| Objective:Colorectal cancer(CRC)is among the most prevalent malignancies globally.Based on global cancer statistics in 2023,CRC holds the third position both in terms of newly diagnosed cases and cancer-related mortality.Distant metastasis,primarily to the liver and lungs,is the primary cause of death in CRC patients,with approximately 20%presenting with distant metastasis at initial diagnosis.Considering that liver metastasis predominantly leads to mortality,its management represents a crucial challenge in CRC treatment.Hence,unraveling the mechanisms underlying CRC liver metastasis and identifying potential diagnostic and therapeutic targets may illuminate new avenues for therapeutic interventions.The liver,being an organ with inherent immune tolerance,frequently serves as a metastatic site for various cancers.The evolution and progression of CRC are dictated not solely by genetic and epigenetic changes but also substantially by the tumor microenvironment(TME),especially the tumor immune microenvironment.The intricate interplay between tumor cells and various elements of the TME,such as immune effectors,blood vessels,fibroblasts,signaling molecules,and extracellular matrix,has gained significant attention owing to its impact on therapeutic outcomes.The tumor microenvironment reciprocally influences tumor development by eliciting extracellular signals that facilitate tumorigenesis and induce peripheral immune tolerance,and immune cells within the TME can affect the growth and evolution of the cancer cells.While the process of cancer cell invasion is extensively studied,the role of liver-specific factors in metastasis and their contribution to metastatic dissemination mechanisms remain largely uncharted.Programmed cell death-ligand 1(PD-L1)expression is a common feature across human cancers.It interacts with Programmed Death 1(PD1)to suppress the activation signal of T cells,thereby promoting immune evasion.Although the recent advent of PD1-PD-L1inhibitors has achieved enduring tumor remission in patients with diverse advanced cancers,their efficacy against CRC,particularly liver metastases,is limited.Therefore,a detailed understanding of the CRC immune escape mechanism in the liver promises to offer fresh mechanistic insights and potential therapeutic targets for mitigating CRC liver metastasis.In our earlier work,we established two CRC cell lines(CRLM-H)with high liver metastasis potential by developing animal models of CRC liver metastases.We identified Alpha-1-antitrypsin(A1AT)as a molecule showing significantly increased expression in liver metastasis through transcriptome sequencing.Building on this foundation,the current study further investigates the molecular mechanism by which A1AT regulates transcription,enhances immune evasion,and promotes CRC liver metastasis.Methods:1.To discern the expression and implications of A1AT in CRC liver metastasis:(1)A1AT expression in 20 primary CRC samples and 19 liver metastasis samples was retrieved from the Gene Expression Omnibus(GEO)database to explore its role in CRC liver metastases.R programming language(gupper)was utilized to analyze A1AT expression differentials between primary and metastatic sites.Real-time quantitative PCR(q RT-PCR)and Western blot(WB)analysis were conducted to determine A1AT m RNA and protein expression in CRC cell lines.Tissues collected from 37 patients with CRC liver metastasis were subjected to immunohistochemistry(IHC)to ascertain A1AT expression.(2)To characterize the genetic attributes of A1AT,flow cytometry analyzed the proportion of A1AT+PD-L1+cells in CRC liver metastasis,and confocal imaging examined the co-localization of A1AT and PD-L1 in metastatic CRC tissues.Western blot assay detected A1AT and PD-L1 expression in CRC cells.2.To define the association between A1AT and tumor immunity,public datasets analyzed the correlation of A1AT with CRC liver metastasis.Colon cancer liver metastasis samples in the GSE49355 dataset were categorized into A1AT high and low expression groups for correlation analysis.(2)To probe the correlation of A1AT with T cells,CIBERSORT evaluated the relationship between A1AT and CD8+T cells.Flow cytometry detected the proliferation and activation of CD8+T cells,expression of CD4+FOXP3+T,and IL-10 expression in human peripheral blood T cells co-cultured with CRC cells.Immunohistochemistry assessed the A1AT and FOXP3 expression in human CRC liver metastases.