Journal: Cell Communication and Signaling : CCS

Article Title: Chemotherapy-induced senescent nasopharyngeal carcinoma cells suppress NK cell-mediated antitumor immunity by upregulating EBI3 via mtDNA-cGAS-STING pathway

doi: 10.1186/s12964-026-02848-6

Figure Lengend Snippet: Chemotherapy-induced senescence of NPC cells recruits NK cells and inhibits the killing of cancer cells mediated by them, thus promoting the progression of NPC cells. A The schematic diagram of co-culturing the culture supernatant of the non-senescent group (Ctrl) and the senescent group (Se) with NK-92MI cells and measuring the chemotaxis of NK-92MI cells using a transwell migration assay. B Quantification of the in vitro migration of NK-92MI cells was performed using a transwell migration assay in the presence of conditioned media containing Ctrl or Se cells ( n = 3 experiments). C Schematic diagram of experimental workflow: NK-92MI cells were pretreated with conditioned medium (CM) from non-senescent (Ctrl) or senescent (Se) NPC cells, followed by CM removal and co-culture with NPC target cell. D and E Flow cytometry was used to detect the expression of degranulated CD107a in NK-92MI cells after co-culture. ( D ) Flow cytometry representative image of degranulated CD107a. ( E ) Quantification of degranulation CD107a ( n = 3 experiments). F and G Flow cytometry was used to detect the expression of GZMB and IFN-γ in NK-92MI cells after co-culture. ( F ) Flow cytometry representative images of GZMB and IFN-γ. ( G ) Quantification of cytokines GZMB and IFN-γ ( n = 3 experiments). H The ELISA kit was used to detect TNF-α, GZMB, and IFN-γ secreted by NK-92MI cells after co-culture ( n = 3 experiments). I The schematic diagram of evaluating the effect of cisplatin-induced senescenc NPC cells on NK cell-mediated cancer cell killing by co-culturing NK-92MI cells with cell culture supernatants from the non-senescent group (Ctrl) and the senescent group (Se), and then co-culturing the co-cultured NK-92MI cells with CFSE-labeled NPC cells at an effector-to-target (E: T) ratio of 5:1. J Use flow cytometry to detect CFSE + PI + NPC cells. K The positive rate of CFSE + PI + NPC cells was calculated ( n = 3 experiments). L Schematic of in vivo experimental design: NKG were subcutaneously co-inoculated with GFP-LUC-HK1 cells and non-senescent Ctrl-HK1 or senescent Se-HK1 cells (GFP-LUC-HK1 cells were not treated with cisplatin; cisplatin treatment was applied only to non-labeled HK1 cells to induce senescence prior to co-inoculation), with or without tail vein injection of NK-92MI cells ( n = 5 per group). M Quantitative analysis of average luciferase fluorescence intensity of subcutaneous GFP-LUC-HK1 tumors at the experimental endpoint. N Representative flow cytometry plots of GFP-LUC-HK1 cell death in tumors from Ctrl-HK1 and Se-HK1 co-inoculation groups, with or without NK-92MI injection. O Quantitative analysis of NK cell activation marker (CD107a + ) expression from flow cytometry. P Quantitative analysis of NK cell activation marker (GZMB + ) expression from flow cytometry. (B, E, G, H, K, M,O, P) Data are presented as mean ± SD; (B, E, G, H, K, O,P) Two-tailed unpaired t-tests were used for statistical analysis. (M)one-way ANOVA was applied

Article Snippet: NK92MI cells were cultured using NK-92MI cell specific culture medium (Procell, China).

