| Background and objectivesColorectal cancer(CRC),the third most frequent cancer in the world,is a serious threat to human life and health due to its high metastasis rate.The clinical treatment methods of CRC are still mainly surgery,chemotherapy,and targeted drugs at present,while the overall therapeutic effect is hardly satisfactory.Early diagnosis and precise intervention are the keys to ensuring a good prognosis for colorectal cancer.Therefore,more in-depth mechanism research is urgently needed to discover biomarkers for the early diagnosis of colorectal cancer and to develop new therapeutic approaches.Tumor microenvironments play a significant role in tumor growth,metabolism,and metastasis.Cancer-associated fibroblasts(CAFs)which are the major stromal cells in tumor microenvironments may promote the invasion and migration of tumor cells in several ways.It has been found that fibroblasts undergo metabolic reprogramming after contact with cancer cells,producing lactate through glycolysis to provide a metabolic substrate for neighboring tumor cells.The exchange of lactate between cancer cells and fibroblasts through the biofilm via monocarboxylate transporters(MCTs)is known as the lactate shuttle in cancer.For one thing,lactate which is an interchangeable intermediate establishes metabolic coupling between tumor cells,immune cells,and stromal cells to maintain the metabolic advantage of tumor cells.For another,as a metabolite with signaling properties,lactate promotes tumor metastasis.Consequently,paying attention to the "shuttle" of lactate in the tumor microenvironment is of great significance to study its role in promoting tumor metastasis.There may be a similar process in the tumor microenvironment of colorectal cancer,and colorectal cancer cells may enhance their abilities to invasion and migration by establishing metabolic coupling with CAFs,but the specific phenomenon and mechanism remain to be studied.Here,we investigate the lactate metabolism coupling between colorectal cancer cells and CAFs in the tumor microenvironment and explore the correlation and possible mechanism between CAFs and the invasion and migration of colorectal cancer cells from the perspective of metabolism.Simultaneously,a holistic approach was proposed to inhibit oxidative stress and lactate shuttle in the microenvironment to provide better theoretical and practical support for anti-invasive and metastasis therapy of colorectal cancer.Methods and Results1.Methods1.1 Study on the expression of key protein MCT1 of lactate shuttle in colorectal cancer(1)Gene expression data and clinical features of cancer patients were obtained from TCGA and GTEx databases,and the expression of MCT1 was analyzed.(2)The expression levels of MCT1 in common colorectal cancer cell lines were analyzed to determine the cell lines used in this study.1.2 Study on the metabolic coupling between colorectal cancer cells and CAFs(1)An indirect co-culture system was established in vitro.Fibroblasts were induced by a conditioned medium of LoVo and SW480 cells,and parallel treatment of HFF-1 cells medium was used as the control group.The morphological changes of fibroblasts after induction were analyzed by immunofluorescence staining and the protein and mRNA expressions of surface markers of CAFs were detected,namely FAP-α,Vimentin,and ACTA.(2)DCFH-DA fluorescence staining was used to analyze the changes in intracellular ROS level of fibroblasts after induction,and HFF-1 cells were treated with Mito-Tracker red CMXROS to analyze the changes in mitochondrial mass.Glucose uptake,lactate production,intracellular LDH activity,and the protein and mRNA expressions of key glycolytic enzymes LDHA and PKM2 were detected.(3)The direct co-culture system of colorectal cancer cells and fibroblasts was established.Colorectal cancer cells were respectively treated with the conditioned medium from LoVo or SW480 cells and HFF-1 cells co-culture,and the single culture medium of LoVo or SW480 cells was used as the control group.The mitochondrial quality of colorectal cancer cells after induction was determined,and the intracellular lactate concentration,the protein and mRNA expression levels of the key enzymes SDH and FH of oxidative phosphorylation,and the key lactate transporter MCT1 in LoVo and SW480 cells were analyzed.1.3 To evaluate the effects of CAFs on the progression of colorectal cancer(1)The effects of CAFs on the invasion and migration ability of LoVo and SW480 cells were investigated by transwell assay,sphere-forming assay,and cell migration experiments.(2)The effect of CAFs on the angiogenesis ability of LoVo and SW480 cells was studied by tubule formation assay.(3)EdU staining and MTT assay were used to analyze the effects of CAFs on the proliferation of LoVo and SW480 cells.1.4 Study on the intervention effect of inhibiting oxidative stress and lactate shuttle under metabolic coupling conditions in colorectal cancer(1)Acetylcysteine(NAC)and AZD3965 were respectively used to inhibit oxidative stress and lactate shuttle to unbind metabolic coupling,and the changes of related metabolic indexes in CAFs and colorectal cancer cells were analyzed.