Journal: Cancer Research

Article Title: Base Editing of TIGIT Reprograms CD155 Signaling in Natural Killer Cells to Enhance Cancer Immunotherapy Efficacy

doi: 10.1158/0008-5472.CAN-25-0733

Figure Lengend Snippet: Cytotoxicity activities of TIGIT BE-NK cells in vitro . A–E, A variety of different cancer cell lines (GFP expressing or red dye labeled) were inoculated into 96-well plates in quantities of 10 4 cells/well and left for 6 hours to allow cells to attach, then 10 4 PB-NK or BE-NK cells were added, and images of the same field of view were recorded at different times. A, NSCLC cell line H1299 (GFP expressing). B , NSCLC cell line A549 (GFP expressing). C , Fibrosarcoma cell line HT-1080 (GFP expressing). D , Hepatocellular cell line Huh7 (red dye labeled). E , Hepatoma cell line PLC/PRF/5 (red dye labeled). Images recorded and data analyzed by IncuCyte S3, and data are shown as mean ± SD.

Article Snippet: Plates were then transferred into the Incucyte S3 (Sartorius, RRID: SCR_023147), and images were captured by the Incucyte S3 system with a × 10 objective lens at 6-hour time intervals.

Techniques: In Vitro, Expressing, Labeling

Journal: Cancer Research

Article Title: Base Editing of TIGIT Reprograms CD155 Signaling in Natural Killer Cells to Enhance Cancer Immunotherapy Efficacy

doi: 10.1158/0008-5472.CAN-25-0733

Figure Lengend Snippet: TIGIT BE-NK cells specifically recognized target cells in a CD155-/CD226-dependent manner. A, The mean fluorescence intensity (MFI) of CD155 on H1299, H1975, A549, and H460 (NSCLC cell line), K652 (chronic myelogenous leukemia cell line), and CEM (acute lymphoblastic leukemia cell line) was assessed by flow cytometry. B, Tumor cells and PB-NK or TIGIT BE-NK cells were inoculated into 48-well plates at a 1:1 ratio, and CD107a expression on NK cells was detected by flow cytometry 4 hours later. The increase in CD107a-positive cells in the TIGIT BE-NK group was normalized to the PB-NK group. C, Tumor cells were coincubated with PB-NK or TIGIT BE-NK cells for 24 hours at an E:T ratio of 1:1, and then NK cell–mediated lysis was evaluated by luminescent cell viability assay. E:T corresponds to E:T cells. D, The mean fluorescence intensity of CD155 on U251-MG (glioblastoma cell line), Huh7 (hepatocellular cell line), HT-1080 (fibrosarcoma cell line), PLC/PRF/5 (hepatoma cell line), BxPC-3 (pancreatic adenocarcinoma cell line), T24 (urinary bladder cancer cell line), U2OS (osteosarcoma cell line), MCF7 (breast cancer cell line), HeLa (cervical carcinoma cell line), SKOV3 (ovarian cancer cell line), HCT116 (colorectal carcinoma cell line), HGC-27 (gastric cancer cell line), KG-1A, OCI-AML3 (acute myeloid leukemia cell line), and MM.1S (multiple myeloma) was assessed by flow cytometry. E, Tumor cells and PB-NK or TIGIT BE-NK cells were inoculated into 48-well plates at a 1:1 ratio, and CD107a expression on NK cells was detected by flow cytometry 4 hours later. The increase in CD107a-positive cells in the TIGIT BE-NK group was normalized to the PB-NK group. F, The correlation analysis of the result is shown in D and E . Simple linear regression analysis. G and H, The NK cell–mediated cytotoxicity was evaluated using a luminescent cell viability assay following coincubation of A549 ( G ) or Huh7 ( H ) cells with NK cells at a 1:1 ratio for 24 hours. I and J, Cytotoxic effects of individual and combined TIGIT, CD96, and PVRIG KO in NK cells. Data recorded and analyzed by IncuCyte S3; data are shown as mean ± SD. K and L, PB-NK, TIGIT BE-NK, or TIGIT/CD226 double–KO NK cells were cocultured with A549 or Huh7 tumor cells. NK cells were harvested at 0, 2, 15, and 30 minutes after stimulation, and p44/42 MAPK (ERK1/2) and phospho-p44/42 MAPK (ERK1/2; Thr202/Tyr204) activity was assessed by Western blotting. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.

Article Snippet: Plates were then transferred into the Incucyte S3 (Sartorius, RRID: SCR_023147), and images were captured by the Incucyte S3 system with a × 10 objective lens at 6-hour time intervals.

Techniques: Fluorescence, Flow Cytometry, Expressing, Lysis, Cell Viability Assay, Activity Assay, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: TSPO acts as an immune resistance gene involved in the T cell mediated immune control of glioblastoma

doi: 10.1186/s40478-023-01550-9

Figure Lengend Snippet: TSPO protects GB cells against T cell-mediated cytotoxicity in both allogeneic and autologous co-culture models. a - d Tetrazolium salt-based XTT assay to determine the impact of TSPO expression in GB cells on T cell-mediated killing. Human GB cell lines ( a ) U-87 MG and ( b ) U-251 MG were transfected with either control siRNA or a pool of four TSPO-specific non-overlapping siRNAs. After 72 h of transfection, the cells were pulsed with 0.01 μg/ml flu-peptide and cultured with FluTC for 4 h. The primary cell lines BTIC13 (mesenchymal, c ) and BTIC129 (proneural, d ) were transduced with either non-targeting control or TSPO-specific shRNA (TSPO-proficient/deficient = TSPO ±). Stable cell lines were seeded on a 96-well plate and co-cultured the next day with ( c ) FluTC after being pulsed with 0.001 μg/ml flu-peptide or ( d ) pre-activated autologous TIL129 for 4 h. Target-cell lysis was determined as described in the Materials and Methods section. e–g Real-time cytotoxicity assay (IncuCyte® SX5 System) to analyze T cell-mediated GB cell killing over 24 h. Incucyte® Caspase-3/7 Green Dye was added as an indicator of apoptosis. The graphs show total apoptotic tumor cell area (green object area) per well (µm 2 /well). Co-culture of TSPO ± ( e ) BTIC13, ( f ) BTIC13 clones and ( g ) BTIC129 cells with ( e , f ) FluTC and ( g ) pre-activated autologous TIL129. a-d Cumulative data of three to five independent experiments, e–g Representative data of at least three independent experiments. Values represent the mean ± SD. P-value was calculated using paired two-tailed Student`s t-test (* = P < 0.05, ** = P < 0.01, *** = P < 0.005, **** = P < 0.001)

Article Snippet: Caspase-3/-7 mediated apoptosis of BTICs was determined by real-time live-cell imaging using Incucyte® SX5 live cell imager (Sartorius).

Techniques: Co-Culture Assay, XTT Assay, Expressing, Transfection, Cell Culture, Transduction, shRNA, Stable Transfection, Lysis, Cytotoxicity Assay, Clone Assay, Two Tailed Test