Techniques: Chemotaxis Assay, Transwell Migration Assay, In Vitro, Migration, Co-Culture Assay, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Labeling, In Vivo, Injection, Luciferase, Fluorescence, Activation Assay, Marker, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: Chemotherapy-induced senescent nasopharyngeal carcinoma cells suppress NK cell-mediated antitumor immunity by upregulating EBI3 via mtDNA-cGAS-STING pathway

doi: 10.1186/s12964-026-02848-6

Figure Lengend Snippet: Chemotherapy-induced senescence of NPC cells promotes the recruitment of NK cells and inhibits the killing of cancer cells mediated by them through the expression of EBI3. A Sort according to the log2FoldChange enrichment multiple and the heat map of GSEA. Take the intersection of the top 20 molecules with log2FoldChange and the top 20 molecules from the GSEA heat map. B RT-qPCR was used to detect the mRNA levels of SLC38A4 , EBI3 , CLDN1 , INHBA , CLTRN , AOX1 , KRT6A , and NUPR1 in NPC cells of the non-senescent group (Ctrl) and the senescent group (Se) ( n = 3 experiments). C RT-qPCR was used to detect the mRNA level of EBI3 in mouse xenograft tumor tissues. D Immunoblotting was used to analyze the protein level of EBI3 in NPC cells of the non-senescent group (Ctrl) and the senescent group (Se) ( n = 3 experiments). E Three sgRNAs of EBI3 were designed using CRISPR-Cas9 technology and analyzed for knockdown efficiency using immunoblotting. F The SA-β-Gal staining method was used to detect the senescence of WT NPC HK1 cells without treatment, WT HK1 cells treated with cisplatin, sgEBI3 HK1 cells without treatment, and sgEBI3 HK1 cells treated with cisplatin. The left image is a representative image of SA-β-gal staining, with a scale bar of 100 μm. The right image is the quantification of SA-β-Gal positive cells ( n = 3 experiments). G and H The culture supernatants of WT NPC cells without treatment, WT NPC cells treated with cisplatin, sgEBI3 NPC cells without treatment, and sgEBI3 NPC cells treated with cisplatin were co-cultured with NK-92MI cells. Flow cytometry was used to detect the expression of degranulated CD107a in NK-92MI cells after co-culture. ( G ) The left image is a representative flow cytometry image of degranulated CD107a in NK-92MI cells after co-culture with the culture supernatant of HK1 cells and NK-92MI cells, and the right image is the quantification of degranulated CD107a ( n = 3 experiments). ( H ) Quantification of degranulated CD107a in NK-92MI cells after co-culture with the culture supernatant of HONE1 cells and NK-92MI cells ( n = 3 experiments). I and J Flow cytometry was used to detect the expression of GZMB and IFN-γ in NK-92MI cells after co-culture. ( I ) The left image is a representative flow cytometry image of GZMB and IFN-γ in NK-92MI cells after co-culture with the culture supernatant of HK1 cells and NK-92MI cells, and the right image is the quantification of GZMB and IFN-γ ( n = 3 experiments). ( J ) Quantification of GZMB and IFN-γ in NK-92MI cells after co-culture with the culture supernatant of HONE1 cells and NK-92MI cells ( n = 3 experiments). K The left image is the quantification of GZMB and TNF-α in NK-92MI cells after co-culture with the culture supernatant of HK1 cells and NK-92MI cells ( n = 3 experiments). The right image is the quantification of IFN-γ and TNF-α in NK-92MI cells after co-culture with the culture supernatant of HK1 cells and NK-92MI cells ( n = 3 experiments). L ELISA was used to detect TNF-α, GZMB, and IFN-γ secreted by NK-92MI cells after co-culture ( n = 3 experiments). M RT-qPCR was used to analyze the mRNA levels of TNF-α , GZMB , and IFN- γ in NK-92MI cells after co-culture with the culture supernatant of HK1 cells and NK-92MI cells ( n = 3 experiments). N The culture supernatants of HK1 cells from WT without treatment, WT treated with cisplatin, sgEBI3 without treatment, and sgEBI3 treated with cisplatin were co-cultured with NK-92MI cells, and then the co-cultured NK-92MI cells were co-cultured with NPC cells. Flow cytometry was used to detect GFSE + PI + NPC cells. The left image is a representative flow cytometry image, and the right image is the positive rate of GFSE + PI + NPC cells ( n = 3 experiments). (B, C, F-N) The data are presented as mean ± SD; A two-tailed unpaired t-test was used for (B, C); A two-tailed unpaired t-test or one-way analysis of variance (ANOVA) and Tukey’s post-hoc test were used for (F-N)

