Journal: bioRxiv

Article Title: Cytokine-bearing Bacterial Outer Membrane Vesicles with Empowered Efficacy in Intratumoral Immunotherapy

doi: 10.64898/2026.04.02.716109

Figure Lengend Snippet: Representative image of H&E staining MPO immunohistochemistry on FFPE sections of the same tumors described in . CT26 tumors were collected 24 hours after a three intratumoral injections of PBS (50 µL) ( A ) or IL-2-OMVs Δ60 + TNFα-OMVs Δ60 (10 µg in 50 µL PBS) ( B ).( C - D - E ) Flow cytometry analysis of tumors – BALB/c mice were challenged with CT26 and when tumors reached a size of approximately 100 mm 3 mice were treated with one or three doses (two days apart) of the following formulations: PBS (control), 1 μg of OMVs Δ60 , 1 μg of CCL3-OMVs Δ60 , 1 μg of Flt3L-OMVs Δ60 . The day after the treatments, two tumors from each group receiving one dose (Post I) and three tumors from each group receiving three doses (Post III) were surgically removed. Tumor cells (1 x 10 6 ) were incubated with the appropriate fluorescent labelled antibodies and subsequently an alyzed by flow cytometry. Frequencies of regulatory T cells ( C ) γδ T cells ( D ) and LY6C/G + CD11b + myeloid cells ( E ) are calculated within the live, non-aggregated total cell populations. The dotted areas within the bars of panel D indicate the percentage of MHC II-positive γδ T cells. In panel E , each bar is subdivided into three portions of different color intensity. The dark tone represents the fraction of LY6C/G HIGH CD11b MEDIUM cells, the intermediate tone represents the fraction of LY6C/G MEDIUM CD11b HIGH cells and the light tone represents the fraction of myeloid cells excluded by the selected gating parameters. Finally, the dotted areas within each bar represent the fraction of each cell population which is MHC II-positive. The statistical analysis (unpaired, two-tailed Student’s t -test) shown in the right-hand graph refers to the populations of LY6C/G MEDIUM CD11b HIGH cells present in each group (indicated by the lines next to the bars).

Article Snippet: B) 25 μL of the following mixture of fluorescent-labeled antibodies were added to the samples: CD3-APC (BioLegend, San Diego, CA, USA), CD4-BV510 (BioLegend, San Diego, CA, USA), CD8a-PECF594 (BD Bioscience, San Jose, CA, USA), TCRγδ-PE (Miltenyi Biotech, Bergisch Gladbach, Germany), TCRαβ-PEVio770 (Miltenyi Biotech, Bergisch Gladbach, Germany) and MHC II (I-Ek)-VioBright (Miltenyi Biotech, Bergisch Gladbach, Germany).

Techniques: Staining, Immunohistochemistry, Flow Cytometry, Control, Incubation, Two Tailed Test