Journal: iScience
Article Title: TAMs-specific ARF1 inhibition reprograms glioma microenvironment and enhances the therapeutic effect of oncolytic adenovirus
doi: 10.1016/j.isci.2025.112696
Figure Lengend Snippet: TAMs specific suppression of ARF1 expression reprograms TAMs (A) Cartoon shows the structure of oncolytic adenovirus carrying macrophage-specific ARF1 inhibition cassette. (B and C) RAW264.7 cell line was infected by oncolytic adenovirus carrying a macrophage-specific ARF1 knockdown element or control virus, the expression of ARF1 (B) and inflammatory genes (C) was analyzed by RT-PCR. (D–F) GL261-OVA-luci cell line was inoculated into the right brain of C57BL6 mice, seven days after cell inoculation, indicated virus or PBS was injected intratumorally every two days for three times. (D and E) The growth of tumor. The size of tumor in every mouse was detected at 21 days after tumor cell inoculation using an in vivo imaging system. (F) The survival of mice with different treatments was analyzed. (G–J) Mice were sacrificed on day 15 post the last treatment, immune cells were isolated, counted and analyzed by flow cytometry. Tumoral tissues were embedded in OCT, sectioned and analyzed by immunofluorescence staining. (G) TAM-specific knockdown of ARF1 increases the infiltration of immune cells. (H and I) TAM-specific knockdown of ARF1 reprograms TAMs into a pro-inflammatory state. Infection of TAMs was verified by immunostaining (H), Scale bars: 100 μm. F4/80+GFP+ cells were sorted from the total immune cells as TAMs, and the expression of inflammatory genes was analyzed by RT-PCR (I). (J) TAMs specific inhibition of ARF1 increases the percentage of CD8 + T cells in tumor infiltrated immune cells. (K) TAMs-specific inhibition of ARF1 enhances tumor antigen-specific immune response. Tumor-infiltrating CD8 + T cells were isolated and co-cultured with dendritic cells pre-loaded with OVA peptides, the secretion of IFN-γ was analyzed by ELISA. All results are presented as means ± SD.
Article Snippet: Rat anti Mouse CD8 α Monoclonal Antibody (53-6.7) , Thermo Fisher (America) , Cat#A15386; RRID: AB_2534400.
Techniques: Expressing, Inhibition, Infection, Knockdown, Control, Virus, Reverse Transcription Polymerase Chain Reaction, Injection, In Vivo Imaging, Isolation, Flow Cytometry, Immunofluorescence, Staining, Immunostaining, Cell Culture, Enzyme-linked Immunosorbent Assay
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