Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Gut microbial metabolite butyrate boosts p53-expressing telomerase-specific oncolytic adenovirus efficacy by enhancing infectivity and activating MHC-I/cGAS-STING

doi: 10.1007/s00262-025-04252-4

Figure Lengend Snippet: Enhancement of viral infectivity by butyrate A CT26, MC38, SW48, and HCT116 cells were treated with Ad-GFP (1000 MOI for CT26 and MC38, 100 MOI for SW48 and HCT116) with or without butyrate (2 mM) for 24 h, and were observed by fluorescence microscopy (n = 3–4). The lower panels show magnified views of the areas outlined by the red squares in the middle panels. Relative GFP intensity in butyrate-treated cells compared with mock-treated cells was measured in a randomly selected field in each well. Scale bar, 500 μm (upper and middle panels); 50 μm (lower panels). *, p < 0.05. **, p < 0.01. B HCT116 subcutaneous tumors were treated with Ad-GFP (5 × 10 7 PFU) intratumoral injection for 2 days with or without butyrate (1 mM) oral administration, starting 2 days prior to Ad-GFP injection (n = 6). Frozen sections of harvested tumors with or without butyrate treatment were observed by fluorescence microscopy, and GFP intensity was measured in three randomly selected fields in each tumor. Scale bar, 200 μm. *, p < 0.05. C CT26, MC38, SW48, and HCT116 cells were treated with butyrate (2 mM) for 2 days, and analyzed with flow cytometry for CAR, αvβ3, and αvβ5 (n = 3). MFI, mean fluorescence intensity. D HCT116 subcutaneous tumors were treated with or without butyrate (1 mM) oral administration for 7 days, and whole-cell lysates of the harvested tumors were analyzed by western blot for CAR and β-actin. Relative intensity of CAR compared with β-actin is indicated below the CAR bands. E MC38 and HCT116 cells were treated with Ad-GFP (1000 MOI for MC38, 100 MOI for HCT116) with or without butyrate (2 mM) for 24 h. Anti-CAR antibody was administered 2 h prior to Ad-GFP infection. These cells were observed by fluorescence microscopy. Scale bar, 50 μm. F SW48 and HCT116 cells were treated with butyrate (2 mM) and/or OBP-702 (10 MOI) for 0, 12, 24, or 48 h, and the whole-cell lysates were analyzed by western blot for p53, E1A, and β-actin. Relative intensity of E1A compared with β-actin is indicated below the E1A bands. G HCT116 subcutaneous tumors were treated with OBP-702 (5 × 10 7 PFU) intratumoral injection for 2, 12, 24, or 48 h with or without butyrate (1 mM) oral administration, starting 2 days prior to OBP-702 injection. Whole-cell lysates of the harvested tumors were analyzed by western blot for p53, E1A, and β-actin. Relative intensity of E1A compared with β-actin is indicated below the E1A bands

Article Snippet: Flow cytometry was performed for CAR (D3W3G, Cell Signaling Technology), αvβ3 (bs-1310R, Bioss antibodies, Woburn, MA, USA), αvβ5 (bs-1356R, Bioss Antibodies), and MHC-I (BE0077, Bio X Cell, Lebanon, NH, USA) using FACS Array and BD FACS Aria (BD Biosciences, San Jose, CA, USA), and analysis was performed using FlowJo software (BD Biosciences).

Techniques: Infection, Fluorescence, Microscopy, Injection, Flow Cytometry, Western Blot