An Experimental Study On Riboflavin Photosensitization Treatment For Inactivation Of HCT116 Cells Mixed In Peripheral Blood

Background:Surgical resection is one of the most common radical treatments for solid tumors.However,tumors may be compressed or the local intravasc?lar pressure may be increased during surgical manip?lation,which can cause the shedding and entry of tumor cells into the blood circ?lation and distant recurrence and metastasis of tumors,and shorten the survival time of patients.Free tumor cells shed before or during the operation into the blood circ?lation are still high risk factors for postoperative recurrence.Post-operative intravenous chemotherapy has limited effect on survival improvement of tumor patients.Tumor cells which are insensitive to chemotherapy can be hardly eliminated or cleared through systemic chemotherapy once they have entered blood circ?lation before or during surgery.As a res?lt,they are very likely to cause distant metastasis and recurrence,as well as failure of surgical treatment.Even if the tumor cells are sensitive to chemotherapy,the patients may have low tolerance to systemic chemotherapy immediately after surgery due to surgical trauma.Generally,the surgical patients need some time to recover before the chemotherapy.However,the recovery stage is the ideal time for colonization and proliferation of CTCs.Based on the successf?l experience of riboflavin photochemical technology(RPT)in inactivation of virus,bacteria,protozoa for whole blood and blood components and inactivation of lymphocyte to prevent blood transfusion related graft versus host disease(TA-GVHD),We propose to use RPT to treat peripheral blood in patients with malignant tumor during perioperative period in order to inactivate CTCs/CTSCs in peripheral blood,and block the way of hematogenous metastasis of malignant tumor.We selected HCT116 colorectal cancer cell line as the research object to sim?late CTCs/CTSCs in patients'peripheral blood,and explored the feasibility,effectiveness and safety of inactivation of CTCs/CTSCs by RPT through in vitro cell test and animal test.Part ?Evaluation of riboflavin photosensitization treatment for inactivation of HCT116Objective:to treat HCT116 cells and lymphocytes with different RF concentrations and UV irradiation doses,and to evaluate the damage caused by RPT to tumor cells and lymphocytes,and to screen the RF concentration and irradiation dose range that can make HCT116 cells lose proliferation ability and minimize lymphocyte damage.Methods:HCT116 cells were placed in the c?lture dish,and different doses of RF solution were added according to different experimental groups,and the corresponding dose of UV was illuminated.Three concentration gradients of RF were designed:1?mol/L,5?mol/L and 15?mol/L.The duration of irradiation was designed to be 5 min and 10 min.There were 6 experimental groups in tatol,and group A:?mol/L RF,5min of irradiation;Group B:5?mol/L RF,5min of irradiation;Group C:15?mol/L RF,5min of irradiation;Group D:1? mol/L RF,UV exposure for 10min(1,10);Group E:5 ?mol/L RF,UV exposure for 10min(5,10);group F:15?mol/L,UV exposure for 10min(15,10).The control group was normal HCT116 cells without RF and UV irradiation.The treatment conditions on lymphocyte were identical to those of HCT116 cells.The apoptosis rate and tumorigenicity of HCT116 cells,lymphocyte apoptosis rate and cytokine secretion under different treatment conditions were evaluated.Res?lts:1.With the increase of RF concentration and UV irradiation dose,the apoptosis rate of both HCT116 cells and lymphocytes gradually increased,lymphocytes were more sensitive to irradiation dose,but not to riboflavin concentration.As can be seen from the res?lts,the damage of RPT to tumor cells was significantly greater than that to lymphocytes,so the inactivation of tumor cells co?old be achieved under the condition of maintaining certain functions of lymphocytes in the experiment.2.Flow cytometry was used to detect the concentrations of IL-2?IL-4?IL-17A and TFN-? in the supernatant of lymphocyte c?lture after RPT treatment.The concentrations of the four cytokines all decreased with the increase of RF concentration and UV irradiation dose,which showed a dose effect on RF concentration and UV irradiation.3.Experimental