The Mechanism Of Myeloid-derived Suppressor Cell Derived Exosomes On The Stemness Of Colorectal Cancer Cells

Colorectal cancer（CRC）is the most common malignant tumor of digestive system.Although cinical treatment technology have improved outcomes,the mortality rate remains high.Studying the mechanism and seeking new therapeutic targets remains the important task.The cancer stem cell（CSC）is a small group of stem cell-like cell populations and has malignant biological characteristics such as self-renewal,multi-differentiation,indefinite proliferation and so on.Basic and clinical studies confirm the role of CSC in the development of CRC.Myeloid derived suppressor cells（MDSC）is a bone marrow derived heterogeneous cell population with immunosuppressive capacity.It is abundant in pathological conditions such as autoimmune and infection,inflammation and malignancy.MDSC participates in the development of tumor by inhibiting anti-tumor immune response,maintaining tumor cell stemness and promoting angiogenesis,and so on.Exosomes（Exo）are vesicles with a diameter of 30-150 nm.They contain the proteins and nucleic acids of the source cells and play an important role in the process of cell-to-cell communication.Exosomes involved many processes of tumor development.Objective:Investigate the relationship between granulocytic MDSC（G-MDSC）derived exosomes and the stemness of CRC cells,and further seek for the key component of GM-Exo which regulate the stemness of CRC cells.Understand the role of hypoxia on GM-Exo secretion.On the basis of above datas,we evaluate the clinic value of the key component on CRC.Methods:1.G-MDSC could promote the stemness of colorectal cancer cells via exosomes secretionCT-26 cells bearing mouse model was constructed.G-MDSC was sorted with immunomagnetic beads and the purity was analyzed by flow cytometry（FCM）.G-MDSC were cultured and the supernatants were collected.GM-Exo was prepared with ultracentrifugation and exosomes isolation kit.The GM-Exo morphology was observed by transmission electron microscopy,and the surface molecules were detected by Western blot,and protein concentration was determined by BCA microprotein assay.Rab27a is a key protein that determines the secretion of exosomes by cells.siRNA was used to inhibit the expression of Rab27a in G-MDSC and GM-Exo.In vivo,the effect of GM-Exo on G-MDSC-mediated colorectal cancer incidence and progression were observed through transferring G-MDSC into tumor-bearing mouse model.In vitro,CT-26 cells were treated with GM-Exo,and the ability of CT-26 cells to form spheres was measured by spheronization assay.The percentage of CD133/CD44 positive CT-26 cells were detected by FCM.The role of GM-Exo in the growth of CT-26 cells was observed via CT-26-bearing mice.2.Look for the main component of GM-Exo that regulate CT-26 cells stemnesssiRNA was used to knock down the level of S100A9 in G-MDSC and GM-Exo(termed GM-Exo^S100A9KD).CT-26 cells were treated with GM-Exo or GM-Exo^S100A9KD.The ability of spheres formation was detected by spheronization assay.The percentage of CD133/CD44 positive CT-26 cells were detected by FCM.The expression of Sox2/Oct4/Nanog/ALDHA1,Nox4,p-Stat3 and p-P65 were detected by Western blot.The ROS production were analyzed by FCM.The proliferation ability was observed by clone formation assay.The migration ability was observed by migration assay and liver metastasis model.Colitis-associated cancer（CAC）mice were induced with AOM/DSS.PKH-26labeled GM-Exo were injected into mice via tail vein.The distribution of GM-Exo in colorectal was observed by immunofluorescence（IF）and FCM.CAC mice was treated with GM-Exo or GM-Exo^S100A9KD,body weight were monitored,colon appearance was observed,tumor infiltration was detected by H（5）E staining,the exosomes distribution and CD133 expression were observed by IF.The proportion of G-MDSC and T-cell subsets in mice were detected by FCM.3.The effect of hypoxia on exosomes secretion by G-MDSCG-MDSC were isolated from tumor