Journal: Nature Communications

Article Title: Reactivating exhausted tumor-infiltrating T cells by a bispecific DC-T cell engager in mice

doi: 10.1038/s41467-026-70876-4

Figure Lengend Snippet: A – C Female C57BL/6 mice ( n = 5) were inoculated with 5 × 10 5 MC38 cells. CD8 + T cells were sorted for single-cell RNA sequencing 19 days after tumor inoculation. A UMAP plot of 2276 CD8 + T cells exhibiting four cell subsets. B Expression of selected markers among CD8 + T cells by 2D UMAP visualization. C Heatmap plot showing average relative expression of selected markers in four CD8 + T cell subsets. D Female C57BL/6 mice ( n = 6) were inoculated with 1 × 10 6 MC38-OVA cells. The tumor tissues were collected 19 days after tumor inoculation. TIM3 expressions on Tetramer + CD8 + T cells in tumors from MC38-OVA tumor-bearing mice. E Female C57BL/6 mice ( n = 6) were inoculated with 5 × 10 5 MC38 cells. The tumor tissues were collected 19 days after tumor inoculation. Bcl-2 expressions on TIM3 - CD8 + T cells or TIM3 + CD8 + T cells in tumors from MC38 tumor-bearing mice. The proliferation and function of OT-I CD8 + T cells primed by spleen DC (sDC) or TIDC ( n = 3/group) were assessed using CFSE dilution ( F ) and IFN-γ production ( G ). H , I MC38-bearing Female C57BL/6 mice ( n = 7/group) were i.p. treated with 200 μg anti-TIM3 and/or 25 μg Fc-IFNα on day 9, 12 and 15 after tumor inoculation. Tumor volume ( H ) and mice weight ( I ) were measured as indicated. Data are shown as mean ± SEM and are representative of at least two independent experiments. P value was determined by paired two-tailed t tests ( D , E ), unpaired two-tailed t tests ( F , G ) or One-way ANOVA with Tukey’s test ( H , I ). Source data are provided as a Source Data file.

Article Snippet: MC38 cell line was purchased from Cytion (#305223).

Techniques: Single Cell, RNA Sequencing, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Reactivating exhausted tumor-infiltrating T cells by a bispecific DC-T cell engager in mice

doi: 10.1038/s41467-026-70876-4

Figure Lengend Snippet: A Schematic diagram of TIM3-IFNα fusion protein in homodimer or heterodimer format. B – D MC38 tumor-bearing female C57BL/6 mice ( n = 11/group) were i.p. treated with BiDT (25 μg), or a mixture of Fc-IFNα and anti-TIM3 (12.5 μg + 12.5 μg), on day 13, 16 and 19 post inoculation. Mice weight ( B ) and tumor volume ( C ) were measured as indicated and the corresponding mouse survival curve was shown in ( D ). E MC38-OVA tumor-bearing female C57BL/6 mice ( n = 6/group) were i.p. treated with 25 μg BiDT on day 13, 16 and 19 post inoculation. At day15 after treatment, splenocytes from different treatment groups were isolated and stimulated with either OT-I peptide or freeze-thawing MC38-OVA tumor cells. Antigen-specific T cells were detected by ELISpot assay. The splenocyte quantification data are shown in ( E ). F , G MC38-bearing female C57BL/6 mice were i.p. treated with 25 μg BiDT on day 13, 16 and 19 post inoculation. At 15 day after treatment, cured mice ( n = 5/group) were re-challenged with 2 × 10 6 MC38 tumor cells ( F ), 2 × 10 7 splenocytes from cured mice were i.v. transferred to MC38-bearing Rag1 −/− mice ( n = 5/group) 10 days after inoculation ( G ). Tumor volume was measured as indicated. H – J Female C57BL/6 mice ( n = 10/group) were subcutaneously inoculated with 3 × 10 5 B16F10 tumor cells and injected with 2 × 10 6 B16F10 tumor cells through the tail vein on day 10. The subcutaneous tumor was intratumorally treated with 5 μg BiDT on days 11, 14, and 17. The mice were euthanized on day 21 post inoculation. The lungs were collected ( H ). The subcutaneous B16F10 tumor growth curve ( I ). The number of B16F10 tumor metastatic nodes in the lung ( J ). Data are shown as mean ± SEM and are representative of at least two independent experiments. P value was determined by One-way ANOVA with Tukey’s test ( B , C ) or unpaired two-tailed t tests ( E – G , I , J ). Survival curve was compared using the log-rank test ( D ). Source data are provided as a Source Data file.

