Journal: Advanced Science
Article Title: Vinburnine Sensitizes Radiotherapy Efficacy in Nasopharyngeal Carcinoma by Triggering Pyroptosis and Immune Responses via Activation of EDAR‐NFκB Pathway
doi: 10.1002/advs.202506139
Figure Lengend Snippet: Vin+IR activates T cell immunity through the EDAR‐NFκB‐CCL5/CX3CL1 pathway. A) The mRNA expression of CCL5 and CX3CL1 in SUNE1 after 5µ m Vin/ 2Gy IR treatment ( n = 3). B) Diagram of human T cell‐related experiments. C). After 24h of 5µ m Vin / 2Gy IR treatment, 1×10^4 SUNE1 was co‐cultured with 2.5×10^4 T cells for 48h. The suspended T cells were washed away with PBS, and the viability of tumor cells was detected by CCK8 ( n = 5). D) T‐cell chemotaxis assay. The supernatant from SUNE1 cells treated with Vin/IR was collected and placed in the lower chamber of the transwell. T cells were then placed in the upper chamber of the transwell. After 48h, the liquid from the lower chamber was collected for T‐cell counting ( n = 3). E) T cells were co‐cultured with treated SUNE1 cell supernatant for 48h, and CD45 + CD3 + CD8 + GZMB + T cells were detected by flow cytometry ( n = 3). F) The ChIP assay was conducted to detect the binding of p65 to the CCL5 and CX3CL1 promoters following treatment with 5µ m Vin / 2Gy IR for 48h ( n = 3). G) Following sh‐EDAR or NFκBi pretreatment, the mRNA expression of CCL5 and CX3CL1 in Vin/IR‐treated SUNE1 was detected using qRT‐PCR ( n = 3). H–J) After sh‐EDAR or NFκBi pretreatment, the proliferation of SUNE1 cells treated with 5µ m Vin / 2Gy IR and co‐cultured with T cells, the number of chemotactic T cells in the supernatant of Vin/IR‐treated SUNE1 cells, and the proportion of CD45 + CD3 + CD8 + GZMB + T cells were measured ( n = 3). K) EDAR and CD8 multiplex immunofluorescence staining was performed on nasopharyngeal carcinoma tissue microarrays. Comparison of mean fluorescence intensity of EDAR in tissues with recurrence ( n = 39) and without recurrence ( n = 45) in NPC patients. L) Correlation between EDAR and CD8 expression in human NPC tissues ( n = 84). M) The patients were divided into EDAR low‐expression ( n = 21) and EDAR high‐expression ( n = 21) groups based on the quartile method. Survival analysis evaluated the association between EDAR expression levels and patient survival. Multiple samples were presented using mean ± standard deviation (SD). A, D–F) Statistical analysis with One‐way ANOVA was used to analyze the statistical differences among multiple groups. C, G–J) Statistical analysis with Two‐way ANOVA was used to analyze the statistical differences among multiple groups. K) The Mann‐Whitney test was used to analyze the expression of EDAR in patients with recurrence and those without recurrence. L) The correlation between EDAR and CD8 expression in tissue microarrays was analyzed using Pearson's and simple linear regression. M) Survival analysis (Kaplan‐Meier) was utilized to examine the correlation between EDAR expression and the prognosis of patients diagnosed with nasopharyngeal carcinoma. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns for non‐significant.
Article Snippet: EDAR knockdown lentiviral plasmids (GeneChem, China) were packaged with PSPAX2 and PMD2G in 293T cells.
Techniques: Expressing, Cell Culture, Chemotaxis Assay, Cell Counting, Flow Cytometry, Binding Assay, Quantitative RT-PCR, Multiplex Assay, Immunofluorescence, Staining, Comparison, Fluorescence, Standard Deviation, MANN-WHITNEY
