Journal: Oncoimmunology

Article Title: Smad7 is a negative regulator of immunogenic cell death in colorectal cancer

doi: 10.1080/2162402X.2025.2490346

Figure Lengend Snippet: Downregulation of Smad7 protein expression enhances calreticulin exposure on the cancer cell surface (ecto-calreticulin). (A) Representative histograms showing the percentage of ecto-calreticulin-expressing DLD1 cells as assessed by flow cytometry. Cells were transfected with either Smad7 as or sense for 36 hours. Mean fluorescence intensity (MFI) values for calreticulin staining in both ecto-calreticulin-negative cells (red bars) and ecto-calreticulin-positive cells (blue bars) are also indicated. Staining with an isotype control antibody for calreticulin is also shown. Right inset: histograms showing the percentage of ecto-calreticulin-expressing DLD1 cells transfected with either Smad7 as or sense for 36 hours. Data are mean ± SD of six separate experiments. Data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. * p < 0.05; *** p < 0.001. (B) Representative western blots for Smad7, calreticulin, and β-actin in total extracts of DLD1 cells transfected with Smad7 as or sense for 24 hours. β-actin was used as a loading control. The right panels show the mean ± SEM of all experiments; the differences were evaluated using a two-tailed Student’s t-test. * p < 0.05. (C) DLD1 cells were either transfected with Smad7 as or sense for 24 hours. Calreticulin RNA transcripts were evaluated by the real-time polymerase chain reaction. Levels were normalized to β-actin. Values indicate the mean ± SD of three separate experiments. Differences were analyzed using a two-tailed Student’s t-test. (D) DLD1 cells were pre-treated or not with TUDCA (50 μM) and then transfected with Smad7 as or sense (both used at 2 µg/ml) for 36 hours. Representative histograms showing the percentage of ecto-calreticulin-expressing DLD1 cells. Results indicate the percentage of ecto-calreticulin-expressing cells as assessed by flow cytometry. Right inset: data indicate mean ± SD of all experiments. Data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. * p < 0.05.

Article Snippet: Membranes were then incubated with the antibodies anti-human/mouse Smad7 (1:1000, #MAB2029, R&D Systems), anti-human/mouse CRT (1:1000, # PA3–900, Thermo Fisher Scientific), anti-human cleaved caspase-3 (1:1000, #9661S, Cell Signaling Technology, Danvers, MA), anti-human HMGB1 (1:1000, #39355, Cell Signaling), anti-human GPX4 (1:1000, #HY- P80692 , Med Chem Express), anti-human p-MLKL Ser358 (1:1000, #91689S, Cell Signaling Technology), anti-human p-RIP3 Ser227 (1:1000, #EPR9627, Abcam, Cambridge, UK), or anti-human/mouse β-actin (1:5000) and, subsequently, with a secondary antibody conjugated to HRP (1:20000, Dako, Santa Clara, CA).

Techniques: Expressing, Flow Cytometry, Transfection, Fluorescence, Staining, Control, Western Blot, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: Oncoimmunology

Article Title: Smad7 is a negative regulator of immunogenic cell death in colorectal cancer

doi: 10.1080/2162402X.2025.2490346

Figure Lengend Snippet: Involvement of apoptotic cell death in the Smad7 suppression-driven induction of ecto-calreticulin (A) Representative density plots showing the percentage of DLD1 cells expressing calreticulin exposed on the cell surface (ecto-calreticulin) and Annexin V. Cells were transfected with either Smad7 as or sense for 36 hours. Results indicate the percentage of annexin V and/or ecto-calreticulin expressing cells as assessed by flow cytometry. Staining with an isotype control antibody for calreticulin is also shown. Upper right inset: histograms showing the percentage of annexin V and/or ecto-calreticulin expressing DLD1 cells transfected with either Smad7 as or sense for 36 hours. Data are mean ± SD of three separate experiments. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Smad7 as-transfected cells vs sense-transfected cells: ** p < 0.01; *** p < 0.001. (B) Representative Western blots for cleaved caspase-3 and β-actin in total extracts of DLD1 cells transfected with Smad7 sense or as for 48 hours. Protein lysates derived from DLD1 cells treated with 1 µg/ml staurosporin for 18 hours (ST) were used as a positive control of caspase 3 cleavage. β-actin was used as a loading control. (C) Histograms showing the percentage of cell death in DLD1 cells pre-treated with either Q-VD-OPh or DMSO (vehicle) for 1 hour and then transfected with Smad7 sense or as for 60 hours. Data indicate mean ± SD of three separate experiments. Annexin V (AV) and/or propidium iodide (pi)-positive cells were evaluated by flow cytometry. Data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. *** p < 0.001. Right inset: Representative density plots showing the percentages of AV- and/or pi-positive cells treated as indicated. (D) Histograms showing the percentage of ecto-calreticulin expressing DLD1 cells. Cells were pre-treated with either Q-VD-OPh or DMSO (vehicle) for 1 hour and then transfected with Smad7 sense or as for 36 hours. Data indicate mean ± SD of three separate experiments. Data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. *** p < 0.001. Right inset: representative histograms showing the percentage of ecto-calreticulin-expressing DLD1 cells treated as indicated as assessed by flow cytometry.

