Journal: MedComm

Article Title: Chromosome 1 Open Reading Frame 35 Drives Colorectal Cancer Progression by Enhancing Tumor‐Intrinsic Proliferation and CD8 + T Cell Suppression

doi: 10.1002/mco2.70707

Figure Lengend Snippet: Expression levels of C1orf35 protein and mRNA in pan‐cancer and CRC compared to paired normal samples. (A and B) Pan‐cancer comparison of C1orf35 mRNA levels between tumor and normal tissues using TCGA data. (C) TCGA analysis showing upregulation of C1orf35 mRNA in CRC ( n = 647) relative to normal mucosa ( n = 51). (D) TCGA analysis showing upregulation of C1orf35 mRNA in CRC relative to matched normal mucosa ( n = 50). (E) ROC analysis of C1orf35 in CRC for diagnostic value. (F–I) Validation in GEO cohorts confirming higher C1orf35 mRNA expression in CRC versus matched normal controls in GSE89076 ( n = 39), GSE22598 ( n = 17), GSE110224 ( n = 17), and GSE113513 ( n = 14). (J and K) IHC staining of C1orf35 in human tumor tissue microarrays (scale bar, 1 mm, 200 µm) (ns indicates no statistical significance, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: To investigate the functional role of C1orf35, established organoids were treated with recombinant human C1orf35 protein (source: Proteintech, Ag20769) at an optimized concentration of 50 ng/mL.

Techniques: Expressing, Comparison, Diagnostic Assay, Biomarker Discovery, Immunohistochemistry

Journal: MedComm

Article Title: Chromosome 1 Open Reading Frame 35 Drives Colorectal Cancer Progression by Enhancing Tumor‐Intrinsic Proliferation and CD8 + T Cell Suppression

doi: 10.1002/mco2.70707

Figure Lengend Snippet: Correlation study of CRC C1orf35 expression with clinical‐pathological parameters and prognosis based on the TCGA database. (A–I) C1orf35 expression was significantly correlated with T stage (C), N stage (D), M stage (E), pathological stage (F), as well as OS (G), DSS (H), and PFI (I). (L) Kaplan–Meier curves showed that upregulated C1orf35 expression was associated with OS, DSS, and PFI (ns indicates no statistical significance, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: To investigate the functional role of C1orf35, established organoids were treated with recombinant human C1orf35 protein (source: Proteintech, Ag20769) at an optimized concentration of 50 ng/mL.

Techniques: Expressing

Journal: MedComm

Article Title: Chromosome 1 Open Reading Frame 35 Drives Colorectal Cancer Progression by Enhancing Tumor‐Intrinsic Proliferation and CD8 + T Cell Suppression

doi: 10.1002/mco2.70707

Figure Lengend Snippet: Knockdown of C1orf35 inhibits the proliferation and migration of CRC cells. (A) C1orf35 RNA levels were determined by qRT‐PCR and normalized to actin RNA levels. (B) Western blotting analysis demonstrates decreased C1orf35 levels in C1orf35‐knockdown HCT116 and SW480 cells compared to control cells. (C) Effect of C1orf35 knockdown on cell proliferation in HCT116 and SW480 cells. (D) Colony formation assay in C1orf35‐knockdown SW480 cells. (E) Effect of recombinant C1orf35 protein on CRC organoids (scale bar, 250 µm). (F) Transwell assays in C1orf35‐knockdown SW480 and HCT116 cells (scale bar, 100 µm). Data are presented as mean ± SD; n = 3; ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; two‐tailed Student's t ‐tests (E); one‐way ANOVA followed by Dunnett's multiple comparisons (A, C, D, and F).

Article Snippet: To investigate the functional role of C1orf35, established organoids were treated with recombinant human C1orf35 protein (source: Proteintech, Ag20769) at an optimized concentration of 50 ng/mL.

