Inhibition Of PDCD4 Deficiency Macrophage To Hepatocellular Carcinoma And Possible Mechanism

Objectives:Cancer immunotherapy is the most important developments in cancer treatment.Despite its impressive successes,the response of patients is often limited and short lived.This is due to factors that hamper the immune response against tumour cells.To target the immune system,the physicians need the antigen and there is three methods of attack.The first generation which was humoral method using monoclonal antibodies to attack cancer.In front of the problems with this method,the second generation of treatment came up,the immune checkpoint inhibitors medication.The new method is the CELL-BASED THERAPY for unmasking the cancer.TAMs are the most components of the hepatocellular carcinoma tumor microenvironment.TAMs play a pivotal role both in cellular mediated immunity and the humoral immunity.It was known that in HCC patients,TAMs are M2 like phenotype macrophages and the new generation of immune-oncology medication are targeting TAMs for more efficacy and effective response strategies for cancers treatments.Using macrophages as cell-based therapy is a promising way for cancer treatment.More interesting,using gene therapy is becoming the new area of the developing immunotherapy.Pdcd4(Programmed cell death 4)is a novel tumor suppressor gene,which has been reported to suppress tumor genesis by regulating tumor cell apoptosis,cell cycle and cell proliferation.Inside of being differentially expressed in many cancers,Pdcd4 is also associated with some chronic inflammatory diseases.Pdcd4 deficiency has been reported to enhance macrophage lipoautophagy and attenuates foam cell formation and atherosclerosis in mice,autoimmune diseases;and another study suggest that Pdcd4 modulates markers of macrophages alternative activation and airway remodelling in antigen induced pulmonary inflammation.Methods to modulate M2 like phenotype macrophage under inflammatory conditions are therefore needed to improve the efficacy of the macrophages cell based therapies in Hepatocellular carcinoma.Many studies demonstrated that the lost or downregulation of Pdcd4 in oncology is associated to cancer progression.Our team is the first who study the effect of Pdcd4 effect in Immunes cells and their role within inflammatory diseases and cancer.In order to study the effects of Pdcd4 knockout macrophages on hepatocellular carcinoma growth,we firstly produced Pdcd4 knockout macrophages that could be used as immune-gene based cell therapy for hepatocellular carcinoma.The effects of polarisation Pdcd4 knockout macrophages onto hepatocellular carcinoma growth has been studies in vitro and in vivo,finding out drive polarisation of Pdcd4 macrophages within inflammatory conditions.The results at present study would provide reliable data and experiment materials to the treatment based on gene cell therapy for cancers in vivo in the future.Methods1.Pdcd4 deficient macrophages suppress tumor cell proliferation derived fromhepatocellular carcinoma in vitro Pdcd4-/-knockout mice and C57BL/6 wild type mice Peritoneal Macrophages(PMs)and Bone Marrow-Derived Macrophages(BMDMs)were harvested and collected then culture.Cells were cultivated with DMEM in 6-well flat-bottom plates and incubated in humidified 5%CO2 at 37? for 2-6 hours.The nonadherent cells were removed.We then co-cultured separately Pdcd4 deficient and WT macrophages with Hepal-6 cell lines for 24h.After co-culture,the Hepal-6 cell lines were then adjust to 1.103 cells by well and culture into 96 wells plate for 48h.The cells assessed to cck8 kit for the cell proliferation within two days.Every hour before read the OD of cells,the cells were removed from the incubator and 10?l of CCK8 add to those wells suggested to be read and replace into the incubator.We the read the OD a Oh,6h,12h,24h and 48h.2.Pdcd4 deficient macrophages suppress Hepatocellular carcinoma growth in mouse liver modelsBMDMs macrophages density of 4 x 106 were mixed with H22 cell lines at density of 1 x 107 and 100 ?L of each were injected intra-subcutaneously into the posterior leg of BALBc mice.Every 3 days,the tumors growth characteristics was measured and after 16 days,the BALBc mice was sacrificed by cervical dislocation and the tumor harvested.At that time the tumors were weighted and pictured then immerged in formaldehyde for paraffinazation.The tumor weight and volume was analysed using student t-test and embedded tissue were load to H&E staining.3.Pdcd4 deficiency impact thepolarization of Ml and M2macrophages in vitroWT and Pdcd4 knockout gene mice 6 to 8 weeks old were use in all the experience.BMDMs and PMs Macrophages were harvest respectively by collecting bone marrow from femur and tibia,then use M-CSF to differentiate into mature macrophages within 