cu cpt4a  (MedChemExpress)

Journal: Redox Biology

Article Title: Amino acid restriction sensitizes lung cancer cells to ferroptosis via GCN2-dependent activation of the integrated stress response

doi: 10.1016/j.redox.2025.103988

Figure Lengend Snippet: F12 medium sensitizes lung cancer cells to iron-dependent lipid peroxidation . ( A, B ) Quantification by flow cytometry of oxidized BODIPY-C11 fluorescence in A549 (A) or H838 (B) cells that were cultured in RPMI or F12 medium and treated with 100 μM BSO or vehicle for 24 h. ( C ) Dose response curves for F12-cultured A549 and H838 cells treated with BSO in combination with 5 μM liproxstatin-1 (LIP-1), 5 μM ferrostatin-1 (FER-1) or 50 μM α-tocopherol (α-toco) for 72 h. ( D ) Dose response curves for F12-cultured A549 or H838 cells treated with BSO in combination with 5 μM deferoxamine (DFO) for A549 cells and 9 μM for H838 cells for 72 h. ( E ) Dose response curves for F12-cultured A549 or H838 cells treated with BSO in combination with 5 μM necrostatin-1 (NEC-1) or 10 μM ZVAD-FMK (ZVAD) for 72 h. ( F ) Dose response curves for F12-cultured A549 or H838 cells treated with BSO in combination with 4 μg/mL certolizumab (CER), 15 μM CU-CPT4a (C4a), 3 μM resatorvid (RST), or 50 μM necrostatin-1 (NEC-1) for 72 h (ND, not done). Dose response curves were normalized against the mean of the untreated samples for each condition. n = 3 replicates for all datapoints, error bars show SEM. ∗∗∗∗P < 0.0001.

Article Snippet: Drugs used were auranofin (A6733; Sigma-Aldrich), α-tocopherol (T3251; Sigma-Aldrich), bafilomycin A1 (SML1661; Sigma-Aldrich), certolizumab pegol (HYP9953; MedChemExpress), chloroquine (C6628; Sigma-Aldrich), CU-CPT4A (HY-108473; MedChemExpress), deferoxamine (D9533; Sigma-Aldrich), erastin (e7781; Sigma Aldrich), FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) (HY-100410; MedChemExpress), ferrostatin-1 (SML0583; Sigma-Aldrich), l -buthionine-sulfoximine (b2515; Sigma-Aldrich), mito-TEMPO (HY–W001187; MedChemExpress), necrostatin-1 (N9037; Sigma-Aldrich), liproxstatin-1 (SML1414; Sigma-Aldrich), oligomycin (HY–N6782; MedChemExpress), puromycin dihydrochloride (A1113803; Gibco), rotenone (HY–B1756; MedChemExpress), RSL3 (SML2234; Sigma-Aldrich), resatorvid (HY-11109; MedChemExpress), and Z-VAD-FMK (V116; Sigma-Aldrich).

Techniques: Flow Cytometry, Fluorescence, Cell Culture

Journal: Journal for Immunotherapy of Cancer

Article Title: Colorectal cancer-specific IFNβ delivery overcomes dysfunctional dsRNA-mediated type I interferon signaling to increase the abscopal effect of radiotherapy

