Tumor Cell-released Autophagosomes (TRAP) Induce Differentiation Of MDSC

Myeloid-derived suppressor cells(MDSC)play critical roles in promoting immunosuppression and tumor microenvironment(TME),including host immunity compromise,tumor angiogensis and tissues remodeling,which threates survival of the patients.As MDSC are heterogeneous population of progenitor or precursor myeloid cells,the conditions and mechanisms facilitate the expansion of MDSC are still incompletely understood.Numerous studies over the past decade demonstrated that cytokines dysfunction(IL-6,GM-CSF)contributed to development of MDSC.However,especially at early stages,’localized’ tiny solid tumors influence the’distant’ hematopoietic compartment via secreting high concentrations of cytokines is unrealistic.Therefore,we speculated that there should be some other tumor-derived mediators modulate the myelopoiesis.Our previous series of studies have verified that tumor cell-released autophagosomes(TRAP)contributed to immunosuppression.TRAP could be taken up by B cells and induce IL-10 production.TRAP could induce M2 polarization as reflected by increased expression of PD-L1 and IL-10 through TLR4-mediated MyD88-p38-STAT3 signaling pathway.Treatment of neutrophils with TRAP promoted the generation of reactive oxygen species through macropinocytosis.Moreover,TRAP influenced CD4+T cells functions via TLR2-IL-6 cascade.In this study,we sought out to unravel the correlation and mechanisms between TRAP and MDSC,as well as providing justification for diagnostic and therapeutic targeting of MDSC in patients.ObjectivesTo reveal whether TRAP could promote MDSC differentiation and its mechanisms;to seek out the potential mechanisms of TRAP-induced MDSC affiliated with T cell inhibition;to prove the correlation between TRAP and MDSC in peripheral blood of cancer patients.Methods1.Mouse bone marrow(BM)cells were obtained and co-cultured with TRAP for 4 days.The phenotype and percentage of these cells were detected in flow cytometric applications and morphology.BM cells pre-treated with TRAP were co-cultured with αCD3/αCD28 antibodies activated spleen cells.Then CD4+T and CD8+T cell proliferation was measured by CFSE dilution.Mice were intravenous injection(i.v.)of DiR labeled-TRAP for 24 hours and observed by in vivo imaging system.Mouse models injected with TRAP or bearing tumor cells decreased Becn1 expression were established to further verify the correlation between TRAP level and MDSC accumulation.Mice were injected with saline solution(NS)or TRAP respectively for 3 days.Then the BM cells were co-cultured with spleen(SP)cells pair-wise separated by 3 μm microporous membrane in transwell system.BM cells differentiation were detected by flow cytometry after 4 days.BM cells were incubated with TRAP-stimulated-SP supernatant.And stattic was subsequently added to the system to verify whether TRAP mediated PMN-MDSC differentiation indirectly.2.BM cells of mice genetically deficient in TLR2,TLR4 or Myd88 were treated with TRAP for 4 days,and then MDSC differentiation was assessed by flow cytometry.TLR2 ligands blocked TRAP or TRAP isolated from Hsp60 KD/NC B16F10 cells were incubated with BM cells in vitro,then MDSC differentiation was assessed by flow cytometry.WT BM cells were sorted into two groups(TLR2+or TLR2-)by flow cytometry and stimulated with TRAP for 4 days,and then MDSC differentiation was assessed by flow cytometry.In vivo,Tlr2-/-mice were injected with TRAP and WT mice were injected with Hsp60 knockdown TRAP,then circulating PMN-MDSC and M-MDSC were detected by flow cytometry.In addition,the phosphorylation of IKKα/β,IκBα and STAT3 was analyzed by western blot after TRAP incubated with BM cells.3.Multiple proportion dilution of TRAP induced MDSC were co-cultured with CFSE labeled SP cells.Hsp60 blocked or Hsp60 knockdown TRAP-pretreated BM cells,and TRAP-pretreated Tlr2-/-or Myd88-/-BM cells were co-cultured with theαCD3/αCD28 antibodies activated SP cells.Then CD4+T and CD8+T cell proliferation was measured by CFSE dilution.Two types of tumor-bearing mouse models adoptive transferred with these MDSC were considered(s.c.or i.v.B16F10 melanoma cells)to assess TRAP induced MDSC in promoting tumor progression in living animals.We scanned the potential suppressive metabolites(Argl)and upregulating inhibitory ligands(PD-L1 and CTLA4)affiliated with T cell inhibition in the co-cultured system.To reveal TRAP induced MDSC inhibited T cells proliferation was cell contact dependent or not,we co-cultured these MDSC withαCD3/αCD28 antibodies activated SP cells separated by 3 μm microporous membrane in transwell system.Lastly,Argl inhibiting in combined with cutting