| BackgroundSkin Cutaneous Melanoma(SKCM)is highly malignant,prone to early lymph node and blood metastasis,and has a poor clinical prognosis.It is very necessary to explore the markers that can evaluate the prognosis of patients with SKCM.Immune checkpoint inhibitor therapy has become the standard treatment option for patients with advanced unresectable stage Ⅲ and Ⅳ melanoma.At present,many studies have confirmed that the effect of immune checkpoint inhibitor therapy is gratifying,but some patients have no response to the treatment or the effectiveness decreases after long-term medication.This may be due to the immunosuppressive characteristics of the tumor microenvironment,resulting in decreased cytotoxic T cell infiltration and the increase of the number of Treg cells,resulting in primary or acquired resistance to immunotherapy.Therefore,combined with other treatment methods(such as tumor vaccine,etc.),it can effectively activate antitumor effect of lymphocytes,weaken immune tolerance and improve the efficiency of treatment.SKCM is the most beneficial tumor in the treatment of immune checkpoint inhibitors,but nearly 50%of patients are still insensitive or ineffective in clinical application.One of the reasons may be the lack of tumor specific T cells in patients.SKCM may produce more tumor associated antigens and new antigens due to a large number of gene mutations,which can be used as the target of immunotherapy.Glycoprotein 100(GP100)is a nonmutated "self" antigen expressed by melanocytes,pigmented retinal cells and most melanoma cells.It is not expressed in other normal tissues or non-melanoma tumors.Compared with normal melanocytes,GP100 is overexpressed at all stages of melanoma progression,and it is one of the tumor associated antigens(TAA)of SKCM.So,is the expression of GP100 related to the prognosis,clinicopathological factors and tumor immune cell infiltration in patients with SKCM?Firstly,this paper analyzes the relationship between GP100 in gene and protein expression and clinical prognosis,and discusses the related clinicopathological factors.At the same time,based on the public database,the correlation between GP100 and lymphocyte immune infiltration level was further demonstrated and analyzed.Therefore,GP100 was recognized as one of the targets of SKCM immunotherapy.Antigen specific CD8+T cells play an important role in clearing tumor cells.Tumor vaccine can clear tumor cells by inducing initial T cells to differentiate into effector cells or expanding pre-existing antigen-specific CD8+T cells in vivo.It is one of the strategies of melanoma immunotherapy.The immune editing theory of "immune clearance,immune balance and immune escape" proposed by Robert Schreiber et al reveals that while tumor cells are immune cleared,they produce new mutations at any time to escape the clearing effect of the immune system.The specific CD8+T cells induced by tumor vaccine targeting a specific target antigen will be difficult to effectively remove tumor cells,which is the bottleneck in the clinical application of tumor vaccine.Epitope expansion refers to the immune response against other epitopes of antigens generated successively in the process of inducing specific response against pathogens and other immune target antigens by the body’s immune system,which may overcome the immune escape phenomenon caused by the heterogeneity of tumor cells and enhance the anti-tumor effect of the body.However,the characteristics of the vaccine causing epitope expansion and its related mechanism are not clear.In the process of inducing the body to produce antigen-specific CD8+T cells,professional antigen presenting cells are required to ingest and process exogenous antigens and present them to CD8+T cells in the form of MHC-I/polypeptide complex to induce cellular immunity.Existing studies have shown that conventional type 1 dendritic cell(cDC1)is considered to have the most powerful antigen cross presentation ability,which is an important consideration in tumor vaccine design and research.The receptor of X-C motif chemokine ligand 1(XCL1),X-C motif chemokine receptor 1(XCR1),is selectively expressed on the surface of cDC1 cells.Taking GP100 as the model antigen,we aim to construct a tumor vaccine that can expand the recognition epitope of T cell antigen,induce the body to produce immune cascade reaction and enhance the anti-tumor effect.Therefore,in this research,XCL1/GP100 fusion protein targeting cDC1 was constructed to analyze the antitumor protective effect of XCL1/GP100 fusion protein with secondary lymphoid tissue targeting to induce specific cellular immunity against immune antigen GP100,as well as the expansion effect and mechanism of antigenic epitope.It is expected that the relevant research results can provide a theoretical basis for improving the effect of immunotherapy in tumor patients.PurposeTo investigate the expression of GP100 in SKCM and its correlation with clinicopathological factors,and to study its feasibility as a prognostic tumor marker of melanoma(Part Ⅰ).GP100 was used as a target antigen in some adoptive T-cell therapy of SKCM.The level of immune infiltration is often an important factor affecting the prognosis and survival of patients with SKCM.Therefore,we tried to explore the correlation between GP100 and the level of immune infiltration in patients with SKCM(Part Ⅱ).Tumor vaccine can enhance the existing anti-tumor response or stimulate primitive T cells by stimulating the immune response of the body,so as to stimulate the function of effector T cells and kill tumor cells.In this study,taking GP100 as the model antigen,we aim to construct a tumor vaccine that can expand the recognition epitope of T cell antigen,induce the immune cascade reaction and enhance the antitumor effect.We try to explore the antitumor effect and functional mechanism of XCL1/GP100 fusion molecule in melanoma(Part Ⅲ).MethodsPart IThe GP100 gene expression data of 350 patients with SKCM and the