Study On Mechanism Underlying Tumoral Microvesicle-mediated Cancer-fibroblast Metabolic Crosstalk In Promoting Progression Of Oral Squamous Cell Carcinoma

As an important part of the tumor microenvironment,tumor stromal cells interact with tumor cells and establish a series of "crosstalk" during tumor development.Through continuous tumor-stroma crosstalk,tumor cells constantly remodel microenvironment to shape a suitable tumor microenvironment for their own progression.As an activated form of fibroblast,cancer associated fibroblasts(CAFs)are the most important stromal cells involved in the tumor-stroma crosstalk.Microvesicles(MVs)are small vesicles secreted by nearly all types of cells,they can mediate intercellular communication through targeted delivery of their rich contents,and play an important regulatory role in tumor-stroma crosstalk.Metabolic reprogramming is one of the important hallmarks of cancer,both tumor cells and stromal cells undergo metabolic reprogramming to form a metabolic state that is conducive to tumor progression.Tumor-stroma metabolic crosstalk helps tumor adapt to tumor microenvironment that is relatively lacking in nutrition and promotes tumor progression.Oral squamous cell carcinoma(OSCC)is one of the most common malignant tumors in the head and neck,which is prone to invasion and cervical lymph node metastasis.The abundant stromal cells in oral squamous cell carcinoma play an important role in the progression of oral squamous cell carcinoma.In this study,we reveal a novel mechanism for the tumoral microvesicle-mediated glycometabolic reprogramming of stromal cells during progression of OSCC.OSCC cells secret and transmit MVs to normal fibroblasts and transform them into CAFs,and cause the degradation of Caveolin1(CAV1)by activating ERK1/2 signaling pathway.CAV1 degradation mediates glycometabolic reprogramming in fibroblasts,allowing fibroblasts to undergo aerobic glycolysis,absorbing more glucose and secreting more lactate.Then lactate is supplied to the tumor through the MCT4 / MCT1 axis-mediated lactate shuttle to meet the energy and anabolic needs of tumor cells and promote the progression of OSCC.Through the MV-mediated "reverse Warburg effect",OSCC establish a "fibroblast-cancer cell metabolic coupling" based on MCT4/MCT1 axis to enable tumor-stroma metabolic crosstalk and eventually promote the progression of OSCC.This study provides a potential new strategy for the treatment of OSCC by targeting tumor microvesicle-mediated CAV1 degradation and the "fibroblast-cancer cell metabolic coupling".Part.Ⅰ: Study on the Role of Tumoral Microvesicles in the Activation and Glycometabolic Reprogramming of Cancer Associated FibroblastsObjectives: To investigate whether glycometabolic reprogramming occurred in stroma of oral squamous cell carcinoma,and explore the role of tumoral microvesicles in the activation and glycometabolic reprogramming of oral squamous cell carcinomaassociated fibroblasts.Methods: Primary human gingival fibroblasts(HGFs),paracancerous normal fibroblasts(PNFs),and cancer associated fibroblasts(CAFs)were isolated from tissues of healthy volunteers and patients with Oral squamous cell carcinoma(OSCC),respectively,and the fibroblasts were identified by immunofluorescence.Tumoral microvesicles(TMVs)were isolated from OSCC and were identified by scanning electron microscopy(SEM),transmission electron microscopy(TEM),and dynamic light scattering(DLS).HGFs were stimulated with TMVs,and the absorption of TMVs by HGFs was observed with a confocal fluorescence microscope.The effects of TMVs on the cell viability of HGFs were measured by CCK-8 and apoptosis assay.q RT-PCR and Western Blot were used to detect the expression of CAF markers FAP and Tn-c in HGFs stimulated by TMVs(TMV-HGFs).The glucose uptake assay and lactate assay were used to detect the glucose uptake and lactate production of primary PNFs,CAFs and TMV-HGFs.TEM was used to observe the number and morphology of mitochondria in TMV-HGFs.Western Blot was used to detect the expression of glycolysis related proteins CAV1,GLUT1,PDK1 and MCT4 in primary PNFs,CAFs and TMV-HGFs.Results: Immunofluorescence results showed that the extracted primary HGFs,PNFs,and CAFs were in accordance with the characteristics of fibroblasts.SEM,TEM,and DLS results showed that the isolated TMVs conformed to the characteristics of microvesicles.Confocal fluorescence microscopy results showed that HGFs could absorb TMVs,and the absorption of TMVs was time-dependent.After the TMVs were absorbed by HGFs,contents of the TMVs were released and then internalized by the HGFs.CCK-8,apoptosis assay showed that the proliferation and apoptosis of TMVHGFs were weakened.q RT-PCR and Western Blot results showed that the expression of FAP and Tn-c in TMV-HGFs were increased,and the phenotype of HGFs were switched to CAF.Glucose uptake assay,lactate assay,TEM and Western Blot showed that CAFs and TMV-HGFs exhibited higher glucose absorption and lactate production than PNFs and control HGFs,respectively.The expression of CAV1 in CAFs and TMVHGFs were decreased,and expression of GLUT1,PDK1 and MCT4 were increased.Besides,swollen mitochondria were observed in TMV-HGFs,and the number of orthodox mitochondria reduced,indicating that aerobic glycolysis were enhanced in CAFs and TMV-HGFs.Conclusions: CAFs of OSCC exhibit high level of aerobic glycolysis,and glycometabolic reprogramming occur in CAFs.OSCC activate fibroblasts by releasing and transmitting microvesicles,and transform fibroblasts to CAFs.TMVs enhance the aerobic glycolysis of fibroblasts and mediate glycometabolic reprogramming in fibroblasts.Besides,microvesicle-mediated glycometabolic reprogramming in fibroblasts may be achieved by targeting CAV1.Part.