Journal: The Journal of Clinical Investigation

Article Title: Aspartate deficiency amplifies cGAS-STING signaling in antitumor immunity

doi: 10.1172/JCI199716

Figure Lengend Snippet: ( A ) Outline of metabolic inhibitors for screening. ( B ) Human hTERT-immortalized foreskin fibroblast (BJ-5ta, left) and mouse fibroblast (L929, right) cells were treated with inhibitors from A followed by 0.5 μg/mL HT-DNA transfection for 5 hours. Cells were harvested for qPCR analysis of human IFNB or mouse Cxcl10 . ( C ) BJ-5ta cells were treated with 1 mM AOA for 1 hour followed by HT-DNA (0.5 μg/mL) transfection for the indicated time. Cells were harvested for qPCR analysis of IFN response gene expression. ( D and E ) L929 ( D ) and MC38 ( E ) were treated with 0.5 mM AOA for 1 hour followed by DMXAA (50 μM) or HT-DNA (0.5 μg/mL) stimulation for the indicated time. Cells were harvested for qPCR analysis of IFN response gene expression. ( F – H ) Western blot detected phosphorylated (p-) p-IRF3 and p-STAT1 levels in BJ-5ta, L929, and MC38 cells treated as in C – E . ( I ) Representative immunofluorescence images of IRF3 in BJ-5ta cells treated with AOA 1 hour followed by HT-DNA (0.5 μg/mL) transfection or not. Scale bars, 5 μm. ( J ) Schematic of the main targets of AOA. ( K ) Western blot detected p-IRF3, p-STAT1, GOT2, and GPT2 levels in BJ-5ta cells treated as indicated. ( L ) The relative IFNB and CXCL10 mRNA expression in the control BJ-5ta cells versus GOT2 and GPT2 siRNA-silenced BJ-5ta cells after indicated treatment. Data are represented as means ± SEM. Representative data are shown from 2 or 3 independent experiments. Statistical analysis was performed by unpaired t test ( C – E , I , and L ). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Six- to 8-week-old C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd, and age-matched Ifnar1 -KO C57BL/6J mice were obtained from Cyagen Biosciences (Suzhou) Inc. BJ-5ta and L929 cells were obtained from ATCC, while B16-F10 and MC38 cells were purchased from the National Collection of Authenticated Cell Cultures.

Techniques: Transfection, Gene Expression, Western Blot, Immunofluorescence, Expressing, Control

Journal: The Journal of Clinical Investigation

Article Title: Aspartate deficiency amplifies cGAS-STING signaling in antitumor immunity

doi: 10.1172/JCI199716

Figure Lengend Snippet: ( A – F ) Tumor volume ( A , C , E ) and Kaplan-Meier survival curves ( B , D , and F ) for C57BL/6 mice inoculated with approximately 2 × 10 5 B16-F10 cells ( A , n = 5), 2 × 10 5 LLC cells ( C , n = 5), or 8 × 10 5 MC38 cells ( E , n = 6). Mice were treated with 3 μg/mouse cGAMP combined with daily injections of AOA (5 mg/kg, i.p.) or PBS on days 7, 10, and 13. ( G ) AOA enhances cGAMP-mediated antitumor response. Quantification analysis of the percentage of CD8 + T cells in gated CD3 + T cells. ( H and I ) TNF-α ( H ) and IFN-γ ( I ) production on CD8 + T cells isolated from tumors of MC38 tumor–bearing mice. FACS analysis was performed 6 hours after the last cGAMP injection ( n = 5). ( J ) MC38 tumor–bearing mice were administered Asp (100 mg/kg) on days 7–13 by intraperitoneal injection, with cGAMP and AOA treatment as mentioned before. Serum IFNB and CXCL10 levels were measured 6 hours after the last cGAMP injection ( n = 4–5). ( K and L ) MC38 cells were subcutaneously transplanted into C57BL/6J mice ( n = 5). AOA (5 mg/kg) and aspartate (100 mg/kg) were administered intraperitoneally daily along with 3 doses of cGAMP as mentioned above. Tumor volumes ( K ) were measured every 2–3 days, and intratumoral aspartate levels ( L ) were quantified. Data are represented as means ± SEM. Representative data are shown from 2 or 3 independent experiments. Statistical analysis was performed by 1-way ANOVA followed by Tukey’s or Bonferroni’s test ( A , C , E , and G – L ) or log-rank test ( B , D and F ). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Six- to 8-week-old C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd, and age-matched Ifnar1 -KO C57BL/6J mice were obtained from Cyagen Biosciences (Suzhou) Inc. BJ-5ta and L929 cells were obtained from ATCC, while B16-F10 and MC38 cells were purchased from the National Collection of Authenticated Cell Cultures.

