Exosomes Educated Hepatic Stellate Cells Regulates Lactate Metabolism Of Hypoxic Colorectal Tumor Cells Via The IL-6/STAT3 Pathway To Confer Drug Resistance

Objective:Colorectal cancer is one of the main malignant tumors in humans,and its morbidity and mortality are on the rise.Liver metastasis is the most common form of distant in colorectal metastasis and the most important factor affecting the prognosis of patients.Irinotecan-based chemotherapy is the most common treatment for patients with recurrent metastasis or unresectable liver metastasis from colorectal cancer.With the deepening of tumor molecular targeting research,the combined treatment of targeted drugs and chemotherapeutic drugs is improved,but chemotherapy resistance is still the most critical factor for tumor recurrence and metastasis.Changes in the metabolic pathways of tumor cells and stromal cells under different nutritional conditions give tumor cells a stronger ability to proliferate and resist.Recent studies have found that exosomes-regulated tumor-associated fibroblasts（CAFs）and metabolic reprogramming between tumor cells played an important role in tumor cell invasion,metastasis,and drug resistance.The purpose of this study is to explore how the exosomes of normoxic colorectal tumors can promote the resistance of irinotecan by activating hepatic stellate cells to regulate the metabolism of hypoxic tumor cells in liver metastatic microenvironment.The results of this study may provide new predictive markers and potential therapeutic targets for colorectal cancer patients with chemoresistance.Methods:1.Identification of exosomes secreted by colorectal tumor cells by electron microscopy,nanoparticle tracking and Western Blot;2.Detection of activation of hepatic stellate cells（HSCs）by immunofluorescence and Western Blot;3.ELISA method was used to detect the ability of activated HSCs to secrete IL-6;4.Flow cytometry was used to detect glucose uptake capacity of exosome-treated HSCs;5.CCK8 cell proliferation assay was used to detect the effect of exosomes-treated HSCs cultured supernatant on the proliferation of hypoxic tumor cells stimulated by SN38（Irinotecan metabolite）;6.Flow cytometry（Annexin V/PI double staining method）and Western Blot method were used to detect the effects of exosome-treated HSCs cultured supernatant on apoptosis of hypoxic tumor cells stimulated by SN38（Irinotecan metabolite）;7.Mann-Whitney U-test and Kaplan–Meier survival curve were used to analyze the relationship between plasma IL-6levels and clinicopathological characteristics;8.The expressions of monocarboxylate transporters 1（MCT1）and lactate dehydrogenase B（LDHB）in the irinotecan-resistant liver metastases of colorectal cancer patients was detected by immunohistochemical method;9.Statistical analysis:The results of each experiment were repeated 3 times,and the data was analyzed using SPSS 19.0 software.P<0.05 was statistically different.Results:1.HSCs take up normoxic tumor cell exosomes and are activated into MFs.Transmission electron microscopy observed the typical goblet morphology of colorectal tumor cell exosomes.The size of colorectal tumor cell exosomes analyzed by nanoparticle tracking method was 30-150 nm.Western Blot detected the expression of exosomal marker proteins（CD9 and TSG101）.In order to explore the ability of HSCs taking up tumor cell exosomes,DiO（green）was used to label exosomes,and Dil（red）was used to label HSCs.After co-culturing for 48 hours,red HSCs were detected by fluorescence microscopy green spots.Immunofluorescence and Western Blot results showed that HSCs treated with normoxic tumor cell exosomes overexpressedα-SMA（a typical marker of MFs）.2.Exosomes-activated HSCs secreted more IL-6 and increased glycolysis.The concentration of IL-6 in the cultured supernatant of MFs was significantly higher than that of HSCs by ELISA（P<0.05）.At the same time,Western Blot detected that the expression of NF-κB in the nucleus of MFs was significantly higher than that of HSCs.When NF-κB inhibitor（BAY11-7082）and exosomes treated HSCs simultaneously,it was found that the IL-6secreted by MFs was significantly reduced（P<0.05）.Colorectal tumor-derived exosomes treated HSCs were incubated with glucose analogue（2-NBDG）for 1 hours,and the fluorescence intensity was significantly higher than that of the untreated group（P<0.001）.At the same time,the lactate concentration of the cultured supernatant of MFs was significantly higher than that of HSCs and the expression of GLUT1 and LDHA increased significantly（P<0.05）.3.P-ERK and p-AKT from colorectal tumor-derived exosomes promoted HSCs to secrete IL-6 and enhance glycolysis.Western Blot detected p-ERK and p-AKT proteins in colorectal tumor-derived exosomes,and the expression of p-ERK and p-AKT in colorectal tumor cell exosomes treated HSCs was significantly