Article Snippet: NK92MI cells were cultured using NK-92MI cell specific culture medium (Procell, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, CRISPR, Knockdown, Staining, Cell Culture, Flow Cytometry, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: Chemotherapy-induced senescent nasopharyngeal carcinoma cells suppress NK cell-mediated antitumor immunity by upregulating EBI3 via mtDNA-cGAS-STING pathway

doi: 10.1186/s12964-026-02848-6

Figure Lengend Snippet: Chemotherapy-induced senescence of nasopharyngeal carcinoma cells upregulates EBI3, promoting IL35 secretion and thereby suppressing NK cell-mediated anti-tumor immunity. A RT-qPCR was used to detect the mRNA levels of IL12a (p35) , IL27 (p28) , and IL23A (p19) in senescent and non-senescent NPC cells ( n = 3 experiments). B ELISA was used to detect the levels of IL-35 in senescent and non-senescent NPC cells ( n = 3 experiments). C ELISA was used to detect the levels of IL-35 in NPC cells of WT without cisplatin treatment, WT treated with cisplatin, and sgEBI3 treated with cisplatin ( n = 3 experiments). D Schematic diagram of co-culturing cisplatin-induced sgEBI3 NPC cells with or without the addition of r-IL-35 and NK-92MI cells ( E - I ). E Flow cytometry was used to detect the expression of the degranulation marker CD107a in NK-92MI cells after co-culture. The left image is a representative flow cytometry image, and the right image is a statistical graph of CD107a ( n = 3 experiments). F Flow cytometry was used to detect the expression of GZMB and IFN-γ in NK-92MI cells after co-culture. The left image is a representative flow cytometry image, and the right image is a statistical graph of GZMB and IFN-γ ( n = 3 experiments). G Flow cytometry was used to detect the expression of TNF-α, GZMB, and IFN-γ in co-cultured NK-92MI cells ( n = 3 experiments). H RT-qPCR was used to detect the mRNA levels of TNF-α , GZMB , and IFN- γ in co-cultured NK-92MI cells ( n = 3 experiments). I ELISA was used to detect the levels of TNF-α, GZMB, and IFN-γ in co-cultured NK-92MI cells ( n = 3 experiments). J Cisplatin-induced sgEBI3 NPC cells were co-cultured with NK-92MI cells with or without r-IL-35, and then co-cultured with CFSE-labeled NPC cells. Flow cytometry was used to detect CFSE + PI + NPC cells. The left image is a representative image of flow cytometry, and the right image shows the positivity rate of CFSE + PI + NPC cells ( n = 3 experiments). A , B , C , E - J data are expressed as mean ± SD; A , B , E - J a two-tailed unpaired t-test was used; C Use a two-tailed unpaired t-test or one-way analysis of variance (ANOVA) and Tukey’s post hoc test

Article Snippet: NK92MI cells were cultured using NK-92MI cell specific culture medium (Procell, China).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Marker, Co-Culture Assay, Cell Culture, Labeling, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: Chemotherapy-induced senescent nasopharyngeal carcinoma cells suppress NK cell-mediated antitumor immunity by upregulating EBI3 via mtDNA-cGAS-STING pathway

