Effects Of Secretion Phenotype Of Senescent Tumor Cells On Tumor Migration, Invasion And Tumor Immune Micro-Environment And Its Mechanism

Objective:In the cancer treatment,including radiotherapy and chemotherapy,it is inevitable to cause fatal injury to some tumor tissues,and subfatal injury to other tumor tissues,due to various factors such as drug concentration gradient inside the tumor.It has been known that subfatal injuries often lead to aging phenotype of tumor cells or stromal cells in tumor microenvironment,that is,treatment-related aging.Therapy-related senescence plays a bidirectional role in the development of tumors.The long-term chronic effect of tumor treatment-induced cell senescence in the tumor microenvironment may eventually lead to the pro-tumor effect greater than the anti-tumor effect,and finally become an important factor in promoting tumor growth,invasion and metastasis.Based on this phenomenon,It has been believed that if the secretion of senescences-associated secretory phenotype（SASP）can be inhibited during the periods of anti-tumor treatment,the tumor-promoting effect caused by the long-term effect of SASP may be partially eliminated.In recent years,some researchers proposed that the concept of"inflammatory aging",points out that in the aging individuals,due to the aging of SASP secrete a large number of microenvironment including interleukin（interlekin,IL）-1 beta,gm-csf,IFN gamma,inflammatory factors such as environment caused by systemic low-level chronic inflammation in gathered themselves together,and is advantageous to the adaptive immune response,thus shaping the immune to inhibiting tumor microenvironment.The purpose of this study was to study and explore the role of senescent tumor cells and SASP in the influence of tumor invasion,migration ability and tumor immune microenvironment,and to preliminaries explore the molecular mechanism.Materials and Methods:（1）BLM and H₂O₂ were used to induce the senescence of MCF-7 breast cancer cells,A549 and NCI-H1299 lung cancer cells,respectively.（2）SA-β-gal staining was applied to analyze the senescence ratios of cancer cell.Flow cytometry was conducted on senescence-induced cells to evaluate the cell cycle distribution.（3）Conditioned medium（CM）from senescent/non-senescent cells from multiple cancer cell lines was used to treat associated non-senescent cells and wound-healing,invasion,cell proliferation,and EMT were evaluated.（4）An antibody array was used to detect cytokines in CM.ELISA was performed to detect IL-6 expression level in CM of senescent A549,NCI-H1299,and MCF-7 cells.（5）Transcriptome analysis of non-senescent cells was applied to analyze associated signaling pathways.Protein and m RNA expression of PD-L1,YAP,CYR61,c-Myc,and CTGF was analyzed by western blotting and quantitative reverse transcriptase PCR.（6）SA-β-gal staining was applied to fresh-frozen tissue sections.We detected YAP,PD-L1,CD3,and CD8 expression by immunohistochemistry in LUAD tissues.Relationships between senescence and clinical characteristics were also analyzed.（7）We searched the Pub Med,EMBASE,Cochrane,Web of Science,China National Knowledge Infrastructure（CNKI）,and Wan fang Databases for papers investigating the prognostic significance of nuclear YAP expression in NSCLC patients.（8）Western blot was performed to detect c GAS and p IRF3＿S396 protein level in senescent NCI-H1299 cells.The correlation between STING signaling,tumor immune microenvironment（TME）,and the overall survival of LUAD patients was investigated utilizing data from The Cancer Genome Atlas（TCGA）.Ss GSEA,CIBERSORT,and ESTIMATE algorithms were applied before gene set enrichment analysis（GSEA）was used to screen Gene Ontology（GO）terms and Kyoto Encyclopedia of Genes and Genomes（KEGG）signaling pathways between low and high STING signaling groups of LUAD.qRT-PCR was used to validate the relationship between immune-related markers and STING-related gene.Results:（1）The percentages of cells with positive SA-β-gal staining were 91.03±2.30%（P<0.001）,94.37±3.047%（P<0.001）,and 97.57±2.43%（P<0.001）for A549,NCI-H1299,and MCF-7 cells after two cycle treatments with 200μM（A549）,100μM（NCI-H1299）H2O2 or 16μM（MCF-7）bleomycin,respectively.