or spleen of CAC mice with immunomagnetic beads.The levels of HIF-1?and HIF-2?mRNA were detected by qRT-PCR.G-MDSC from tumor or spleen were cultured in vitro.The quantity of GM-Exo were detected by ExoELISA.Splenic G-MDSC were cultured under hypoxia or normal oxygen conditions,supernatants were collected,and GM-Exo were counted by ExoELISA.HIF-1?in G-MDSC was inhibited by HIF-1?inhibitor YC-1 and cultured under hypoxia or normal oxygen conditions in vitro.GM-Exo were counted by ExoELISA and the expression of Rab27a protein was detected by Western blot.4.Assess the clinical application value of plasma exosomal S100A9 in CRCPlasma from different CRC patients and healthy subjects was collected and exosomes were isolated.CD11b⁺exosomes CD11b^-exosomes were sorted by FCM.The levels of S100A9 were detected by ELISA.The levels of exosomal S100A9 from various plasma were detected by ELISA.MDSC from tumor tissues of CRC patients were sorted by FCM.MDSC-derived exosomes（M-Exo）were prepared.MDSC were treated with siRNA and S100A9 level in M-Exo was inhibited(M-Exo^S100A9KD).SW480 cells were treated with M-Exo or M-Exo^S100A9KD,and the ability of spheres formation was measured by spheronization assay.Tumor-bearing mice were constructed with treated SW480 cells,and tumor incidence and tumor growth were monitored.Results:1.Rab27a siRNA treated G-MDSC secreted less exosomes（P＜0.01）.When the exosomes secretio was suppressed,G-MDSC lost partly ability that promote CT-26cells to form spheres,and lost partly ability that promote CD133/CD44 expression（P＜0.01）,and lost partly ability that promote tumor growth（P＜0.05）.Exogenous GM-Exo could be taken up by CT-26 cells.GM-Exo promote the development CT-26cells bearing mice.2.S100A9 was abundant in G-MDSC and GM-Exo.When the S100A9 was decreased,GM-Exo lost partly ability that promote CT-26 cells to form spheres,lost partly ability that promote CD133/CD44 expression（P＜0.01）,lost partly ability that promote the expression of Sox2/Oct4/Nanog/ALDHA1/Nox4/p-Stat3/p-P65 and ROS in CT-26cells（P＜0.05）,and lost partly ability that promote CT-26 cells tproliferative（P＜0.01）and migration（P＜0.05）.When exogenous GM-Exo were injected into mice via tail vein,they distributed to colorectal tissue.Compared with PBS or GM-Exo^S100A9KD100A9KD treated CAC mice,there were slower weight gain and shorter colons（P＜0.05）,more nodules（P＜0.01）,severer tumor infiltration,more percentage of CD133 positive cells（P＜0.01）,more G-MDSC in peripheral blood and tumor tissue（P＜0.01or P＜0.05）),less CD4⁺T cells（P＜0.05）and CD8⁺T cells（P＜0.05）in GM-Exo treated CAC mice.3.The ability of tumoral G-MDSC to secrete exosomes was stronger than splenic G-MDSC（P＜0.01）.The level of HIF-1?mRNA in tumoral G-MDSC was higher than that in splenic G-MDSC（P＜0.01）.Under hypoxia,splenic G-MDSC secrete more exosomes than that under normal oxygen（P＜0.01）,and express more HIF-1?and Rab27a（P＜0.01）.In the presence of hypoxia,HIF-1?inhibitor（YC-1）treated G-MDSC secrete less exosomes than untreated G-MDSC（P＜0.01）,and express less Rab27a（P＜0.01）.4.CD11b⁺exosomes in human plasma accounted for approximately 10%of total exosomes.The level of S100A9 in CD11b⁺exosomes was higher than that in CD11b^-exosomes.The level of plasma exosomal S100A9 in CRC patients were higher than that in healthy people（P＜0.001）,and the level of plasma exosomal S100A9 in postoperative recurrence patients were higher than that in postoperative patients without recurrence（P＜0.001）.Compared with the PBS treated group,the ability of spheres formation in M-Exo treated SW480 was increased（P＜0.01）,and tumor growth was faster（P＜0.01）.When the S100A9 was decreased,GM-Exo lost partly ability that promote SW480 cells to form spheres（P＜0.01）,and tumor growth was slower（P＜0.01）.Conclusion:G-MDSC enhances colorectal cancer stemness by exosomal S100A9.Hypoxic environment is an important factor that promote G-MDSC to secret exosomes.The change of plasma exosomal S100A9 level could provide clue for CRC recurrence.