doi: 10.1186/s12964-026-02848-6

Figure Lengend Snippet: Chemotherapy-induced senescence of nasopharyngeal carcinoma cells upregulates EBI3 to promote IL35 secretion, thereby activating the STAT1-NKG2A signaling axis and affecting NK cell-mediated anti-tumor immunity. A The supernatants of NPC cells without cisplatin treatment, cisplatin-treated WT, and cisplatin-treated sgEBI3 were co-cultured with NK-92MI cells. RT-qPCR was used to analyze the mRNA levels of activating and inhibitory receptors in NK-92MI cells ( n = 3 experiments). B Cisplatin-induced sgEBI3 NPC cells were co-cultured with NK-92MI cells in the presence or absence of r-IL-35. RT-qPCR was used to analyze the mRNA levels of activating and inhibitory receptors in NK-92MI cells ( n = 3 experiments). C Cisplatin-induced sgEBI3 NPC cells were co-cultured with NK-92MI cells in the presence or absence of r-IL-35. Flow cytometry was used to detect the expression of NKG2A in NK-92MI cells. The upper figure is a representative image of flow cytometry, and the lower figure is a statistical chart ( n = 3 experiments). D Cisplatin-induced sgEBI3 NPC cells were co-cultured with NK-92MI cells in the presence or absence of r-IL-35. Flow cytometry was used to detect the expression of NKG2A in NK-92MI cells. The upper figure is a representative image of flow cytometry, and the lower figure is a statistical chart ( n = 3 experiments). E Cisplatin-induced sgEBI3 NPC cells were co-cultured with NK-92MI cells with or without IL-35. RT-qPCR was used to analyze the mRNA levels of IL35R in NK-92MI cells ( n = 3 experiments). F Flow cytometry was used to detect the expression of NKG2A in NK-92MI cells treated with control, r-IL-35, r-IL-35 plus STAT1 inhibitors, and r-IL-35 plus STAT4 inhibitors. The left image is a representative flow cytometry image, and the right image is a statistical graph ( n = 3 experiments). G RT-qPCR was used to detect the mRNA levels of NKG2A in NK-92MI cells treated with control, r-IL-35, r-IL-35 + STAT1 inhibitors, and rIL35 + STAT4 inhibitors ( n = 3 experiments). H Schematic diagram of STAT1 binding in the NKG2A promoter region. I ChIP was used to detect the binding of STAT1 to the NKG2A promoter region ( n = 3 experiments). J Schematic diagram of the molecular mechanism by which IL-35 upregulates NKG2A expression in NK cells via activating the STAT1 signaling pathway through its receptor ( A - D , F - H , J ) data are expressed as mean ± SD; B , F , J A two-tailed unpaired t-test was used; A , C , D , G , H A two-tailed unpaired t-test or one-way analysis of variance (ANOVA) and Tukey’s post hoc test was used

Article Snippet: NK92MI cells were cultured using NK-92MI cell specific culture medium (Procell, China).

Techniques: Cell Culture, Quantitative RT-PCR, Flow Cytometry, Expressing, Control, Binding Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: Cholangiocarcinoma derived exosomes attenuate the anti-tumor functions of NK cells

doi: 10.1038/s41598-026-36706-9

Figure Lengend Snippet: Characteristics of isolated exosomes and internalization by NK-92 cells. ( A ) Morphologies of exosomes isolated from RBE cells, CCA patient serum and healthy people serum respectively under TEM. Yellow arrows indicate the exosomes. ( B ) NTA of exosomes. ( C ) Identification of exosomes by western blot analysis for HSP70 and CD63. ( D ) Tracing and internalization of CCA-Exos by NK-92 cells. Green, blue, and red fluorescence represent exosomes, nucleus and cytoskeleton of NK-92 cells. The three figures were merged into the fourth showing exosomes entered the cells.

Article Snippet: NK-92 cells were cultured in NK-92 cell specific culture medium (Procell, Wuhan, China) with 200 U/mL IL-2 (Procell, Wuhan, China).

