Journal: Molecular Therapy

Article Title: Modulating the Tumor Microenvironment via Oncolytic Viruses and CSF-1R Inhibition Synergistically Enhances Anti-PD-1 Immunotherapy

doi: 10.1016/j.ymthe.2018.11.010

Figure Lengend Snippet: Triple Treatment Is Also Effective in Mouse MC38 Colon Cancer

Article Snippet: Mouse MC38 colon cancer cell line was provided by Innovent Biologics (Suzhou, Jiangsu, China).

Techniques:

Journal: Nature Communications

Article Title: Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

doi: 10.1038/s41467-021-27331-3

Figure Lengend Snippet: a Schematic overview of clonal mutation profiling of MC38 cell line-derived tumors grown in host mice harboring different levels of T cell immunity. b Heatmap showing allele frequency (AF) of hotspot mutations enriched (AF > 0.1) in at least two different tumors from the immunocompetent group ( n = 10 mice for immunodeficient, n = 13 for immunocompetent, and n = 8 for immunotherapy group). A total of 59 mutations in 53 genes were categorized to PD-1–dependent and –independent groups as indicated. Tumor-infiltrating lymphocytes were calculated by mMCP counter. See also Supplementary Fig. . Source data are provided as a source data file.

Article Snippet: MC38 cell line was provided by WuXi AppTec and cultured using DMEM (Gibco) with 10% fetal bovine serum (FBS), respectively.

Techniques: Mutagenesis, Derivative Assay

Journal: Nature Communications

Article Title: Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

doi: 10.1038/s41467-021-27331-3

Figure Lengend Snippet: a Schematic overview of in vivo CRISPR screen to validate candidates from immune-selected mutations. b Tumor growth curves of MC38 tumors in nude mice ( n = 8), and WT mice treated with rat IgG2a and IgG2b isotype ( n = 8), PD-1 antibody ( n = 10), PD-L1 antibody ( n = 10), or CD4 and CD8 antibodies ( n = 7). Data are represented as mean ± s.e.m., **** P < 0.0001, significance was determined using two-way analysis of variance (ANOVA). c – e Distribution histograms of log Fold-change (FC) for all 10 sgRNAs targeting Cd274 ( c ), Pdcd1 ( d ) or Ankrd52 ( e ) as indicated in red lines, overlaid on gray gradient depicting the overall distribution (Cut-off: |FC | > 1.5, P < 0.05 for enrichment or depletion, analyzed by edgeR). f , Volcano plot for selected top guides for Cd274 and Ankrd52 (Cut-off: |FC | > 1.5, P < 0.05, analyzed by MAGeCK). g In vivo competition assay with equal number mixture of MC38 cells infected with sgRNA for non-targeting control (NT) or Ankrd52 ( An ) in WT mice ( n = 5) and nude mice ( n = 5). Data are represented as mean ± s.e.m., *** P = 0.0004, **** P < 0.0001, significance was determined using two-tailed unpaired Student’s t -test to compare sg Ankrd52 vs sgNT #2. h , i , Flow cytometry analysis of CD4 + ( h ) and CD8 + ( i ) T cell populations from NT and Ankrd52 knockout ( An KO) tumors ( n = 5 per group). Data are representative of two independent experiments and represented as mean ± s.e.m., significance was determined using two-tailed unpaired Student’s t -test. See also Supplementary Fig. – . Source data are provided as a source data file.

Article Snippet: MC38 cell line was provided by WuXi AppTec and cultured using DMEM (Gibco) with 10% fetal bovine serum (FBS), respectively.

Techniques: In Vivo, CRISPR, Competitive Binding Assay, Infection, Control, Two Tailed Test, Flow Cytometry, Knock-Out

Journal: Nature Communications

Article Title: Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

doi: 10.1038/s41467-021-27331-3

Figure Lengend Snippet: a Schematic overview of CRISPR screen using co-culture of OT-I T cell with MC38-OVA cells. b Cell viability of MC38-OVA cells co-cultured with OT-I T cells during 2.5 days ( n = 3 per group per timepoint). Data are represented as mean ± s.d., **** P < 0.0001, significance is determined using multiple two-tailed Student’s t -test. c – e Distribution histograms of log 2 FC values for all ten sgRNAs targeting Jak1 ( c ), B2m ( d ), or Ankrd52 ( e ) (Cut-off: FC > 1.4, P < 0.05 for enrichment, analyzed by edgeR). f Killing of MC38-OVA cells with indicated sgRNAs by OT-I T cells at indicated ratio in co-culture. Data are representative of three independent experiments and represented as mean ± s.e.m., ** P < 0.01, *** P < 0.001, significance was determined using multiple two-tailed Student’s t -test (sgNT #1 vs sg Ankrd52 #1, P = 3.72×10 −3 for 1:3, P = 1.74×10 −3 for 1:5; sgNT #2 vs sg Ankrd52 #1, P = 3.64×10 −3 for 1:3, P = 1.67×10 −3 for 1:5; sgNT #1 vs sg Ankrd52 #2, P = 2.28 × 10 −4 for 1:3, P = 4.43 × 10 −3 for 1:5; sgNT #2 vs sg Ankrd52 #2, P = 2.7 × 10 −4 for 1:3, P = 4.57 × 10 −3 for 1:5). See also Supplementary Figs. and . Source data are provided as a source data file.