(2)The ability of invasion,migration,and angiogenesis of colorectal cancer cells was investigated after the uncoupling of metabolism with NAC and AZD3 965.1.5 Study on metabolic coupling mechanism between colorectal cancer cells and CAFs(1)TCGA and GTEx databases were used for single gene enrichment and pathway analysis of MCT1,and related metabolically active pathways of MCT1 were clarified.(2)Immune infiltration analysis was performed on the microenvironment of colorectal cancer to study the proportion of fibroblasts and the expression of MCT4 in the microenvironment.(3)Western blot assay was used to analyze the regulatory mechanism of NF-κB/HIF-1α in CAFs affecting invasion and migration of colorectal cancer cells.1.6 In vivo(1)Colorectal cancer transplant tumor model was established in Balb/c nude mice.LoVo cells were injected into the control group alone,and LoVo cells co-cultured with HFF-1 cells were injected into the other groups.The Balb/c nude mice were respectively treated with blank solvent,AZD3965,NAC,and combined AZD3965 and NAC.(2)The expression of EMT-related molecules E-cadherin and N-cadherin in colorectal cancer tissues was presented by immunohistochemistry,and the distribution of MCT1 and MCT4 in tumor stroma was also detected.(3)The concentration of lactate in colorectal cancer tissues was tested.(4)Western blot was used to detect the expression of E-cadherin,N-cadherin,and MCT1 to analyze the role of lactate metabolizing coupling in the progression of colorectal cancer.2.Results2.1 MCT1 is overexpressed in colorectal cancer tissues(1)The expression of MCT1 was analyzed by TCGA and GTEx databases,and it was found that the expression level of MCT1 in tissues of CRC was higher than that in normal tissues.The analysis of the colorectal cancer metastasis database showed that the gene expression level of MCT1 in metastatic tissues of the liver,ovary,and peritoneal of colorectal cancer was higher than that in colorectal carcinoma in situ,and the gene expression level of MCT1 in lung metastasis tissues of CRC was higher than that in normal lung tissues.(2)CCLE database was used to analyze the relative expression levels of MCT1 in common colorectal cancer cell lines,and we selected LoVo and SW480 cells with relatively high expression levels as the objects of subsequent studies.2.2 Colorectal cancer cells establish metabolic coupling with CAFs through oxidative stress(1)Colorectal cancer cells induced phenotypic transition from normal fibroblasts to CAFs.Immunofluorescence was used to identify the morphology of the induced fibroblasts,and we found that the morphology of the induced fibroblasts was greatly changed.The cells were monopole and short without induction,while the cells were long spindle and radial after induction.Then,the expression of key surface markers of CAFs was detected by Western blot and RT-qPCR,and the protein and mRNA expression levels of FAP-α,ACTA,and Vimentin were increased compared with those before induction.(2)The metabolic pattern of fibroblasts partially shifted to a glycolytic phenotype after induction.Flow cytometry and a microplate reader were used to detect ROS levels in fibroblasts before and after induction,and the results showed that ROS levels in fibroblasts increased after induction.Then the mitochondrial quality of fibroblasts was marked by fluorescent dye,and it was found that the mitochondrial quality of fibroblasts decreased after induction,suggesting the metabolic pattern may be changed.Further analysis of glucose uptake,lactate production,and intracellular LDH activity in fibroblasts before and after induction.We found that the glucose uptake and lactate production of fibroblasts was increased after induction,and the intracellular LDH activity was also signally improved.Meanwhile,the expressions of two key enzymes in glycolysis were detected by Western blot and RT-qPCR.The results showed that the protein and mRNA expression levels of PKM2 and LDHA in the induced fibroblasts were observably increased.(3)The metabolic level of oxidative phosphorylation was partially restored in colorectal cancer cells after establishing metabolic coupling with fibroblasts.Firstly,the co-culture system of colorectal cancer cells and fibroblasts was established in vitro,and the two cells labeled with DiI fluorescent dye showed a good growth status.Then,the changes in mitochondrial quality and concentration of intracellular lactate in LoVo and SW480 cells were tested.The experimental results showed that the mitochondrial quality and concentration of intracellular lactate of colorectal cancer cells were greatly increased after induction.Western blot and RT-qPCR were used to analyze the related indexes of MCT1 and oxidative phosphorylation.The protein expression levels of MCT1,SDH,and FH in LoVo and SW480 cells were significantly increased after induction treatment.2.3 CAFs enhance the ability of invasion,migration,and angiogenesis of colorectal cancer cells(1)The results of the Transwell assay showed that the invasion ability of LoVo and SW480 cells was increased after induction treatment,and the sphere-forming assay showed that the tumor stem cells’ pellet-forming ability was significantly improved.Transwell assay(without Matrigel)showed that the migration ability of LoVo and SW480 cells was highly improved compared with that before induction.The detection of key proteins of EMT indicated the expression of E-cadherin decreased and that the expression of N-cadherin and Vimentin increased in the induction group.