Ⅱ: Study on Molecular Mechanisms of Tumor Microvesicles Mediating Glycometabolic Reprogramming in Cancer Associated FibroblastsObjectives: To investigate the role of ERK1/2-CAV1 pathway in the regulation of glycometabolic reprogramming in fibroblasts and the role of tumoral microvesicles in the activation of ERK1/2 signaling pathway.Methods: CAV1 of HGFs was knocked down using lentiviral particles containing short hairpin RNA(sh RNA)targeting CAV1 and Western Blot was used to detect the expression of glycolysis related proteins GLUT1,PDK1 and MCT4 in CAV1-konckdown HGFs,and the activation of ERK1/2 signaling pathway was also detected.HGFs were pretreated with ERK1/2 signaling pathway inhibitor U0126 before stimulated with TMVs.Western Blot was used to detect the inhibitory effect of U0126 on ERK1/2 and its effect on the expression of CAV1 and glycolysis related proteins GLUT1,PDK1 and MCT4;and the activation of ERK1/2-CAV1 pathway in primary PNFs and CAFs was also detected.Western Blot was used to detect the expression of p-ERK1/2 in cancer cells and TMVs,and the direct transmission of p-ERK1/2 protein between cancer cells and HGFs via TMVs was detected by flow cytometry.Results: Western Blot results showed that CAV1-knockdown increased the expression of PDK1 and MCT4 in HGFs,but the expression of GLUT1 showed no significant difference,indicating that CAV1 targets PDK1 and MCT4 to regulate aerobic glycolysis of HGFs.After inhibiting ERK1/2 signaling pathway with U0126,the decreased CAV1 expression and the increased PDK1 and MCT4 expression induced by TMVs were reversed,indicating that inhibition of ERK1/2 pathway can reverse the decreased CAV1 expression and the enhanced aerobic glycolysis it causes.Flow cytometry showed that TMVs can directly transfer p-ERK1/2 protein to HGFs and activate ERK1/2 signaling pathway.Conclusion: OSCC can directly transfer the p-ERK1/2 protein through the intercellular transmission of TMVs and activate the ERK1/2 signaling pathway;the activated ERK1/2 signaling pathway enhances aerobic glycolysis and mediates glycometabolic reprogramming in fibroblasts by down-regulating the expression of CAV1.Part.Ⅲ: Study on the Mechanism of Cancer-Fibroblast Metabolic Crosstalk in Promoting the Progression of Oral Squamous Cell CarcinomaObjectives: To investigate the role of TMV-mediated glycometabolic reprogramming in the progression of oral squamous cell carcinoma,and explore the molecular mechanism underlying TMV-mediated cancer-fibroblast crosstalk.Methods: Establish a cancer-fibroblast co-culture model in vitro and detect the migration and invasion ability of OSCC cells co-cultured with TMV-pretreated HGFs by wound healing test and cell invasion assay.The expression of MMPs,MCT1 and SDHA of co-cultured OSCC cells were detected by Western Blot.Cancer-fibroblast coculture model in vivo were established in nude mice,a mixture of OSCC cells and HGFs was inoculated subcutaneously into the flanks of the BALB/c nude mice to form xenografts.TMVs were injected regularly and the growth rate of the tumors was measured.MCT1 expression in OSCC cells was knocked down using lentiviral particles containing sh RNA targeting MCT1,and the influence of MCT1-knockdown on the migration and invasion abilities of OSCC cells co-cultured with TMV-pretreated HGFs was also detected.The expressions of CAV1,MCT4 and MCT1 in xenografts and OSCC tissues were detected by immunohistochemical staining and their correlations were analyzed.Results: Wound healing test and cell invasion assay showed that TMV-pretreated HGFs enhanced the migration and invasion abilities of OSCC cells in vitro.Western Blot results showed that the expression of the invasion associated MMPs,the protein MCT1 that absorbs lactate,and the key enzyme of mitochondrial respiration SDHA were increased in OSCC cells co-cultured with TMV-pretreated HGFs.Cancer-fibroblast coculture model in vivo established in nude mice showed that TMV-pretreated HGFs promoted the growth of OSCC.MCT1-knockdown in OSCC can reverse the promoted migration and invasion abilities of OSCC cells co-cultured with TMV-pretreated HGFs.The results of immunohistochemical staining showed that TMVs-stimulated xenografts and OSCC tissues exhibited lower expression of CAV1 and higher expression of MCT4 in stromal cells,while exhibited higher expression of MCT1 in cancer cells.And CAV1 expression and MCT4 expression in stromal cells were negative correlated,MCT1 expression in cancer cells and MCT4 expression in stromal cells was positively correlated.Besides,MCT1 expression was heterogeneous in OSCC tissues,MCT1 expression in peripheral tumor cells was significantly lower than that in central tumor cells.Conclusion: OSCC secret TMVs to mediate glycometabolic reprogramming in fibroblasts and provide lactate to OSCC cells via the MCT4/MCT1 axis,meeting the energy and material needs of OSCC and promoting their progression.TMVs mediate the "fibroblast-cancer cell metabolic coupling" by MCT4/MCT1 axis and promote the progression of OSCC.