Article Snippet: MC38 cell line was provided by WuXi AppTec and cultured using DMEM (Gibco) with 10% fetal bovine serum (FBS), respectively.

Techniques: CRISPR, Co-Culture Assay, Cell Culture, Two Tailed Test

Journal: Nature Communications

Article Title: Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

doi: 10.1038/s41467-021-27331-3

Figure Lengend Snippet: a Hallmark gene sets enriched for commonly down-regulated genes in Ankrd52 -null (both An KO1 and An KO2, 1.5-fold-change cut-off, P < 0.05, analyzed by edgeR) MC38 cells compared to control cells after IFNγ treatment. b , c Enrichment of genes associated with IFNγ response in Ankrd52 -null cells exposed to IFNγ. d Heatmap showing down-regulated IFNγ responsive gene expression in Ankrd52 -null cells by RNA-seq analysis ( n = 2 per group per condition). e , f Cxcl9 ( e ) and Cxcl10 ( f ) mRNA level in control and ANKRD52 -null MC38 cells treated with IFNγ ( n = 3 per group). Data are representative of two independent experiments and represented as mean ± s.e.m., significance was determined using two-tailed unpaired Student’s t -test. g Abundance of IFNγ signaling proteins in control and Ankrd52 -null MC38 cells treated with IFNγ. Data are representative of five independent experiments. h Abundance of membrane MHC-I expression in control and Ankrd52 -null MC38 cells after treatment with IFNγ. MFI (mean fluorescence intensity) of H2-K b was normalized by the responding group without IFNγ treatment ( n = 3 per group per condition). Data are representative of four independent experiments and represented as mean ± s.d., significance was determined using two-tailed unpaired Student’s t -test. i Presentation of OVA-derived peptide (SIINFEKL) in OVA-treated control and Ankrd52 -null MC38 cells. MFI of SIINFEKL-H2K b was normalized by the responding group without IFNγ treatment ( n = 3 per group per condition). Data are representative of three independent experiments and represented as mean ± s.d., significance was determined using two-tailed unpaired Student’s t -test. See also Supplementary Figs. and . Source data are provided as a source data file.

Article Snippet: MC38 cell line was provided by WuXi AppTec and cultured using DMEM (Gibco) with 10% fetal bovine serum (FBS), respectively.

Techniques: Control, Gene Expression, RNA Sequencing, Two Tailed Test, Membrane, Expressing, Fluorescence, Derivative Assay

Journal: Nature Communications

Article Title: Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

doi: 10.1038/s41467-021-27331-3

Figure Lengend Snippet: a Localization of clinical hotspot ANKRD52 mutations from combined COSMIC and OncoWuXi database. b Protein abundance of p-STAT1 and p-STAT3 in Ankrd52 -null MC38 cells expressing WT or indicated mutant ANKRD52 after IFNγ treatment. Data are representative of two independent experiments. c Membrane MHC-I level in Ankrd52 -null MC38 cells expressing WT or indicated mutant ANKRD52 after IFNγ treatment. MFI of H2-K b was normalized by the responding group without IFNγ treatment ( n = 5 per group per condition). Data are representative of two independent experiments and represented as mean ± s.d., significance was determined using two-tailed unpaired Student’s t -test. d Killing of OVA-treated Ankrd52 -null MC38 cells expressing WT or mutant ANKRD52 by OT-I T cells ( n = 3 per group per condition). Data are representative of two independent experiments and represented as mean ± s.d., significance was determined using two-tailed unpaired Student’s t -test. e Bar plot showing the percentage of patients with mutations in ANKRD52 across cancer patients receiving ICB therapies (anti-CTLA-4 or anti-PD-1) reported by Van Allen et al., Science 2015 and Riaz et al., Cell 2017. The non-response group is composed of SD and PD. f Bar plot showing the percentage of patients with mutations in ANKRD52 across multiple cancer types. Source data are provided as a source data file.

Article Snippet: MC38 cell line was provided by WuXi AppTec and cultured using DMEM (Gibco) with 10% fetal bovine serum (FBS), respectively.

