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: The following antibodies were used: cGAS (ab224144, Abcam, Cambridge, UK), STING (#13647, Cell Signaling Tech., California, USA), GAPDH (Ab9485, Abcam), RIG1 (A0550, Abclonal, Massachusetts, USA), TLR3 (ab62566, Abcam), MDA5 (A2203, Abclonal), MAVS (A5764, Abclonal), beta-actin (IR2-7, iREAL Biotech.), p-IRF3 S388 (AP1333 and AP0263, Abclonal, IRF3 (#41075, Cell Signaling Tech.), p-STAT1 T701 (bs-1657R, Bioss antibodies and AP0054, Abclonal) and STAT1 (sc-464, Santa Cruz, California, USA).

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

Journal: Molecular Oncology

Article Title: Arachidonic acid, a clinically adverse mediator in the ovarian cancer microenvironment, impairs JAK‐STAT signaling in macrophages by perturbing lipid raft structures

doi: 10.1002/1878-0261.13221

Figure Lengend Snippet: Inhibition of cytokine‐induced STAT and JAK signaling in monocyte‐derived macrophages (MDMs) by polyunsaturated fatty acids (PUFAs). (A) Inhibition of IFNβ‐induced phosphorylation of STAT1 (Y701) by different PUFAs. The p‐STAT1 antibody recognizes both the STAT1α and β isoforms. (B) Inhibition of IFNγ‐induced phosphorylation of STAT1 by different PUFAs. (C) Inhibition of IL‐6 induced phosphorylation of STAT3 (Y705) by different PUFAs. AA, arachidonic acid; LA, linoleic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; ETYA, 5,8,11,14‐eicosatetraynoic acid. (D) Inhibition of IFNβ‐induced phosphorylation of JAK1 (Y1034/1035) by AA. (E) Inhibition of IFNγ‐induced phosphorylation of JAK2 (Y1007/Y1008) by AA. In each case, MDMs were pretreated with 50 µ m of the indicated PUFA for 30 min prior to stimulation with the IFNβ, IFNγ or IL‐6 for 30 min. A representative immunoblot and the quantification of n = 7 (A–C) or n = 5 (D–E) independent experiments (different donors; indicated by different symbols) are shown in each panel. Statistical significance was analyzed by paired t test (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant). Horizontal lines indicate the median.

Article Snippet: The following antibodies were used: p‐p38 (T180/Y182; #4511; Cell Signaling, Frankfurt, Germany); p38 (#9228; Cell Signaling), p‐STAT1 (T701; #612132; BD Bioscience, Franklin Lakes, NJ, USA); Stat1 (9172, Cell Signaling); p‐STAT3 (Y705; #9145; Cell Signaling); STAT3 (#9139; Cell Signaling); p‐JAK1 (T1034/1035; #66245; Cell Signaling); JAK1 (50996; Cell Signaling); p‐JAK2 (Y1007/1008; #8082S; Cell Signaling); JAK2 (#3230; Cell Signaling); Flotillin‐1 (#74566; Santa Cruz Technologies, Dallas, TX, USA); CD71 (#65882; Santa Cruz); IκB‐α (#371; Santa Cruz); IκBβ (#8635; Cell Signaling); β‐actin (#A5441; Sigma); Phospho‐SMAD2 (Ser465/467, #3108S; Cell Signaling); SMAD2 (#sc‐393312; Santa Cruz); GAPDH (#G9545; Sigma), α‐rabbit IgG HRP‐linked AB (#27; Cell Signaling) and α‐mouse IgG HRP‐linked AB (#32; Cell Signaling).

