Research On The Affection Of DAMP Induced By Chemotherapy To The Killing Effect Of CIK On Lung Adenocarcinoma Cells

Introduction:Tumor microenvironment is immunosuppression.Tumor microenvironment may lead to immune escape of tumor cells.Tumor chemotherapy not only kill cancer cell non-specifically,but also cause the body’s immune suppression.Tumor chemotherapy could further aggravate the immune escape of tumor cells.In some cancer chemotherapy,tumor cells go into a special form of cell death.The form is immunogenicity cell death(immunogenic cell death,ICD).In the ICD,a series of medium expressed highly on the surface of tumor cells,a series of medium were released by the tumor cells.The medium can be recognized by toll-like receptors(Toll-like receptor,TLR),Nod receptors(Nod-like receptor,NLR)and induced the body’s natural immune response and the specific immune response.This is known as the medium damage associated molecular patterns(damage-associated molecular patterns,DAMP).The medium mainly included CRT,HSPs HMGB1 and ATP[1,2].DAMP may become key molecular targets in the combination of tumor chemotherapy and immunotherapy.Lung cancer is the most common malignant tumor in our country.The surgery therapy,radiation therapy,chemotherapy and targeted therapy are the main treatment methods.Epidermal growth factor receptor inhibitor:EGFR tyrosine kinase inhibitors(EGFR-TKI)makes a significant breakthrough in the treatment of lung cancer.However,the patients with K-ras mutation are resistant to TKI drugs,the overall survival of the patients was also significantly shortened[3].Due to the resistance,chemotherapy is still the main treatment of lung cancer.In recent years,cell immunotherapy becomes another important way of tumor therapy[4,5],following surgery,radiation,chemotherapy and targeted therapy.The therapy of cytokine induced killer cells(cytokine-induced killer,CIK)associated with chemotherapy was applied more and more in the anti-tumor treatment.CIK cells can kill cancer cells directly in the body,but the clinical curative effect of combined application of chemotherapy is not satisfactory.The chemotherapy can kill immune cells.The damage may be induce immunosuppression in vivo[6].This study combination therapy of chemotherapy with CIK cells in vitro and in vivo.This cisplatin and doxorubicin were used to treat lung adenocarcinoma cancer A549 cells with RAS mutation,the supernatant of the damage A549 cells was obtained.DAMP was included in the supernatant.The CIK cells were simulated with the supernatant.The effect and the possible mechanism of DAMP on immune cells were studied in vitro and in vivo.The effect and the possible mechanism of combination therapy of chemotherapy with CIK cells was studied in vitro and in vivo.Objective:To study the effect and the possible mechanism of combination therapy of chemotherapy with CIK cells in vitro and in vivo.To study the effect and the possible mechanism of DAMP induced by cisplatin and doxorubicin treated on lung adenocarcinoma cancer A549cells with RAS mutation in vitro and in vivo.To study the effect and the possible mechanism of DAMP on CIK cells in vitro and in vivo.This study may provide the theoretical basis for the combination therapy of chemotherapy with CIK cells in further clinic.Methods:1.Isolating the peripheral blood mononuclear cells(PBMCs)of healthy people and the PBMCs were cultured with cell factors to CIKs.2.A549 lung adenocarcinoma cancer cell line with RAS mutation were Cultured in Vitro.3.In vitro assay:multiple concentrations of chemotherapy drugs were used on A549 cells.The morphology of A549 cells treated by chemotherapy drugs was observed under a microscope.Next,the supernatant of A549 cells was added to CIK cells for co-culture.The immunophenotype of CIK cell after co-culture was detected by Flow cytometry.MTT assay detected the inhibitory rate of A549 lung adenocarcinoma cells induced by killing A549 cell supernatants.ELISA assays detected the concentration of CRT,ATP,and HMGB1 in A549 cells induced by various concentrations of chemotherapeutic agents.4.In vivo assay:establishing a tumor-bearing model in nude mice,chemotherapy and immunotherapy were given to study changes in tumor volume,detect tumor apoptosis,proliferation index,HE staining and immunohistochemical staining of tumor slices,DAMP molecules of serum by ELISA.Our research team collected serum from a healthy volunteer and 5 patients in the clinic.All 5 patients were treated with immunocytochemistry after chemotherapy.Serum concentrations of DAMP were measured by ELISA.Results:The cell morphology after killing A549 cells with low concentration of chemotherapeutic drugs showed more immunogenic death characteristics;after adding the supernatant of killing A549 cells into CIK cells for co-culture,the immunophenotype of CIK cells was significantly changed,and the proportion of CD8~+and CD56~+was significantly higher.The proportion of CIK cells in the same chemotherapeutic drug was significantly increased[CD8+:cisplatin group(46.2±1.14)%vs(16.7±1.77)%,doxorubicin group(51.5±1.86)%vs(38.8±0.94)%,cisplatin+doxorubicin group(50.2±1.07)%vs.