| BackgroundHead and neck squamous cell carcinoma(HNSC)is one of the most common cancer types in the world.Due to cognitive deficiency,most patients have entered the middle and late stage when diagnosed,and more than half of them will have recurrence or distant metastasis,resulting in a five-year survival rate of less than 40%.In recent years,immunotherapy has been regarded as the most promising method for the treatment of advanced or aggressive cancer and has become the first-line drug for HNSC.Immune checkpoint blockades are an important part of immunotherapy,the most representative of which are PD-1/PD-L1 blockades.The mechanism of action of PD-1/PD-L1 blockades is to block the activity of immune checkpoint molecules,release the immune brake in the tumor microenvironment,and reactivate the killing effect of T cells on tumors,thereby achieving anti-tumor therapeutic effects.Although immunotherapy represented by PD-1 blockade can significantly improve the treatment effect of HNSC patients,its overall objective response rate in unselected patients is only about 20%,which may also be related to the presence of an inhibitory immune microenvironment in some patients.Therefore,it is necessary to establish a gene signature that can predict the prognosis and the efficacy of immunotherapy in HNSC patients,so as to enrich the population benefiting from immunotherapy.In addition,screening for active ingredients that can improve the immune status of patients for use in combination with immunotherapy will help to further improve the objective response rate.Promoting precision medicine has become the development direction of cancer treatment,and the use of big biological data to mine genetic information is an important method.With the rise of precision medicine,gene signature-based drug discovery has shown promising potential in screening combination immunotherapy drugs.Ginseng is a representative of traditional Chinese medicine with immunomodulatory effects,and its main active ingredient,ginsenosides,has dual effects of anti-tumor and immune regulation.A large number of studies have shown that ginsenosides can regulate the tumor immune microenvironment by stimulating lymphocyte proliferation,regulating immune cell function and immunoglobulin production,etc.,thereby enhancing the anti-tumor effect.Therefore,the active ingredients can be screened from ginsenosides by gene signature,and the possibility of their combined use with immunotherapy deserves further exploration.ObjectiveIn view of the low objective response rate of immunotherapy in the treatment of HNSC,this study aimed to construct a gene signature that can predict the prognosis of patients and the efficacy of immunotherapy.Then to screen active ingredients that could reverse the gene signature from the ginsenosides,and confirm that the candidate compound combined with anti-PD-1 antibody has a synergistic effect in the treatment of HNSC.Methods1.Based on the transcriptome data and clinical information of HNSC patients,the LASSO regression algorithm was used to construct the optimal gene signature.Its ability to predict patient outcomes was assessed by K-M survival curves and time-dependent ROC curves in multiple datasets.The association between the gene signature and clinicopathological factors and overall survival was assessed by univariate and multivariate Cox regression analysis,and the nomogram was constructed on this basis.In addition,the correlation between the gene signature and immune cell infiltration,anticancer immune response status,and immunotherapy response were assessed.2.The effects of various ginsenosides on the proliferation of head and neck squamous cell carcinoma and other tumor cells were explored through cytotoxicity experiments,and the effects of these components on the activity of immune cells were also explored.Based on the gene signature,RT-qPCR experiments were used to screen out candidate ginsenoside that could reverse the gene signature.3.The effect of candidate ginsenoside on the killing ability of immune cells on tumor cells was explored through a real-time dynamic imaging system,lymphocyte transformation experiment,and co-culture system of PBMC and tumor cells.And the effects of candidate ginsenoside on the proportions of various immune cell subsets were explored by flow cytometry.In addition,a tumor-bearing mouse model was constructed to explore the effect of the combined use of candidate ginsenoside and anti-PD-1 antibodies on tumor growth and tumor immune microenvironment by monitoring tumor weight and tumor volume,and detecting tumor-infiltrating lymphocytes.4.The correlations between gene signature-related genes affected by candidate ginsenoside and PD-1 were evaluated by Pearson correlation analysis and gene set enrichment analysis(GSEA).The effect of candidate ginsenoside on the expression level of PD-1 in lymphocytes was explored by flow cytometry and Western Blot experiments.Finally,the binding of candidate ginsenoside to PD-1 was evaluated by molecular docking and molecular dynamics simulations.Results1.In this study,a gene signature composed of 13 immune-related genes was constructed,which has good potential to predict the prognosis of HNSC patients and can be used as an independent prognostic factor.The nomogram constructed based on the gene signature and multiple clinicopathological factors can be used to quantify the survival probability of individual HNSC patients clinically.In addition,the gene signature is associated with a variety of immune cell infiltration levels,can better reflect the state of the tumor immune microenvironment,and has good potential in predicting immunotherapy responses.2.Ginsenosides have broad-spectrum anti-tumor effects.Rg3,Rh2,and CK have significant inhibitory effects on five tumor cells,among which the inhibition effect on tongue squamous cell carcinoma Cal-27 cells is the most significant.At the same concentration,ginsenosides did not damage the activity of immune cells.Besides,since ginsenoside Rh2 is the main metabolite of Rg3,the pharmacological effects of the two are similar,but ginsenoside Rh2 has the most potential in reversing gene expression associated with gene signature.3.The combined use of ginsenoside Rh2 and anti-PD-1 antibody can significantly enhance the killing effect of PBMC on tumor cells,and can also promote the proliferation of NK cells and CD8+T cells.In HNSC tumor-bearing mice,ginsenoside Rh2 can significantly promote the inhibitory effect of anti-PD-1 antibody on the tumor,indicating that ginsenoside Rh2 has a synergistic effect with anti-PD-1 antibody.The combination of the two drugs can ameliorate the state of spleen enlargement and thymus atrophy,increase the proportion of T cells and the proportion of B cell activation in the spleen,and significantly increase the infiltrating abundance of CD8+T cells and the proportion of B cell activation in the tumor microenvironment,thereby enhancing the anti-tumor immune response.4.The gene signature-related genes affected by ginsenoside Rh2 were significantly positively correlated with PD-1,and were involved in regulating the pathway "cancer immunotherapy by PD-1 blockade".In addition,ginsenoside Rh2 can down-regulate the expression of PD-1 on PBMC and can stably bind to PD-1 protein.The above results indicated that ginsenoside Rh2 might inhibit the PD-1 signaling pathway by down-regulating the level of PD-1 protein,thereby having a synergistic effect with anti-PD-1 antibodies.ConclusionThis study established a robust gene signature that can predict prognosis and immunotherapy response in HNSC patients,which can help predict the survival probability of patients in the clinic and enrich the immunotherapy effective population.At the same time,potential candidate compounds for combined immunotherapy were screened based on this gene signature.Among them,ginsenoside Rh2 can promote the killing effect of lymphocytes on tumor cells and enhance the inhibitory effect of anti-PD-1 antibody on HNSC,which may be associated with binding and inhibiting PD-1 protein.This combination therapy has important clinical application value,and this research strategy will provide new ideas for the discovery of combination immunotherapy drugs from traditional Chinese medicine. |
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