Techniques: Inhibition, Derivative Assay, Western Blot

Journal: Molecular Oncology

Article Title: Arachidonic acid, a clinically adverse mediator in the ovarian cancer microenvironment, impairs JAK‐STAT signaling in macrophages by perturbing lipid raft structures

doi: 10.1002/1878-0261.13221

Figure Lengend Snippet: Inhibition of the cytokine‐induced nuclear translocation of STAT1 and STAT3 by arachidonic acid (AA). (A) Monocyte‐derived macrophages (MDMs) were pretreated with 50 µ m AA or solvent for 30 min prior to stimulation with IFNγ for 30 min as in Fig. and the subcellular localization of STAT1 was analyzed by immunofluorescence (green). Nuclei were visualized by staining with 4′,6‐diamidin‐2‐phenylindol (DAPI). (B) Stimulation of MDMs with IL6 and staining of STAT3 as in panel A. The figure shows representative images. The experiments were performed with three different donors, which all showed a > 90% inhibition of the nuclear translocation of STAT1 and STAT3, respectively. Scale bars indicate 50 µm.

Article Snippet: The following antibodies were used: p‐p38 (T180/Y182; #4511; Cell Signaling, Frankfurt, Germany); p38 (#9228; Cell Signaling), p‐STAT1 (T701; #612132; BD Bioscience, Franklin Lakes, NJ, USA); Stat1 (9172, Cell Signaling); p‐STAT3 (Y705; #9145; Cell Signaling); STAT3 (#9139; Cell Signaling); p‐JAK1 (T1034/1035; #66245; Cell Signaling); JAK1 (50996; Cell Signaling); p‐JAK2 (Y1007/1008; #8082S; Cell Signaling); JAK2 (#3230; Cell Signaling); Flotillin‐1 (#74566; Santa Cruz Technologies, Dallas, TX, USA); CD71 (#65882; Santa Cruz); IκB‐α (#371; Santa Cruz); IκBβ (#8635; Cell Signaling); β‐actin (#A5441; Sigma); Phospho‐SMAD2 (Ser465/467, #3108S; Cell Signaling); SMAD2 (#sc‐393312; Santa Cruz); GAPDH (#G9545; Sigma), α‐rabbit IgG HRP‐linked AB (#27; Cell Signaling) and α‐mouse IgG HRP‐linked AB (#32; Cell Signaling).

Techniques: Inhibition, Translocation Assay, Derivative Assay, Solvent, Immunofluorescence, Staining

Journal: Molecular Oncology

Article Title: Arachidonic acid, a clinically adverse mediator in the ovarian cancer microenvironment, impairs JAK‐STAT signaling in macrophages by perturbing lipid raft structures

doi: 10.1002/1878-0261.13221

Figure Lengend Snippet: Inhibition of STAT phosphorylation by arachidonic acid (AA) is independent of p38 MAPK. (A) Monocyte‐derived macrophages (MDMs) were stimulated with IFNγ after preincubation with solvent, AA, the p38 inhibitors SB203580 or BIRB796, or combinations of these (details as in Fig. ). Cell extracts were analyzed for changes in STAT1 (Y701) and p38 (T180/Y182) phosphorylation. The panel shows a representative immunoblot and a quantification for n = 7 different donors (represented by different symbols). (B) MDMs were stimulated with IL‐6 after preincubation as in panel A ( n = 6). Cell extracts were analyzed for changes in STAT3 (Y705) and p38 (T180/Y182) phosphorylation. Statistical significance was analyzed by paired t test (** P < 0.01; *** P < 0.001; **** P < 0.0001). Horizontal lines indicate the median.