increased.In order to verify tumor-derived exosomes transmit p-ERK and p-Akt to activate the signal pathway of HSCs,1μM p-AKT inhibitor（MK226）and 10μM p-ERK inhibitor（U0126）were used to inhibit the p-AKT and p-ERK in tumor cells.It was found that the expression of p-ERK and p-AKT was decreased in tumor-derived exosomes and p-ERK and p-AKT were no longer activated in HSCs.To further verify the regulatory role of p-AKT and p-ERK in MFs,MK226 and U0126 were used to inhibit p-AKT and p-ERK of MFs,it was found that MK226 and U0126 can inhibit the secretion of IL-6.MFs treated with MK226and U0126 significantly reduced the uptake of 2-NBDG and the production of lactate（P>0.05）.4.Cultured supernatant of MFs promoted SN38 resistance of hypoxic tumor cells by secreting IL-6.Firstly,CoCl₂ was used to simulate the hypoxic microenvironment.LoVo and HCT116 cells stimulated by SN38 were cultured in the supernatant of MFs and HSCs.CCK-8 showed that the cell viability in the supernatant of MFs was significantly higher than that in the supernatant of HSCs（P<0.05）.Flow cytometry revealed that apoptosis of SN38 stimulated hypoxic LoVo and HCT116 in MFs cultured supernatants were significantly reduced（P<0.001）.Western Blot results showed that compared with HSCs group,the expression of pro-apoptotic protein（Bax）was decreased and the expression of anti-apoptotic protein（Bcl-2）was increased in tumor cells of MFs group.In order to explore whether MFs promote the resistance of hypoxic tumor cells by secreting IL-6,IL-6 neutralizing antibodies were used to treat hypoxic LoVo and HCT116 stimulated by SN38.The results showed that the proliferation of hypoxic tumor cells in the cultured supernatant of MFs was significantly reduced（P<0.001）,and apoptosis was significantly increased（P<0.001）.5.IL-6 promoted SN38 resistance by regulating lactate metabolism of hypoxic tumor cells.Western Blot detected that MFs cultured supernatant could increase the expression of MCT1 and LDHB in SN38-stimulated hypoxic tumor cells.ATP energy measurement results showed that compared with HSCs,the cultured supernatant of MFs treated SN38 stimulated hypoxic LoVo and HCT116 could release more ATP.After MCT1siRNA and LDHB siRNA were used to treat LoVo,cell viability and ATP production were significantly reduced（P<0.001）,and apoptosis was significantly increased in the cultured supernatant of MFs（P<0.001）.After IL-6 neutralizing antibody treatment,the expression of MCT1 and LDHB in hypoxic tumor cells in the cultured supernatant of MFs was significantly reduced.Similarly,IL-6 neutralizing antibodies could reduce the production of ATP in hypoxic tumor cells in the cultured supernatant of MFs（P<0.05）.6.Cultured supernatants of MFs enhanced lactate metabolism in hypoxic tumor cells by activating the IL-6/STAT3 pathway.Western Blot detected that the expression of p-STAT3 in hypoxic LoVo and HCT116 cells stimulated by SN38 in cultured supernatant of MFs was significantly increased.After Stattic treated hypoxic LoVo and HCT116 stimulated by SN38,the proliferation of tumor cells in the cultured supernatant of MFs was significantly reduced（P<0.001）,and apoptosis was significantly increased（P<0.001）.Stattic and STAT3 siRNA could reduce the expression of LDHB and MCT1 in hypoxic tumor cells stimulated by SN38 in MFs medium,and Stattic and STAT3 siRNA could reduce the production of ATP in cultured supernatant of MFs（P<0.001）.7.The increased level of plamsa IL-6 in colorectal cancer patients with liver metastasis was significantly related to the irinotecan resistance and poor prognosis.The clinicopathological characteristics of 78patients with liver metastasis from colorectal cancer were collected.It was found that the level of plamsa IL-6 in patients with irinotecan resistance was significantly higher than sensitive patients（P<0.0001）.Further analysis showed that elevated IL-6 levels in patients with advanced colorectal cancer were significantly associated with high CA199,extrahepatic metastases,and increased liver metastases（P<0.05）.Immunohistochemical analysis showed that the expression of MCT1 and LDHB in liver metastases of irinotecan-resistant colorectal cancer patients is significantly increased（P<0.05）.Conclusion:1.Exosome of normoxic colorectal tumor can activate HSCs into MFs.2.Colorectal tumor-derived exosomes promote HSCs to secrete IL-6 and enhance glycolysis by transmitting p-ERK and p-AKT.3.Culture supernatant of MFs promotes SN38resistance by activating the IL-6/STAT3 pathway to enhance lactate metabolism in hypoxic tumor cells.4.The increased level of plamsa IL-6 in colorectal cancer patients with liver metastasis is significantly related to the irinotecan resistance and prognosis.5.The expression of MCT1 and LDHB in liver metastasas of colorectal cancer patients with irinotecan resistance was significantly increased.