389/fcell.2023.1240159. eCollection 2023.
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. 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. 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. 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.
口腔鳞状细胞癌（OSCC）是头颈癌最常见的形式，其5年生存率约为50%。迫切需要新的治疗策略来提高生存率，而要制定这些策略，需要更好地了解肿瘤发生的潜在机制。肿瘤微环境（TME）的作用在肿瘤进展和转移中日益被认为至关重要。TME的主要成分之一，癌症相关成纤维细胞（CAFs），在影响肿瘤的生物学行为中起关键作用。多种机制促成CAF的激活，如TGFβ信号传导，但细胞外囊泡（EVs）在OSCC中CAF激活中的作用了解甚少。评估口腔癌来源的EVs对CAF激活的影响将有助于更好地阐明OSCC的病理生理学，并可能推动新治疗方案的开发。使用差速离心法从OSCC细胞系（Cal 27、SCC-9、SCC-25）中分离出EVs。纳米颗粒跟踪分析用于EV的表征，蛋白质免疫印迹法用于确认EV蛋白标志物的存在。将口腔成纤维细胞与富集的EVs、TGFβ或PBS共培养72小时以评估激活情况。流式细胞术用于评估CAF标志物。提取从成纤维细胞收集的RNA并对转录组进行测序。用细胞因子阵列分析评估共培养的条件培养基。OSCC来源的EVs可将口腔成纤维细胞激活为与TGFβ激活的不同的CAFs，提示激活机制不同且功能特性不同。基因集富集分析显示，暴露于OSCC来源的EVs的那些CAFs中有几种炎症途径上调。炎症性CAF亚型的标志物基因也上调，但在TGFβ激活的CAFs中未上调。最后，对分泌蛋白的细胞因子阵列分析显示，来自EV激活的CAFs的几种促炎细胞因子水平升高，例如IL-8和CXCL5。我们的结果揭示了OSCC来源的EVs将成纤维细胞激活为CAFs的能力。这些CAFs似乎具有独特的特性，与TGFβ激活的CAFs不同。了解EVs与CAFs等基质细胞之间的相互作用可能会进一步深入了解OSCC的肿瘤发生及潜在的新疗法。