Article Snippet: The following antibodies were used: p‐p38 (T180/Y182; #4511; Cell Signaling, Frankfurt, Germany); p38 (#9228; Cell Signaling), p‐STAT1 (T701; #612132; BD Bioscience, Franklin Lakes, NJ, USA); Stat1 (9172, Cell Signaling); p‐STAT3 (Y705; #9145; Cell Signaling); STAT3 (#9139; Cell Signaling); p‐JAK1 (T1034/1035; #66245; Cell Signaling); JAK1 (50996; Cell Signaling); p‐JAK2 (Y1007/1008; #8082S; Cell Signaling); JAK2 (#3230; Cell Signaling); Flotillin‐1 (#74566; Santa Cruz Technologies, Dallas, TX, USA); CD71 (#65882; Santa Cruz); IκB‐α (#371; Santa Cruz); IκBβ (#8635; Cell Signaling); β‐actin (#A5441; Sigma); Phospho‐SMAD2 (Ser465/467, #3108S; Cell Signaling); SMAD2 (#sc‐393312; Santa Cruz); GAPDH (#G9545; Sigma), α‐rabbit IgG HRP‐linked AB (#27; Cell Signaling) and α‐mouse IgG HRP‐linked AB (#32; Cell Signaling).

Techniques: Inhibition, Derivative Assay, Solvent, Western Blot

Journal: Molecular Oncology

Article Title: Arachidonic acid, a clinically adverse mediator in the ovarian cancer microenvironment, impairs JAK‐STAT signaling in macrophages by perturbing lipid raft structures

doi: 10.1002/1878-0261.13221

Figure Lengend Snippet: Inhibition of lipopolysaccharide (LPS)‐induced STAT1 signaling in monocyte‐derived macrophages (MDMs) by arachidonic acid (AA). (A) Inhibition of LPS‐induced phosphorylation of STAT1 (Y701) by AA or 5,8,11,14‐eicosatetraynoic acid (ETYA). MDMs were pretreated with 50 µ m of AA or ETYA for 30 min prior to stimulation with 100 ng·mL −1 LPS for 60 min. A representative immunoblot and quantification of six replicates are displayed. (B) RT‐qPCR analysis showing inhibition of CXCL10 by AA and verification of CXCL10 as a STAT1 target gene ( n = 6 donor; represented by different symbols). MDMs were pretreated with 50 µ m AA or the 0.5 µ m of the STAT1 inhibitor Ruxolitinib for 30 min prior to stimulation with 100 ng·mL −1 LPS for 3 h. Statistical significance was analyzed by paired t test (**** P < 0.0001). Horizontal lines indicate the median.

Article Snippet: The following antibodies were used: p‐p38 (T180/Y182; #4511; Cell Signaling, Frankfurt, Germany); p38 (#9228; Cell Signaling), p‐STAT1 (T701; #612132; BD Bioscience, Franklin Lakes, NJ, USA); Stat1 (9172, Cell Signaling); p‐STAT3 (Y705; #9145; Cell Signaling); STAT3 (#9139; Cell Signaling); p‐JAK1 (T1034/1035; #66245; Cell Signaling); JAK1 (50996; Cell Signaling); p‐JAK2 (Y1007/1008; #8082S; Cell Signaling); JAK2 (#3230; Cell Signaling); Flotillin‐1 (#74566; Santa Cruz Technologies, Dallas, TX, USA); CD71 (#65882; Santa Cruz); IκB‐α (#371; Santa Cruz); IκBβ (#8635; Cell Signaling); β‐actin (#A5441; Sigma); Phospho‐SMAD2 (Ser465/467, #3108S; Cell Signaling); SMAD2 (#sc‐393312; Santa Cruz); GAPDH (#G9545; Sigma), α‐rabbit IgG HRP‐linked AB (#27; Cell Signaling) and α‐mouse IgG HRP‐linked AB (#32; Cell Signaling).

Techniques: Inhibition, Derivative Assay, Western Blot, Quantitative RT-PCR

Journal: Molecular Oncology

Article Title: Arachidonic acid, a clinically adverse mediator in the ovarian cancer microenvironment, impairs JAK‐STAT signaling in macrophages by perturbing lipid raft structures

doi: 10.1002/1878-0261.13221

Figure Lengend Snippet: Impact of arachidonic acid (AA) incorporated into phospholipids versus free AA on lipid rafts. (A–D) Analysis of the effect of Triacsin C, an inhibitor of long fatty acyl CoA synthetase, on the AA‐mediated inhibition of STAT1 phosphorylation induced by INFβ (panel A, B) or IFNγ (panel C, D) in monocyte‐derived macrophages (MDMs). Experimental details were as in Fig. . Quantifications are shown for of n = 5 independent experiments (five different donors; represented by different symbols) in panel B and n = 4 donors in panel D. (E) Mass‐spectrometry‐based analysis of concentrations of free AA in n = 5 independent preparations of lipid rafts from MDMs treated with solvent or 50 µ m AA for 1 h. Statistical significance was analyzed by paired t test (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant). Horizontal lines indicate the median.