Techniques: Isolation, Injection

Journal: The Journal of Clinical Investigation

Article Title: Aspartate deficiency amplifies cGAS-STING signaling in antitumor immunity

doi: 10.1172/JCI199716

Figure Lengend Snippet: ( A ) MC38 cells were transduced with control sgRNA or sgRNA targeting cGAS . Whole cell lysates (WCLs) were analyzed by immunoblotting with the indicated antibodies. ( B and C ) MC38 cells as described in A were subcutaneously transplanted into C57BL/6J mice and administered according to the protocol mentioned above. Tumor volume ( B ) and tumor photos ( C ) were recorded. ( D and E ) MC38 cells described in A were subcutaneously transplanted into C57BL/6J mice and administered according to the protocol mentioned above. The percentage of CD8 + T cells in gated CD3 + T cells ( D ) and IFN-γ production ( E ) on CD8 + T cells. ( F ) MC38 cells were transduced with control sgRNA or sgRNA targeting Zbp1 . WCLs were analyzed by immunoblotting with the indicated antibodies. ( G and H ) MC38 cells as described in F were subcutaneously transplanted into C57BL/6J mice and administered according to the protocol mentioned above. Tumor volume ( G ) and tumor photos ( H ). ( I and J ) MC38 cells described in G were subcutaneously transplanted into C57BL/6J mice and administered according to the protocol mentioned above. The production of IFN-γ ( I ) and TNF-α ( J ) on isolated CD8 + T cells. ( K ) MC38 cells were transduced with control shRNA or shRNA targeting Ripk1 , and the stable cells were further transduced with control shRNA or sh Ripk3 to generate double-knockdown (double-KD) cells. WCLs were analyzed by immunoblotting with the indicated antibodies. ( L and M ) MC38 cells described in K were subcutaneously transplanted into C57BL/6J mice and administered according to the protocol mentioned above. Tumor volume ( L ) and tumor photos ( M ). ( N and O ) The production of IFN-γ ( N ) and TNF-α ( O ) on isolated CD8 + T cells. ( P ) MC38 cells were transduced with control shRNA or shRNA targeting Got2 , and the stable cells were further transduced with control shRNA or sh Gpt2 to generate double-KD cells. WCLs were analyzed by immunoblotting with the indicated antibodies. ( Q and R ) MC38 cells described in P were subcutaneously transplanted into C57BL/6J mice and administered according to the protocol above. Tumor volume ( Q ) and tumor photos ( R ). ( S and T ) The percentage of CD8 + T cells in gated CD3 + T cells ( S ) and IFN-γ production ( T ) on CD8 + T cells. Statistical analysis was performed by 2-way ANOVA and Tukey’s or Bonferroni’s test ( B – T ). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Six- to 8-week-old C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd, and age-matched Ifnar1 -KO C57BL/6J mice were obtained from Cyagen Biosciences (Suzhou) Inc. BJ-5ta and L929 cells were obtained from ATCC, while B16-F10 and MC38 cells were purchased from the National Collection of Authenticated Cell Cultures.

Techniques: Transduction, Control, Western Blot, Isolation, shRNA, Knockdown

Journal: The Journal of Clinical Investigation

Article Title: Aspartate deficiency amplifies cGAS-STING signaling in antitumor immunity