Techniques: Knockdown, Migration, Quantitative RT-PCR, Western Blot, Control, Colony Assay, Recombinant, Two Tailed Test

Journal: MedComm

Article Title: Chromosome 1 Open Reading Frame 35 Drives Colorectal Cancer Progression by Enhancing Tumor‐Intrinsic Proliferation and CD8 + T Cell Suppression

doi: 10.1002/mco2.70707

Figure Lengend Snippet: PYCR2 is involved in the oncogenic function of C1orf35 in CRC. (A) Heatmap showing the top 30 genes positively correlated with C1orf35 in CRC. High and low expressions are indicated in red and blue, respectively. (B) Scatter plot of the relationship between C1orf35 and PYCR2 expression levels. (C) TCGA analysis showing upregulation of PYCR2 mRNA in CRC ( n = 647) relative to normal mucosa ( n = 51). (D) TCGA analysis showing upregulation of PYCR2 mRNA in CRC relative to matched normal mucosa ( n = 50). (E) Kaplan–Meier curve showing that upregulation of PYCR2 expression is associated with decreased OS. (F) GO analysis of upregulated and downregulated differentially expressed genes (DEGs). (G–I) Hallmark, GO, and KEGG pathway enrichment analyses of C1orf35‐related genes (ns indicates no significance, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: To investigate the functional role of C1orf35, established organoids were treated with recombinant human C1orf35 protein (source: Proteintech, Ag20769) at an optimized concentration of 50 ng/mL.

Techniques: Expressing

Journal: MedComm

Article Title: Chromosome 1 Open Reading Frame 35 Drives Colorectal Cancer Progression by Enhancing Tumor‐Intrinsic Proliferation and CD8 + T Cell Suppression

doi: 10.1002/mco2.70707

Figure Lengend Snippet: C1orf35 promotes colorectal cancer progression via the c‐Myc/PYCR2 axis. (A) Quantitative RT‐qPCR analysis of c‐Myc and PYCR2 expression in control (shCtrl) and C1orf35‐knockdown (shC1orf35) CRC cells. (B) Western blot analysis of c‐Myc and PYCR2 expression in control (shCtrl) and C1orf35‐knockdown (shC1orf35) CRC cells. (C) Western blotting analysis demonstrates decreased PYCR2 level in PYCR2‐knockdown SW480 cells compared to control cells. (D) PYCR2 is essential for the oncogenic functions of C1orf35. Cell migration was assessed by transwell assay in four groups: control (Ctrl), C1orf35 overexpression (OE), PYCR2 knockdown (shPYCR2), and combined C1orf35 OE + PYCR2 knockdown. Representative images (scale bar, 100 µm) and quantification are shown. (E) Cell proliferation was measured by CCK‐8 assay in the same four groups over 72 h. (F–H) c‐Myc is required for C1orf35‐mediated upregulation of PYCR2. CRC cells were transfected with vector or C1orf35 overexpression plasmid and treated with DMSO or a c‐Myc inhibitor (10058‐F4). Representative RT‐qPCR analysis and western blot of c‐Myc and PYCR2 expression. Data are mean ± SD; n = 3; ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two‐way ANOVA).

Article Snippet: To investigate the functional role of C1orf35, established organoids were treated with recombinant human C1orf35 protein (source: Proteintech, Ag20769) at an optimized concentration of 50 ng/mL.

Techniques: Quantitative RT-PCR, Expressing, Control, Knockdown, Western Blot, Migration, Transwell Assay, Over Expression, CCK-8 Assay, Transfection, Plasmid Preparation

Journal: MedComm

Article Title: Chromosome 1 Open Reading Frame 35 Drives Colorectal Cancer Progression by Enhancing Tumor‐Intrinsic Proliferation and CD8 + T Cell Suppression

doi: 10.1002/mco2.70707

Figure Lengend Snippet: Associations between C1orf35 expression and immune parameters, immune‐cell infiltration, and the tumor microenvironment in colorectal cancer (CRC). (A–C) Correlation of C1orf35 expression with ESTIMATE, stromal, and immune scores. (D) The relationship between C1orf35 expression and immune cell infiltration in CRC based on EPIC analysis. (E) The relationship between C1orf35 expression and immune cell infiltration in CRC based on MCPCOUNTER analysis. (F) The relationship between C1orf35 expression and immune cell infiltration in CRC based on QUANTISEQ analysis. (G) The relationship between C1orf35 expression and immune cell infiltration in CRC based on TIMER analysis. (H) XCELL‐based associations between C1orf35 expression and the estimated abundance of specific infiltrating immune and stromal cell populations. (I) TIP analysis showing the relationship between C1orf35 expression and functional anti‐tumor immune processes and immune‐cell recruitment in CRC (ns indicates no significance, *p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: To investigate the functional role of C1orf35, established organoids were treated with recombinant human C1orf35 protein (source: Proteintech, Ag20769) at an optimized concentration of 50 ng/mL.