Article Snippet: The following antibodies were used: p‐p38 (T180/Y182; #4511; Cell Signaling, Frankfurt, Germany); p38 (#9228; Cell Signaling), p‐STAT1 (T701; #612132; BD Bioscience, Franklin Lakes, NJ, USA); Stat1 (9172, Cell Signaling); p‐STAT3 (Y705; #9145; Cell Signaling); STAT3 (#9139; Cell Signaling); p‐JAK1 (T1034/1035; #66245; Cell Signaling); JAK1 (50996; Cell Signaling); p‐JAK2 (Y1007/1008; #8082S; Cell Signaling); JAK2 (#3230; Cell Signaling); Flotillin‐1 (#74566; Santa Cruz Technologies, Dallas, TX, USA); CD71 (#65882; Santa Cruz); IκB‐α (#371; Santa Cruz); IκBβ (#8635; Cell Signaling); β‐actin (#A5441; Sigma); Phospho‐SMAD2 (Ser465/467, #3108S; Cell Signaling); SMAD2 (#sc‐393312; Santa Cruz); GAPDH (#G9545; Sigma), α‐rabbit IgG HRP‐linked AB (#27; Cell Signaling) and α‐mouse IgG HRP‐linked AB (#32; Cell Signaling).

Techniques: Inhibition, Derivative Assay, Mass Spectrometry, Solvent

Journal: Molecular Oncology

Article Title: Arachidonic acid, a clinically adverse mediator in the ovarian cancer microenvironment, impairs JAK‐STAT signaling in macrophages by perturbing lipid raft structures

doi: 10.1002/1878-0261.13221

Figure Lengend Snippet: Abrogation of the inhibitory effect of arachidonic acid (AA) on STAT1 phosphorylation by water‐soluble cholesterol/methyl‐β‐cyclodextrin (Ch/MCD). Monocyte‐derived macrophages (MDMs) were pretreated with 50 µ m AA for 30 min and 50 µ m Ch/MCD prior to stimulation with IFNβ (A) or IFNγ (B) for 30 min. Representative immunoblots and quantification for MDMs from n = 7 different donors (represented by different symbols) are shown. Statistical significance was analyzed by paired t test (*** P < 0.001; **** P < 0.0001). Horizontal lines indicate the median.

Article Snippet: The following antibodies were used: p‐p38 (T180/Y182; #4511; Cell Signaling, Frankfurt, Germany); p38 (#9228; Cell Signaling), p‐STAT1 (T701; #612132; BD Bioscience, Franklin Lakes, NJ, USA); Stat1 (9172, Cell Signaling); p‐STAT3 (Y705; #9145; Cell Signaling); STAT3 (#9139; Cell Signaling); p‐JAK1 (T1034/1035; #66245; Cell Signaling); JAK1 (50996; Cell Signaling); p‐JAK2 (Y1007/1008; #8082S; Cell Signaling); JAK2 (#3230; Cell Signaling); Flotillin‐1 (#74566; Santa Cruz Technologies, Dallas, TX, USA); CD71 (#65882; Santa Cruz); IκB‐α (#371; Santa Cruz); IκBβ (#8635; Cell Signaling); β‐actin (#A5441; Sigma); Phospho‐SMAD2 (Ser465/467, #3108S; Cell Signaling); SMAD2 (#sc‐393312; Santa Cruz); GAPDH (#G9545; Sigma), α‐rabbit IgG HRP‐linked AB (#27; Cell Signaling) and α‐mouse IgG HRP‐linked AB (#32; Cell Signaling).

