The Study On Mechanisms Of Tumor-derived Exosome GDF-15 Regulating Cancer Cachexia

Background Cancer cachexia is the main complication observed at the late stage of several kinds of malignant tumors.It is a kind of metabolic syndrome,which leads to serious body weight loss characterized with skeletal muscle atrophy mainly（with or without lipolysis）and influences the survival and life quality of patients seriously.And it is also a huge financial burden to family,society and health care.Exosomes,as a kind of small extracellular vesicles,have been involved in the development of cancer cachexia by transferring nucleic acids and proteins.The aim of this study was to search the key protein of exosome regulating cancer cachexia based on related quantitation proteomics.To make further study on the mechanisms of skeletal muscle atrophy in cancer cachexia which is induced by abnormal increased GDF-15 protein level observed in exosomes.Methods（1）Different kinds of cancer cachexia models including cell and animal level were established to compare the ability of different tumor cells in inducing skeletal muscle atrophy,including MC38 colon cancer cell（non-cachectic）,C26 colon cancer and Lewis lung cancer cells（cachectic）.（2）The exosomes of C26 and MC38 cells were collected to detect their effects of inducing C2C12 myotube diameter atrophy.GW4869,an inhibitor of exosome excretion,was used to further verify the effects of C26 exosomes on skeletal muscle atrophy.（3）Relative quantitation proteomics was conducted to analyze the profiles of proteins in C26 and MC38 exosomes.（4）GDF-15 protein was selected for further study after a systematic review of the literature,and the levels of GDF-15 in the two kinds of cells and exosomes were verified by western blots.（5）Database of ualcan.path.uab.edu and genomicscape.com were used to analyze the expression of GDF-15 in pan-cancers including colon cancer and the influence on the overall survival in colon patients.（6）Many kinds of cachectic tumor cells and their exosomes were collected to analyze the broad-spectrum of GDF-15 increase in these cells and exosomes.（7）Single clone cells of MC38 GD15 over-expressed（MC3 8-GDF15-OE）and C26 GDF15-knockdown（C26-GDF15-SH）were constructed by lentivirus for the further study.（8）Exosomes were detected in MC38 and C26 cells,as well as MC38-TB,C26-TB serum.And the GDF-15 expression in skeletal muscle was also analyzed including MC38-TB,C26-TB,LLC-TB.（9）Two different ways were made to study the effects of GDF-15 protein on the atrophy of C2C12 myotubes.Such as recombinant GDF-15 protein or GDF15 over-expressed in myotubes.（10）Collecting the conditioned medium and exosomes of MC38-GDF15-OE and C26-GDF15-SH respectively to confirm the effects of GDF-15 protein on the atrophy in C2C12 myotubes.（11）The roles of GDF-15 on the cancer cachexia skeletal atrophy was also studied in animal experiment.（12）The apoptosis ratio of mouse skeletal tissue from health and C26-TB group was also conducted by TUNEL experiment.（13）The effects of MC38-GDF15-OE and C26-GDF15-SH conditioned medium and exosomes on the expression of apoptotic pathway relevant proteins in C2C12 myotubes were conducted through western blots.Results（1）In comparison with MC38 cells,C26 cells conditioned medium can induce C2C12 myotubes atrophy significantly（P<0.0001）;In mouse model of cachectic of C26 colon cell,body weight increased 8.2%in health group while decreased 6.9%in C26-TB group at the end of experiment.What’s more,the average food intake per day decreased in C26-TB group（P<0.05）,mass of skeletal muscle declined（P<0.05）,the myofiber area of muscle cells decreased（P<0.001）and the grip strength also declined（P<0.01）compared with health group.And these results were also observed in the mouse cachectic model of Lewis lung carcinoma（LLC）,a universally-recognized cachexia-inducible mouse model.On the contrary,in MC38-TB non-cachectic mouse model,body weight increased 10.7%in health group and added 6.2%in MC38-TB group.And there is no difference in food intake,weight of muscle,myofiber area of muscle cells and grip strength.