Figure 1. Antitumor activity of PEDD of nelitolimod on LM was enhanced by checkpoint inhibitors irrespective of delivery route. (A) Schematic representation of the experimental procedures. 8–12-week-old C57/BL6 mice were challenged with 106 MC38-Luc cells/mouse and delivered via spleen followed by splenectomy. After 7 days mice were treated with nelitolimod via PEDD in combination with α-PD-1 administered Sys or SQ on days mentioned in the schema. (B) Bioluminescence values were determined by IVIS on D0, D2, D4, D7 and D10. Phosphate-buffered saline delivered via PV (Portal Vein) using PEDD served as Veh control. Fold change of the tumor burden was calculated based on D0 baseline bioluminescence (total flux/sec). Two-way ANOVA with Tukey’s multiple comparison test was performed to compare the tumor progression among the groups. (C) Mice were sacrificed on D10, and representative images (n=3–4 per group) depicting the bioluminescence at D10 and gross images of the harvested livers. (D) i Representative H&E images show (a–d) the morphology of tumor-bearing liver tissue followed by treatment (n=3); (e–h) depicting portal tract inflammation (white arrowheads) (n=3); (i–l) demonstrating lobular inflammation (black arrowheads) (n=3). ii Graphical representation of tumor burden data, iii table showing the presence or absence of portal tract or lobular inflammations within tumor-bearing liver treated with nelitolimod±α-PD-1, respectively. One-way ANOVA was performed to determine statistical differences among multiple groups. Data presented as the presence and absence of inflammation in the tissue obtained from representative mice per group are mentioned in the figure. Results are representative of at least three independent experiments with the cumulative n reported in the respective figures. Data is presented as mean±SEM; and p value is mentioned in the graph. ANOVA, analysis of variance; IVIS, in vivo imaging system; PEDD, pressure-enabled drug delivery; PD-1, programmed cell death-1; Veh, vehicle.