(42.4±1.02)%,P<0.05;CD56+:cisplatin group(16.9±1.20)%vs(8.5±1.69)%doxorubicin group(14.6±1.74))%vs(11.2±2.46)%,cisplatin+doxorubicin group(17.5±0.91)%vs(13.0±1.18)%,P<0.05];The inhibitory rate of A549 lung adenocarcinoma cells was markedly changed by CIK cells induced by A549 cell killing supernatant.The inhibitory rate of CIK induced by A549 cell killing supernatant on A549 cells was significantly higher than that of CIK cells treated by the same concentration of chemotherapeutic drugs on A549 cells.Cisplatin group(31.34±1.51)%vs(5.97±1.74)%,doxorubicin group(45.46±1.78)%vs(6.22±1.34)%,cisplatin+doxorubicin group(45.78±1.14)%vs(11.94±3.11)%,P<0.05].In addition,the inhibitory rate of CIK induced by the supernatant of low-concentration chemotherapy drug-killed A549 cells on A549 cells was higher than that of CIK induced by the supernatant of higher-concentration chemotherapy drug-killed A549 cells on A549 cells[Cisplatin group(11.47±1.41)%vs(8.39±0.74)%vs(5.28±.87)%,the doxorubicin group(8.94±0.28)%vs(6.50±1.80)%vs(3.95±0.64)%,cisplatin+doxorubicin group(10.54±0.28)%vs(9.18±1.80)%vs(8.73±1.32)%,P<0.05];the concentration of immunogenic death-associated molecules CRT,ATP,and HMGB1 in the supernatants of A549 cell were significantly different among various concentrations of chemotherapeutic drug groups,higher concentrations of CRT,ATP,and HMGB1 were detected in the supernatant of the low concentration group[CRT,cisplatin group(11.79±0.86)ng/ml vs.(10.88±1.32)ng/ml vs.(6.24±0.94)ng/ml,doxorubicin group(15.21±1.12)ng/ml vs.(16.59±0.85)ng/ml vs.(8.28±0.57)ng/ml,cisplatin+doxorubicin group(12.91±0.98)ng/ml vs(12.41±0.34)ng/ml vs(2.86±0.20)ng/ml,P<0.05;HMGB1,cisplatin group(4.03±0.74)ng/ml vs(3.45±0.25)ng/ml vs(2.27±0.29)ng/ml,doxorubicin group(7.82±0.57)ng/ml vs(6.33±0.25)ng/ml vs(1.68±0.14)ng/ml,cisplatin+doxorubicin group(8.19±0.77)ng/ml vs(6.81±0.65)ng/ml vs(1.89±0.17)ng/ml,P<0.05;ATP cisplatin group(9.29±0.23)ng/ml vs(8.92±0.39)ng/ml vs(1.93±0.19)ng/ml,doxorubicin group(5.13±0.46)ng/ml vs(4.47±0.41)ng/ml vs(3.76±0.10)ng/ml,cisplatin+doxorubicin group(3.54±0.44)ng/ml vs(3.18±0.16)ng/ml vs(1.29±0.17)ng/ml P<0.05].In addition,the immunophenotype of CIK cells and the inhibition rate of A549 lung adenocarcinoma cells improved with the increase of CRT,ATP,and HMGB1 concentrations.The immune phenotype and the inhibition rate of CIK cells was related with the concentration of CRT,ATP,HMGB1.The higher the concentration of CRT,ATP,HMGB1was,the higher the immune phenotype and the inhibition rate was.At the end of the animal experiment,tumors were removed.Results showed that the tumor growth rate in the low-dose combination treatment group was the smallest.The number of cancer cells per unit area in the tumor section of the low-dose combination therapy group was the least.After chemotherapy,the content of CRT in tumors was detected by immunohistochemistry.Results showed that the content of low dose chemotherapy group was the highest.The low-dose chemotherapy group had a higher Caspase-3 level than the high-chemotherapy group and a lower Ki67 level than the high-chemotherapy group.The ELISA assay measured the serum CRT,ATP,and HMGB1 levels in mice.The experimental results showed that the concentrations of CRT,ATP,and HMGB1 in serum were higher in nude mice after a lower concentration of chemotherapy.The follow-up of clinical blood samples was collected.The results showed that patients with no elevation of DAMP after the chemotherapy on the 1st,2nd,and 3rd were in a state of progression,and patients with DAMP on the 4th and 5th after chemotherapy were in stable condition.Conclusions:Chemotherapeutic drugs can induce immunogenic death.Less than standard concentration chemotherapy or combination of lower than standard concentration chemotherapy is more likely to cause immunogenic death of A549cells,release higher concentrations of DAMP molecules.In vitro experiments confirmed that the CD8+and CD56+phenotypes of CIK cells and the inhibition rate of A549 cells were positively correlated with the concentration of DAMP molecules in the immunogenic supernatant.Tip:When combined with chemotherapy and immunotherapy to reduce the dosage can achieve the same or even higher efficacy.In vivo experiments confirmed that the weight reduction of the nude mice in the clinical standard concentration chemotherapy group and the clinical standard concentration combined immunotherapy group was the most significant,and the difference in the weight gain between the standard concentration chemotherapy group and the substandard concentration chemotherapy combined immunotherapy group and the blank group was not significant.The inhibition rate of tumor xenografts in nude mice was positively correlated with the concentration of DAMP molecules in nude mice.The H-Score of CRT and Caspase-3 in nude mice was negatively correlated with the concentration of chemotherapy,and the negative correlation between the concentration of CD8 H-Score and the concentration of chemotherapy.The H-Score of Ki67 was positively correlated with the concentration of chemotherapy.Tip:DAMP can stimulate the maturation and immune response of immune cells in the micro-environment of chemotherapy damage in vivo,so that immunotherapy and chemotherapy can produce synergistic effects in inhibiting cancer cells.When the combination of chemotherapy and immunotherapy is used,decreasing the dosage can achieve higher efficacy and reduce toxic and side effects.Clinical results suggest that DAMP may be used as a molecular indicator to predict the effect of chemotherapy combined with immune cells.