Expression Of CD276 In Esophageal Squamous Cell Carcinoma And Its Effect On The Efficacy Of CAR-T Cell Therapy

IntroductionEsophageal cancer(EC)is one of the most common upper gastrointestinal tract malignancies worldwide and the sixth leading cause of cancer-related deaths globally.Current main treatments for esophageal cancer include surgery,chemotherapy,radiotherapy,targeted therapy and combined treatment.However,due to the lack of effective detection methods,almost 30% of diagnosed esophageal cancer patients are already in the advanced stages,and accompanied with distant metastasis,so that the therapeutic effect of traditional treatments is not as effective as expected.Despite some progress in very recent years,the overall 5-year survival remains poor at 16.9%.New active therapies and treatment approaches for esophageal cancer are needed.As a new therapeutic modality,immunotherapy has been widely utilized in the tumor therapy,CAR-T cell therapy is among the most effective current therapy for cancer.Currently,the success of CAR-T-cell therapy is by now confined to hematological malignancies,but,the treatment effect on solid tumor patients is not ideal.CAR T cell therapy for solid tumors can be hampered by tumor microenvironment,like PD-1/PD-L1.The antibody of PD-1/PD-L1 has demonstrated promising clinical outcomes.The B7 family is one of the most important and most likely the best characterized network,the upregulation of B7 inhibited molecules is associated with tumor immune evasion in tumor microenvironment.CD276(also known as B7-H3),a newly identified member of the B7 family,is involved in several signaling pathways,such as MAPK and NF-k B pathways.It is reported that the expression of CD276 in microenvironment leads to inhibition of T-cell proliferation and functions,like IFN-γproduction.CD276 is abnormally overexpressed in diverse human cancers,including lung,colorectal and breast cancers and associated with poor prognosis.CD276 and regulatory T cells(Tregs)were identified as having potential cooperative role in the immune evasion of tumor cells,and the resulting poor outcomes.Similarly,co-expression of CD276 and CD133 was evidently associated with progression of CD133+ colorectal cancer.High CD276 expression was found in human breast cancer tissues and play an important role in tumor progression and invasiveness.This expression appeared to increase the secretion ability of the immunosuppressive cytokine IL-10.The features of CD276 make it a potential candidate for cancer therapy.It is reported that CAR-T cells target CD276 showed comparable antitumor activity.There were no reports of CD276 in the progression of esophageal cancer.In the light of existing research,CD276 may play a greater role in promoting esophageal cancer progression.To investigate the underlying mechanism of CD276 provide a theoretical basis for the immunotherapy in esophageal cancer.This study investigated the regulatory mechanism of CD276 in esophageal squamous cell carcinoma from several aspects.Based on analyses of TCGA database and clinical data,CD276 was overexpressed in esophageal squamous cell carcinoma(ESCC)and was associated with poor prognosis of patients.Then,the glycolysis and lactate secretion of ESCC cell lines were significantly increased when CD276 was overexpressed.Regulation of glycolysis in ESCC cell lines by CD276 was mediated largely through its phosphorylation of PKM2.Finally,a tumor-bearing mouse model was established and then received CAR-T cell infusion.The results showed that high expression of CD276 promoted tumor progression and promoted the secretion of lactic acid by tumor cells.The accumulation of local lactic acid inhibited T cell function,resisted cell killing and achieved immune escape.New mechanisms of CD276 which promoted tumor progression in ESCC was found in our study,and it will provide a theoretical basis for the treatment of esophageal squamous cell carcinoma.1 Association between CD276 expression and disease progress as well as T cell infiltration in ESCC1.1 ObjectiveThe expressions of CD276 in ESCC specimens and TGCA database were analyzed,to further investigate the effect and prognostic value of CD276.Association between CD276 expression and lymphocytes infiltration in ESCC were analyzed,to further investigate the function of CD276 in immune infiltration.1.2 Methods(1)The different gene expressions of CD276 in ESCC tissues and normal esophageal specimens were evaluated by TGCA database.(2)The different expressions of CD276 in ESCC tissues and normal esophageal specimens were evaluated by RT-PCR and immunohistochemistry.(3)The prognostic efficacy of CD276 in ESCC patients was analysed.(4)Correlation analysis was conducted between CD276 and lymphocytes infiltration in tumor microenvironment using the TIMER2.0.(5)Correlation analysis was conducted between CD276 and T cell infiltration in tumor microenvironment using immunofluorescence.1.3 Results(1)Analysis of TCGA database revealed that tissues from the ESCC patients had higher expression of CD276,compared with normal tissues.(2)Results of RT-PCR showed that tissues from the ESCC patients had higher gene expression of CD276,compared with normal tissues.(3)Results of immunohistochemistry showed that tissues from the ESCC patients had higher protein expression of CD276,compared with normal tissues.