Effect Of CALD1 On Immune Cell Infiltration In Malignant Melanoma

Objective:Malignant melanoma(SKCM)is a solid tumor that is difficult to diagnose and treat effectively because it is prone to invasion in the early stages and its 5-year overall survival is only 10%,which seriously threatens the prognosis of patients.caldesmon1(CALD1)gene expression affects smooth muscle cell contraction,cytoskeletal depolymerization and rearrangement,and the composition of the extracellular matrix.Based on transcriptomic data of malignant melanoma in the TCGA database,we found that patients with primary malignant melanoma with high expression of CALD1 gene have poor survival prognosis and reduced CD8+ T cell infiltration.By analyzing the local immune cell infiltration of tumors in different tumor-bearing mice using a B16 cell line with stable knockdown of CALD1 gene and tumor-bearing mice as models,we investigated the role of CALD1 molecule in the immune infiltration of melanoma.Methods:Frist,survival analysis of the relationship between CALD1 expression and clinical information in cutaneous melanoma tissues by TCGA database,using eight algorithms such as ESTIMATE algorithm,CIBERSORT algorithm,and X-cell algorithm to estimate and compare the immune infiltration at different CALD1 expression levels.Differential analysis,KEGG pathway enrichment analysis and GO gene function enrichment analysis,as well as weighted gene co-expression network analysis(WGCNA)were performed for CALD1 high expression group as well as low expression group.Second,the stable knockout B16 cell lines were constructed using CRISPR technology,and the knockout status was identified using WB method and gene sequencing,and the stable knockout lines were subjected to functional studies at the cytological level,including cell proliferation,migration,invasion,and apoptosis.The cells were used to perform the experiments in tumor-bearing mice to detect tumor volume changes,final tumor weight and knockdown of CALD1 in tumor tissues of animal models.The local immune cell infiltration of the tumor in different hormonal mice was analyzed by detecting the differences in HE staining,immunohistochemistry,and transcriptome expression.Third,the expression clustering and correlation analysis of CALD1 were performed using the HPA database,and the CAF scores of primary melanoma patients were calculated by the ss GSEA algorithm,and the ROC curve was used to detect whether the expression of CALD1 gene could be used as a basis for judging the CAF scores.Combined with the prebiotic letter results and literature to screen the physical barrier-related proteins affecting local immune infiltration of tumors,the correlation of these physical barrier-related proteins with CALD1 protein was evaluated bioinformatically,and then the changes of m RNA expression levels of physical barrier-related proteins were detected by RT-q PCR method and transcriptome analysis.Results:1.CALD1 has prognostic value in primary SKCM,where high CALD1 expression leads to poor patient prognosis.In the analysis of eight immune infiltration algorithms,a higher percentage of CD8+ T cells was observed in the CALD1 low expression group.Subsequently,the m RNA expression profiles of CALD1 high expression patients and CALD1 low expression patients were differentially analyzed,and a total of 2338 differential genes were screened,which revealed that the up-regulated genes were mainly enriched in matrix-related functions and pathways such as extracellular matrix formation,cell adhesion,and regulation of the actin backbone,and the down-regulated genes were mainly enriched in adaptive immune responses,lymphocyte migration,T cell receptors,and other immune The down-regulated genes are mainly enriched in immune-related functions and pathways such as adaptive immune response,lymphocyte migration and T-cell receptor.In the WGCNA analysis,genes in the blue module showed the highest positive correlation with CALD1 expression,and most of their core genes were associated with the matrix.The magenta module had the highest negative correlation with CALD1 expression,and most of its core genes were immune-related.We speculate that CALD1 may affect the stromal component of TME and is associated with the immune infiltration of malignant melanoma.2.To test whether CALD1 affects local immune infiltration in malignant melanoma,we constructed CRISPR/Cas9-CALD1-px459 plasmid and obtained B16-F0 and B16-F10 cells with stable knockdown of CALD1 gene.B16-F0-CALD1-Sg1 and B16-F10-CALD1-Sg2 were identified as stable knockdown cell lines using WB method and gene sequencing,and these cells were used as the experimental group.Then,we investigated the effect of CALD1 gene on tumor cells at the cytological level,and we found that the growth of B16-F10 cells was inhibited after knockdown of CALD1 gene in terms of proliferation,migration,invasion,apoptosis and other cytological abilities,while there was no significant change in the phenotype of B16-F0 cells.We used the two groups of cells to perform the lotus mouse experiment,and then performed HE staining,immunohistochemistry and transcriptome analysis on the tumor tissues,and found that the melanoma in the experimental group had more local lymphocyte infiltration and higher content of CD8+ T cells,NK cells and neutrophils.In addition,we found that B16-F0 cells in the experimental group grew more slowly in C57 mice and the final tumor size was smaller,while B16-F10 cells in the experimental group did not grow significantly differently in mice compared with the control group.It indicates that the low expression of CALD1 in tumors can promote the infiltration of local immune cells in tumors and slow down the growth of primary melanoma cells in mice in vivo,while it has little effect on the growth of this cell in vitro.3.In the clustering of expression in single cells and tissues,CALD1 was found to be associated with ECM tissues such as smooth muscle cells and fibers,respectively.Based on the characteristic gene sets associated with CAF such as collagen fibers and extracellular matrix,the CAF score of each primary melanoma patient was evaluated using the ss GSEA algorithm,and the AUC value obtained using the ROC curve was 0.76(>0.7),indicating that the expression level of CALD1 gene can determine the degree of CAF infiltration,i.e.,the correlation between the two is high.After screening,the physical barrier-related proteins(DDR1,NCKAP1,FAP)affecting local immune infiltration of tumors were obtained,among which NCKAP1 belonged to the hub gene of the blue module in WGCNA analysis.The correlations of physical barrier-related proteins with CALD1 gene were 0.10,0.61 and 0.30 by raw signal analysis,indicating that the level of CALD1 protein could potentially affect NCKAP1 protein expression and rise with the increase of CALD1 expression.Finally,the expression of physical barrier-related genes was examined by RT-q PCR and mouse tumor transcriptome data,and it was found that in B16-F0 cells,low expression of CALD1 had little effect on DDR1 and FAP,but the expression level of NCKAP1 gene in this cell was down-regulated,while in B16-F0 tumor-bearing mice,low expression of CALD1 appeared as DDR1 and NCKAP1 gene was down-regulated in B16-F0 tumor-bearing mice.This is basically consistent with the correlation analysis of raw letter,and we speculate that the CALD1 gene in B16-F0 melanoma cells promotes local immune cell infiltration of the tumor by causing changes in the physical barrier-related gene NCKAP1,which ultimately leads to the slow growth of subcutaneously injected primary melanoma in mice.Conclusion:The results of the present study suggest that low expression of CALD1 in the growth and development of B16-F0 cells in mice may lead to reduced expression levels of the physical barrier-related gene NCKAP1,causing altered immune infiltration status in the tumor microenvironment,which in turn leads to increased infiltration of local immune cells in primary melanoma and inhibits tumor development.Our findings reveal the possible factors of CALD1 regulation of local immune infiltration in malignant melanoma,and CALD1 is expected to be a candidate therapeutic target for patients with primary melanoma.