doi: 10.1136/jitc-2023-008515

Figure Lengend Snippet: Radiotherapy (RT) enhanced cytosolic dsRNA accumulation for TLR3-mediated type I IFN production. (A) HCT116 cells were irradiated for 24 hours, and then RNA was extracted for electrophoresis (7.5% polyacrylamide gel electrophoresis). HCT116 cells were irradiated for 24 and 48 hours, and the content of cytosolic dsRNA was measured by dsRNA ELISA kit (n=3). *p<0.05, **p<0.01. One-way ANOVA test. (B) SW480 cells were irradiated with 5 Gy for 24 hours, and the level of dsRNA was observed by immunofluorescence staining. *p<0.05 Unpaired t-test. (C) SW480 cells were treated with conditioned medium for 24 hours and then examined by qRT-PCR (n=3). The conditioned medium was collected from irradiated cells and then incubated with RNase A and RNase III for 2 hours. The level of IFNβ1 was analyzed by qRT-PCR (n=3). ***p<0.001. One-way ANOVA test. (D) SW480 cells were treated with conditioned medium for 24 hours and then examined by qRT-PCR (n=3). The conditioned medium was collected from irradiated cells and then incubated with RNase A and RNase III for 2 hours. The level of CXCL10 was analyzed by qRT-PCR (n=3). **p<0.01. One-way ANOVA test. (E) Representative images of cytosolic dsRNA in pre-neoCRT biopsies and post-neoCRT surgical tissues. (F) The correlation between cytosolic dsRNA and tumor IFNβ expression was measured (non-linear regression model, p=0.0012, r =0.2794, n=131). (G) SW480 cells were infected with lentivirus carrying shRNA against RIG1, MDA5 and MAVS. The knockdown efficacy was examined by western blotting. (H) SW480 shNC , SW480 shRIG1 , SW480 shTLR3 , SW480 shMDA5 and SW480 shMAVS cells were irradiated with 5 Gy. After 24 hours, the cells were harvested, and the mRNA levels of IFNβ1 and CXCL10 were examined by qRT-PCR (n=3). *p<0.05, **p<0.01. One-way ANOVA test. (I) HCT116 cells were infected with lentivirus carrying shRNA against TLR3. The knockdown efficacy was measured by immunoblotting. HCT116 shNC , HCT116 shTLR3#1 and HCT116 shTLR3#2 cells were irradiated with 5 and 10 Gy. After 24 hours, the cells were harvested, and the mRNA levels of IFNβ1 and CXCL10 were examined by qRT-PCR (n=3). **p<0.01, ***p<0.001. One-way ANOVA test. (J) HCT116 shNC and HCT116 shTLR3 cells were with or without irradiation with 5 Gy. After 24 hours, CFSE-labeled SupT1 T cells were seeded in the upper wells, and the migrated T cells were examined by flow cytometry (n=3). **p<0.01, ***p<0.001. One-way ANOVA test. (K) HCT116 isogenic TLR3 knockout cells (HCT116-TLR3 KO ) constructed with TLR3 gRNA and HCT115-WT cells constructed with non-targeting (NT) gRNA. HCT116-WT and HCT116-TLR3 KO cells were irradiated with 5 Gy and harvested after 24 hours. The levels of IFNβ1 were analyzed by qRT-PCR (n=3). *p<0.05. One-way ANOVA test. ANOVA, analysis of variance; CFSE, Carboxyfluorescein succinimidyl ester; qRT-PCR, quantitated by real-time PCR.

Article Snippet: HCT116 cells were treated with 2.5 μg/mL poly (I:C) (Sigma, Missouri, USA) or 10 μM selective TLR3 inhibitor CU-CPT4a (Tocris, Pennsylvania, USA) for 24 hours.

Techniques: Irradiation, Electrophoresis, Polyacrylamide Gel Electrophoresis, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Incubation, Expressing, Infection, shRNA, Knockdown, Western Blot, Labeling, Flow Cytometry, Knock-Out, Construct, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: Colorectal cancer-specific IFNβ delivery overcomes dysfunctional dsRNA-mediated type I interferon signaling to increase the abscopal effect of radiotherapy

doi: 10.1136/jitc-2023-008515

Figure Lengend Snippet: TLR3 and IFNAR1 are indispensable for irradiation-induced type I IFN and CXCL10 production. (A) SW480 cells were irradiated and treated with 2.5 µg/mL poly (I:C) and 10 µM TLR3 inhibitor together for 24 hours. The mRNA levels of TLR3 , IFNβ1, CXCL10 and MX1 were analyzed by qRT-PCR (n=3). *p<0.05, **p<0.01 and ***p<0.001. One-way ANOVA test. (B) SW480 cells were irradiated and treated with 2.5 µg/mL poly (I:C) and 10 µM TLR3 inhibitor together for 24 hours. The protein levels of p-IRF3, IRF3, p-STAT1, and STAT1 were examined by immunoblotting. *p<0.05, **p<0.01, ***p<0.001. One-way ANOVA test. (C) SW480 cells were transfected with pCMV6-vector, pCMV-TLR3-WT, or pCMV-TLR3-L412F for 24 hours and then irradiated with 5 Gy. After 24 hours, we analyzed the protein level by immunoblotting. (D) The quantification of p-IRF3/IRF3 and p-STAT1/STAT1 is shown. *p<0.05, **p<0.01. One-way ANOVA test. (E) HCT116 cells were infected with lentivirus carrying shRNA against IFNAR. The knockdown efficacy was measured by immunoblotting. HCT116 shNC , HCT116 shIFNAR1#1 and HCT116 shIFNAR1#2 cells were irradiated with 5 and 10 Gy. After 24 hours, the cells were harvested, and the mRNA level of CXCL10 was examined by qRT-PCR. ***p<0.001. One-way ANOVA test. (F) HCT116 shNC and HCT116 shIFNAR1#2 cells were irradiated with 5 Gy. After 24 hours, conditioned medium was harvested to analyze the level of CXCL10 by ELISA. ***p<0.001. One-way ANOVA test. ANOVA, analysis of variance; qRT-PCR, quantitated by real-time PCR.