Article Snippet: MC38 cell line was purchased from Cytion (#305223).

Techniques: Isolation, Enzyme-linked Immunospot, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Reactivating exhausted tumor-infiltrating T cells by a bispecific DC-T cell engager in mice

doi: 10.1038/s41467-026-70876-4

Figure Lengend Snippet: A MC38-bearing female Rag1 −/− mice ( n = 6/group) were i.p. treated with 25 μg BiDT on day 13, 16 and 19 after tumor inoculation. Tumor volume was measured as indicated. B MC38-bearing female C57BL/6 mice ( n = 7/group) were i.p. treated with 25 μg BiDT on day 13, 16 and 19 after tumor inoculation. Anti-CD4 or anti-CD8 was administrated twice a week for 2 weeks. Tumor volume was measured as indicated. C MC38-bearing female C57BL/6 mice ( n = 7/group) were i.p. treated with 25 μg BiDT on day 13, 16 and 19 after tumor inoculation. Anti-CSF1R or anti-NK1.1 was administrated twice a week for 2 weeks. D , E Female C57BL/6 mice were lethally irradiated (10 Gy). Bone marrow was isolated from CD11c-DTR donor mice. Irradiated C57BL/6 mice as recipient were i.v. transferred with 2 × 10 6 CD11c-DTR bone marrow cells. CD11c-DTR chimeric mice were rested for 7 weeks and then were inoculated with MC38 cells. MC38-bearing CD11c-DTR chimeric mice ( n = 5/group) were i.p. treated with 25 μg BiDT on day 18, 21 and 24 after tumor inoculation. DT was administrated at 200 ng two days before treatment initiation and then every other day for 2 weeks ( D ). Tumor volume was measured as indicated ( E ). F MC38-bearing female C57BL/6 mice ( n = 7/group) were i.p. treated with 25 μg BiDT on day 13, 16 and 19 after tumor inoculation. FTY720 was administrated at 20 μg every other day for 2 weeks. G Female C57BL/6 mice were inoculated with 5 × 10 5 MC38 cells. The mice were euthanized on day 16 after tumor inoculation. 120 mm 3 MC38 tumor tissues were collected and s.c. transferred to Rag1 −/− mice ( n = 6/group). The subcutaneous tumor was intratumorally treated with 25 μg BiDT on days 0, 3, and 6 after tumor transplanting. Data are shown as mean ± SEM and are representative of at least two independent experiments. P value was determined by One-way ANOVA with Tukey’s test ( B , C , E , F ) or unpaired two-tailed t tests ( A , G ). Source data are provided as a Source Data file.

Article Snippet: MC38 cell line was purchased from Cytion (#305223).

Techniques: Irradiation, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: Reactivating exhausted tumor-infiltrating T cells by a bispecific DC-T cell engager in mice

doi: 10.1038/s41467-026-70876-4

Figure Lengend Snippet: A Female C57BL/6 mice ( n = 6/group) were inoculated with 1 × 10 6 Ifnar1 −/− MC38 cells and treated with 25 µg BIDT on day 13, 16 and 19 after tumor inoculation. Tumor volume was measured as indicated. B MC38-bearing female Ifnar1 −/− mice ( n = 6/group) were i.p. treated with 25 µg BiDT on day 10, 13 and 16 after tumor inoculation. C , D 3 × 10 6 CD3 + T cells from spleen of female C57BL/6 mice or Ifnar1 −/− mice were adoptively transferred to MC38-bearing Rag1 −/− mice ( n = 6/group) 2 days after tumor inoculation. 25 µg BiDT was i.p. administrated on day10, 13 and 16 after tumor inoculation ( C ). Tumor volume was measured as indicated ( D ). E Female Ifnar1 fl/fl mice and Zbtb46 - Cre Ifnar1 fl/fl mice ( n = 8/group) were inoculated with 5 × 10 5 MC38 tumor cells and i.p. treated with 25 µg BiDT on day 13, 16 and 19 after tumor inoculation. F Female Havcr2 fl/fl mice, Zbtb46 - Cre Havcr2 fl/fl mice or Lyz2 - Cre Havcr2 fl/fl mice ( n = 6/group) were inoculated with 5 × 10 5 MC38 tumor cells and i.p. treated with 25 µg BiDT on day 13, 16 and 19 after tumor inoculation. G Female Havcr2 fl/fl mice or Cd4-Cre Havcr2 fl/fl mice ( n = 6/group) were inoculated with 5 × 10 5 MC38 tumor cells and i.p. treated with 25 µg BiDT on day 13, 16 and 19 after tumor inoculation. H MC38-bearing female C57BL/6 mice ( n = 6/group) were treated with 25 µg BiDT on day 16. Tumor tissues were collected 72 h after injection. The number of intratumoral total CD8 + T cells/TIM3 + CD8 + T cells and TIM3 - CD8 + T cells ( H ) were measured by FACS. I MC38-OVA tumor-bearing female C57BL/6 mice ( n = 6/group) were treated with 25 µg BiDT on day 16. Tumor tissues were collected 72 h after injection. The number of total Tetramer + , TIM3 + Tetramer + CD8 + T cells and TIM3 - Tetramer + CD8 + T cells ( I ) were measured by FACS. Data are shown as mean ± SEM and are representative of at least two independent experiments. P value was determined by unpaired two-tailed t tests ( A , B , H , I ) or One-way ANOVA with Tukey’s test ( D – G ). Source data are provided as a Source Data file.

