Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Electrolyte imbalance causes suppression of NK and T cell effector function in malignant ascites

doi: 10.1186/s13046-023-02798-8

Figure Lengend Snippet: Malignant ascites causes dysfunction during in vitro activation of NK and T cells. (A) Expression of surface markers on stimulated NK cells in presence of ascites. Isolated healthy donor NK cells were coincubated with IL2 (400 U/ml) either in medium or 25%ascites-supplemented media. After 48 h expression of DNAM-1, CD69 and TIGIT was measured by FACS. (B) NK cytotoxicity of stimulated NK cells in presence of ascites. Percentage of CD107a-positive NK cells was assessed by FACS after 6-hours of stimulation with PMA (50ng/ml) and Ionomycin (1 µg/ml) either in media or 25% ascites-supplemented media. (C-F) Stimulation of T cells in presence of ascites. Isolated healthy donor T-cells were prestained with Cell Proliferation Dye EFluor 450 and stimulated using CD2/CD3/CD28 activator complex (1:40) either in medium or 25%ascites-supplemented media for four days. (C) Graph showing T cells proliferation. (D) Representative experiment showing T cell proliferation. (E) Secreted IFNγ [ng/ml] during T cell proliferation determined by ELISA. (F) Percentage of CD25-positive (left column) and TIM3-positive T cells (right column). (G + H + I) Intracellular Calcium-flux during T cell stimulation in presence of ascites. T cells were exposed to CD2/CD3/CD28 activator either in presence of ascites or medium. Intracellular Ca2 + flux was monitored via Fluo-4 dye. (G) Fold increase of 480/25 absorbance at peak of calcium flux. (H) Representative experiment of calcium flux after T cell stimulation in presence of ascites. (I) ROC analysis shows significant difference between calcium flux curves in suppressive ascites environment (red and green line) compared to medium (dotted red line). Data are presented as individual values with mean value as center of error bar ± standard deviation. Each datapoint represents one healthy donor. The normalization was done according to normal medium control. For significance testing ordinary one-way (2.A-C, E-F) and paired (2.G) ANOVA or ROC analysis (2.I) were performed, followed by Dunnett’s multiple comparison posthoc test between ascites and medium control group where appropriate. * ( p < 0.05), ** ( p < 0.01), *** ( p < 0.001), **** ( p < 0.0001)

Article Snippet: T cells were stimulated for four days with CD2/CD3/CD28 activator complex diluted at 1:40 (StemCell Technologies).

Techniques: In Vitro, Activation Assay, Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Cell Stimulation, Standard Deviation, Comparison

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Electrolyte imbalance causes suppression of NK and T cell effector function in malignant ascites

doi: 10.1186/s13046-023-02798-8

Figure Lengend Snippet: Ascites dialysis partially reverses inhibitory properties caused by non-protein and heat resistant components. (A) Graphical illustration of protein depletion in ascites. Ascites sample was processed via ultracentrifugal filtration and protein-rich retentate and protein-less permeate are collected. (B) Composition of ascites before and after protein depletion. Heatmap showing altered composition of protein-depleted ascites samples and protein-rich retentates. (C) Electrophoresis of ascites sample. Representative electrophoresis blot of ascites sample 16 and corresponding permeate. (D) Proliferation of activated T cells in presence of protein-depleted ascites. Prestained donor T-cells were stimulated using CD2/CD3/CD28 activator complex (1:40) in protein-less ascites permeate. (E) NK ADCC in presence of ascites permeate. Resting NK cells were coincubated in 1:1 ratio with IGROV1 cells with addition of ADCC-inducing anti-EGFR antibody Cetuximab in presence of unmodified or protein-less ascites permeate. (F) NK ADCC in presence of heat inactivated ascites permeate. Resting NK cells were coincubated in 1:1 ratio with IGROV1-cells and ADCC-inducing anti-EGFR antibody Cetuximab with protein-depleted ascites permeate which was heat inactivated at 90 °C for 1 h. (G) Graphical illustration of ascites dialysis in cell culture medium. Ascites samples were processed in medium overnight using 1 kDa or 25 kDa cutoff dialysis columns. (H) Composition of ascites before and after dialysis. Heatmap showing altered composition of ascites samples processed by dialysis. (I) NK ADCC in presence of dialyzed ascites. Resting NK cells were coincubated in 1:1 ratio with IGROV1 cells with addition of ADCC-inducing anti-EGFR antibody Cetuximab in presence of unmodified or dialyzed ascites. After 6 h expression of CD107a on NK cells was determined by flow cytometry. Each datapoint represents one healthy donor. For significance testing ordinary one-way ANOVA followed by Dunnet posthoc test (4.D) and two-way ANOVA followed by Sidak posthoc test was used (4.E, F and I). ns (non-significant), * ( p < 0.05), ** ( p < 0.01), **** ( p < 0.0001)

Article Snippet: T cells were stimulated for four days with CD2/CD3/CD28 activator complex diluted at 1:40 (StemCell Technologies).