Article Snippet: Membranes were then incubated with the antibodies anti-human/mouse Smad7 (1:1000, #MAB2029, R&D Systems), anti-human/mouse CRT (1:1000, # PA3–900, Thermo Fisher Scientific), anti-human cleaved caspase-3 (1:1000, #9661S, Cell Signaling Technology, Danvers, MA), anti-human HMGB1 (1:1000, #39355, Cell Signaling), anti-human GPX4 (1:1000, #HY- P80692 , Med Chem Express), anti-human p-MLKL Ser358 (1:1000, #91689S, Cell Signaling Technology), anti-human p-RIP3 Ser227 (1:1000, #EPR9627, Abcam, Cambridge, UK), or anti-human/mouse β-actin (1:5000) and, subsequently, with a secondary antibody conjugated to HRP (1:20000, Dako, Santa Clara, CA).

Techniques: Expressing, Transfection, Flow Cytometry, Staining, Control, Western Blot, Derivative Assay, Positive Control

Journal: Oncoimmunology

Article Title: Smad7 is a negative regulator of immunogenic cell death in colorectal cancer

doi: 10.1080/2162402X.2025.2490346

Figure Lengend Snippet: Downregulation of Smad7 protein expression in DLD1 cells enhances the secretion of ATP and HMGB1 and the activation of co-cultured dendritic cells. (A) Histograms showing the concentrations of ATP secreted in the culture medium of DLD1 cells transfected with Smad7 as or sense for 48 hours and measured by a luciferase assay. Data are expressed as mean ± SD of all experiments. Data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. ** p < 0.01, *** p < 0.001. (B) Histograms showing the concentration of HMGB1 secreted in the culture medium of DLD1 cells transfected with Smad7 as or sense for 60 hours and measured by ELISA. Data are expressed as mean ± SD of all experiments. Data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. * p < 0.05. (C) Representative western blots for Smad7, HMGB1, and β-actin in DLD1 cells transfected with either Smad7 as or sense. β-actin was used as a loading control. (D) Representative density plots showing the percentage of CD11c + that have phagocytized cell-trace labeled DLD1 cells transfected with Smad7 as or sense. Staining with an isotype control antibody for CD11c is also shown. The right panel indicates the mean ± SD of all experiments. Data were analyzed using a two-tailed Student’s t-test. * p < 0.05. (E) Representative density plots showing the percentage of HLA-DRII + cells gated as indicated. Staining with an isotype control antibody for HLA-DRII is also shown. The right panel indicates the mean ± SD of all experiments. Data were analyzed using a two-tailed Student’s t-test. ** p < 0.01.

Article Snippet: Membranes were then incubated with the antibodies anti-human/mouse Smad7 (1:1000, #MAB2029, R&D Systems), anti-human/mouse CRT (1:1000, # PA3–900, Thermo Fisher Scientific), anti-human cleaved caspase-3 (1:1000, #9661S, Cell Signaling Technology, Danvers, MA), anti-human HMGB1 (1:1000, #39355, Cell Signaling), anti-human GPX4 (1:1000, #HY- P80692 , Med Chem Express), anti-human p-MLKL Ser358 (1:1000, #91689S, Cell Signaling Technology), anti-human p-RIP3 Ser227 (1:1000, #EPR9627, Abcam, Cambridge, UK), or anti-human/mouse β-actin (1:5000) and, subsequently, with a secondary antibody conjugated to HRP (1:20000, Dako, Santa Clara, CA).