Techniques: Expressing, Functional Assay

Journal: MedComm

Article Title: Chromosome 1 Open Reading Frame 35 Drives Colorectal Cancer Progression by Enhancing Tumor‐Intrinsic Proliferation and CD8 + T Cell Suppression

doi: 10.1002/mco2.70707

Figure Lengend Snippet: Elevated C1orf35 in human CRC tumor tissues is negatively associated with CD8 + T cells. (A) Multiplex immunofluorescence of human tumor tissues (scale bar, 1 mm, 200 µm). (B) Comparison of C1orf35 expression levels in patients with CRC at different stages. (C) Relationship between C1orf35 expression levels and PYCR2 expression levels. (D) Relationship between C1orf35 expression levels and CD8 + T‐cell content. (E) C1orf35 impairs CD8 + T cell cytotoxicity in a manner dependent on c‐Myc activity. CD8+ T cells were co‐cultured for 24 h with control MC38 cells or MC38 cells overexpressing C1orf35 (OE‐C1orf35) in the presence or absence of the c‐Myc inhibitor 10058‐F4 (10 µM). Tumor cell killing was assessed by crystal violet staining of the remaining adherent MC38 cells. (Left) Representative images. (Right) Quantitative analysis of crystal violet absorbance. Data are mean ± SD ( n = 3). Statistical significance was determined by two‐way ANOVA (ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: To investigate the functional role of C1orf35, established organoids were treated with recombinant human C1orf35 protein (source: Proteintech, Ag20769) at an optimized concentration of 50 ng/mL.

Techniques: Multiplex Assay, Immunofluorescence, Comparison, Expressing, Activity Assay, Cell Culture, Control, Staining

Journal: MedComm

Article Title: Chromosome 1 Open Reading Frame 35 Drives Colorectal Cancer Progression by Enhancing Tumor‐Intrinsic Proliferation and CD8 + T Cell Suppression

doi: 10.1002/mco2.70707

Figure Lengend Snippet: Knockdown of C1orf35 inhibits tumor growth in mice. (A–F) Tumor progression generated by orthotopic injection of HCT116 cells into nude mice ( n = 7–8/group). (A) Subcutaneous tumor formation experiments with shNC cells, shC1orf35‐1 cells, and shC1orf35‐2 HCT116 cells. (B) Measurement of tumor volume after subcutaneous injection. (C) Immunohistochemical staining results of tumor tissues in the three groups. (D–F) Statistical analysis of immunohistochemical results. Scale bars, 200 µm. (G–I) Tumor progression generated by orthotopic injection of MC38 cells into C57BL/6 mice (n = 7–8/group). (G) Subcutaneous tumor formation experiments with shNC cells, shmmtag2 MC38 cells. (H) Measurement of tumor weight after subcutaneous injection. (I) CD8 immunohistochemistry in murine tumors. Scale bars, 200 µm. Data are presented as mean ± SEM; * p < 0.05; *** p < 0.001; one‐way ANOVA followed by Dunnett's multiple comparisons.

Article Snippet: To investigate the functional role of C1orf35, established organoids were treated with recombinant human C1orf35 protein (source: Proteintech, Ag20769) at an optimized concentration of 50 ng/mL.

Techniques: Knockdown, Generated, Injection, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Oncogenesis

Article Title: Teriflunomide modulates the PD-1/PD-L1 axis and enhances antitumor immunity in colorectal cancer

doi: 10.1038/s41389-026-00607-3

Figure Lengend Snippet: A The viability of hPD-1 Jurkat-T cells and hPD-L1 CHO cells following treatment with the indicated concentrations of TER for 24 h. B Luciferase activity measured using a PD-1/PD-L1 blockade bioassay. hPD-1 Jurkat-T cells (effector cells) were co-cultured with hPD-L1-expressing aAPC/CHO-K1 cells (target cells) in the presence of indicated concentrations of TER. The luminescence signal indicates the level of TCR signaling activation. αPD-L1 was used as a positive control. * <0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with the respective control.

Article Snippet: Recombinant hPD-L1 (#71104, BPS Bioscience) was coated onto 96-well plates (Corning Inc., New York, NY, USA) at a concentration of 1 mg/mL in PBS and incubated overnight.