Techniques: Filtration, Electrophoresis, Cell Culture, Expressing, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Electrolyte imbalance causes suppression of NK and T cell effector function in malignant ascites

doi: 10.1186/s13046-023-02798-8

Figure Lengend Snippet: Malignant ascites causes transcriptional and translational changes of signaling proteins and electrolyte channels in immune cells. (A) Heatmap showing altered transcriptional gene expression in immune cells in presence of ascites. Resting NK cells were incubated with ascites for 6 h, and IL2 activated NK cells for 24 h. Resting T cells were incubated with ascites for 24 h and the activation complex was treated for 48 h. Total RNA was isolated from NK and T cells. Quantitative real-time PCR was conducted with annealing temperature of 62 °C for all primers. Primers are listed in Supplementary Table (B) Correlation between NK effector function, electrolyte content in ascites and altered mRNA-expression of selected genes. Correlation matrix depicts significant gene expression changes (p < 0,05) in resting and activating NK cells (left) and resting and activated T cells (right). (C) Correlation between electrolyte content in ascites and altered mRNA-expressions of selected genes. Significant Pearson correlations of gene expression changes and electrolyte content in ascites. Sodium correlation to PI3K in IL2-activated NK cells (first panel). Chloride correlation to Granzyme B (third panel) and SLC24A2 (fourth panel) in resting T cells. Correlation of CLIC1 to sodium (second panel) and KCNMB3 to chloride (fifth panel) in complex activated T cells. (D) Clustering of mRNA-transcripts of stimulated NK cells in presence of ascites. Hierarchical clustering done according to gene expression data from ascites treated activated NK cells (left). t-SNE plot demonstrating unbiased grouping of ascites samples according to most affected genes (right). (E) Graphical illustration of crucial transcriptional changes in NK and T cells after ascites exposure. Portrayed genes were affected by ascites exposure significantly as shown by both ANOVA and Pearson correlation. Lines connecting genes show Pearson correlation relationships. Every gene affected by specific electrolyte was marked with appropriate symbol (Na-red, Cl-Green and K-Purple). (F, G, H and I) Protein expression of electrolyte channels and immune effector molecules in NK and T cells affected by ascites. Both NK and T cells were incubated in medium or 25% ascites supplemented medium for 24 h (resting cells) or 48 h (activated cells). NK cells were treated with 50 ng/ml of PMA, 1 µg/ml of Ionomycin, while T cells were activated by adding 5 µl of CD2/CD3/CD28 activator. Protein expression was determined by densitometric analysis of western-blots and normalized to GAPDH. (F) Heatmap overview showing protein expression comparison between the healthy donor (C), two inhibitory (No.16, 21), and one non-inhibitory (No.29) ascites- treated NK and T cell samples. (G) p85 expression in samples from resting and activated T cells as determined by western blot. (H) Bar graph depicting different Granzyme B expression in samples from activated T and NK cells. (I) Western blot bands from representative experiment. (J, K and L) Ascites-mediated inhibition of p85 membrane recruitment in T cells. Isolated T cells were activated for 5 min with CD2/CD3/CD28 activator in either medium or ascites 21 supplemented medium. Surface localization and expression of p85 was determined by confocal microscopy. (J) Violin plot depicting p85 mean intensity on T cell surface. (K) Violin plot depicting the maximum of p85 intensity of each cell. (L) Representative confocal microscopy images showing T cell surface expression of p85 (red) in different conditions. Data are presented as individual values with mean value as center of error bar ± standard deviation. For significance testing two-tailed Pearson correlation were used (5.B, C), paired t-test was used to assess the significance (5. G, H), ordinary one-way ANOVA and Dunnett posthoc (5.J. K). *( p < 0.05), **( p < 0.01), ****( p < 0.0001)

Article Snippet: T cells were stimulated for four days with CD2/CD3/CD28 activator complex diluted at 1:40 (StemCell Technologies).