Article Snippet: HCT116 cells were treated with 2.5 μg/mL poly (I:C) (Sigma, Missouri, USA) or 10 μM selective TLR3 inhibitor CU-CPT4a (Tocris, Pennsylvania, USA) for 24 hours.

Techniques: Irradiation, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Infection, shRNA, Knockdown, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: Colorectal cancer-specific IFNβ delivery overcomes dysfunctional dsRNA-mediated type I interferon signaling to increase the abscopal effect of radiotherapy

doi: 10.1136/jitc-2023-008515

Figure Lengend Snippet: TLR3 signaling is important for type I IFN and CXCL10 production to recruit intratumoral TILs in vivo. (A) Tumor growth of CT26-driven colon carcinoma established in BALB/c mice (n=8/group) that were treated with radiotherapy (5 Gy for two fractions) or radiotherapy/TLR3 inhibitor (0.4 mg/kg TLR3 inhibitor 1 hour before and after irradiation). Tumor volume was recorded every 3 days (mean±SE). *p<0.05. The resected tumor tissues were harvested on day 25 for further analysis. (B) The representative image of cytosolic dsRNA in resected tumor tissues which was observed by anti-dsRNA (J2 clone) immunofluorescence staining (n=3). The cytosolic dsRNA fluorescence intensity was examined. ***p<0.001. One-way ANOVA test. (C) Resected tumors were extracted, and the mRNA levels of Ifnβ1 and Cxcl10 were analyzed by qRT-PCR (n=4). *p<0.05. One-way ANOVA test. (D) Resected tumors were extracted, and the mRNA levels of Mx1 and Cxcr3 were analyzed by qRT-PCR (n=4). *p<0.05. One-way ANOVA test. (E) The representative images of CD3 + TILs and CD8a + TILs which were analyzed by immunohistochemistry. The density of CD3 + TILs and CD8a + TILs was counted under high-power-field microscopy (n=6). **p<0.01, ***p<0.001. One-way ANOVA test. (F) The representative images of GzmB + CD8a + TILs which were analyzed by immunofluorescence staining. The density of GzmB + CD8a + TILs was counted under high-power-field microscopy (n=6). **p<0.01, ***p<0.001. One-way ANOVA test. (G) The representative images of Cxcr3 + CD8a + TILs and Cxcr3 + CD11c + DCs and dsRNA which were by immunofluorescence staining. The density of Cxcr3 + CD8a + TILs and Cxcr3 + CD11c + DCs was counted under high-power-field microscopy (n=6). **p<0.01, ***p<0.001. One-way ANOVA test. (H) The resected tumors were extracted and analyzed by immunoblotting (n=3). Quantitative analysis of the immunoblotting results. ***p<0.001. One-way ANOVA test. ANOVA, analysis of variance; qRT-PCR, quantitated by real-time PCR; TIL, tumor-infiltrating lymphocyte.

Article Snippet: HCT116 cells were treated with 2.5 μg/mL poly (I:C) (Sigma, Missouri, USA) or 10 μM selective TLR3 inhibitor CU-CPT4a (Tocris, Pennsylvania, USA) for 24 hours.

Techniques: In Vivo, Irradiation, Immunofluorescence, Staining, Fluorescence, Quantitative RT-PCR, Immunohistochemistry, Microscopy, Western Blot, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: Colorectal cancer-specific IFNβ delivery overcomes dysfunctional dsRNA-mediated type I interferon signaling to increase the abscopal effect of radiotherapy