Techniques: Quantitative Proteomics, Expressing, Mutagenesis, Membrane, Two Tailed Test

Journal: Nature Communications

Article Title: Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

doi: 10.1038/s41467-021-27331-3

Figure Lengend Snippet: a p-STAT1 and p-STAT3 abundance in MC38 cells treated with IFNγ and increasing Longdaysin (0, 50, 100 μM, a CK1α inhibitor). Data are representative of three independent experiments. b Heatmap showing commonly upregulated gene expression of IFNγ signaling by RNA-seq analysis of Ankrd52 -null MC38 cells ( n = 2 replicates per group). c SOCS1 mRNA level in control and ANKRD52 -null 293 T cells overexpressing miR-155 ( n = 3 per group per condition). Data are representative of two independent experiments and represented as mean ± s.e.m., significance was determined using two-tailed unpaired Student’s t -test. d Activity of WT and mutant SOCS1 3’UTR (Rluc/Fluc) in a dual-luciferase reporter in 293 T cells overexpressing miR-155 ( n = 3 per group per condition). Data are representative of three independent experiments and represented as mean ± s.e.m., significance was determined using two-tailed unpaired Student’s t -test. e Schematic overview of qPCR test targeting mutant region to check Socs1 knockout efficiency. f Socs1 mRNA level tested by qPCR targeting mutant region in Ankrd52 -null MC38 cells with inactivated SOCS1 ( n = 5 per group). Data are represented as mean ± s.e.m., significance was determined using two-tailed unpaired Student’s t -test. g p-STAT1 and p-STAT3 abundance in Ankrd52 -null MC38 cells with inactivated SOCS1 after IFNγ treatment. Data are representative of three independent experiments. h Membrane MHC-1 expression in Ankrd52 -null MC38 cells with inactivated SOCS1 after IFNγ treatment. MFI of H2-K b was normalized by the responding group without IFNγ treatment ( n = 3 per group per condition). Data are representative of two independent experiments and represented as mean ± s.d., significance was determined using two-tailed unpaired Student’s t -test. i Killing of OVA-treated Ankrd52 -null MC38 cells with inactivated SOCS1 by OT-I T cells ( n = 3 per group per condition). Data are representative of two independent experiments and represented as mean ± s.d., significance was determined using two-tailed unpaired Student’s t -test. j Volcano plot showing the Spearman’s correlation and estimated significance of ANKRD52 with SOCS1 mRNA levels across all TCGA cancer types. Each dot represents a cancer type, blue dots indicate significant negative correlations ( P < 0.05, TIMER). See also Supplementary Figs. and . Source data are provided as a source data file.

Article Snippet: MC38 cell line was provided by WuXi AppTec and cultured using DMEM (Gibco) with 10% fetal bovine serum (FBS), respectively.

Techniques: Gene Expression, RNA Sequencing, Control, Two Tailed Test, Activity Assay, Mutagenesis, Luciferase, Knock-Out, Membrane, Expressing

Journal: Nature Communications

Article Title: Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

doi: 10.1038/s41467-021-27331-3

Figure Lengend Snippet: a Diagram showing the Spearman’s correlation of top co-dependent proteins with ANKRD52 or PPP6C in CRISPR (Avana) Public 20Q3 database. Solid lines depict significant positive correlations (Correlation > 0.25, P < 0.001) and dashed lines depict weak correlation (Correlation > 0.1, P < 0.01). b , c Volcano plot showing the Spearman’s correlation and estimated significance of DICER1 ( b ) or XPO5 ( c ) with SOCS1 mRNA levels from RNA-seq data across all TCGA cancer types. Each dot represents a cancer type in TCGA; blue dots indicate significant negative correlations ( P < 0.05, TIMER). d SOCS1 mRNA level in MC38 cells with targeted sgRNAs ( n = 3 per group). Data are representative of three independent experiments and represented as mean ± s.e.m., significance was determined using two-tailed unpaired Student’s t -test. e Killing of OVA-treated MC38 cells with targeted sgRNAs by OT-I T cells ( n = 3 per group per condition). Data are representative of two independent experiments and represented as mean ± s.d., significance was determined using two-tailed unpaired Student’s t -test. f Tumor growth curves of Ago2 -null or control MC38 tumors in WT mice treated with PD-1 antibody or not ( n = 5 for NT tumor, n = 6 for NT with anti-PD-1 and n = 7 for Ago2 -null with or without anti-PD-1). Data are represented as mean ± s.e.m., significance was determined using two-tailed unpaired Student’s t -test. g Heatmap showing the Spearman’s correlation of ANKRD52 , AGO2 , DICER1 , XPO5 , DROSHA , PPP6C , or SOCS1 mRNA levels with CD4 + and CD8 + T cell abundance in tumors across all TCGA cancer types. h Model of miRNA machinery in regulation of cancer-intrinsic evasion from T cell attack. See also Supplementary Figs. – . Source data are provided as a source data file.

Article Snippet: MC38 cell line was provided by WuXi AppTec and cultured using DMEM (Gibco) with 10% fetal bovine serum (FBS), respectively.

Techniques: CRISPR, RNA Sequencing, Two Tailed Test, Control