Techniques: Derivative Assay, Western Blot

Journal: Molecular Oncology

Article Title: Arachidonic acid, a clinically adverse mediator in the ovarian cancer microenvironment, impairs JAK‐STAT signaling in macrophages by perturbing lipid raft structures

doi: 10.1002/1878-0261.13221

Figure Lengend Snippet: Model of arachidonic acid (AA) regulated signal transduction pathways triggered by pro‐inflammatory mediators. AA interferes with the lipid‐raft association of pro‐inflammatory cytokine receptors, receptor‐associated JAK protein kinases and STAT proteins. This mislocalization impairs the cytokine‐triggered phosphorylation and activation of JAK1/2 and STAT1/3, and thereby induction of their target genes. EX, extracellular space; PM, plasma membrane; CYT, cytosol; NUC, nucleus.

Article Snippet: The following antibodies were used: p‐p38 (T180/Y182; #4511; Cell Signaling, Frankfurt, Germany); p38 (#9228; Cell Signaling), p‐STAT1 (T701; #612132; BD Bioscience, Franklin Lakes, NJ, USA); Stat1 (9172, Cell Signaling); p‐STAT3 (Y705; #9145; Cell Signaling); STAT3 (#9139; Cell Signaling); p‐JAK1 (T1034/1035; #66245; Cell Signaling); JAK1 (50996; Cell Signaling); p‐JAK2 (Y1007/1008; #8082S; Cell Signaling); JAK2 (#3230; Cell Signaling); Flotillin‐1 (#74566; Santa Cruz Technologies, Dallas, TX, USA); CD71 (#65882; Santa Cruz); IκB‐α (#371; Santa Cruz); IκBβ (#8635; Cell Signaling); β‐actin (#A5441; Sigma); Phospho‐SMAD2 (Ser465/467, #3108S; Cell Signaling); SMAD2 (#sc‐393312; Santa Cruz); GAPDH (#G9545; Sigma), α‐rabbit IgG HRP‐linked AB (#27; Cell Signaling) and α‐mouse IgG HRP‐linked AB (#32; Cell Signaling).

Techniques: Transduction, Activation Assay, Membrane

Journal: bioRxiv

Article Title: In vivo CRISPR screen identifies KRAS-induced COX-2 as a driver of immune evasion and immunotherapy resistance in lung cancer

doi: 10.1101/2023.04.13.536740

Figure Lengend Snippet: (A) Flow cytometry analysis showing surface expression of IFN-β receptor subunit β on KPAR cells and Ifngr2 -/- cells. (B) Immunoblot for pSTAT1 and STAT1 in KPAR cells and Ifngr2 -/- cells treated for 24h with 100ng/ml IFNγ. (C) Incucyte analysis showing growth rate of KPAR cells and Ifngr2 -/- cells in vitro . (D) Kaplan-Meier survival of immune-competent or Rag2 -/- ; Il2rg -/- mice following orthotopic transplantation with KPAR cells or Ifngr2 -/- (clone 2), n=5-10 per group. Analysis of survival curves was carried out using log-rank (Mantel-Cox) test; * P<0.05. (E) Incucyte analysis showing growth of KPAR cells in the presence of 200ng/ml IFNα, 200ng/ml IFN′3 or 100ng/ml IFNγ. (F) Heatmap showing mRNA expression of IFN-response genes in KPAR cells and Ifngr2 -/- cells treated with 100ng/ml IFNγ. (G) Flow cytometry analysis showing surface expression of H2-Db (left) and PD-L1 (right) on KPAR cells and Ifngr2 -/- cells treated for 24h with either 200ng/ml IFNα, 200ng/ml IFN′3 or 100ng/ml IFNγ. (H) Surface expression of the IFNγ-receptor ′3 chain on KPAR cells treated with 10nM trametinib, 1µM linsitinib and 40nM everolimus for 24h.