6 days in cell culture incubator(at 37 ?,5%CO2)or by injecting 2 mL of starch solution intraperitoneal to each mouse then 3 days after the mice were killed by cervical dislocation then the PMs collected using DMEM in 20 mL syringe.Macrophages were separated in 3 grouped and plated at a density of 4 x 106 cell/well in 6 well plates for 4 to 6 hours.After,the medium was discarded and the cell stimulated with LPS for M1,IL-4 for M2 or without for Mo.The supernatant were collected for ELISA to determine the cytokines productions and the mRNA extracted to explore the expression of macrophages markers.4.Pdcd4 deficient macrophages resist M2 polarization induced by supernatants of tumor cells cultured Briefly,isolated BMDMs or PMs were incubated,stimulated or co-cultured with Hepal-6 cells for 24h in 6 well plates then plated at density of 1 x 103 cells/well in 96-well plates for 24h,48h and 72h.After every 12h,10 ?L of ccK8 was added to the well in position to be read and incubated into the cell incubator for 1h,then the cell proliferation OD was read and the data analysed using ANOVA.In parallel,the supernatant collected from the stimulation,the incubation or the co-culture were assessed to ELISA analysis for cytokines production.The mRNA from macrophages was also extract and suggest to qRT-PCR.5.Pdcd4 deficient macrophages inhibits the macrophages alternatively activation and re-educate TAMs in tumor microenvironment in liver mouse modelsBMDMs macrophages density of 4 × 106 were mixed with H22 cell lines at density of l x 107 and 100 ?L of each were injected intra-subcutaneously into the posterior leg of BALBc mice.After 16 days,the tumors were dissected and load to flow cytometry analysis for macrophages subtypes'population phenotyping.In parallel,another tumors embedded were used for IHC staining targeting F4/80,CD206,PD-L1.Results1.Pdcd4 deficient macrophages suppress tumor cell proliferation derivedfromhepatocellular carcinoma in vitro Many studies indicatethat Pdcd4 deficiency is association with cancer progression,but there is no study which evaluate the impact of Pdcd4 knockout on immune cells.In order to study the effect of Pdcd4 deficient macrophages in anti-tumor,especially in anti-hepatocellular carcinoma,peritoneal macrophages(PMs)and bone marrow-derived macrophages(BMDMs)from Pdcd4-/-knockout mice and C57BL/6 wild type mice were harvested and then co-cultured withtumor cell of Hepal-6 cell lines for 24h and 48h.The cell proliferation was analyzed by cck8 method.Results showed that Pdcd4 knockout macrophages inhibited strongly Hepal-6 cell proliferation and survival compared with the control.This result suggests that endogenous PDCD4 inhibits the anti-tumor ability of macrophage.2.Pdcd4 deficient macrophages suppress Hepatocellular carcinoma growth in mouse liver modelsThe Inorder to further investigate the impact of PDCD4 deficiency on anti-tumor of macrophages,BMDM from wild type mice or from PDCD4 knockout mice were mixed with murine Hepatocellular carcinoma cell line,H22 cells,and injected subcutaneously into BALB/c mice and were allowed to develop for tumors.After 16 days,mice were sacrificed and tumors characteristics were taken.The tumor growth curve characteristic was determined using a graduate digital calliper to measure tumor length and wide every three days till mice were sacrificed.Results showed thatPdcd4 knockout macrophages considerably suppressed tumor growth in vivo compared with the WT macrophages and the control.The tumor size and tumor weight from Pdcd4 knockout macrophages was significantly smaller than the tumor from the WT macrophages control or untreated cells.Furthermore,the tissue was load to HE staining and the results showed that there are more necrosis tissue within tumor with Pdcd4 knockout macrophagesthan the tumor with WT macrophages.3.Pdcd4 deficiency impact the polarization of Ml and M2 macrophages phenotype in vitroPrevious researches have shown that M1 macrophages play an anti-tumor role but M2 macrophages promote tumor proliferation.In order to investigate the mechanism that Pdcd4 deficiency macrophages inhibit proliferation of hepatocellular carcinoma,we first explore the effect of Pdcd4 on macrophages polarization.The peritoneal macrophages(PMs)and bone marrow derived macrophages(BMDMs)were collected from Pdcd4-/-and WT mice and then induced for 24h to differentiate to M1 in presence of LPS stimulation and to M2 in presence of IL-4.The phenotypes of M1 and M2 were detected by qRT-PCR and ELISA.The results from qRT-PCR showed that Pdcd4 deficient macrophages slightly increased the expression of IL-6 but not IL-12,TNF-a and iNOS(Ml markers)compared with the WT in condition of M1 polarization.However,Pdcd4 deficiency markedly inhibited M2 maker,including Arg-1,CD206 and IL-10,in condition of M2 