Techniques: Expressing, Incubation, Activation Assay, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Comparison, Inhibition, Membrane, Confocal Microscopy, Standard Deviation, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Electrolyte imbalance causes suppression of NK and T cell effector function in malignant ascites

doi: 10.1186/s13046-023-02798-8

Figure Lengend Snippet: Malignant ascites-mediated immunosuppression can partially be reversed by inhibitors of sodium channels. (A, B and C) Sodium influx in T cells in presence of ascites. T cells were exposed to 25%-ascites supplemented media or PBS and media control, respectively. After 25 min. influx of sodium was measured using fluorescent CoroNa Green dye. (A) Representative experiment showing sodium influx in T cells in presence of different malignant ascites samples. (B) Relative increase of intracellular sodium in T cells in presence of different ascites samples. Comparison is made between experiment endpoint values. (C) ROC curve analysis shows significant difference between measured sodium flux curves in suppressive ascites environment (red and green line) compared to medium (dotted red line). (D) Sodium influx in T cells in presence of ascites and inhibitors of sodium channels. CoroNa Green prestained resting T-cells were exposed to different samples of 25%-ascites-supplemented media (left: ascites 21, right: ascites 29) and media control, respectively. Different inhibitors of sodium channels were added (150 µM). Results were normalized to starting fluorescence. Representative experiments are shown. (E and F) Calcium influx in activated T cells in presence of ascites and inhibitors of sodium channels Fluo-4 prestained resting T cells were activated using CD2/CD3/CD28 complex either in normal media or high sodium malignant ascites sample No.16. Sodium channel inhibitor were added with activator complex simultaneously. (E) Representative experiment of calcium influx in activated T cells in presence of amiloride and lidocaine and ascites 16 (middle panel) and media control (left panel). ROC curve analysis (right panel) shows significant differences between measured calcium flux curves in ascites 16 with addition of amiloride (cyan line) or lidocaine (purple line), compared to vehicle control (dotted red line). (F) Representative experiment of calcium influx in activated T cells in presence of cariporide and digitoxin and ascites 16 (middle panel) and media control (left panel). ROC curve analysis (right panel) shows significant difference between measured calcium flux curves in ascites 16 with addition of cariporide (orange line) or digitoxin (yellow line), compared to vehicle control (dotted red line). (G) NK cell degranulation in presence of ascites and sodium channel inhibitors. Isolated NK cells were pretreated with water (vehicle control) or 15 µM addition of amiloride or lidocaine for one hour. Washed NK cells were exposed to 25%-ascites supplemented media and media control, respectively. After 6 h NK cell degranulation was measured using FACS. (H) NK-TC conjugation in presence of ascites and amiloride. Isolated NK cells were pretreated with water (vehicle control) or 1,5, 15 or 150 µM addition of amiloride for one hour. NK cells and IGROV1-cells were mixed in 4:1 effector to target ratio in presence of 1 µg/ml Cetuximab and in presence or absence of 25%-ascites-supplemented media. (I) Expression of NK cell signal transduction and effector molecules in presence of ascites 21 and amiloride. Resting and activated NK cells were incubated with ascites sample No. 21 and water (vehicle control) or 150 µM addition of amiloride for 3 h. Fold change of PIK3CD, AKT1, PRKCQ and GZMB gene expressions. Gene expression was normalized to housekeeping gene and respective ascites treated controls. (J) p85 membrane recruitment in activated T cells in presence of ascites 21 and amiloride. Isolated T cells were activated with CD2/CD3/CD28 activator in either medium or ascites 21 supplemented medium with addition of 150 µM of Amiloride for five minutes. Surface localization and expression of p85 was determined by confocal microscopy. Violin plot depicts p85 mean intensity on T cell surface. (K and L) Phosphorylation of signal transduction protein p85-PI3K in NK cells in presence of ascites 16 and amiloride. (K) Isolated NK cells were exposed to were exposed to 25%-ascites supplemented media and media control. During the incubation, NK cells were treated with 50 ng/ml of PMA, 1 µg/ml of Ionomycin and/or 150 µM of amiloride for five minutes. Phosphorylation was determined by densitometry and normalized to GAPDH and respective ascites treated controls. (L) Western blot bands from representative experiment and GAPDH control. Each datapoint represents one healthy donor. For significance testing RM one-way ANOVA followed by Dunnett’s multiple comparison posthoc test (6.B and J), two-way ANOVA followed by Dunnett’s multiple comparison posthoc test (6.H) and paired t-test were used (6.G, I and K). ns (non-significant), * ( p < 0.05), ** ( p < 0.01), ****(p < 0.0001).

Article Snippet: T cells were stimulated for four days with CD2/CD3/CD28 activator complex diluted at 1:40 (StemCell Technologies).

Techniques: Comparison, Fluorescence, Isolation, Conjugation Assay, Expressing, Transduction, Incubation, Membrane, Confocal Microscopy, Western Blot