（2）Senescent MCF-7 cells induced by BLM mainly arrested at S phase（P<0.001）and G2/M phase（P<0.001）,while H₂O₂ treatment caused NCI-H1299 and A549 cells to arrest at S（P<0.001）and G0/1（P<0.001）phases,respectively.（3）After 24 h and 48 h of cultivation,the percentages of healed areas for A549,NCI-H1299,and MCF-7 cells remarkably increased under senescent CM treatment compared with non-senescent CM treatment（all P<0.001）.（4）Senescent CM of A549,NCI-H1299,and MCF-7 cells significantly promoted cell invasion compared with non-senescent CM（P<0.001 for all）.Senescent CM of A549,NCI-H1299 and MCF-7 cells did not promote cell proliferation compared with non-senescent CM（P（29）0.05 for all）.（5）Senescent CM of A549 and NCI-H1299 cells significantly downregulated the protein level of E-cadherin（P=0.04 and P=0.004）,while remarkably upregulated the protein level of Vimentin（P=0.001 and P=0.008）,Snail（P=0.022 and P=0.003）and Slug（P=0.007 and P<0.001）compared with non-senescent CM（P=0.04 and P=0.004）.（6）181 cytokines were upregulated>2-fold in senescent CM compared with the181 cytokines levels in non-senescent CM,including inflammatory cytokines,extracellular matrix proteins,growth factors,and chemokines.（7）IL-6 levels were significantly increased in senescent CM of A549 cells（P?<?0.01）compared with non-senescent CM.（8）IL-6 levels were remarkably increased in senescent CM of NCI-H1299 cells（P?<?0.01）compared with non-senescent CM.（9）IL-6 levels were significantly increased in senescent CM of MCF-7 cells（P=?0.001）compared with non-senescent CM.（10）We identified 1497 genes that were differentially transcribed（P<0.05）in NCI-H1299 cells after treatment with senescent CM compared with the expression level of 1497 genes in the non-senescent CM-treated cells.Transcriptome analysis confirmed significant enrichment of m RNA expression of the Hippo pathway（FDR<0.05）.The highly-enriched GO terms associated with differentially-expressed genes were remarkably related to immune response,regulation of cell proliferation,and adhesion.（11）NCI-H1299 cells had higher m RNA（P?<?0.001）and protein（P=0.025）expression levels of PD-L1 than that of A549 cells.（12）Further mechanistic investigations verified that the YAP（P<0.001）and PD-L1（P<0.001）protein and YAP（P<?0.01）,PD-L1（P<?0.01）,CYR61（P<?0.05）,c-Myc（P<?0.01）,and CTGF（P<?0.001）m RNA expression level were significsntly upregulated after treatment with senescent CM.（13）The positive rate of SA-β-Gal staining in the tumor tissues from the retreatment lung cancer patients was significantly higher than that in the cancer tissues from the primary lung cancer patients（P<0.001）.The expression level of YAP in primary tumor tissues from the aging lung adenocarcinoma patients was significantly higher than that in the primary tumor tissues from non-aging lung adenocarcinoma patients（P<0.001）.The expression level of PD-L1 in the primary tumor tissues from the aging lung adenocarcinoma patients was significantly higher than that in the primary tumor tissues from the non-aging lung adenocarcinoma patients（P<0.001）.（14）In the clinical lung adenocacinoma cancer specimens,the infiltration degree of CD3+CD8+T lymphocytes in the primary tumor tissues from the senescent lung adenocarcinoma patients was significantly higher than that in the primary tumor tissues from the non-senescent lung adenocarcinoma patients（P=0.008）.（15）The expression level of PD-L1 in aging lung adenocarcinoma cancer tissues was positively correlated with the expression level of YAP in the nucleus from the cancer tisshues（r=0.414,P=0.026）.The clinicopathological characteristics of the lung adenocarcinoma patients,including gender（P=1）,smoking history（P=0.753）,tumor status（P=0.508）,lymph node metastasis（P=1）,TNM stage（P=1）,and tumor differentiation degree（P=0.317）,were not significantly correlated with the immunohistochemical score of senescent lung cancer tissues.（16）High nuclear YAP1 expression was negatively correlated with their OS in the414 NSCLC patients（HR=1.52;95%CI:1.11 2.08;P=0.01）;The high expression level of YAP1 in the nucleus was negatively correlated with PFS（HR=2.11;95%CI:1.52 2.93;P<0.001）.