doi: 10.1172/JCI199716

Figure Lengend Snippet: ( A and B ) Quantitative analysis of intracellular cGAMP in MC38 cells with or without gemcitabine or oxaliplatin treatment by ELISA. ( C and D ) MC38 tumor tissues were treated with gemcitabine ( C ) or oxaliplatin ( D ) combined with or without AOA for 24 hours, and the tissular protein expression of p-IRF3 and p-STAT1 was then detected by immunoblotting. ( E and F ) MC38 tumor tissues were treated as in C and D and the tissular mRNA expression of Ifnb , Ifit1 , Ifi44 , Isg15 , and Ccl5 was detected. ( G and H ) Tumor volume ( G ) and Kaplan-Meier survival curves ( H ) for C57BL/6 mice inoculated with approximately 8 × 10 5 MC38 cells. Mice were treated with gemcitabine (50 mg/kg, i.p.) combined with daily injections of AOA (5 mg/kg, i.p.) or PBS on days 7, 10, 13, and 16 ( n = 6). ( I and J ) Tumor volume ( I ) and Kaplan-Meier survival curves ( J ) for C57BL/6 mice inoculated with approximately 8 × 10 5 MC38 cells. Mice were treated with oxaliplatin (5 mg/kg, i.p.) combined with daily injections of AOA (5 mg/kg, i.p.) or PBS on day 8 and 12. Tumor volume and mouse survival were measured 2–3 times a week ( n = 8). ( K – N ) MC38-bearing mice were treated as in G and I ; lymphocyte infiltration was measured by FACS. Quantification analysis of the percentage of CD8 + T cells in living cells from MC38 tumors after indicated treatment ( K and M ) ( n = 4–5). Representative data and quantification analysis of the percentage of TNF-α + CD8 + T cells from MC38 tumors after indicated treatment ( L and N ) ( n = 4–5). Statistical analysis was performed by 1-way ANOVA followed by Tukey’s test ( A , B , E – G , I , and K – N ) or log-rank test ( H and J ). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Six- to 8-week-old C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd, and age-matched Ifnar1 -KO C57BL/6J mice were obtained from Cyagen Biosciences (Suzhou) Inc. BJ-5ta and L929 cells were obtained from ATCC, while B16-F10 and MC38 cells were purchased from the National Collection of Authenticated Cell Cultures.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: iScience

Article Title: Targeting USP14 enhances immunotherapy response by reprogramming tumor-associated macrophages in colon cancer

doi: 10.1016/j.isci.2026.115362

Figure Lengend Snippet: Targeting USP14 inhibits tumor growth and induces local anti-tumor immunity in vivo (A) Representative images of MC38 tumors harvested on day 21 post-inoculation from mice treated with IU1 (20 mg/kg, i.p., on days 6, 9, 12, and 15) or vehicle control. (B) Statistics of MC38 tumor growth rate following IU1 or vehicle treatment in vivo . The intraperitoneal injection dose of IU1 was 20 mg/kg, administered on days 6, 9, 12, and 15. Data are presented as the mean ± SEM ( n = 6 per group). (C) Spider diagram of the tumor volume growth in each mouse from the IU1 group and the PBS group. (D) Gating strategy for the detection of the TAMs by flow cytometry. We first obtained live cells, and then identified cells that were positive for CD45, CD11b, F4-80, and CD206 as M2 macrophages. (E–P) Proportions of neutrophil (E), M2 macrophage (F), M1 macrophage (G), MDSC (H), activated DCs (I), CD4 T cell (J), Treg cells (K), CD8 T cell (L), IFN-γ + CD8 T cell (M), precursor exhausted T cells (TCF-1 + ) (N), effective CD8 T cells (PD-1 + ) (O) and activated CD8 T cell (CD69 + ) (P) in the TME of the IU1 group and the control group by using flow cytometry. (Q–U) Cytokines IFN-γ (Q), TNF-α (R), IL-2 (S), IL-10 (T), and IL-12 (U) in the TME of each group were detected by Mul-Analyte Flow Assay Kit. (V) Schematic illustration of the proposed mechanism of action of IU1 in reprogramming the tumor microenvironment. Data are presented as the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, and ns: not significant.

Article Snippet: Mouse colon cancer cells MC38 , Procell , CL-0972.

