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Additional file 8. 3FR BiTE treatment triggers wide-ranging increases in immunomodulatory cytokines and chemokines. Cell-free supernatants from two ascites samples treated (or not) for five days with 3Ctrl or 3FR BiTE were assessed using a 13-plex immunoassay. Fold-increases in cytokine/chemokine levels were calculated relative to “Mock”-treated samples. Data show mean ± SD of biological triplicates.

Additional file 8. 3FR BiTE treatment triggers wide-ranging increases in immunomodulatory cytokines and chemokines. Cell-free supernatants from two ascites samples treated (or not) for five days with 3Ctrl or 3FR BiTE were assessed using a 13-plex immunoassay. Fold-increases in cytokine/chemokine levels were calculated relative to “Mock”-treated samples. Data show mean ± SD of biological triplicates.

Additional file 4. CD206- and FRβ BiTE-induced T cell-mediated killing of MDMs is dependent on the perforin pathway. T cells were co-cultured with autologous CFSE-stained MDMs and treated (or not) with the indicated BiTEs for 96 h, at which point % Live cells were calculated with Celigo image cytometry. For inhibition of perforin, T cells were pre-treated for 2 h with Concanamycin A (100 ng/mL), or an equivalent concentration of vehicle control (DMSO), then washed prior to BiTE addition. Inhibitors of Fas/FasL (anti-Fas antibody, clone ZB4, 2 μg/mL) and TRAIL (TRAIL-R1-Fc, 1 μg/mL) were added at the point of BiTE treatment and not removed. Data show mean ± SD of biological triplicates. Statistical significance was assessed by two-way ANOVA followed by Bonferroni post-hoc analysis, with each treatment being compared to the relevant “Mock” condition (*, P

Additional file 1. Dot blot analysis of BiTE/TriTEs. A-C, Two-fold serial dilutions of BiTE/TriTE-containing supernatants from transfected HEK293A cells were concentrated and applied to a nitrocellulose membrane, alongside a 10xHis-tagged protein of known concentration. Membranes were probed with anti-His primary antibody and HRP-conjugated anti-mouse secondary antibody. A, An exemplary dot blot of a 10xHis-tagged protein of known concentration. B, An exemplary standard curve to determine BiTE/TriTE concentration, as generated by measuring dot intensity with ImageJ software. C, Dot blot analyses BiTE/TriTE-containing supernatants.

Additional file 7. Representative flow cytometric dot plots of CD206 and FRβ expression by whole ascites cells from five cancer patients.

Additional file 11. Expression of M1 macrophage markers on ascites from individual patient samples following virus treatment. Total unpurified ascites cells from different patients were infected with 100 vp/cell parental or BiTE-expressing EnAd for 5 days in the presence or absence of autologous fluid. Cells were stained with anti-CD11b, anti-CD64, anti-CD80 and anti-CD86 antibodies, as well as a LIVE/DEAD fixable stain, then analysed by flow cytometry. Fold-changes in geometric mean fluorescence intensity (MFI) values of CD64, CD80 and CD86 on live CD11b+CD64+ ascites cells were calculated relative to “Mock”-treated samples. Data show mean ± SD of biological triplicates. Statistical significance was assessed by two-way ANOVA followed by Bonferroni post-hoc analysis, with each treatment being compared to either “EnAd” alone or, in the case of EnAd, “Mock”. (*, P

Additional file 9. A schematic representation of the EnAd genome encoding a BiTE transgene under the control of the CMV promoter.

Additional file 13. Estimates of virus particles (vp)/mL for purified virus stocks, as quantified by Picogreen and HPLC.

Additional file 10. Supernatants from cells infected with BiTE-armed EnAd trigger T cell-mediated cytotoxicity of macrophages. A,B, DLD-1 cells were infected with the indicated viruses at a dose of 100 virus particles/cell. 72 h later, supernatants were harvested and applied at the indicated dilutions to co-cultures of PBMC-derived T cells and CFSE-stained monocyte-derived macrophages (MDM). A, After four days’ co-culture, MDM killing was assessed with propidium iodide staining and Celigo image cytometry. B, T cell activation was assessed by measuring CD25 expression with flow cytometry. Data show mean ± SD of biological triplicates (A,B). Statistical analysis was performed by two-way ANOVA with Bonferroni post-hoc tests comparing with the relevant “Mock” condition (A), or with one-way ANOVA followed by Dunnett’s post-hoc analysis compared with “Mock”-treated cells (B). (*, P

Additional file 7. Representative flow cytometric dot plots of CD206 and FRβ expression by whole ascites cells from five cancer patients.

Additional file 15. Patient indications for malignant ascites samples used in this study.

Additional file 14. Cellular composition of malignant ascites samples used in Figs. 6 and 7.

Additional file 13. Estimates of virus particles (vp)/mL for purified virus stocks, as quantified by Picogreen and HPLC.

Additional file 11. Expression of M1 macrophage markers on ascites from individual patient samples following virus treatment. Total unpurified ascites cells from different patients were infected with 100 vp/cell parental or BiTE-expressing EnAd for 5 days in the presence or absence of autologous fluid. Cells were stained with anti-CD11b, anti-CD64, anti-CD80 and anti-CD86 antibodies, as well as a LIVE/DEAD fixable stain, then analysed by flow cytometry. Fold-changes in geometric mean fluorescence intensity (MFI) values of CD64, CD80 and CD86 on live CD11b+CD64+ ascites cells were calculated relative to “Mock”-treated samples. Data show mean ± SD of biological triplicates. Statistical significance was assessed by two-way ANOVA followed by Bonferroni post-hoc analysis, with each treatment being compared to either “EnAd” alone or, in the case of EnAd, “Mock”. (*, P

Additional file 15. Patient indications for malignant ascites samples used in this study.

Additional file 14. Cellular composition of malignant ascites samples used in Figs. 6 and 7.

Additional file 2. CD206 and FRβ expression by MDMs. MDMs from healthy donor PBMCs were polarised as indicated and stained with PE-conjugated anti-CD206 or anti-FRβ antibodies, or isotype controls, then analysed by flow cytometry. Representative histograms are displayed.

Additional file 9. A schematic representation of the EnAd genome encoding a BiTE transgene under the control of the CMV promoter.

Additional file 1. Dot blot analysis of BiTE/TriTEs. A-C, Two-fold serial dilutions of BiTE/TriTE-containing supernatants from transfected HEK293A cells were concentrated and applied to a nitrocellulose membrane, alongside a 10xHis-tagged protein of known concentration. Membranes were probed with anti-His primary antibody and HRP-conjugated anti-mouse secondary antibody. A, An exemplary dot blot of a 10xHis-tagged protein of known concentration. B, An exemplary standard curve to determine BiTE/TriTE concentration, as generated by measuring dot intensity with ImageJ software. C, Dot blot analyses BiTE/TriTE-containing supernatants.

Additional file 2. CD206 and FRβ expression by MDMs. MDMs from healthy donor PBMCs were polarised as indicated and stained with PE-conjugated anti-CD206 or anti-FRβ antibodies, or isotype controls, then analysed by flow cytometry. Representative histograms are displayed.