doi: 10.1136/jitc-2023-008515

Figure Lengend Snippet: Patients with the TLR3 loss-of-function variant (L412F/rs3775291) were associated with less infiltration of CD8 + TILs, poor therapeutic response and survival outcome. (A) Kaplan-Meier curves for 5-year DFS in individuals with high or low CD8 + TILs density in the post-neoCRT surgical tissues of LARC patient who received neoCRT in a retrospective study (log-rank p=0.042, n=111). (B) Kaplan-Meier curves for 5-year DFS in individuals with wild-type or defective TLR3 variant (L412F/rs3775291) in the cohort receiving neoCRT treatment (log-rank p=0.011, n=118). (C) Representative image of CD8 + TILs within the TME in wild-type and mutant (L412F/rs3775291) TLR3 before and after neoCRT treatment. The density of CD8 + TILs within the TME in the post-neoCRT tissues is shown (unpaired t-test, p=0.0216, n=100). (D) Kaplan-Meier curves for 5-year DFS in individuals with TLR3 variant (L412F/rs3775291) and high/low CD8 + TILs density in the cohort receiving neoCRT treatment (log-rank p=0.0063, n=100). (E) The forest plot of multivariate analysis including pN stage, TRG, TLR3 variant, CD8 counts and TLR3/CD8 (n=100). Multivariate Cox analysis. (F) Scheme of advanced CRC patient enrolment (n=24). T0: diagnosis day; T1: patients received 1 week of treatment; T2: surgery day. (G) The level of type I signatures including IFNα2, IFNβ1 and CXCL10 was measured in the serum of patients with advanced CRC carrying wild-type TLR3 (n=9, paired t-test). *p<0.05 and **p<0.01. (H) The density of CD8 + TILs within TME was measured in the patients with advanced CRC carrying wild-type TLR3 (n=9, paired t test). *p<0.05. (I) The level of type I signatures including IFNα2, IFNβ1 and CXCL10 was measured in the serum of patients with advanced CRC carrying mutant TLR3 (n=15, paired t-test). (J) The density of CD8 + TILs within TME was measured in the patients with advanced CRC carrying mutant TLR3 (n=15, paired t test). (K) The association between the level of tumor-intrinsic CXCL10 mRNA level and the response to neoCRT was evaluated in the external dataset retrieved from NCBI GEO dataset (GSE46862). MI: minimal response (TRG1); MO: moderate response (TRG2); NT: near complete response (TRG3) and TO: total response (TRG4). (n=64, one-way ANOVA test). (L) Kaplan-Meier curves for DFS in patients with colorectal cancer with high or low CXCL10 mRNA (cut-off=median TPM), which was retrieved form the GEPIA database (Gene Expression Profiling Interactive Analysis, http://gepia.cancer-pku.cn/ ). Log-rank test (n=270). ANOVA, analysis of variance; CRC, colorectal cancer; DFS, analysis of variance; TILs, tumor-infiltrating lymphocytes; TME, tumor microenvironment.

Article Snippet: HCT116 cells were treated with 2.5 μg/mL poly (I:C) (Sigma, Missouri, USA) or 10 μM selective TLR3 inhibitor CU-CPT4a (Tocris, Pennsylvania, USA) for 24 hours.

Techniques: Variant Assay, Clinical Proteomics, Mutagenesis, Biomarker Discovery, Gene Expression

Journal: Journal for Immunotherapy of Cancer

Article Title: Colorectal cancer-specific IFNβ delivery overcomes dysfunctional dsRNA-mediated type I interferon signaling to increase the abscopal effect of radiotherapy

doi: 10.1136/jitc-2023-008515

Figure Lengend Snippet: Local IFNβ1 overcomes the impact of TLR3 dysfunction and significantly enhances the therapeutic efficacy of radiotherapy. (A) Tumor growth of CT26-driven colon carcinoma established in BALB/c mice (n=5/group) that were treated with local radiotherapy (5 Gy for two fractions), radiotherapy/TLR3 inhibitor (0.4 mg/kg TLR3 inhibitor 1 hour before and after irradiation), and radiotherapy/TLR3 inhibitor/IFNβ (10,000 U/mouse). (B) Tumor growth was recorded as the mean tumor volume±SEM (n=5). *p<0.05. (C) The survival time was recorded every 3 days, and the mice were sacrificed if the average tumor diameter reached 20 mm. (D) Diagram of the novel engineered AAV-IFNβ with a CRC-specific promoter and the administration protocol. A total of 3×10 5 CT26 cells were subcutaneously injected into the right leg, and 2×10 5 CT26 cells were subcutaneously injected into the left back 2 days later. Local radiotherapy was given on days 10 and 13, and AAV (1×10 8 vg in 50 µL PBS, intramuscular injection) was administered on days 9, 13, 16 and 20. AAV-Vec: empty AAV vector with CRC-specific CEA promoter; CEAp: CEA promoter; gray box: inverted terminal repeats (ITR). (E) CT26 tumor-bearing mice (n=5–6/group) received AAV and local radiotherapy (5 Gy for two fractions) on the indicated days. Tumor volume was measured every 3 days, and tumors were harvested on day 31. **p<0.01, ***p<0.001. CR: complete response. (F) The resected irradiated tumors were weighed (n=3–5, variable due to CR). **p<0.01, ***p<0.001. One-way ANOVA test. (G) The tumor-infiltrating DCs and CD8 + immune cells in resected primary tumors were evaluated by immunofluorescence staining. (H) Quantification of tumor-infiltrating CD8 + immune cells in resected primary tumors. *p<0.05, **p<0.01. One-way ANOVA test. (I) Quantification of tumor-infiltrating CD11c + DCs in resected primary tumors. *p<0.05, **p<0.01. One-way ANOVA test. (J) The tumor volume of non-irradiated tumors (abscopal tumors) was measured every 3 days, and the tumors were harvested on Day 31. *p<0.05, **p<0.01. (K) The resected abscopal tumors were weighed (n=4–5, variable due to CR). **p<0.01, ***p<0.001. One-way ANOVA test. (L) The tumor-infiltrating DCs and CD8 + immune cells in resected abscopal tumors were evaluated by immunofluorescence staining. (M) Quantification of tumor-infiltrating CD8 + immune cells in resected abscopal tumors. *p<0.05, **p<0.01. One-way ANOVA test. (N) Quantification of tumor-infiltrating CD11c + DCs in resected abscopal tumors. *p<0.05 and **p<0.01. One-way ANOVA test. ANOVA, analysis of variance.