Techniques: Expressing, Activation Assay, Cell Culture, Transfection, Luciferase, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Labeling, Staining, Two Tailed Test

Journal: Oncoimmunology

Article Title: Smad7 is a negative regulator of immunogenic cell death in colorectal cancer

doi: 10.1080/2162402X.2025.2490346

Figure Lengend Snippet: SMAD7-deficient CT26 cells engrafted in mice induce an anti-cancer CD8 + T cell-dependent immune response. (A-B) Representative images and relative graphs showing the volume of CT26-derived tumors in BALB/c mice. CT26 cells were transfected with either Smad7 as or sense for 48 hours and subcutaneously injected into the left flank of mice (day 0). Tumor growth was monitored until sacrifice (day 14). Each point in the graph indicates the tumor volume in each mouse. ** p < 0.01. (C) Representative density plots and histograms (right panels) showing the frequency of CD3 + CD8 + and CD3 + CD8- cells isolated from tumors taken from mice injected with CT26 cells as indicated in A. Data were analyzed using a two-tailed Student’s t-test. ** p < 0.01. (D-E) immunofluorescence images of CD8 (red, D) or CD4 (green, E) expression in sections derived from tumors extracted from mice injected with CT26 cells as indicated in A. Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). (F) Representative images and the relative graph (right panel) showing the volume of CT26-derived tumors. CT26 were transfected with either Smad7 as or sense for 48 hours and subcutaneously injected into the left flank of BALB/c mice (day 0). CD8 + cell depletion was obtained upon intraperitoneal injections of anti-mouse CD8 antibody (every 4 days starting from day − 2 until day 10). Tumor growth was monitored until sacrifice (day 14). Each point in the graph represents the value of tumor volume in each mouse.

Article Snippet: Membranes were then incubated with the antibodies anti-human/mouse Smad7 (1:1000, #MAB2029, R&D Systems), anti-human/mouse CRT (1:1000, # PA3–900, Thermo Fisher Scientific), anti-human cleaved caspase-3 (1:1000, #9661S, Cell Signaling Technology, Danvers, MA), anti-human HMGB1 (1:1000, #39355, Cell Signaling), anti-human GPX4 (1:1000, #HY- P80692 , Med Chem Express), anti-human p-MLKL Ser358 (1:1000, #91689S, Cell Signaling Technology), anti-human p-RIP3 Ser227 (1:1000, #EPR9627, Abcam, Cambridge, UK), or anti-human/mouse β-actin (1:5000) and, subsequently, with a secondary antibody conjugated to HRP (1:20000, Dako, Santa Clara, CA).

Techniques: Derivative Assay, Transfection, Injection, Isolation, Two Tailed Test, Immunofluorescence, Expressing, Staining

Journal: Oncoimmunology

Article Title: Smad7 is a negative regulator of immunogenic cell death in colorectal cancer

doi: 10.1080/2162402X.2025.2490346

Figure Lengend Snippet: Smad7 protein downregulation induces immunogenic cell death. (A-B) Representative image and the relative graph (right panel) showing the volume of CT26-derived tumors in immunocompetent BALB/c mice vaccinated with mitomycin C-treated CT26 cells or Smad7 as transfected CT26 cells. Each point in the graph represents the tumor volume in each mouse. *** p < 0.001. (C-D) Percentage of tumor-free mice vaccinated with mitomycin C-treated CT26 cells or SMAD7 as transfected CT26 cells and challenged after 7 days with CT26 (C) or TS/A (D) cells. Tumor growth was monitored until sacrifice (day 14).

Article Snippet: Membranes were then incubated with the antibodies anti-human/mouse Smad7 (1:1000, #MAB2029, R&D Systems), anti-human/mouse CRT (1:1000, # PA3–900, Thermo Fisher Scientific), anti-human cleaved caspase-3 (1:1000, #9661S, Cell Signaling Technology, Danvers, MA), anti-human HMGB1 (1:1000, #39355, Cell Signaling), anti-human GPX4 (1:1000, #HY- P80692 , Med Chem Express), anti-human p-MLKL Ser358 (1:1000, #91689S, Cell Signaling Technology), anti-human p-RIP3 Ser227 (1:1000, #EPR9627, Abcam, Cambridge, UK), or anti-human/mouse β-actin (1:5000) and, subsequently, with a secondary antibody conjugated to HRP (1:20000, Dako, Santa Clara, CA).

Techniques: Derivative Assay, Transfection