clinical data and prognosis data of corresponding patients were downloaded from TCGA database,and the GP100 gene expression data of normal skin were collected from GTEx data set.At the same time,the expression of GP100 protein in tissue microarray of 50 patients with SKCM with prognostic information was analyzed by immunohistochemistry.To analyze the correlation between GP100 gene and protein expression and clinicopathological features,as well as the relationship with prognosis.Part ⅡThe GP100 expression was evaluated in SKCM via Tumor Immune Estimation Resource,Oncomine and Gene Expression Profiling Interactive Analysis(GEPIA).We also evaluate the influence of GP100 on overall survival via GEPIA,PrognoScan,and immunohistochemistry in human tissue microarray.The correlation between GP100 expression level and immune cell or gene markers of immune infiltration level was explored on Tumor Immune Estimation Resource and GEPIA.Part ⅢIn this study,XCL1/GP100 fusion molecule(DNA nucleic acid vaccine)was constructed by connecting the carboxyl end of chemokine XCL1 with the amino end of mutated GP100.The molecule was injected into subcutaneous tissue of tumorigenic mice by gene gun to observe its effect on the survival of mice and explore its possible mechanism.This study is mainly divided into the following steps:1)Constructing the plasmids expressing XCL1/GP100 and GP100;2)Experimental exploration of B 16-ova tumorigenesis;3)To explore the effect of XCL1/GP100 on the growth and survival of SKCM(C57 mice);4)To explore the amplification effect of XCL1/GP100 on pre-exist T cells(PMEL mice);5)To explore whether XCL1/GP100 can induce anti-tumor immune cascade under immune conditions;6)To explore the application value of XCL1/GP100 combined with PD-1 in anti-SKCM.ResultsPart I1.Data analysis of 350 patients with TCGA and 50 patients with TMA in SKCM confirmed that GP100 was highly expressed in SKCM.2.Cox multivariate regression analysis of TCGA data showed that GP100 expression(P=0.002),age(P=0.039)and tumor load(P<0.0001)were independent risk factors affecting the prognosis and survival of patients with melanoma.3.Cox multivariate regression analysis of TMA data showed that GP100 protein expression intensity(P=0.002),distant metastasis(P=0.002)and AJCC stage(P<0.0001)were independent risk factors affecting the prognosis and survival of patients with melanoma.4.TMA analysis showed that GP100 expression was significantly correlated with Clark grade,ulcer and AJCC stage in patients with SKCM(all P<0.05).5.In the above two groups of studies,GP100 is an independent risk factor affecting the prognosis and survival of patients with melanoma(P=0.002).Part Ⅱ1.The data in TCGA and GTEx were analyzed based on GEPIA database.It was found that the GP100 expression level in SKCM(461 samples)was significantly higher than that in normal tissues(558 samples),with statistical difference(P<0.05).2.TIMER and GEPIA database confirmed that high expression of GP100 often indicates poor prognosis in patients with SKCM.These results suggest that GP100 can be used as a prognostic marker in patients with SKCM.3.TIMER database analysis confirmed that there was a significant negative correlation between GP100 expression and tumor immune cells and gene markers of immune infiltration level.Part Ⅲ1.XCL1/GP100 fusion molecule and XCL1/mCherry fusion indicator molecule were successfully constructed.2.XCL1/GP100 fusion protein can effectively chemotaxis to human XCR1+CD141+DC cells.3.XCL1 fusion protein can effectively promote the uptake of antigen by native CD8+DC.4.XCL1/GP100 can effectively inhibit tumor growth and prolong the survival time of melanoma mice.5.XCL1/GP100 fusion molecule can induce specific cellular immunity against nonimmune target antigens and expands T cell antigen recognition epitopes.6.XCL1/GP100 fusion molecule can enhance the anti-SKCM effect of PD-1 antibody.Conclusion1.High expression of GP100 in patients with SKCM often indicates poor prognosis,so GP100 can be used as a prognostic tumor marker in patients with SKCM.Advanced age(>60 years),distant metastasis,AJCC stage(Ⅲ,Ⅳ)and tumor bearing status also predict the poor prognosis of patients with melanoma.2.The high expression of GP100 often indicates that the infiltration level of immune cells such as CD8+T cells,macrophages,neutrophils and dendritic cells in patients is low.There is a significant negative correlation between GP100 expression and tumor immune cells and gene markers of immune infiltration level.3.Compared with GP100 alone,XCL1/GP100 fusion protein can effectively chemotaxis to human CD141+ DC cells,which indicates that the fusion protein still maintains the chemotactic activity of XCL1 and ensures that the fusion protein can target the professional antigen cross presenting cell CD141+ DC expressing XCR1.4.XCL1 fusion protein can effectively promote the continuous and massive entry of antigen into local drainage lymph nodes and uptake by CD8+DC cells,which is conducive to antigen cross presentation and sensitization of Naive CD8+T cells.5.In the melanoma mouse implanted tumor model,the XCL1/GP100 fusion molecule can effectively inhibit the growth of mouse melanoma and prolong the survival time of mice.XCL1/GP100 fusion molecule can not only induce the de novo anti-tumor immune response,but also amplify the pre-existing anti-tumor response in vivo,so as to inhibit tumor growth.6.XCL1/GP100 anti-melanoma DNA vaccine can not only induce specific cellular immunity against immune target antigen GP100,but also induce specific cellular immunity against non-immune target antigen OVA,expand T cell epitope spread,and then enhance the killing effect of tumor.7.Compared with single XCL1/GP100 or PD-1 antibody,XCL1/GP100 combined with PD-1 antibody can significantly slow down the growth rate of tumor and prolong the survival time of mice.It is proved that XCL1/GP100 fusion molecule can significantly enhance the antitumor effect of PD-1 antibody. |