（17）The c GAS expression level in lung caner cell line NCI-H1299 treated with100μm H₂O₂ was significantly higher than that in the control group（P<0.001）.The expression level of PIRF3＿S396 in lung cancer cell line NCI-H1299 treated with 100μm H₂O₂ was significantly higher than that in the control NCI-H1299 cells（P<0.0001）.（18）Among 497 lung adenocarcinoma patients from TCGA,the proportion of tumor cells in the group with high STING signal rating（HS）was significantly lower than that in the group with low STING signal rating（LS）（P<0.0001）.The proportion of interstitial and lymphocyte in HS group was significantly higher than that in LS group（all P<0.0001）.Immune score（P<0.0001）,interstitial score（P=0.0011）,and ESTIMATE score（P<0.0001）in HS group were significantly higher than those in LS group.The ratio of activated CD4/CD8 T cells in HS group was significantly higher than that in LS group（P<0.0001）.There was a significant positive correlation between the expression scores of anti-tumor immune cells and promotion tumor immune cells in HS and LS groups（P<0.0001,R2=0.7294）.（19）The relative ratio of M1 macrophages andγδT cells in HS group was significantly higher than that in LS group（all P<0.0001）.There was no statistically significant difference in the relative proportion of the majority of immune cells between the HS and LS groups.（20）The level of immune checkpoint expression,including PD-1,PD-L1,CTLA4,IDO1,LAG3 and ICOS in HS group was significantly higher than that in LS group（all P<0.0001）.The expression levels of 20 cytokines/chemokines in lung adenocarcinoma patients in the HS group were significantly higher than those in the LS group（all P<0.05）.（21）The most significant biological processes in HS group included positive regulation of biological stimulus response,positive regulation of innate immune response,activation of innate immune response,regulation of innate immune response and regulation of response to biological stimulus（FDR<0.001）.The top five cell components in HS group were most significantly enriched,including cytoplasmic processing bodies,plasma membrane signal receptor complex,early intosomal membrane,exogenous components of membrane and phagocytes（FDR<0.05）.The top five GO items with the most significant enrichment in HS group were positive regulation of innate immune response,regulation of biological stimulus response,positive regulation of biological stimulus response,regulation of innate immune response and activation of innate immune response（FDR<0.0001）.The top five KEGG pathways in HS group were T cell response,GIR class I receptor,B cell receptor signal,NK-mediated cytotoxicity and apoptosis signal（FDR<0.05）.（22）The OS of CD8^Hi group was significantly higher than that of CD8^Lo group（P=0.0423）.OS in HS group(STING^Hi)was significantly higher than that in LS group(STING^Lo)（P=0.0315）.The OS in the STING^Hi+CD8^Hi group was significantly higher than that in the STING^Lo+CD8^Higroup,the STING^LoCD8^Hi group,and the STING^Lo+CD8^Lo group（P=0.001,P=0.009 and P=0.005,respectively）.（23）The m RNA expression of PD-L1（r=0.886,P=0.019）,IFNG（r=0.984,P=0.000）,CXCL9（r=0.961,P=0.002）,CXCL11（r=0.929,P=0.007）,and CD8（r=0.943,P=0.005）was positively correlated with that of CXCL10 in patients with LUAD.Conclusions:（1）SASP in senescent epithelial cancer cells promotes invasion,migration and EMT in non-senescent epithelial tumor cells,which may be mediated in part via activation of the Hippo/YAP pathway and further induction of PD-L1 expression.（2）The lung adenocarcinoma tissues with high senescence index had higher PD-L1 and YAP expression,as well as a higher proportion of CD3+CD8+T lymphocyte infiltration than those in the lung adenocarcinoma tissues with low senescence index.（3）The high level of YAP expression in the nucleus of NSCLC cancer tissues was negatively correlated with their poor prognosis.（4）Activation of STING signaling pathway in lung adenocarcinoma may be the molecular mechanisms leading to cancer cell senescence and tumor immune microenvironment change.