Experimental Study On The Induction Of Tumor Cells Immunogenic Death And Enhancement Of Adoptive T Cell Therapeutic Effect By IR-780

Background and objective:Immunogenic cell death(ICD)converts dying cancer cells into a therapeutic vaccine and stimulates a systemic antigen-specific antitumor immune response,which can effectively subvert the immunosuppressive TME and enhance the efficiency of immune responses,relative to conventional immunotherapeutic regimens.However,the application of traditional inducers of ICD in anti-cancer immunotherapy has been limited because of low levels of ICD induction and a lack of tumor-targeting accumulation.Therefore,it is of great significance to find strong ICD inducers that specifically target tumors.At present,adoptive T cell therapy(ACT),which is very effective for patients with hematological malignancies,has been widely used in clinical practice.However,ACT has no obvious effect on solid tumors,due to the inefficiency of the immune response and the immunosuppressive tumor microenvironment(TME),and it is of great significance to develop new effective adjuvant therapeutic agents in combination with ACT to increase its therapeutic effect on solid tumors.This study aims to investigate the role of IR-780,a small molecule of heptamethine cyanine as a tumor-targeting ICD inducer,in enhancing the therapeutic effect of ACT on solid tumors.Methods:In vitro experiments,the effects of different concentrations of IR-780 on the proliferation of colorectal cancer cell line CT26 were measured by CCK-8 experiments.AnnexinⅤand 7-AAD staining were used to detect the apoptosis of CT26 cell lines after treated IR-780.The near-infrared fluorescence characteristics of IR-780 and co-localization with Mito-tracker Green,a mitochondrial-specific fluorescent probe,were used to determine the target site of IR-780.Detection of ICD-related markers with ATP detection kit,high mobility group box protein 1(HMGB1)ELISA kit,flow cytometry and cellular immunofluorescence staining.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect tumor cell antigen-presentation-related gene expression,and immunofluorescence staining was used to detect the mitochondrial-associated protein HSP90 exposure level.Bone marrow-derived dendritic cells(BMDC)were cultured with IR-780-treated tumor cells for 24hours,and the BMDC maturation was detected by flow cytometry.The animal experiment includes three parts:(1)A subcutaneous xenograft tumor model of colorectal cancer CT26 in mice was established with BALB/c mice,and the mice were treated with IR-780 and PBS,respectively.The tumor diameter was measured,and the volume was calculated according to the formula.After the treatment,the mice were sacrificed by cervical dislocation and the tumor tissue was removed and weighed.One part of the tumor tissue was digested into a single cell suspension to detect the infiltrating immune cells in the tumor tissue,and another part of the tumor tissue was fixed for tissue sectioning.Immunofluorescence staining was used to detect ICD-related markers CRT and CD3~+,CD8~+T lymphocyte infiltration.Utilizing the near-infrared fluorescence characteristics of IR-780,near-infrared imaging of living small animals was performed on the mice of IR-780 group to detect the distribution of IR-780 in vivo.(2)Vaccine experiment validates IR-780 as a new ICD inducer.CT26 cells were pretreated with IR-780,mitoxantrone(MTX),and cisplatin(CIS)and injected into the left back of BALB/c mice,and PBS was used as a control group.Live CT26 cells were injected into the right back,the tumor formation rate on the right back was observed,and the size of the tumor was measured.Similarly,CT26 cells pretreated with IR-780 were injected into the right back of BALB/c mice,and live CT26 cells were injected through tail vein one week later.The tumor metastasis of the mice was observed 20 days later.(3)B16-OVA mouse xenograft model was established and divided into Control group,IR-780treatment group,OT-1T cell treatment group and OT-1+IR-780 combination treatment group,the IR-780 treatment group and the combination treatment group were administered IR-780 intraperitoneally on days-3,-1,1,3,and 5respectively.The OT-1T cell treatment group and the combination treatment group were administration OT-1T lymphocyte via tail vein.Mice were sacrificed by cervical dislocation after observation at day 10,tumor tissues were removed and weighed,immune cells in tumor tissue and tumor draining lymph nodes in mice were detected by flow cytometry.Detection of CD8~+T cell effect-related gene expression levels in tumor tissues by qRT-PCR.Results:1.Targeted killing effect of IR-780 on colorectal cancer:IR-780inhibits the proliferation of CT26 cells and induces apoptotic cells in a concentration dependent manner.Near-infrared fluorescence microscopy detected the specific accumulation of IR-780 in tumor cells,and confocal microscopy revealed that IR-780 was localized to the mitochondria of the cells.In animal experiments,IR-780 can significantly inhibit tumor growth.The Kodak In-Vivo FX professional Imaging System was used to image the IR-780treated tumor-bearing mice,and IR-780 was specifically accumulated in tumor tissue.2.IR-780 induces immunogenic cell death of colorectal cancer in mice:The amount of ATP and HMGB1 released extracellularly was higher than that of the control group,and the cell membrane expression level of CRT was also higher than that of the control group.The expression levels of HSP90 and antigen-presenting genes in IR-780 treated group were higher than those in control group.After co-cultured with IR-780 treated CT26 cells,the maturation ratio of BMDC increased significantly.IR-780 was proved to be a new inducer of ICD through vaccine experiments,and increased infiltration of T lymphocytes in tumors in the IR-780 treatment group was found by flow cytometry and immunofluorescence staining.And it can inhibit the formation of lung metastatic nodules in mice with colorectal cancer through the effect of vaccine.3.IR-780 enhances the role of ACT in B16-OVA tumor model:The combination of IR-780 and OT-1T cells can significantly inhibit tumor growth.Flow cytometry results showed that the proportion of CD8~+T lymphocytes with killing effect in tumor tissue and draining lymph nodes was higher in the combined treatment group than other groups.In addition,qRT-PCR results also obtained similar results,that is,the expression of CD8~+T cell effect-related genes was highest in the combination treatment group.Conclusion:1.IR-780 can effectively inhibit the proliferation and induce apoptosis of colorectal cancer cells,and it can significantly inhibit tumor growth in the mouse tumor model of colorectal cancer cell line CT26.IR-780specifically accumulates in tumor cells and targets tumor cells’mitochondria.2.IR-780 can induce immunogenic cell death of colorectal cancer in mice and increase the infiltration of T lymphocytes in tumors.And it can inhibit the formation of lung metastatic nodules in mice with colorectal cancer through the effect of vaccine.3.IR-780 can enhance the inhibitory effect of ACT on tumor growth in B16-OVA xenograft tumor model,and can enhance the function of CD8~+T lymphocytes.