off cell-contact was used to restore T cells division.4.136 freshly peripheral blood samples were collected from cancer patients not undergoing therapy.Firstly,the phenotype and frequency of circulating MDSC were analyzed by multicolor flow cytometry.Then extracellular vesicles(EVs)in plasma were stained with LC3B and EpCAM.The concentration of LC3+EVs was detected and its correlation with the percentage of PMN-MDSC/M-MDSC was analyzed.Results1.TRAP treated BM cells exhibited the same phenotype and percentage of CD11b+Gr-1+cells as IL-6 and GM-CSF cytokines induced MDSC.TRAP treated BM cells resulted in a profound expansion of CD11b+Ly6Chi monocytic MDSC(M-MDSC),whereas IL-6 and GM-CSF cytokines mostly induced CD11b+Ly6G+polymorphonuclear MDSC(PMN-MDSC),both in flow cytometric applications and morphology.A markedly accumulation of TRAP could be observed in mouse models’ bone marrow after i.v.TRAP for 24 hours in vivo.Intravenous injection of TRAP induced the similar levels of MDSC accumulation as tumor bearing mice in peripheral blood.In mice bearing Becnl knockdown B16F10(Becn1 KD),the frequency of CD11b+Ly6Chi cells as well as the CD11b+Ly6G+cells decreased significantly compared with the control tumor bearing mice(Becn1 NC).TRAP-treated SP cells could mediate BM cells differentiate into CD11b+Ly6G+subset.Stattic could silence PMN-MDSC induction in both TRAP-stimulated-SP supernatant and IL-6/GM-CSF treated group.2.MDSC differentiation did not occur in BM cells from Tlr2-/-and Myd88-/-mice.While Tlr4-deficient did not impact TRAP induced MDSC as well as the wild-type(WT)controls.All these genes deficient had no significant effect on IL-6/GM-CSF stimulation.TLR2+BM cells could be induced to significantly amount of M-MDSC by TRAP compared with TLR2-BM cells.Tlr2-/-mice injected with TRAP could induce CD11b+Ly6G+PMN-MDSC subset accumulation only.Blocking Hsp60 on the surface of TRAP dramatically diminished MDSC differentiation.TRAP down-expression of Hsp60 could rarely induced MDSC differentiation compared with the control TRAP.In mice bearing Hsp60 KD B16F10 models,the frequency of circulating M-MDSC markedly decreased compared with the mice bearing Hsp60 NC B16F10.While tumor cells expressed different levels of Hsp60 had no effect to PMN-MDSC induction in vivo.Additionally,compared to intact TRAP,breaking down the structure of TRAP significantly decreased MDSC differentiation.The level of IKKα/β and IκBαphosphorylation in relation to the timing of TRAP stimulation could be significantly observed.But no phosphorylated STAT3 signal was detected.TRAP combined with TRAP-stimulated-SP supernatant could robustly initial both NF-κB and STAT3 signaling activation.3.TRAP-induced M-MDSC had stronger suppressive capacity than cytokines-induced PMN-MDSC on per cell basis.TRAP treated Tlr2-/-or Myd88-/BM cells lose the suppressive function to T cell division.Hsp60 KD TRAP rarely endowed BM cells with suppressive functionality.In subcutaneous injected B16F10 mouse model,adoptive transfering of TRAP-induced MDSC significantly enhanced tumor growth compared with NS or BM cells control groups.And in i.v.B16F10 mouse model,adoptive transfering of TRAP-induced MDSC promoted lung metastasis remarkably.TRAP-induced MDSC expressed high levels of PD-L1 and CTLA-4 as well as Arg1.In the transwell system,a significant reduction of T cells proliferation was observed in TRAP-induced MDSC group,while IL-6/GM-CSF induced MDSC restored T cells division.Inhibiting Arg1 in the co-cultured system restored majority of T cells division.Inhibiting Arg1 in combined with cutting off cell-contact completely reversed T cells suppression.4.The percentages of CD11b+HLA-DR-/low CD15+PMN-MDSC and CD11b+HLA-DR-/low CD 14+M-MDSC in peripheral blood were significantly elevated in cancer patients compared with those in healthy donors.Up to 80%LC3+extracellular vesicles expressed high levels of EpCAM in patients in contrast with those in healthy donors,indicating most of the patients’circulating LC3+extracellular vesicles were tumor cell-released(TRAP).Concentration of LC3+extracellular vesicles were significant higher in cancer patients compared to controls and positive correlated with the percentage of M-MDSC.Conclusions1.TRAP could flow into bone marrow cavity and directly induce M-MDSC formation in vitro and in vivo.2.TRAP triggered mouse bone marrow cells differentiate to M-MDSC through TLR2-Myd88-NF-κB signaling pathway.Hsp60 on intact TRAP surface played critical roles in M-MDSC induction.3.TRAP-induced M-MDSC inhibited T cells division mainly in an Argl dependent manner.4.TRAP level was correlated with circulating M-MDSC accumulation in cancer patients.