Techniques: Isolation, Western Blot, Fluorescence

Journal: Scientific Reports

Article Title: Cholangiocarcinoma derived exosomes attenuate the anti-tumor functions of NK cells

doi: 10.1038/s41598-026-36706-9

Figure Lengend Snippet: Apoptosis of NK-92 cells induced by CCA-Exos. ( A ) Flow cytometry assays showing apoptosis of NK-92 cells induced by RBE-Exos. ( B ) Comparison of apoptosis rate of NK-92 cells induced by RBE-Exos and PBS. ( C ) Flow cytometry assays showing apoptosis of NK-92 cells induced by CCA serum-Exos and healthy serum-Exos. ( D ) Comparison of apoptosis rate of NK-92 cells induced by CCA serum-Exos and healthy serum-Exos. Data are presented as the mean ± SEM from n = 3 independent experiments. ( E ) Protein levels of BCL-2 in NK-92 cells treated with RBE-Exos or PBS. ( F ) Protein levels of BCL-XL in NK-92 cells treated with RBE-Exos or PBS. ( G ) Protein levels of BAX in NK-92 cells treated with RBE-Exos or PBS. ( H ) Protein levels of BAD in NK-92 cells treated with RBE-Exos or PBS. ( I ) Relative transcriptional levels of BCL-2 , BCL-XL , BAX , and BAD of NK-92 cells treated with RBE-Exos or PBS. Data were presented as mean ± SEM for three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: NK-92 cells were cultured in NK-92 cell specific culture medium (Procell, Wuhan, China) with 200 U/mL IL-2 (Procell, Wuhan, China).

Techniques: Flow Cytometry, Comparison

Journal: Scientific Reports

Article Title: Cholangiocarcinoma derived exosomes attenuate the anti-tumor functions of NK cells

doi: 10.1038/s41598-026-36706-9

Figure Lengend Snippet: Proliferation, production of effectors and killing activity of NK-92 cells. ( A ) Proliferation of NK-92 cells treated with RBE-Exos or PBS. CCK8 detecting showed no significance between proliferation of NK-92 cells treated with RBE-Exos or PBS. ( B ) Proliferation of NK-92 cells treated with serum exosomes. CCK8 detecting showed no significance between proliferation of NK-92 cells treated with CCA serum-Exos or healthy serum-Exos. Data are presented as the mean ± SEM from n = 3 independent experiments. ( C ) Production of effectors. ELISA showed that RBE-Exos decreased the production of IFN-γ and granzyme B obviously but not TNF-α and perforin. ( D ) Cytotoxicity of NK-92 cells treated with RBE-Exos or PBS. LDH detection displayed the cytotoxicity of NK-92 cells was suppressed by RBE-Exos. ( E ) Cytotoxicity of NK-92 cells treated serum exosomes. LDH detection displayed the cytotoxicity of NK-92 cells was suppressed by CCA serum-Exos. Data were presented as the mean ± SEM from n = 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: NK-92 cells were cultured in NK-92 cell specific culture medium (Procell, Wuhan, China) with 200 U/mL IL-2 (Procell, Wuhan, China).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Cholangiocarcinoma derived exosomes attenuate the anti-tumor functions of NK cells

doi: 10.1038/s41598-026-36706-9

Figure Lengend Snippet: Effect of RBE-Exos on the adhesion of NK-92 cells. ( A ) Growth status of NK-92 cells. NK cells treated with RBE-Exos lost the normal cluster growing. ( B ) CD11a protein levels. ( C ) CD18 protein levels. ( D ) CD54 protein levels. Western blot showed that the protein levels of CD11a, CD18 and, CD54 were obviously decreased in NK-92 cells treated with RBE-Exos. ( E ) Relative transcriptional levels of CD11a , CD18 , CD54 and, CD2 in NK-92 cells. qRT-PCR displayed that RBE-Exos downregulated the transcriptional levels of CD11a , CD18 , CD54 , but not CD2 . Data were presented as mean ± SEM for three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: NK-92 cells were cultured in NK-92 cell specific culture medium (Procell, Wuhan, China) with 200 U/mL IL-2 (Procell, Wuhan, China).

Techniques: Western Blot, Quantitative RT-PCR