Journal: Journal for Immunotherapy of Cancer

Article Title: In situ endoscopic photodynamic therapy combined with immature DC vaccination induces a robust T cell response against peritoneal carcinomatosis

doi: 10.1136/jitc-2024-009752

Figure Lengend Snippet: PDT induces ferroptosis and immunogenic cell death. ( A–F ) CT26 colorectal carcinoma and AB12 mesothelioma two-dimensional (2D) cell cultures and 3D spheroids were treated with the following drugs: ferrostatin-1 (10 µM), liproxstatin-1 (10 µM), necrostatin-1 (20 µM for 2D and 30 µM for 3D), Zvad-fmk (25 µM for 2D or 40 µM for 3D). After 48 hours pretreatment, cell cultures or spheroids were incubated with PS-OR141 in the dark for 1 hour (2D) or 4 hours (3D) at the indicated concentrations, followed by a washing step. Photoactivation was performed using an LED light source for 60 min (2D) or 90 min (3D) and incubation was continued after the renewal of cell death inhibitors. ( A ) PDT-induced cytotoxicity over time in CT26 spheroids (cytotox green dye, Incucyte) in the presence of different cell death inhibitors. ( B, C ) Dose-response curves depicting the viability of CT26 and AB12 ( B ) 3D spheroid (propidium iodide measurement by flow cytometry) and ( C ) 2D cell monolayers (PrestoBlue) 48 hours and 24 hours post-PDT, respectively, in the presence of 10 µM ferrostatin-1. ( D ) Representative COX2, HSP70, MDA and β-actin immunoblots from CT26 spheroids collected 20 hours post-PDT. ( E ) Extracellular ATP release from CT26 spheroids (expressed as fold change) was determined 30 min post-PDT. ( F ) Heat map of LEGENDplex proinflammatory chemokines (13-plex) released by CT26 spheroids 3 hours post-PDT. N=2. ( G, H ) Prophylactic vaccination model. Mice were injected s.c. with 1×10 6 luc + AB1 2D cells exposed in vitro to photoactivated 100 nM PS-OR141 (or PBS as control) and challenged 1 week later with i.p. injection of live 1×10 6 luc + AB1 cells. ( G ) Tumor burden monitored by bioluminescence (expressed as photons/s/cm 2 /sr) and ( H ) corresponding survival curves. (**p≤0.01, N=8 mice per group). In vitro data ( B, C, E ) are plotted as the means±SEM from 3 independent experiments performed with ≥3 technical replicates (***p≤0.001; ****p≤0.0001; ns, p>0.05); when spheroids are involved, minimum of 6 spheroids were pooled together per condition. Significance was determined by one-way ANOVA with Tukey’s multiple comparison test. Log-rank (Mantel-Cox) test was used for survival curves. ANOVA, analysis of variance; i.p., intraperitoneal; PDT, photodynamic therapy.

Article Snippet: Bead-based immunoassays were analyzed using the NovoCyte Quanteon Flow Cytometer System (four lasers) with LEGENDplex Data Analysis Software Suite (Qognit).

Techniques: Incubation, Flow Cytometry, Western Blot, Injection, In Vitro, Control, Comparison