(4)Survival analysis showed that high expression of CD276 was associated with worse survival of ESCC patients.(5)TIMER2.0 analysis showed that the expressions of CD276 in ESCC were negatively correlated with the infiltration of various immune cells,and the negative correlation with T cells was the most obvious.(6)Results of immunofluorescence showed that the expression level of CD276 was negatively correlated with T cell infiltration in ESCC.1.4 SummaryCompared with normal tissues,tissues from the ESCC patients had higher expression of CD276 and associated with worse survival.The expression of CD276 in ESCC was negatively correlated with the infiltration of various immune cells,and the negative correlation with T cells was the most obvious.2 Expression of CD276 in ESCC cell lines promoted glycolysis and lactic acid accumulation2.1 ObjectiveEvaluated the expression levels of CD276 in ESCC tissues.To investigate the underlying mechanism of CD276 promoting tumor progression.To investigate the underlying mechanism of CD276 inhibiting T cell infiltration.2.2 Methods(1)CD276 expressions in ESCC cell lines were measured by flow cytometry.(2)To generate cell lines with stable knockdown or overexpression of CD276 using lentiviral transduction.(3)Correlation between CD276 and ECAR in ESCC cell lines was determined with Seahorse instrument.(4)Lactate secretion of ESCC cell lines with different CD276 expression was determined by lactate kit.(5)Differential expressions of critical rate-limiting enzymes in ESCC glycolysis were evaluated by TCGA database.(6)Correlation between CD276 and critical rate-limiting enzymes in glycolysis were evaluated by TCGA database.(7)The gene expressions of critical rate-limiting enzymes in glycolysis were determined by RT-PCR when the expression of CD276 was changed.(8)Correlation between CD276 and PKM2 as well as Ki67 in ESCC tissues were determined by immunohistochemistry.(9)The gene expressions of PKM2 and STAT3 were determined by WB when the expression of CD276 was changed.(10)Small molecule inhibitors were employed in order to investigate the roles of CD276/PKM2/ STAT3 in ESCC.2.3 Results(1)CD276 was highly expressed in ESCC cell lines except EC109.(2)CD276-knockdown was determined in KYSE150 and KYSE510.CD276-overexpression was determined in EC109.(3)Overexpression of CD276 promoted glycolysis of ESCC cell lines.(4)Overexpression of CD276 promoted lactate secretion of ESCC cell lines.(5)In TCGA anaylsis,PKM2,HK2,HK3 and PFKP were highly expressed in ESCC tissues than normal tissues.(6)In TCGA analysis,significant correlation between CD276 and PKM2 in ESCC tissues was determined.(7)The corresponding changes of PKM2 and Ki67 were found when CD276 was knockdown or overexpression in different cell lines.(8)Significant correlation between CD276 and PKM2 as well as Ki67 in ESCC tissues was determined by immunohistochemistry.(9)CD276 appeared to positively regulate PKM2 and STAT3 by affecting PKM2 and STAT3 phosphorylation.(10)Small molecule inhibitors showed that the promotion of CD276 on esophageal squamous cell lines was dependent on the expression and activation of PKM2 and STAT3.2.4 SummaryOverexpression of CD276 promoted glycolysis in ESCC cell lines.The regulation of CD276 in glycolysis of ESCC relyed on the expression of PKM2.Overexpression of CD276 promoted lactate secretion in ESCC cell lines.3 Expression of CD276 in ESCC cell lines was resistant to CAR-T cell therapy3.1 ObjectiveTo investigate the influence of CD276 expression in ESCC on tumour progression.To investigate the influence of CD276 expression in ESCC on CAR-T cell therapy.3.2 Methods(1)The sequence of CAR construct was searched from NCBI.(2)A second-generation CAR construct(p CDH-EF1a-CAR.HER2-GFP)was generated in a p CDH lentiviral vector.(3)293FT cells were transfected with the lentiviral expression plasmids.(4)CD8+ T cells were infected with lentiviruses expressing CAR to construct CAR.HER2-T cells.KYSE150 cells and KYSE150-CD276-SH were infected with lentiviruses expressing luciferase to construct KYSE150-Luc and KYSE150-Luc-CD276-SH cell line.(5)Subcutaneous tumor bearing model of mice was established by injecting KYSE150-Luc and KYSE150-Luc-CD276-SH cell lines.(6)CAR.HER2-T cells were injected into mice via tail vein infusion.(7)Tumor-bearing mice were imaged live by small animal imaging.3.3 Results(1)Significant reduced tumour progression was seen in KYSE150-Luc-CD276-SH tumour-bearing mice.(2)KYSE150-Luc-CD276-SH tumour-bearing mice treated with CAR.HER2-T cells were more likely to show significant tumor regression.(3)CAR.HER2-T cells treatment significantly improved survival in KYSE150-Luc-CD276-SH tumour-bearing mice compared to controls.3.4 SummarySignificant enhanced tumour progression was seen in tumour-bearing mice with high expression of CD276.The high expression of CD276 on tumor cells is a new mechanism of resistance to CAR-T cell therapy.3.5 ConclusionsHigh expression of CD276 in ESCC was associated with poor prognosis as well as immune cell infiltration.By upregulating PKM2 expression and activation,CD276 promoted glycolysis and lactate secretion in ESCC cells,thereby promoting tumor progression and resisting CAR-T cell therapy.