Article Snippet: MC38 cell line was purchased from Cytion (#305223).

Techniques: Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Reactivating exhausted tumor-infiltrating T cells by a bispecific DC-T cell engager in mice

doi: 10.1038/s41467-026-70876-4

Figure Lengend Snippet: A MC38 tumor-bearing female C57BL/6 mice ( n = 6/group) were treated with 25 µg BiDT on day 16. Tumor tissues were collected 72 h after injection. Expression of CD80 in tumor-infiltrating DCs was measured by FACS. B MC38-bearing female C57BL/6 mice ( n = 7/group) were treated with 25 μg BiDT on days 13, 16 and 19 after tumor inoculation. For blocking CD80/86, anti-CD80 and anti-CD86 were administrated twice a week for 2 weeks. C – G MC38 tumor-bearing female C57BL/6 mice ( n = 6/group) were treated with 25 µg BiDT on day 16 after tumor inoculation. For blocking CD80/86, anti-CD80 and anti-CD86 were administrated on day 16 after tumor inoculation. Tumor tissues were collected on day 19 after tumor inoculation. The percentage of active caspase3 + in TIM3 + CD8 + T cells ( C ), the percentage of Bcl-2 + in TIM3 + CD8 + T cells ( D ), the percentage of CD25 in total CD8 + T cells/TIM3 + CD8 + T cells and TIM3 - CD8 + T cells ( E ), the percentage of IL-2 + in TIM3 + CD8 + T cells ( F ), the percentage of IFN-γ + in TIM3 + CD8 + T cells ( G ) were measured by FACS. H MC38-bearing female C57BL/6 mice ( n = 7/group) were treated with BiDT, or BiDT + anti-CD122 on days 13, 16 and 19 after tumor inoculation. I The female C57BL/6 mice ( n = 6/group) were euthanized, and tumor tissues were collected 72 h after BiDT treatment. Expression of PD-L1 in DC was measured by FACS. J , K B16F10-bearing female C57BL/6 mice ( n = 10/group) were i.p. treated with 50 μg BiDT and/or 150 μg anti-PD-L1 on days 9, 12 and 15 after tumor inoculation. Tumor volume ( J ) was measured as indicated, and the mouse survival curve was shown in ( K ). L 30 days after treatment with BiDT and anti-PD-L1, cured B16F10-bearing female mice ( n = 5/group) were re-challenged with 2 × 10 6 B16F10 cells. Data are shown as mean ± SEM and are representative of at least two independent experiments. P value was determined by unpaired two-tailed t tests ( A , I , L ) or One-way ANOVA with Tukey’s test ( B – G , H , J ). Survival curve was compared using the log-rank test ( K ). Source data are provided as a Source Data file.

Article Snippet: MC38 cell line was purchased from Cytion (#305223).