(3)To explore the relationship between A1AT and macrophages,GSEA analyzed A1AT’s associated functions.TIMER evaluated the correlation of A1AT with macrophages,and the correlation of A1AT with CD206-expressing macrophages in patient-derived single cells was analyzed using a CRC liver metastasis single-cell database.3.To elucidate the mechanism of A1AT-mediated promotion of CRC liver metastasis:(1)To investigate how A1AT recruits M2-type macrophages,ELISA analyzed the influence of A1AT on inflammatory factors in CRC cell supernatants.q RT-PCR evaluated CCL5expression in CRC cells,and confocal imaging studied the co-expression of A1AT and CCL5 and M2-type macrophage expression in CRC liver metastases.Macrophages co-cultured with CRC cells underwent flow cytometry and Transwell migration assays to study the effects of CCL5 and A1AT on macrophage migration.(2)To further investigate A1AT’s role in promoting M2 macrophage polarization,q RT-PCR analyzed m RNA expression of IL-6,IL-8,and IL-10 post macrophage and CRC cell co-culture.Western blot assessed the effects of IL-6,Stattic,and NF-κB-IN-1 on PD-L1 expression in CRC cells.Co-immunoprecipitation(Co-IP)experiments investigated the interaction between A1AT and STAT3,and the effects of A1AT and STAT3 under inflammatory conditions.(3)To verify the correlation between A1AT and PD-L1 and their roles in CRC liver metastasis.Results:1.A1AT was overexpressed in colorectal cancer(CRC)liver metastases and exhibited a positive correlation with PD-L1 expression:(1)The GSE49355 dataset analysis revealed that A1AT expression in CRC liver metastases was significantly elevated compared to primary colon cancer lesions.Findings from q RT-PCR,Western blot,and immunohistochemistry showed a high expression of A1AT in CRC liver metastatic cells.(2)Flow cytometry identified a proportion of A1AT+PD-L1+cell population in CRC liver metastases.Confocal imaging revealed conspicuous co-localization of A1AT and PD-L1 in human CRC liver metastases,and Western blot analysis demonstrated a positive correlation between A1AT and PD-L1 expression in CRC liver metastatic cells.2.A1AT was found to potentiate the immunosuppression of CRC:(1)The GSE49355dataset indicated that the development of CRC liver metastasis is associated with the tumor microenvironment.(2)CIBERSORT analysis demonstrated a negative correlation between A1AT and CD8+T cell activation in CRC liver metastatic cells.Flow cytometry revealed A1AT significantly suppressed the proliferation and activity of CD8+T cells,while promoting the proliferation of FOXP3~+Treg cells and IL-10 expression.Immunohistochemical findings presented a positive correlation between A1AT and FOXP3 expression in CRC liver metastases.(3)GSEA analysis indicated A1AT is closely linked to antigen presentation,cytokine-receptor interactions,and cell adhesion molecules.TIMER analysis results demonstrated a positive correlation between A1AT and macrophages.Analysis of a single-cell database from colon cancer liver metastasis showed high expression of A1AT and CD206-expressing macrophages.3.A1AT facilitated CRC liver metastasis by modulating PD-L1 transcription via nuclear STAT3:(1)ELISA and q RT-PCR indicated a positive correlation between A1AT and CCL5 expression in CRC liver metastatic cells.Confocal imaging analysis depicted co-expression of A1AT and CCL5,and presence of M2-polarized macrophages in human CRC liver metastases.(2)Flow cytometry and Transwell migration assays demonstrated that A1AT in CRC liver metastases recruited macrophages through CCL5 and enhanced the polarization of M2-type macrophages.(3)Western blot results showed IL-6,acting on STAT3 in CRC cells,facilitated PD-L1 expression.GEO database analysis indicated a positive correlation between PD-L1 and IL-6.Co-immunoprecipitation(Co-IP)experiments showed that IL-6 could promote the interaction between A1AT and STAT3in the nucleus of CRC cells.(4)A1AT and PD-L1 were shown to promote CRC liver metastasis in mice.Conclusion:1.In cells representative of hepatic metastasis from colorectal cancer,the expression of A1AT was notably elevated and demonstrated a positive correlation with PD-L1.2.A1AT bolsters the proliferation of FOXP3~+Treg cells and the polarization of M2macrophages by suppressing the proliferation of CD8~+T cells,thus augmenting immune escape and facilitating the onset of CRC liver metastases.3.Through the derivatives of CCL5,A1AT fosters macrophage migration,regulates M2macrophage polarization,and promotes IL-6 secretion.This process stimulates CRC cells to allow nuclear translocation of A1AT and STAT3 for PD-L1 transcription,thereby promoting CRC liver metastasis. |
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