Techniques: Luciferase, Activity Assay, Bioassay, Cell Culture, Expressing, Activation Assay, Positive Control, Control

Journal: Oncogenesis

Article Title: Teriflunomide modulates the PD-1/PD-L1 axis and enhances antitumor immunity in colorectal cancer

doi: 10.1038/s41389-026-00607-3

Figure Lengend Snippet: A The viability of hPD-L1 MC38 cells following treatment with the indicated concentrations of TER for 72 h. B CD8 + T cells were isolated from tumors of hPD-1 knock-in mice bearing hPD-L1 MC38 tumors. These tumor-infiltrating CD8 + T cells were co-cultured with hPD-L1 MC38 cells as target cells in the presence of TER for 72 h. Cell viability measured using the CCK assay is depicted. C PD-L1 expression in hPD-L1 MC38 cells, as assessed by western blot analysis using protein lysates from co-culture conditions. GAPDH was used as a loading control. D The levels of immune-related factors, including GrB, IL-2, and IFN-γ, measured in the co-culture supernatant by ELISA. * <0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with the respective control.

Article Snippet: Recombinant hPD-L1 (#71104, BPS Bioscience) was coated onto 96-well plates (Corning Inc., New York, NY, USA) at a concentration of 1 mg/mL in PBS and incubated overnight.

Techniques: Isolation, Knock-In, Cell Culture, Expressing, Western Blot, Co-Culture Assay, Control, Enzyme-linked Immunosorbent Assay

Journal: Oncogenesis

Article Title: Teriflunomide modulates the PD-1/PD-L1 axis and enhances antitumor immunity in colorectal cancer

doi: 10.1038/s41389-026-00607-3

Figure Lengend Snippet: A Body weight of hPD-1 knock-in mice during the treatment period. The mice were treated with vehicle or TER (10 or 30 mpk) for the indicated time. B Spleen weight of mice at the endpoint of the experiment. C Tumor volume was measured over time in hPD-1 knock-in mice bearing hPD-L1 MC38 tumors treated with vehicle or TER (10 or 30 mpk). Representative images of excised tumors from each group are shown. D Tumor weight at the endpoint of the experiment. E Flow cytometry analysis of CD8 + T-cell populations in tumors from each treatment group. F PD-L1 expression in tumors from each group, as assessed by western blot analysis. GAPDH was used as a loading control. G IHC staining of tumor sections for immune-related markers, including CD8 + T cells and GrB. Representative images from each group are shown, and the quantitation of marker-positive cells per field is presented. * <0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with the respective control.

Article Snippet: Recombinant hPD-L1 (#71104, BPS Bioscience) was coated onto 96-well plates (Corning Inc., New York, NY, USA) at a concentration of 1 mg/mL in PBS and incubated overnight.

Techniques: Knock-In, Flow Cytometry, Expressing, Western Blot, Control, Immunohistochemistry, Quantitation Assay, Marker

Journal: Oncogenesis

Article Title: Teriflunomide modulates the PD-1/PD-L1 axis and enhances antitumor immunity in colorectal cancer

doi: 10.1038/s41389-026-00607-3

Figure Lengend Snippet: A Body weight of hPD-1 knock-in mice during the treatment period. The mice were treated with vehicle or TER (30 mpk) and received either an isotype control or a CD8 depletion antibody. B Spleen weight of mice at the endpoint of the experiment. C Tumor volume was measured in hPD-1 knock-in mice bearing hPD-L1 MC38 tumors over time following treatment with vehicle or TER (30 mpk) with or without CD8 depletion. Representative images of excised tumors from each group are shown. D Tumor weight at the endpoint of the experiment. E Flow cytometry analysis confirming CD8 + T-cell depletion in tumors from each treatment group. The proportion of CD8 + cells among total live cells was quantified. F IHC staining of tumor sections for CD8 + T cells and GrB. Representative images from each treatment group are shown, and the quantitation of marker-positive cells per field is presented. * <0.05, ** p < 0.01, and **** p < 0.0001 compared with the respective control.

Article Snippet: Recombinant hPD-L1 (#71104, BPS Bioscience) was coated onto 96-well plates (Corning Inc., New York, NY, USA) at a concentration of 1 mg/mL in PBS and incubated overnight.

Techniques: Knock-In, Control, Flow Cytometry, Immunohistochemistry, Quantitation Assay, Marker