| Objective: Microsatellite instability-high(MSI-H)is a hypermutation pattern that occurs in genomic microsatellites,which occurs due to functional defects in DNA mismatch repair in tumor tissues,namely mismatch repair deficient(d MMR).Colorectal cancer(CRC)originates from normal colon epithelium,and genomic instability is a key factor driving its occurrence.There are two types of genomic instability in CRC,including chromosomal instability and microsatellite instability.MSI-H/d MMR has occurred in different types of cancer,with a high incidence in CRC,accounting for about 15% of the overall and 5% of metastatic CRC.This subtype of patients is the dominant population in immune checkpoint blockade(ICB)treatment.Nonetheless,several studies have found that only about 40% of MSI-H/d MMR CRC patients benefit from ICB therapy,and a substantial majority of patients do not respond to treatment or do not benefit consistently.This suggests the heterogeneity of MSI-H/d MMR CRC.Therefore,it is crucial to further stratify MSI-H/d MMR CRC patients,to screen the characteristic genes of those who benefit from immunotherapy,and to explore their molecular mechanism.In this study,the key gene of MSI-H/d MMR CRC was screened through data analysis of single-cell transcriptome sequencing,and its functions and potential mechanisms in ICB treatment were explored in depth through experiments.Methods: 1.The bibliometrix R package was used to analyze bibliometric data,including the number of publications and citations,international partnerships,national and journal contributions,highly cited articles,highly contributing journals,and historical direct citation networks.2.Cite Space software was used to analyze the references with the strongest citation bursts.3.VOSviewer was used for the visualization of bibliometric network data,such as keywords,terms,authors and institutions,etc.The links between nodes represent relationships such as co-occurrence and coupling.4.The single-cell transcriptome data GSE144735 was analyzed in this study,and quality control and batch effect correction were performed.5.The Seurat R package was used for cell clustering analysis,and the t-distribution random neighbor embedding method was used to visualize the cell clustering.6.Cell type annotations were performed by Single R R package and Cell Marker database.7.Single cell pseudotime trajectories were constructed with Monocle 2 for intestinal cancer epithelial cells to infer the occurrence and development of cancer cells and analyze the gene expression changes in the pseudotime trajectories.8.Screening of differentially expressed genes in cell clusters by R package Seurat.9.Conduct pathway enrichment analysis for each cell through single sample gene enrichment analysis(single sample GSEA)and annotate biological functions through the Hallmark database gene sets.10.The relationship between genes and various clinicopathological parameters was analyzed in GSE39582 and GSE72970 datasets.11.Tumor purity of colorectal cancer samples was estimated by R package Estimate.12. Western blotting was used to detect protein expression levels.13.Detect m RNA expression levels by quantitative real-time polymerase chain reaction(q RT-PCR).14.siRNA and c DNA plasmid transfection technology were used to knock down and overexpress the target gene.15.Construct stably transfected cell lines by lentiviral infection and drug screening.16.Peripheral blood mononuclear cells(PBMC)were extracted from healthy human peripheral blood by density gradient centrifugation.17.Co-culture tumor cells and PBMC in 96-well plates and detect the survival rate of tumor cells by MTS and inverted fluorescence microscope.18.The expression of HLA-DR on tumor cells was detected by flow cytometry staining.19.R software and Graph Pad Prism9 were used for the above analysis.All statistical tests were double-tailed and P < 0.05 was considered statistically significant.Results: 1.The bibliometric study included 11,971,191,and 335 documents in the dataset of PD-1 and PD-L1 molecule,randomized clinical trials as well as meta-analyses of anti-PD-1 and anti-PD-L1 antibodies,respectively.2.The literature on PD-1 and PD-L1 molecules was mainly divided into 3 research topics.The biomarkers used to predict the efficacy of PD-1 and PD-L1 monoclonal antibodies,including MSI-H,were important components of cancer and survival topics.In randomized clinical trials of PD-1and PD-L1 monoclonal antibodies,researchers focused on the selection of treatment population,while in the meta-analyses,more attention was paid to immune-related adverse events and the impact of demographic characteristics on the efficacy of ICB.3.The research focus changed from molecular mechanism to clinical applications including strategies such as ICB combined with chemotherapy or molecular targeted therapy.The biomarkers of immunotherapy covering MSI,immune-related adverse events,the characters of patients’ responses to anti-PD1/PDL1 treatment such as sensitivity,resistance and tumor heterogeneity might warrant sustained attention in the future.4.Single-cell clustering analysis of colorectal epithelial cells suggested the diversity of colorectal tumor cells.This study identified 10 epithelial cell subsets,including left colon-specific caner cell subsets,mixed cancer cell subsets,advanced cancer cell subsets and MSI-H cancer cell subsets.5.The analysis of single cell pseudotime trajectories found that epithelial-mesenchymal transition and angiogenic function enrichment gradually increased in the trajectory from left colon-specific cell subsets to advanced cancer cells.6.In the branch of MSI-H cancer cells,the enrichment of immune-related pathways gradually increased,such as inflammatory response,tumor necrosis factor,interferon response.7.ANXA10 is a characteristic gene of the MSI-H cancer cell subset with the highest enrichment of immune-related functions.The expression of ANXA10 in MSI-H cancer branch gradually increased with the development of the trajectory.The expression of ANXA10 in d MMR CRC was higher than that in p MMR type.In addition,ANXA10 was also highly expressed in proximal and right colon cancers and associated with poorer response to 5-fluorouracil-based chemotherapy(P<0.05).8.The expression of ANXA10 was higher in the MSI-H colorectal cancer cell line HCT15,and lower in the MSI-H colorectal cancer cell line HCT116 and RKO.9.ANXA10 increased the survival rate of MSI-H CRC cells under the action of PBMC(P<0.05).10.In MSI-H CRC cell lines,ANXA10 could significantly increase the sensitivity of cancer cells to anti-PD-1therapy(P<0.001).11.ANXA10 significantly increased the proportion of positive HLA-DR expression on the cell surface of MSI-H CRC cells(P<0.01)and promoted constitutive and IFN-γ-inducible expression of HLA-DR dimers.12.The m RNA levels of HLA-DRα and HLA-DRβ were not up-regulated after ANXA10 was overexpressed.13.ANXA10 knockdown significantly reduced CD74 protein and m RNA expression levels(P<0.01).Conclusions: 1.The focus of PD-1 and PD-L1 research had gradually changed from molecular mechanisms to clinical applications.The biomarkers of immunotherapy covering MSI,immune-related adverse events,sensitivity and resistance of anti-PD-1and anti-PD-L1 treatment,and tumor heterogeneity might warrant sustained attention in the future.2.ANXA10 was a characteristic gene of the MSI-H cancer cell subset with the highest enrichment of immune-related functions.It was highly expressed in d MMR colorectal cancer and might play an important biological function in this population.3.In MSI-H CRC cell lines,ANXA10 could significantly increase the sensitivity of cancer cells to anti-PD-1 therapy.4.ANXA10 might promote HLA-DR expression on the cell surface by increasing the expression of CD74,and ultimately improved the sensitivity of MSI-H CRC cells to anti-PD-1 therapy. |
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