Article Snippet: Proteins were detected by Western blotting using the following primary antibodies against: Flag (M2, Sigma), ERK1/2 (3A7, Cell Signalling), p-ERK1/2 (Thr202/Tyr204) (9101, Cell Signalling), Myc (Y69, Abcam), STAT1 (#9172, Cell Signaling), p-STAT1 (T701) (58D6, Cell Signaling) STAT2 (D9J7L, Cell Signaling) COX-2 (D5H5, Cell Signalling), Vinculin (VIN-11-5, Sigma) and β-Actin (8H10D10, Cell Signaling).

Techniques: Flow Cytometry, Expressing, Western Blot, In Vitro, Transplantation Assay

Journal: bioRxiv

Article Title: In vivo CRISPR screen identifies KRAS-induced COX-2 as a driver of immune evasion and immunotherapy resistance in lung cancer

doi: 10.1101/2023.04.13.536740

Figure Lengend Snippet: (A) Kaplan-Meier survival of immune-competent or Rag2 -/- ; Il2rg -/- mice following orthotopic transplantation with KPAR cells or Ptgs2 -/- cells (n=5-10 per group). Analysis of survival curves was carried out using log-rank (Mantel-Cox) test; **** P<0.0001. (B) Kaplan-Meier survival of mice treated with 200µg anti-NK1.1 and/or 200µg anti-CD8 or corresponding isotype control (n=5-7 per group) after orthotopic transplantation of Ptgs2 -/- cells. Treatment was initiated 1 day before transplantation and was administered once weekly until endpoint. Analysis of survival curves was carried out using log-rank (Mantel-Cox) test; * P<0.05, ** P<0.01. (C) Frequency of tumour-infiltrating T cell populations and NK cells in KPAR and Ptgs2 -/- orthotopic tumours. (D) Quantification and representative immunohistochemistry staining for NKp46 + NK cells. Scale bar represents 100µm. (E) Stacked bar plots showing frequency of central memory (CD62L + CD44 + ), effector memory (CD44 + CD62L - ) and naïve (CD62L - CD44 - ) CD8 + (left) and CD4 + (right) T cells. (F) Surface expression of CD44 on CD8 + (left) and CD4 + (right) T cells. (G) Surface expression of CD86 (left) and MHC-II (right) on CD11b + macrophages and CD11c + macrophages. (H) Percentage of Arg1 + CD11b + macrophages. (I) Quantification and representative immunohistochemistry staining for the immunosuppressive macrophage marker Arg1. Scale bar represents 100µm. (J) Representative flow cytometry plots of CD206 and MHC-II surface expression on CD11b + macrophages (left) and quantification of M1/M2 ratio based on the gated populations (right). (K) Heatmap showing hierarchical clustering of KPAR and Ptgs2 -/- tumours based on mRNA expression of anti-tumour immunity genes assessed by qPCR. Data are mean ± SEM for (C-J), n=6-9 per group. Symbols represent pooled tumours from individual mice. Statistics were calculated by paired, two-tailed Student’s t-test (C-D and F-J) or two-way ANOVA, FDR 0.05 (E); * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

Article Snippet: Proteins were detected by Western blotting using the following primary antibodies against: Flag (M2, Sigma), ERK1/2 (3A7, Cell Signalling), p-ERK1/2 (Thr202/Tyr204) (9101, Cell Signalling), Myc (Y69, Abcam), STAT1 (#9172, Cell Signaling), p-STAT1 (T701) (58D6, Cell Signaling) STAT2 (D9J7L, Cell Signaling) COX-2 (D5H5, Cell Signalling), Vinculin (VIN-11-5, Sigma) and β-Actin (8H10D10, Cell Signaling).