Techniques: In Vivo, Control, Injection, Flow Cytometry

Journal: iScience

Article Title: Targeting USP14 enhances immunotherapy response by reprogramming tumor-associated macrophages in colon cancer

doi: 10.1016/j.isci.2026.115362

Figure Lengend Snippet: Identification of the mechanism underlying IU1-mediated reprogramming of M2 macrophages (A and B) Verification of the efficiency of i.v. injection of clodronate liposome (Clo) in depleting TAMs of the blood (A) and the TME (B). (C and D) Statistics of tumor size monitored for 21 days in MC38 tumor-bearing mice after various indicated treatments. (E) Statistic of the percentage of CTLs in the TME after the indicated treatment. (F) Volcano plots of the differentially expressed genes between the PBS and the IU1 group. Red dots show significantly up-regulated genes in the IU1 group, and green dots show significantly down-regulated genes. (G) Heatmap illustrates the differentially expressed M1-and M2-related genes in TAMs in the IU1 group and the PBS group based on RNA sequencing results. (H) KEGG analysis identifies the 17 most enriched pathways based on the differentially expressed genes of the two groups. (I) Western blotting of p -JNK, p -ERK, p-p38, and GAPDH in IL-4/13-BMDM M2 cells treated with IU1 at the indicated time points. (J) RT-PCR to verify the typical M1/M2 polarization-related genes in M2 macrophages after treatment with IU1. (K) Flow cytometry analysis of CD206 expression on the IL-4/13-induced BMDM M2 cells from various indicated treatments. Treatments indicated: DMSO stimulation, IU1 (10 μM) stimulation, IU1 (10 μM) stimulation in the presence of inhibitors of p38 (SB203580, 10 μM), JNK (SP600125, 10 μM), Erk1/2 (U0126-EtOH, 10 μM). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test. Data are presented as the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, and ns: not significant.

Article Snippet: Mouse colon cancer cells MC38 , Procell , CL-0972.

Techniques: Injection, RNA Sequencing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Expressing, Comparison

Journal: iScience

Article Title: Targeting USP14 enhances immunotherapy response by reprogramming tumor-associated macrophages in colon cancer

doi: 10.1016/j.isci.2026.115362

Figure Lengend Snippet: Combined blockade of USP14 and PD-1 exerts a synergistic anti-tumor effect in vivo (A) Gating strategy for distinguishing tumor cells, DC cells, and macrophages. (B) Flow cytometry was used to analyze the differences in PD-L1 expression in tumor cells, DC cells, and macrophages in the indicated treatment groups. (C) Flow cytometry was used to statistically analyze the differences in PD-L1 expression in tumor cells, DC cells, and macrophages in the specified groups. (D) Treatment schedule of the combination of IU1 and PD-1 antibody for the MC38 tumor-bearing mice. The intraperitoneal injection dose of IU1 was 20 mg/kg, administered on days 6, 9, 12, and 15. The intraperitoneal injection dose of anti-PD-1 antibody was 7.5 mg/kg, administered on days 7, 9, 11, and 13. (E and F) Tumor growth was monitored for 21 days in MC38 tumor-bearing mice after various indicated treatments. (G) Kaplan-Meier survival plot shows the survival of mice after the indicated treatments ( n = 10). (H–I) Multicolor immunofluorescence detection of M2 macrophage (H) and CTLs (I) in the TME of the indicated treatment group. Scale bar is 100 μm. Data are presented as the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, and ns: not significant.

Article Snippet: Mouse colon cancer cells MC38 , Procell , CL-0972.

Techniques: In Vivo, Flow Cytometry, Expressing, Injection, Immunofluorescence

Journal: Biomaterials Research

Article Title: A Peptide Inhibitor of Lymphocyte Activation Gene-3 Interaction with Fibrinogen-like Protein 1 Synergizes with Programmed Death-Ligand 1 Blockade to Restore T Cell Activity and Inhibit Tumor Growth

doi: 10.34133/bmr.0364

Figure Lengend Snippet: Combined treatment with LAG3pep-2 and anti-programmed death-ligand 1 (anti-PD-L1) antibody restores T cell activity against tumor cells. (A) Experimental schemes. CD8+ T cells were isolated from the spleen of MC38 tumor-bearing mice and incubated for 48 h with anti-CD3/CD28 beads (activation) and interleukin-2 (IL-2)/interleukin-15 (IL-15) (proliferation). The activated T cells were co-cultured with MC38 cells in the absence or presence of LAG3pep-2 and anti-mouse PD-L1 antibody alone or in combination. Created with BioRender.com . (B) Activated CD8+ T cells were stained with carboxyfluorescein succinimidyl ester (CFSE) dye and co-cultured with tumor cells for 24 h. The population of CD3+/CFSE− cells was measured. (C to E) After co-culturing for 24 h, the culture medium was collected, and the percentage of cell death (lactate dehydrogenase [LDH] release) (C) and the concentrations of interferon-γ (IFN-γ) (D) and granzyme B (E) were measured. Data are presented as the mean ± SD of 3 independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant by one-way analysis of variance (ANOVA).

