Fig 2: In vitro analyses of IRE-treated cancer cells and their impacts on dendritic cells. a Annexin V-FITC/PI staining of KRAS* and B16F10 cells. Cells were suspended in phosphate buffered saline (PBS) and electroporated in a cuvette with a 4-mm gap. The parameters for electroporation were: voltage = 200 or 960 V, pulse duration = 100 µs, pulse repetition frequency = 1 Hz, number of pulses = 20. Cells were stained and analyzed within 30 min of treatment. b ATP and HMGB1 concentrations in cell supernatants of untreated control (black square), 200 V (red square), and 960 V (blue square) groups (n = 3). c Relative expression of DC activation markers, including CD40, MHC-II, CCR7, and CD86, on bone marrow-derived DCs (CD11c+) after incubation with live or IRE (960 V)-treated KRAS* cells for 24 h. Expression was quantified via geometry mean fluorescence intensity (MFI; n = 3). Data are representative of 3 independent experiments and presented as mean ± standard error of mean (SEM). Significance was determined using two-sided Student’s t-test. ****p < 0.0001, n.s. not significant. Source data are provided as a Source Data file

Fig 3: IL-6 induces CD40 expression through Stat3 and HIF-1a.a BM-derived M?s were isolated from mice and treated with 50 ng/ml IL-4 and IL-6 for 2 days, followed by RNA-seq analysis (n = 3 mice). Shown are top upregulated transcriptional factors induced by IL-6. Left, heatmap. Right, means of fold expression of control. b Human monocytes were transfected with siRNA targeting NF-?B2, Stat3, or control sequence and treated with IL-6 or control medium. Cell lysates were immunoblotted. This experiment was repeated independently twice with similar results. c–e Human monocytes were treated with IL-6 or control medium under d normoxia or e hypoxia. Nuclei protein was immunoprecipitated with d anti-Stat3 or e anti-HIF-1a antibody, or IgG, and subjected to ChIP analysis with different primers. c Results shown are from quantitative real-time polymerase chain reaction (RT-PCR) analysis (n = 3 human samples, means ± SEM). Statistical analysis by two-way ANOVA with Tukey’s test. f Human monocytes were treated with IL-6 or control medium under normoxia or hypoxia, followed by immunoblot analysis. This experiment was repeated independently twice with similar results. g Human monocytes were pretreated with siRNA targeting HIF-1a or control sequence, and treated with IL-6 or control medium under hypoxia. Cell lysates were immunoblotted. This experiment was repeated independently twice with similar results. Source data are provided as a Source data file.

Fig 4: High IL-6 expression and low CD40 expression correlate with poor survival in human GBM patients.a, b Correlation of CD40 expression with a IL-6 and b IL-4 expression was subjected to linear regression analyses using GlioVis/TCGA GBM-RNA-seq (n = 160 patients) and low-grade glioma (n = 513 patients) data sets. Statistical analysis by linear regression analysis. c, d Correlation of IL-6 and CD40 expression (high/low cutoff of 40%) with overall survival was analyzed using TCGA-Firehose data set. Statistical analysis by two-sided log-rank test. e A schematic model. IL-6 induces anti-inflammatory and pro-inflammatory functions in M?s, through IL-10 and CD40 expression, respectively. Combination therapy by anti-IL-6 neutralization and CD40 activation reverses M?-mediated tumor immunosuppression and promotes T-cell infiltration and activation, sensitizing tumor to checkpoint inhibition treatment.

Fig 5: Comparison between radiation and IRE in the enhancement of anti-PD1 therapy. a Annexin V-FITC/PI staining of KRAS* suspended in phosphate buffered saline (PBS) after 10 Gy of radiation, or electroporated using 960-V, 100-µs electric pulses at 1 Hz frequency for 20 pulses in a 4 mm-gap cuvette. b, c ATP and HMGB1 concentrations in the cell supernatants of control (black bar), 10 Gy (red bar), and IRE (blue bar) (n = 3). Cells were analyzed within 30 min of treatments. d Representative expression of DC activation markers, including CD40, MHC-II, CCR7, and CD86, on bone marrow-derived DCs (CD11c+) after incubation with KRAS* cells for 24 h. Expression was quantified via geometry mean fluorescence intensity (MFI; n = 3). e Kaplan–Meier survival curves of mice in sham control (black solid circle, n = 8), 10 Gy + anti-PD1 (red diamond, n = 10), and IRE + anti-PD1 groups (blue triangle, n = 11). f–i Tumor weight, frequencies of intratumoral CD8+ T cells and Treg, and CD8-to-Treg ratios from 4 to 6 tumors per group collected at 9 days after radiation or IRE. j Representative hematoxylin–eosin staining of tumor sections and quantification of tumor necrosis. Scale bar = 2 mm. Data are presented as mean ± standard error of mean (SEM). Significance was determined using 1-way ANOVA followed by Tukey post hoc analysis or Student’s t-test. *p < 0.05, **p < 0.01, ****p < 0.0001, n.s. not significant. Source data are provided as a Source Data file