group 1 and experimental group 2 showed no tumor formation in NOD/SCID mice,but positive control group showed tumor formation in NOD/SCID mice,which indicated that RPT can effectively inactivate HCT116 cells and make them lose the ability of proliferation.Conclusion:There was a difference in damage caused by RPT to HCT116 cells and lymphocytes,and HCT116 cells are more susceptible to RPT damage.Therefore,it is possible to inactivate HCT116 cells under the condition that the lymphocytes maintain adequate immune function.Part IIEffect evaluation of inactivation of RPT in sim?lating CTCs(HCT116 cells)in peripheral bloodObjective:we adopted RPT to inactivate HCT116 cells that were mixed in sim?lated peripheral blood,and looked for possible RPT technical conditions,which can not only make HCT116 tumor cell mixed in sim?lated peripheral blood lose proliferation,and keep the red blood cell damage,coag?lation function and immune function control in a body's acceptable range.Methods:Whole blood samples from healthy donors were respectively mixed with HCT116 cells at a certain proportion within 2h after sample collection.The final concentration of the HCT116 cells was 5×105/ml.Then 50?mol/L RF was added and mixed well,and the mixture was transferred to an EVA high UV transmission storage bag.The sample mixture was irradiated by the alternating UVA(365nm)/UVB(310nm)source at the temperature of(6±2)C.Depending on the irradiation doses,two experimental groups were set up,experimental group 1(7.2J/cm2)and experimental group 2(10.8J/cm2).The control group was also set up,with neither addition of RF nor UV irradiation.CD326(EpCAM)MicroBeads were used to separate and enrich HCT116 tumor cells in whole blood,and the apoptosis rate and tumorigenicity of HCT116 tumor cells were detected.Lymphocytes in whole blood treated with RPT were isolated using Ficoll to evaluate their apoptosis rate and cytokine secretion.TEG technique was used to evaluate the coag?lation function of whole blood after RPT treatment.The concentration of free hemoglobin in whole blood after RPT treatment was detected,and the hemolysis rate of red blood cells was calc?lated,and the red blood cell injury was evaluated.Res?lts:1.CD326(EpCAM)MicroBeads were used to separate HCT116 tumor cells in whole blood,and the purity was(97.20±2.36)%,which co?ld effectively complete the enrichment of HCT116 cells in peripheral blood of healthy donors.2.The apoptosis rate of HCT116 cells in the control group,experimental group land experimental group 2 were 6.88%,37.99%and 41.98%,respectively.The apoptosis rate increased significantly in the two experimental groups as compared with the control group.Moreover,as the irradiation dose increased,the apoptosis rate further increased3.6 NOD/SCID mice in the control group all developed tumors,and the tumor volume was(0.996±0.42)cm3;6 NOD/SCID mice in experimental group1 all developed tumors,and the tumor volume was(0.234±0.19)cm3;6 NOD/SCID mice in experimental group 2 all had no tumors.The res?lts of HE staining and immunohistochemical detection showed that the tumor stripped from the control group and experimental group 1 was HCT116 colon tumor tissue,while the stripped tissue from group 2 was normal connective tissue,and no HCT116 cells were found.4.The survival rates of lymphocytes in the control group,experimental group 1and experimental group 2 were(84.63±6.30)%,(81.66±6.73)%and(81.25±7.77)%,respectively.Although the survival rate of lymphocytes decreased as the irradiation dose increased,there was no significant difference between the experimental groups and control group(experimental group 1 vs.control group P=0.24;experimental group 2 vs.control group P=0.22)5.After RPT,the cytokine-secreting abilities of lymphocytes in the experimental groups decreased slightly,but showing no significant difference as compared with the control group(P>0.05).6.The thermal effect caused by RPT wo?ld cause damage to peripheral blood coag?lation.The experimental conditions were adjusted by controlling the ambient temperature at(6±2)?,which preserved acceptable coag?lation function.7.The hemolytic rates in the control group,experimental group land experimental group 2 were(0.02±0.02)%,(0.07±0.03)%and(0.10±0.04)%,respectively.The hemolytic rate of peripheral blood