Article Snippet: HEK 293T (human embryonic kidney) cells, Jurkat T (human acute T cell leukemia) cells, THP-1 (acute monocytic leukemia) cells, HepG2 (human liver cancer) cells, and MC38 (C57BL/6 murine colon tumor) cells were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Activity Assay, Isolation, Incubation, Activation Assay, Cell Culture, Staining

Journal: Biomaterials Research

Article Title: A Peptide Inhibitor of Lymphocyte Activation Gene-3 Interaction with Fibrinogen-like Protein 1 Synergizes with Programmed Death-Ligand 1 Blockade to Restore T Cell Activity and Inhibit Tumor Growth

doi: 10.34133/bmr.0364

Figure Lengend Snippet: LAG3pep-2 synergizes with anti-programmed death-ligand 1 (anti-PD-L1) antibody to inhibit tumor growth and enhance anti-tumor immunity. (A) Experimental schemes. Mice bearing subcutaneous MC38 tumor were treated with intravenous (iv) administration of LAG3pep-2 and intraperitoneal (ip) administration of anti-mouse PD-L1 and anti-mouse lymphocyte activation gene-3 (LAG-3) antibodies (10 mg/kg body weight for single treatment and 5 mg/kg body weight for combined treatment). Created with BioRender.com . (B) Tumor volumes (mm 3 ) after treatments. Data are presented as mean ± SD ( n = 5/group). * P < 0.05 ** P < 0.01 from 2-way analysis of variance (ANOVA) including all treatment groups. (C) Tumor volumes (mm 3 ) of each treatment group after tumor inoculation. (D to G) Tumor tissue cell suspensions were prepared and gated for CD45+CD3+ lymphocytes. The populations of CD4+ T cells (D), CD8+ T cells (E), and FoxP3+ T cells (F) and the ratio of CD8+ T cells/FoxP3+ T cells (G) were measured. Data are presented as mean ± SD ( n = 4/group). * P < 0.05; *** P < 0.01; ns, not significant by one-way ANOVA.

Article Snippet: HEK 293T (human embryonic kidney) cells, Jurkat T (human acute T cell leukemia) cells, THP-1 (acute monocytic leukemia) cells, HepG2 (human liver cancer) cells, and MC38 (C57BL/6 murine colon tumor) cells were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Activation Assay

Journal: Biomaterials Research

Article Title: A Peptide Inhibitor of Lymphocyte Activation Gene-3 Interaction with Fibrinogen-like Protein 1 Synergizes with Programmed Death-Ligand 1 Blockade to Restore T Cell Activity and Inhibit Tumor Growth

doi: 10.34133/bmr.0364

Figure Lengend Snippet: LAG3pep-2 does not induce immunotoxicity in mice. (A) LAG3pep-2 was administered intraperitoneally to keyhole limpet hemocyanin (KLH)-challenged C57BL/6 mice at the indicated doses (0.01 to 100 mg/kg body weight). Cyclophosphamide (CPA) was used as a positive control. Mice were sacrificed, and spleen weights were measured on day 7 posttreatment. (B and C) Serum levels of total IgG2a (B) and total IgE (C). (D to F) The population of CD4+IFN-γ+ T cells (D), CD4+IL-4+ T cells (E), and CD4+IL-17A+ T cells (F) in the spleen. Data are presented as mean ± SD ( n = 5/group).

Article Snippet: HEK 293T (human embryonic kidney) cells, Jurkat T (human acute T cell leukemia) cells, THP-1 (acute monocytic leukemia) cells, HepG2 (human liver cancer) cells, and MC38 (C57BL/6 murine colon tumor) cells were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Positive Control