（2）Compared to MC38 exosomes,C26 exosomes induced C2C12 myotubes atrophy significantly（P<0.001）.GW4869,an inhibitor of exosome excretion,can relieve C2C12 myotubes atrophy induced by C26 exosomes with dose-dependent.And the expression level of MHC increased,Atrogin-1 decreased.（3）1599 proteins were detected by relative quantitation proteomics.54 protein levels were increased and 24 protein concentrations decreased significantly in C26 exosomes compared with MC38 exosomes（P<0.01 and Fold change>3）.（4）GDF-15 protein was selected for further study after a systematic review of the literature,and the levels of GDF-15 in the two kinds of cells and exosomes in line with the proteomics data confirmed by western blots.（5）Database analysis confirmed that GDF-15 ranked 51st on the top 250 over-expressed genes in colon adenocarcinoma.And the expression of GDF-15 also increased significantly in pan-cancers.The overall survival time of high GDF-15 group decreased compared with low GDF-15 group in colon patients（P<0.01）.（6）The concentrations of GDF-15 increased significantly in many kinds of cachectic tumor cells and their exosomes.（7）Single clone cells of MC38 GDF-15 over-expressed（MC38-GDF15-OE）and C26 GDF15 knockdown（C26-GDF15-SH）were constructed successfully compared with Vector group.And GDF-15 was verified to secrete mainly through exosomes.（8）The concentrations of exosomes in C26-TB serum increased significantly compared with health group（P<0.05）.There is no significant different in the contains of exosomes between MC38-TB and health group serum.On the contrary,the exosomes contained in C26-TB serum increased significantly compared to MC38-TB（P<0.0001）.What’s more,the contains of GDF-15 increased both in cachectic mouse serum exosomes and skeletal muscle,and there is no difference in non-cachectic mouse skeletal muscle.（9）GDF-15 recombinant protein can induce C2C12 myotubes atrophy significantly with dose-dependent compared with control group（P<0.05）.And the diameter of C2C12 myotubes over-expressed with GDF-15 decreased obviously in comparison with Vector group（P<0.001）.（10）The conditioned medium or exosomes of MC38-GDF15-OE induce C2C12 myotubes atrophy significantly compared with MC38-Vector（P<0.001）.On the contrary,the conditioned medium or exosomes of C26-GDF15-SH alleviate C2C12 myotubes atrophy in comparison with C26-Vector（P<0.001）.（11）Body weight decreased（P<0.05）,mass of skeletal muscle decreased（P<0.01）and average food intake per day decreased significantly（P<0.05）in MC38-GDF15-OE group versus MC38-Vector group.（12）TUNEL detection suggested that ratio of apoptosis increased significantly in skeletal muscle of C26-TB group versus health group（P<0.001）.（13）Western Blot results confirmed that conditioned medium or exosomes of MC38-GDF15-OE can activate the apoptotic pathway in C2C12 myotubes versus MC3 8-Vector.Such as ratio of Bcl-2/Bax decreased（P<0.05）,Cleaved caspase-3/caspase-3 increased（P<0.05）;On the contrary,the conditioned medium or exosomes of C26-GDF15-SH can alleviate the activation of apoptotic pathway in C2C12 myotubes in comparison with C26-Vector.Such as ratio of Bcl-2/Bax increased（P<0.05）,Cleaved caspase-3/caspase-3 decreased（P<0.05）.Conclusions The secretion of exosomes in tumor cells increased significantly and participate in the process of cancer cachexia induced skeletal muscle atrophy through contained active molecules.The abnormal increased GDF-15 levels in C26 exosomes involved in inducing skeletal muscle atrophy by activating apoptotic pathway.Our results illuminate that GDF-15 taking part in the process of cancer cachexia via exosomes for the first time,and enrich the effects of exosomes in cancer cachexia.What’s more,these findings providing new strategies for understanding the mechanisms and treatment of cancer cachexia via targeting GDF-15 protein.