polarization.In addition,the expressions of IL-6,TNF-a and IL-10 were further confirmed by ELISA these results suggest that Pdcd4 deficiency promote slightly M1 macrophage polarization but markedly inhibits M2 macrophage polarization.4.Pdcd4 deficient macrophages resist M2 polarization induced by supernatants of tumor cells in vitroTo further address the impact of Pdcd4 deficiency on macrophage polarization in tumour nicroenvironment,we designed an experimental system in vitro,where Pdcd4 deficient and WT macrophages were incubated with supernatantof Hepal-6 cell cultured for 24h and 48h and the polarization of macrophagewas investigated via analysing the M1 and M2 marker using qRT-PCR and ELISA.In line with the results form macrophage polarization stimulation,Pdcd4 deficiency significantly increased IL-6 mRNA expression of macrophageswhileexpression of other M1 markers,IL-12,TNF-a and iNOS,had no significant changes in the presence of tumor-cultured supernatant Considerably,we found a significant decreased in the mRNA expression of M2 markers(Arg-1,CD206,TGF-?1 and IL-10)in the Pdcd4 deficient macrophages compared with the WT macrophages.In addition,the resultsfrom ELISA showedthat Pdcd4 deficiency also increased of IL-6 secretion(co-cultured with tumor-cultured supernatant for 24h and 48h)and TNF-?(co-cultured with tumor-cultured supernatant for 48h Interestingly,we also noted the significant decreased of IL-10 in the Pdcd4 deficient macrophages.All this data suggested that Pdcd4 deficient macrophages resist to M2-like phenotype polarization while tends to differentiate to M1 phenotype in tumor inflammatory condition.In sum,Pdcd4 deficient macrophages significantly inhibited the M2-like phenotype macrophages polarization in tumor inflammatory conditions in vitro.We then forward to the in vivo experimentation of the Pdcd4 effect onto HCC.5.Pdcd4 deficient macrophages inhibits the macrophages alternatively activation and re-educate TAMs in tumor microenvironment in liver mouse modelsWT and Pdcd4 deficient BMDMs were injected simultaneously with H22 cells into BALB/c mice.After 16 days the tumors were dissected,then digested using percoll density procedure(describe previously)and analysed for F4/80,CD206 and CMHII macrophages phenotype by FACs.All events were gated in P2 windows and the dead cells gated in Zombie windows.The control immune macrophages express an immunosuppressive profile.However the Pdcd4 deficiency macrophages from the tumor microenvironment show a significant decrease in the F4/80+/CD206+M2macrophages phenotypes.ConclusionImmunotherapy,through gene based-cell therapy under inflamed tumor microenvironments,may just be the novelty therapy to the poor prognosis of the cancers pathologies.Pdcd4 deficiency was thought to be poor prognostic within cancer since the never study the effect of Pdcd4 deficiency in immune cells.Our results showed that Pdcd4 deficiency macrophage to Hepatocellular carcinoma tumor microenvironment resulted in a significant suppression of the tumor invasion and metastasis and potentially increased the substantial survival of the mice with HCC.TAMs are major contributors to the suppressive tumour microenvironments and immune modulators that selectively mitigate these cells are needed.In HCC,TAMs are alternatively activated.Our data demonstrated that Pdcd4 switch off M2-like phenotype activated macrophage in TME and increased tumor infiltrating effectors immune cells.Additionally,cancer immunotherapy is hindered by the short-lived anti-tumour effect due to the exhaustion of tumour-specific effector CD8 T-cells.The possible mechanisms can be,phenotype and differentiation status of CD8 T-cells have major influences on their function;probable phagocytosis of cancer cells by Pdcd4 deficiency macrophages or the more convincing the re-education of TAMs in the Tumor microenvironment.Pdcd4 deficient macrophages demonstrate their interest in manipulating immune modulators to maximise their cytotoxicity and enhance their longevity as part of hepatocellular carcinoma immunotherapy.This could be clinically tried as a potential therapy for the patients with HCC condemned because of the lack of liver donors for transplantation.Combined with others methods and discoveries,Pdcd4 deficiency macrophage would not only be used as palliative therapy,but would also act as curative immunotherapy for several cancers.Pdcd4 is a great way,because some mice tumor were completely suppressed.While systemic therapy in HCC has been disappointing in the past,we are at the dawn of new area.More studies that utilize genomic profiling,cellular related-matched molecularly targeted therapy as well as immunotherapy,and combinations of the above,are needed in order to improve the prognosis of patients suffering from HCC and advance the field...