Journal: Scientific Reports

Article Title: Evaluating a combination treatment of NK cells and reovirus against bladder cancer cells using an in vitro assay to simulate intravesical therapy

doi: 10.1038/s41598-024-56297-7

Figure Lengend Snippet: Viability of bladder cancer cell lines to reoviruses infection, RC402 and RP116. ( A , B ) JAM-A surface expression on BCC lines was assessed via flow cytometry, and the results are presented as the mean fluorescence intensity (MFI) ratio based on triplicate values in a representative experiment (n = 3). BCC lines were incubated in the presence of reovirus RC402 and RP116 at different MOI values. ( C ) Following a 48 h incubation, cell survival was analyzed by MTS assay, and the mean cell viability was calculated for each group at 100 MOI (right). ( D ) RP116 was treated on BCC lines for 24, 48 and 72 h at various MOI values. All data are shown as means from triplicate independent experiments (5637, HT-1376, n = 3; 253J-BV, n = 2). Statistical significance was calculated by Mann–Whitney test ( A , B ) and one-way ANOVA with Turkey’s multiple comparisons test in ( C ). MOI multiplicity of infection. *p < 0.1; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Cytotoxicity of eNK cells against BCC lines (5637, HT-1376, 253J-BV) was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies)-based assay for 4 h. Target cells were stained with 0.5 μM CFSE in FACS buffer for 10 min at 37 °C and washed twice with complete media.

Techniques: Infection, Expressing, Flow Cytometry, Fluorescence, Incubation, MTS Assay, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Evaluating a combination treatment of NK cells and reovirus against bladder cancer cells using an in vitro assay to simulate intravesical therapy

doi: 10.1038/s41598-024-56297-7

Figure Lengend Snippet: Scheme of an intravesical therapy-simulating in vitro assay and measuring the oncolytic activity of reovirus. 5637, HT-1376, and 253J-BV cell lines were treated with RP116 at an MOI of 10 or 100 MOI for 2 h, followed by a washing step with PBS. To evaluate the anti-tumor effect of RP116, the remaining live target cells were assessed after an additional 48 h of culture. ( A ) Schematic representation of the intravesical therapy-simulating in vitro assay. The supernatant was collected after an additional 2 h to measure the remaining viral particles, which were compared to those in the samples without washing. ( B ) The reduction in viral titer resulting from PBS washing was determined by quantifying the average concentration of viral particles (sized 65–75 nm). (n = 7–11). ( C ) The number of surviving target cells was observed using counting beads after RP116 treatment. (n = 6–10). ( D ) The relative viability of each cell line was analyzed. (n = 6–8) Statistical significance was calculated by Mann–Whitney test ( B ) and one-way ANOVA with Turkey’s multiple comparisons test in ( D ). **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Cytotoxicity of eNK cells against BCC lines (5637, HT-1376, 253J-BV) was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies)-based assay for 4 h. Target cells were stained with 0.5 μM CFSE in FACS buffer for 10 min at 37 °C and washed twice with complete media.

Techniques: In Vitro, Activity Assay, Concentration Assay, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Evaluating a combination treatment of NK cells and reovirus against bladder cancer cells using an in vitro assay to simulate intravesical therapy

doi: 10.1038/s41598-024-56297-7

Figure Lengend Snippet: Effect of eNK cells on bladder cancer cell lines in an in-vitro assay emulating intravesical therapy. PBMC-derived NK cells were utilized in the experiments. Cytokine-pretreated eNK cells were cultured with 50 ng/mL of IL18 and 10 U/mL of IL-21 overnight before cytotoxicity assay. ( A ) eNK cell cytotoxicity against 5637, 253J-BV and HT-1376 was analyzed after 2 h after treatment with the indicated E:T ratios (n = 3) ( B ) Compiled data from three donors showing the percentage of CD107a expression (n = 3). ( C ) NK cell mediated ligands on BCC lines was detected via flow cytometry, and the results are presented as the mean fluorescence intensity (MFI) ratio based on triplicate values in a representative experiment (n = 3). ( D ) The remaining number of eNK cells was quantified after PBS washing. (n = 6) ( E ) The remaining target cell population was quantified using counting beads after eNK cell treatment (n = 6–10). ( F ) The relative viability of each cell line was analyzed (n = 6–10). Statistical significance was calculated using Mann–Whitney test ( B – D ) and one-way ANOVA with Turkey’s multiple comparisons test ( F ). *p < 0.1; **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Cytotoxicity of eNK cells against BCC lines (5637, HT-1376, 253J-BV) was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies)-based assay for 4 h. Target cells were stained with 0.5 μM CFSE in FACS buffer for 10 min at 37 °C and washed twice with complete media.

Techniques: In Vitro, Derivative Assay, Cell Culture, Cytotoxicity Assay, Expressing, Flow Cytometry, Fluorescence, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Evaluating a combination treatment of NK cells and reovirus against bladder cancer cells using an in vitro assay to simulate intravesical therapy

doi: 10.1038/s41598-024-56297-7

Figure Lengend Snippet: Combinational therapy of RP116 and IL-18/-21-pretreated eNK cells on bladder cancer cells. 253J-BV and HT-1376 were treated with mono-treatment of RP116 and eNK cells, as well as their combination, to evaluate the cell lysis effect. For low doses, E:T ratio of 0.25:1 and MOI 10 were utilized. For high doses, E:T ratio of 1:1 and MOI 100 were employed. ( A ) The number of living residual target cells was analyzed by counting beads after PI staining (n = 6–10). ( B ) The relative viability of each cell line was analyzed (n = 6–10). Statistical significance was calculated using one-way ANOVA with Turkey’s multiple comparisons test in ( B ). *p < 0.1; **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Cytotoxicity of eNK cells against BCC lines (5637, HT-1376, 253J-BV) was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies)-based assay for 4 h. Target cells were stained with 0.5 μM CFSE in FACS buffer for 10 min at 37 °C and washed twice with complete media.

Techniques: Lysis, Staining