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: Nucleic Acids Research

Article Title: UNG-initiated base excision repair is the major repair route for 5-fluorouracil in DNA, but 5-fluorouracil cytotoxicity depends mainly on RNA incorporation

doi: 10.1093/nar/gkr563

Figure Lengend Snippet: Verification of siRNA knockdown of UNG , SMUG1 , TDG and MSH2 and their effect on FdUrd DNA incorporation. ( A ) Quantification of siRNA knockdown by western blots from whole-cell extracts of SW480 and HeLa MSH2 , UNG , SMUG1 , TDG siRNA silenced cells harvested 48 h post-transfection. β-actin was used as loading control. ( B ) Quantification of glycosylase knockdown by specific enzyme activity assays from whole-cell extracts 48 h after transfection. UNG excision activity was measured by the release of [ 3 H]uracil from labelled calf thymus DNA (U:A substrate). SMUG1 and TDG activity were measured using a U:G oligomer substrate in the presence of either Ugi and neutralizing TDG antibodies, or Ugi and neutralizing SMUG1 antibodies, respectively. ( C ) Quantification by LC/MS/MS of incorporated FdUrd per nucleotide DNA after 24 h FdUrd exposure of MSH2 , UNG , SMUG1 and TDG silenced SW480 (40 µM) and HeLa (4 µM) cells 48 h after transfection. Cells were harvested, and DNA isolated, hydrolysed and analysed for FdUrd content. FdUrd levels are normalized to the measured total number of normal deoxynucleosides in each sample. The data points represent fold change compared to control as is the mean ± SD of two to four parallel experiments.

Article Snippet: Nucleic acids were isolated from fluoropyrimidine-treated cells by the DNeasy Blood and cell culture DNA isolation kit (Qiagen) or by the mirVana RNA-isolation kit (Ambion).

Techniques: Knockdown, Western Blot, Transfection, Control, Activity Assay, Liquid Chromatography with Mass Spectroscopy, Isolation

Journal: Nucleic Acids Research

Article Title: UNG-initiated base excision repair is the major repair route for 5-fluorouracil in DNA, but 5-fluorouracil cytotoxicity depends mainly on RNA incorporation

doi: 10.1093/nar/gkr563

Figure Lengend Snippet: Quantification of FU accumulation in RNA and DNA, relative TS-inhibition by FdUrd, FUrd and FU and reversal of their cytotoxicities and RNA incorporation by nucleosides/nucleotides. ( A ) Quantification of incorporated FUrd per nucleotide RNA and FdUrd per nucleotide DNA after 24 h FU or FdUrd exposure of SW480 and HeLa cells. Cells were harvested and RNA or DNA isolated, hydrolysed and analysed for FUrd or FdUrd content by LC/MS/MS. FUrd and FdUrd levels are normalized relative to the total number of normal nucleosides or deoxynucleosides measured. ( B ) TS activity measured by adding 1 µCi [5- 3 H]deoxyuridine and counting [ 3 H]H 2 O released into the growth medium after 90 min in HeLa or SW480 cells pretreated with indicated concentrations of FdUrd, FUrd or FU. Measurements are plotted relative to the activity of untreated samples. ( C ) Urd reversal of FUrd incorporation into RNA. HeLa or SW480 cells were treated with 2.5 µM FUrd for 24 h in the presence or absence of Urd at indicated concentrations. RNA was isolated, hydrolysed and FUrd levels quantified by LC/MS/MS. The data represent the mean ± SD of at least two parallel measurements. ( D ) Effect on survival after exposure to FdUrd, FU and FUrd by concurrent treatment with various concentrations of nucleosides (dThd, dUrd or Urd). Increasing concentrations of nucleosides were added to SW480 or HeLa cells simultaneously treated with a fixed dose of either FdUrd (SW480: 25 µM, HeLa: 0.25 µM), FU (SW480: 15 µM, HeLa: 50 µM), or FUrd (2.5 µM). Survival was measured by the MTT assay after four days exposure. ( E ) Effect on FdUrd, FU and FUrd survival by concurrent treatment with nucleotides (dTMP, dUMP, UMP). Increasing concentrations of nucleotides were added to SW480 or HeLa cells simultaneously treated with either FdUrd (SW480: 50 µM, HeLa: 5 µM), FU (50 µM), or FUrd (5 µM). Survival was measured by the MTT assay after four days exposure. The survival data represent the mean ± SD of at least two parallel measurements.

Article Snippet: Nucleic acids were isolated from fluoropyrimidine-treated cells by the DNeasy Blood and cell culture DNA isolation kit (Qiagen) or by the mirVana RNA-isolation kit (Ambion).

Techniques: Inhibition, Isolation, Liquid Chromatography with Mass Spectroscopy, Activity Assay, MTT Assay

Journal: Nucleic Acids Research

Article Title: UNG-initiated base excision repair is the major repair route for 5-fluorouracil in DNA, but 5-fluorouracil cytotoxicity depends mainly on RNA incorporation

doi: 10.1093/nar/gkr563

Figure Lengend Snippet: Overview of metabolism of 5-fluoropyrimidines and correlations between survival and RNA incorporation or TS-inhibition. ( A ) Schematic overview of 5-fluoropyrimidine metabolism the three main routes to FU cytotoxicity: RNA incorporation of FUTP (red), TS-inhibition by FdUMP (blue) and DNA incorporation of FdUTP and dUTP (green). FdUTP incorporated into DNA end up as FU:A (mainly) or FU:G base pairs, which are predominantly repaired by the BER pathway initiated by UNG, with minor contributions from SMUG1, TDG and MMR for repair of FU:G. FU and FUrd cytotoxicity is predominantly mediated through RNA incorporation (red) and FdUrd through dTTP depletion (blue), while misincorporated 5-FdUTP and dUTP (green) contribute negligibly to overall 5-fluoropyrimidine cytotoxicity. DUT, deoxyuridine triphosphatase; DPD, dihydropyrimidine dehydrogenase; OPRT, orotic acid phosphoribosyl transferase; RR, ribonucleotide reductase; TK, thymidine kinase; TP, thymidine phosphorylase; UK, uridine kinase; UP, uridine phosphorylase. ( B ) Analysis of correlation of RNA incorporation versus survival and TS-inhibition versus survival after FdUrd, FUrd and FU treatment. Values for FUrd RNA levels ( A and C) or TS-inhibition {100% – [TS-activity ( B)]} are plotted against cell survival after treatment with the nearest corresponding 5-fluoropyrimidine concentration. Lines represent linear trend lines in each plot.

Article Snippet: Nucleic acids were isolated from fluoropyrimidine-treated cells by the DNeasy Blood and cell culture DNA isolation kit (Qiagen) or by the mirVana RNA-isolation kit (Ambion).

Techniques: Inhibition, Activity Assay, Concentration Assay