Techniques: Transplantation Assay, Control, Immunohistochemistry, Staining, Expressing, Marker, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: In vivo CRISPR screen identifies KRAS-induced COX-2 as a driver of immune evasion and immunotherapy resistance in lung cancer

doi: 10.1101/2023.04.13.536740

Figure Lengend Snippet: (A) Surface expression of CD86 (left) and MHC-II (right) on CD11b + macrophages and CD11c + macrophages in KPAR tumours treated for 7d with 30mg/kg celecoxib. (B-D) Percentage of Arg1 + CD11b + macrophages (B), quantification of M1/M2 macrophages (C), and frequency of CD69 + CD8 + T cells (D) in KPAR tumours treated as in (A). (E) Kaplan-Meier survival of mice treated intraperitoneally with 200µg anti-PD-1 and/or daily oral gavage of 30mg/kg celecoxib after orthotopic transplantation of KPAR cells. Daily celecoxib treatment was initiated on day 7 and anti-PD-1 began on day 10 and was administered twice weekly for a maximum of 3 weeks. Data from two independent experiments, n=15-16 per group. Analysis of survival curves was carried out using log-rank (Mantel-Cox) test; * P<0.05, *** P<0.001, **** P<0.0001. (F) Quantification of CD8 + T cells by immunohistochemistry in KPAR tumours treated for 7d with celecoxib and/or anti-PD-1. (G) CD8 + T cell phenotypes in KPAR tumours treated as in (F). (H) mRNA expression by qPCR of anti-tumour immunity genes in KPAR tumours treated as in (F). Data are mean ± SEM for (A-D and F-H), n=5-10 per group. Samples were analysed using unpaired, two-tailed Student’s t-test (A-D), one-way ANOVA, FDR 0.05 (F and H) or two-way ANOVA, FDR 0.05 (G); ns, not significant, * P<0.05, ** P<0.01, *** P<0.001.

Article Snippet: Proteins were detected by Western blotting using the following primary antibodies against: Flag (M2, Sigma), ERK1/2 (3A7, Cell Signalling), p-ERK1/2 (Thr202/Tyr204) (9101, Cell Signalling), Myc (Y69, Abcam), STAT1 (#9172, Cell Signaling), p-STAT1 (T701) (58D6, Cell Signaling) STAT2 (D9J7L, Cell Signaling) COX-2 (D5H5, Cell Signalling), Vinculin (VIN-11-5, Sigma) and β-Actin (8H10D10, Cell Signaling).

Techniques: Expressing, Transplantation Assay, Immunohistochemistry, Two Tailed Test

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: The following antibodies were used: cGAS (ab224144, Abcam, Cambridge, UK), STING (#13647, Cell Signaling Tech., California, USA), GAPDH (Ab9485, Abcam), RIG1 (A0550, Abclonal, Massachusetts, USA), TLR3 (ab62566, Abcam), MDA5 (A2203, Abclonal), MAVS (A5764, Abclonal), beta-actin (IR2-7, iREAL Biotech.), p-IRF3 S388 (AP1333 and AP0263, Abclonal, IRF3 (#41075, Cell Signaling Tech.), p-STAT1 T701 (bs-1657R, Bioss antibodies and AP0054, Abclonal) and STAT1 (sc-464, Santa Cruz, California, USA).

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: 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: The following antibodies were used: cGAS (ab224144, Abcam, Cambridge, UK), STING (#13647, Cell Signaling Tech., California, USA), GAPDH (Ab9485, Abcam), RIG1 (A0550, Abclonal, Massachusetts, USA), TLR3 (ab62566, Abcam), MDA5 (A2203, Abclonal), MAVS (A5764, Abclonal), beta-actin (IR2-7, iREAL Biotech.), p-IRF3 S388 (AP1333 and AP0263, Abclonal, IRF3 (#41075, Cell Signaling Tech.), p-STAT1 T701 (bs-1657R, Bioss antibodies and AP0054, Abclonal) and STAT1 (sc-464, Santa Cruz, California, USA).

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