Department of Stomatology North Sichuan Medical College, Affiliated Hospital of North Sichuan Medical College, Nanchong, China.
Department of Stomatology, Nan Chong Central Hospital, Second Clinical Medical College of North Sichuan Medical College, Nanchong, China.
BMC Cancer. 2021 Dec 14;21(1):1329. doi: 10.1186/s12885-021-09049-z.
Glucose metabolism in cancer associated fibroblasts (CAFs) within the tumor microenvironment is a material and energy source for tumorigenesis and tumor development. However, the characteristics and important regulatory mechanisms of glucose metabolism in fibroblasts associated with oral squamous cell carcinoma (OSCC) are still unknown.
We successfully isolated, cultured, purified and identified CAFs and normal fibroblasts (NFs). Cell culture, immunohistochemistry (IHC) and CCK8, flow cytometry, Seahorse XF Analyzer, MitoTracker assay, western blotting (WB), transmission electron microscope, Quantitative real-time PCR (qPCR), immunofluorescence (IF), and Label-free quantitative proteomics assay, animal xenograft model studies and statistical analysis were applied in this study.
We demonstrated that the proliferation activity of CAFs was significantly enhanced as compared to NFs, while the apoptosis rate was significantly decreased. CAFs in OSCC preferentially use oxidative phosphorylation (OXPHOS) rather than glycolysis. Moreover, CAFs showed stronger maximal respiration, a larger substantial mitochondrial spare respiratory capacity (SRC) and higher adenosine triphosphate (ATP) production capacity than NFs. The results of mitotracker green fluorescence staining showed that compared with NFs, CAFs exhibited stronger green fluorescence. The results of WB showed the expression level of Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) obviously increased in CAFs compared to NFs. These results confirmed that CAFs have greater mitochondrial activity and function than NFs. Furthermore, Label-free quantitative proteomics assays showed that both ATP synthase subunit O (ATP5O) and tumor necrosis factor receptor-associated protein 1 (TRAP1) are important differentially expressed proteins in the mitochondria of CAFs/NFs. Overexpression of TRAP1 in CAFs increased basal oxygen consumption rate (OCR), maximal respiration, ATP production and SRC. In vivo, overexpression TRAP1 expression in CAFs suppress tumor growth.
Taken together, the results indicated that TRAP1 is an important regulatory molecule of CAFs glucose metabolism and promotes OSCC progression by regulating the OXPHOS of CAFs.
肿瘤微环境中的癌相关成纤维细胞（CAFs）中的葡萄糖代谢是肿瘤发生和发展的物质和能量来源。然而，与口腔鳞状细胞癌（OSCC）相关的成纤维细胞的葡萄糖代谢特征及其重要调节机制尚不清楚。
我们成功地分离、培养、纯化和鉴定了 CAFs 和正常成纤维细胞（NFs）。应用细胞培养、免疫组织化学（IHC）和 CCK8、流式细胞术、 Seahorse XF 分析仪、MitoTracker 检测、Western blot（WB）、透射电子显微镜、实时定量 PCR（qPCR）、免疫荧光（IF）和无标记定量蛋白质组学检测、动物异种移植模型研究和统计分析等方法进行研究。
我们证明，与 NFs 相比，CAFs 的增殖活性显著增强，而凋亡率显著降低。OSCC 中的 CAFs 优先利用氧化磷酸化（OXPHOS）而不是糖酵解。此外，CAFs 表现出比 NFs 更强的最大呼吸能力、更大的实质性线粒体备用呼吸能力（SRC）和更高的三磷酸腺苷（ATP）产生能力。Mitotracker 绿色荧光染色的结果表明，与 NFs 相比，CAFs 表现出更强的绿色荧光。WB 的结果表明，与 NFs 相比，CAFs 中过氧化物酶体增殖物激活受体 γ 共激活因子 1α（PGC-1α）的表达水平明显增加。这些结果证实 CAFs 比 NFs 具有更强的线粒体活性和功能。此外，无标记定量蛋白质组学检测表明，ATP 合酶亚基 O（ATP5O）和肿瘤坏死因子受体相关蛋白 1（TRAP1）都是 CAFs/NFs 线粒体中重要的差异表达蛋白。在 CAFs 中过表达 TRAP1 增加了基础耗氧率（OCR）、最大呼吸、ATP 产生和 SRC。在体内，过表达 CAFs 中的 TRAP1 表达抑制肿瘤生长。
综上所述，结果表明 TRAP1 是 CAFs 葡萄糖代谢的重要调节分子，通过调节 CAFs 的 OXPHOS 促进 OSCC 进展。