(2)At the same time,HUVECs were treated with the conditioned medium from colorectal cancer cells co-cultured with fibroblasts.The conditioned medium of colorectal cancer cells cultured alone served as the control group.The tube-forming ability of HUVECs treated with a conditioned medium for 6 hours was dramatically improved.(3)Finally,the results of the EdU and MTT assay showed that the proliferation ability of colorectal cancer cells was improved after induction.2.4 Targeting oxidative stress and the lactate shuttle process can effectively inhibit the invasion and migration of colorectal cancer cells(1)It was found that the glucose uptake and lactate production of colorectal cancer cells were decreased after induction,while the intracellular lactate concentration was increased after the use of AZD3965.The level of glycolysis in colorectal cancer cells was reduced by detecting the protein expression of key glycolysis enzymes.Meanwhile,the experimental results showed that the ROS level in fibroblasts decreased after the use of NAC,and the glucose uptake and lactate production returned to normal levels.(2)The ability of invasion,migration,and angiogenesis of colorectal cancer cells was observably reduced after using the combination of NAC and AZD3965.2.5 NF-κB/HIF-1α affects metabolic reprogramming between colorectal cancer cells and CAFs(1)It was found that the common active pathways of MCT1 included monocarboxylic acid transport,pyruvate metabolism,and tricarboxylic acid cycle through the gene enrichment and pathway analysis,indicating that the high expression of MCT1 may be closely linked to metabolic coupling.(2)Immune infiltration analysis of the tumor microenvironment of colorectal cancer showed that the proportion of CAFs in colorectal cancer tissues was significantly increased,and the expression level of MCT4 in CAFs was also markedly higher than that of normal fibroblasts.(3)Western blot results showed that the expression level of NF-κB/HIF-1α in LoVo and SW480 cells was increased after induction,while the expression level of NFκB/HIF-1α was decreased after NAC and AZD3965 treatment.The expression levels of MCT1 and MCT4 showed a similar trend,observably increased after induction,but decreased after the use of the inhibitors.Detection of proteins related to the EMT process showed that the expression of E-cadherin was increased and that the expression of N-cadherin was decreased in colorectal cancer cells after induction with the inhibitors treatment.2.6 Co-administration of AZD3965 and NAC can effectively inhibit the growth,invasion,and migration of subcutaneously transplanted tumors in nude mice.(1)The results showed that the tumor volume and tumor weight of the group of LoVo cells which were co-cultured with HFF-1 cells in vitro were higher than that of the LoVo cells group alone,and the efficacy of the combined administration group was better than that of single administration group.In the meantime,the concentration of lactate in tumor tissue in the combined administration group was higher than that in the single administration group.(2)According to the results of immunohistochemistry,MCT1 was mainly distributed in epithelial cancer cells and did not exist in the surrounding stromal matrix,while MCT4 was distributed in the surrounding stromal fibroblasts.The results also showed that the expression of E-cadherin in the group of LoVo cells which were cocultured with HFF-1 cells in vitro was lower than that in the group of LoVo cells alone,while the expression of N-cadherin was improved,and this trend was reversed after combined administration.(3)Western blot results of tumor tissue showed that the expression levels of MCT1 and N-cadherin in LoVo cells co-cultured with HFF-1 cells were higher than those in the LoVo cells group alone,while the expression of E-cadherin was lower than that LoVo cells group alone.The trend was also reversed after the combined administration.Conclusions1.Colorectal cancer cells establish metabolic coupling with fibroblasts through the oxidative stress effect,which triggers their metabolic reprogramming and those of fibroblasts.2.Colorectal cancer cells enhance their ability to invasion,migration,and angiogenesis by establishing metabolic coupling with stromal fibroblasts.3.Simultaneously,targeting oxidative stress and the lactate shuttle process can effectively inhibit the invasion and migration of colorectal cancer cells.4.NF-κB/HIF-1α affects the metabolic reprogramming between colorectal cancer cells and CAFs. |
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