increased after RPT,but it was still far lower than the quality control bottom limit at the end of the storage period.Therefore,RPT damage to red blood cells is also acceptable.Conclusion:The feasibility of RPT(RF50?mol/L,UV irradiation dosel0.8 J/cm2)for inactivation of HCT116 CTCs in tumor patients was demonstrated through in vitro cell?lar assays and animal experiments.RPT is expected to make tumor cells lose the ability of proliferation in the peripheral blood of tumor patients,while minimizing the damage to the blood cells,coag?lation function and immune function of patients.Part ?An experimental study on riboflavin photosensitization treatment for sim?lated inactivation of HCT116 cells mixed with salvaged blood from the surgical fieldObjective:This study was designed to evaluate the inactivation effect of riboflavin photochemical treatment(RPT)on HCT116 cells mixed in blood salvaged during operation and the superimposed damage to red blood cells,and to explore a new technique for intraoperative blood salvage(IBS)in patients with malignant solid tumor.Methods:Whole blood from healthy donors was mixed with HCT116 cells in a certain proportion,so that the final concentration of HCTI16 cells was 5×105/ml,and the mixed blood was transferred to EVA transparent blood storage bag after adding RF with the final concentration of 50?mol/L.The mixture of blood,tumor cells and riboflavin is alternately irradiated in UVA(365nm)and UVB(310nm)light sources,and the ambient temperature was controlled at(6±2)?.All samples were divided into three experimental groups and one control group according to the irradiation dose.Experimental groups included group 1(18J/cm2),group 2(23.4J/cm2)and group 3(28.8J/cm2).No RF or UV treatment was used as the control group.The whole blood of RPT was treated by Cell Saver Elite machine and matching consumables to simulate the blood salvage program during operation.The inactivation effect of HCT116 cells and the damage to red blood cells under different RPT conditions were evaluatedRes?lts:1?The apoptosis rates of HCT116 in control group,experimental group 1,group 2 and group 3 were(7.52±0.69)%,(52.94 ± 1.12)%,(61.46±2.79)%and(66.14±4.57)%,respectively.P values in group 1 vs.control group,group 2 vs.control group and group 3 vs.control group were 2.2E-12,2.8E-10 and 6.2E-09,respectively.2?Tumor growth was found in six mice of the control group,and the tumor volume was(0.608±0.38)cm3.There was no tumor growth in 18 mice of group1,group 2 and group 3.3?The contents of free hemoglobin in control group,experimental group 1,group 2 and group 3 were(0.14±0.09)g/L,(0.32±0.09)g/L,(0.36±0.08)g/L,(0.41±0.07)g/L,respectively.P values in group 1 vs.control group,group 2 vs.control group and group 3 vs.control group were 0.00081,0.00007 and 0.000002,respectively.4?The hemolysis rates of red blood cells in control group,experimental group1,group2 and group 3 were(0.07±0.04)%,(0.15±0.04)%,(0.17±0.04)%and(0.20±0.03)%,respectively.P values in group 1 vs.Control group,group 2 vs.Control group and group 3 vs.control group were 0.00081,0.00007 and 0.000002,respectively.5?The concentrations of Ca,Cl,CO2 and Na in the supernatant of erythrocytes in each group were not significantly different from those in the control group(P>0.05),but the concentrations of K in experimental group 2 and group 3 were significantly higher than those in the control group(P<0.05).The concentration of LDH increased with the increase of irradiation dose,but there was no significant difference compared with the control group(P>0.05).6?The contents of ATP in erythrocytes of control group,experimental group 1,group 2 and group 3 were(2.78±1.99)?mol/gHb,(2.64±1.40)?mol/gHb,(2.89?2.11)? mol/gHb,and(3.54± 1.95)?mol/gHb,respectively.P values in group 1 vs.control group,group 2 vs.Control group and group 3 vs.control group were 0.87,0.91 and 0.43,respectively.Conclusion:The final concentration of 50?mol/L RF and 18J/cm2 UV dose can effectively inactivate HCT116 tumor cells mixed the sim?lated salvage blood during the operation and make them lose the ability to proliferate.Under this condition,the effect on the structure and function of red blood cells is relatively limited.Therefore,RPT is expected to lead to routine IBS in patients with malignant solid tumors.