Article Snippet: HCT116 cells were treated with 2.5 μg/mL poly (I:C) (Sigma, Missouri, USA) or 10 μM selective TLR3 inhibitor CU-CPT4a (Tocris, Pennsylvania, USA) for 24 hours.

Techniques: Drug discovery, Irradiation, Injection, Plasmid Preparation, Immunofluorescence, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Colorectal cancer-specific IFNβ delivery overcomes dysfunctional dsRNA-mediated type I interferon signaling to increase the abscopal effect of radiotherapy

doi: 10.1136/jitc-2023-008515

Figure Lengend Snippet: Cancer-intrinsic IFNβ1 expression by AAV transduction significantly reshaped cancer immunogenicity to enhance RT-induced immunity. (A) The tumor-infiltrating CD8 + CD3 + CD45 + T cells were analyzed by flow cytometry (one-way ANOVA test, n=3–5). *p<0.05, **p<0.01. One-way ANOVA test. (B) The tumor-infiltrating CD44 + CD62L - CD8 + CD3 + CD45 + T EM cells were analyzed by flow cytometry (one-way ANOVA test, n=3–5). *p<0.05, **p<0.01. One-way ANOVA test. (C) The tumor-infiltrating CD11c + MHC-II+ DCs were analyzed by flow cytometry (one-way ANOVA test, n=3–5). *p<0.05, **p<0.01. One-way ANOVA test. (D) The tumor-infiltrating IFNγ + CD8 + CD3 + CD45 + T cells were analyzed by flow cytometry (one-way ANOVA test, n=3–5). *p<0.05. One-way ANOVA test. (E) The tumor-infiltrating IFNγ + CD49b + CD45 + NK cells were analyzed by flow cytometry (one-way ANOVA test, n=3–5). *p<0.05. One-way ANOVA test. (F) The ratio of IFNγ + CD8 + T cells/Foxp3 + Treg cells. *p<0.05. One-way ANOVA test. (G) Gene expression in whole tumors from mice on day 30 after the indicated treatments. Fold-change over isotype control values represented. The results are representative of two independent experiments (n=2 mice per group). (H) Diagram of AAV-IFNβ1, local RT and anti-mouse CD8a antibodies administration protocol. A total of 3×10 5 CT26 cells were subcutaneously injected into the right leg. Local RT was given on days 10 and 13, and AAV (1×10 8 vg in 50 µL PBS, intramuscular injection) was administered on days 9, 13, 16 and 20. Anti-mouse CD8a antibodies were given by intraperitoneal injection (100 µg/mouse). Tumor volume was measured every 2–3 days, and tumors were harvested on day 28 (n=5). **p<0.01, ***p<0.001. (I) The proposed mechanism by which engineered CRC-specific AAV-IFNβ therapy overcomes dysfunctional TLR3-mediated IFN-I attenuation to enhance the response to RT. AAV, adenovirus-associated virus; ANOVA, analysis of variance; RT, radiotherapy.

Article Snippet: HCT116 cells were treated with 2.5 μg/mL poly (I:C) (Sigma, Missouri, USA) or 10 μM selective TLR3 inhibitor CU-CPT4a (Tocris, Pennsylvania, USA) for 24 hours.

Techniques: Expressing, Transduction, Immunopeptidomics, Flow Cytometry, Gene Expression, Control, Injection, Virus