Fig. 6: An OV targeting PD-L1 via extracellular vesicle delivery of shPD-L1 molecules has enhanced therapeutic activity in vivo.

a Immunoblot analysis showing protein expression levels of PD-L1, GAPDH (loading control), and total proteins in B16-F10 cells subjected or not to VSV∆51-amiR-NTC or VSV∆51-shPD-L1 infection (n = 3). b, c Schematic representation (b) of transwell coculture assays designed to assess the transfer of shPD-L1 via infected cell-derived SEVs to uninfected cells. c Immunoblot analysis of PD-L1, GAPDH and VSV protein levels in producer cells (upper compartment) and receiving cells (lower compartment) after 48 h of education by cell-secreted factors derived from VSV∆G-shPD-L1-infected cells (upper compartment). Both uninfected and VSVΔ51-infected cell lysates are included as controls. Immunoblots are representative of three biological replicates. d Kaplan–Meier survival curves of mice bearing subcutaneous (SC) B16-F10 tumours and treated as indicated with vehicle control (PBS) or with VSVΔ51-amiR-NTC control or VSV∆51-shPD-L1 (n = 10 mice per group). Dotted vertical lines indicate virus treatments. Log-rank (Mantel–Cox) test, ****P < 0.0001. e Mice that had completely cleared B16-F10 tumours upon VSV∆51-shPD-L1 treatment (in d) were re-challenged SC with 5 × 10⁵ B16-F10 cells on day 93 (n = 4). Arrows indicate initial VSVΔ51-shPD-L1 treatments. f B16-F10 and TH04 cells were infected with indicated OVs (VSVΔ51-amiR-NTC, VSVΔ51-shPD-L1, VSVΔ51-amiR-4, VSVΔ51-amiR-4-shPD-L1) at MOIs of 0.01 and 0.001, respectively. Cell lysates were collected 48 hpi and prepared for immunoblotting with antibodies against ARID1A, PD-L1, and β-actin (loading control), n = 3. g Schematic showing the bystander effects of VSVΔ51-amiR-4-shPD-L1. In our model, cell death occurs in a two-pronged attack against cancer cells; following OV infection of cancer cells and via synthetic lethal interactions in target cells receiving ARID1A-targeting amiR-4 delivered by SEVs from neighbouring OV-infected cells and systemic administration of the EZH2 methyltransferase inhibitor GSK126. In addition, another bystander effect is induced by shPD-L1-containing SEVs derived from VSVΔ51-shPD-L1-infected cells. While OV infection induces PD-L1 protein levels, expression of an shRNA targeting PD-L1 from an OV backbone decreases PD-L1 to baseline levels and enhances T cell-mediated death of cancer cells. Source data are provided as a Source data file.