Techniques: Injection, Expressing, Blocking Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Reactivating exhausted tumor-infiltrating T cells by a bispecific DC-T cell engager in mice

doi: 10.1038/s41467-026-70876-4

Figure Lengend Snippet: A Schematic diagram of Pro-BiDT. B , C MC38-bearing female C57BL/6 mice ( n = 6/group) were treated i.p. with equal molar amounts (600 pmol) of BiDT or Pro-BiDT on days 13, 16 and 19 post-inoculation. Tumor volumes ( B ) and Body weights ( C ) were measured as indicated. D – F MC38-bearing female mice ( n = 6/group) were treated i.p. with equal molar amounts (600 pmol) of BiDT or Pro-BiDT on day 13 after tumor inoculation. Blood samples were collected 24 h after the first injection. Serum MCP-1 levels were measured by cytometric bead assay ( D ). Complete blood count was measured ( E , F ). G B16F10-bearing female C57BL/6 mice ( n = 6/group) were i.p. treated with equal molar amounts (600 pmol) of BiDT or Pro-BiDT on days 8, 11 and 14 post-inoculation. Tumor volumes were measured as indicated ( G ). Data are shown as mean ± SEM and are representative of at least two independent experiments. P value was determined by One-way ANOVA with Tukey’s test ( B – G ). Source data are provided as a Source Data file.

Article Snippet: MC38 cell line was purchased from Cytion (#305223).

Techniques: Injection

Journal: Cell reports

Article Title: HRS mediates tumor immune evasion by regulating proteostasis-associated interferon pathway activation

doi: 10.1016/j.celrep.2023.113352

Figure Lengend Snippet: (A and B) Tumor growth (A) and survival curves (B) of C57BL/6J mice inoculated with control or HRS KO B16F10 melanoma cells. (C and D) Tumor growth (C) and survival curves (D) of athymic nude mice inoculated with control or HRS KO B16F10 melanoma cells. (E and F) Tumor growth (E) and survival curves (F) of C57BL/6J mice inoculated with control or HRS KD MC38 colon cancer cells. (G and H) Tumor growth (G) and survival curves (H) of athymic nude mice inoculated with control or HRS KD MC38 colon cancer cells. (I and J) Tumor growth (I) and survival curves (J) of C57BL/6J mice inoculated with control or HRS KO B16F10 cells, and the mice were intraperitoneally injected with isotype or anti-CD8 antibodies. (K and L) Tumor growth (K) and survival curves (L) of C57BL/6J mice inoculated with control, HRS KO B16, B2M KO, or HRS B2M double KO (dKO) B16F10 cells. Data represent the mean ± SEM; n = 7 or 8 immunocompetent C57BL/6J mice per group, n = 5 or 7 athymic nude mice per group. The p values were determined by two-way ANOVA (A, C, E, G, I, and K) or log-rank test (B, D, F, H, J, and L). See also and .

Article Snippet: Mouse cell line: MC38 , Serge Y. Fuchs, Penn Veterinary School of Medicine , PMID: 32807917; RRID:CVCL_B288.

Techniques: Control, Injection

Journal: Cell reports

Article Title: HRS mediates tumor immune evasion by regulating proteostasis-associated interferon pathway activation

doi: 10.1016/j.celrep.2023.113352

Figure Lengend Snippet: (A and B) Immunohistochemistry (A) with quantification (B) of CD45 + immune cells (left) and CD8 + T cells (right) in control and HRS KO B16F10 tumors. (C) The infiltration of CD4 + (left) and CD8 + (right) lymphocytes (tumor-infiltrating lymphocytes [TILs]) from control and HRS KO B16F10 tumors in C57BL/6J mice. (D) The expression of Ki-67 (left) and GzmB (right) by CD8 + TILs from control and HRS KO B16F10 tumors. (E) The infiltration of CD4 + (left) and CD8 + (right) TILs from control and HRS KD MC38 colon tumors in C57BL/6J mice. (F) The expression of Ki-67 (left) and GzmB (right) by CD8 + TILs from control and HRS KD MC38 colon tumors. Data represent the mean ± SD; n = 7 or 8 mice per group. The p values were determined by unpaired Student’s t test (B–F). See also .

Article Snippet: Mouse cell line: MC38 , Serge Y. Fuchs, Penn Veterinary School of Medicine , PMID: 32807917; RRID:CVCL_B288.

Techniques: Immunohistochemistry, Control, Expressing

Journal: Cell reports

Article Title: HRS mediates tumor immune evasion by regulating proteostasis-associated interferon pathway activation

doi: 10.1016/j.celrep.2023.113352

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse cell line: MC38 , Serge Y. Fuchs, Penn Veterinary School of Medicine , PMID: 32807917; RRID:CVCL_B288.

Techniques: Virus, Recombinant, SYBR Green Assay, Staining, shRNA, Plasmid Preparation, Software, CRISPR

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