Abstract No. 207 Damage-associated molecular patterns released by irreversible electroporation–treated cancer cells skew macrophages to M2 phenotype

Abstract

To investigate DAMPs released from tumor cells after IRE and their impact on macrophage polarization Mouse bladder cancer cell line (MB49) was treated with IRE (2000 V/cm, 64 pulses, 70 microsecond duration) in a 4mm gap cuvette. DAMPs released into the supernatant by IRE were quantified by measurement of ATP with Cell Titer-Glo assay, and HSP70 and HMGB1 by western blotting. Mouse macrophage cell line (RAW 264.7) was incubated with supernatant from IRE treated cancer cells. Macrophage phenotype was assessed by surface markers for CD11b (pan macrophage), CD80 (M1 macrophage), and CD206 (M2 macrophage) evaluated at 4hours, 24hours and 48 hours. Macrophage survival, proliferation, and motility were assessed using trypan blue staining, CCK-8 and Transwell migration assay, respectively. Macrophage treated with IL-4 and lipopolysaccharide were used as positive controls of M1 and M2 phenotypes, respectively. Mean MB49 cell viability after IRE was up to 1.2% in CCK-8 and 1.4% with cell counting. ATP in the supernatant was significantly higher in IRE group (5.9μM) compared with the untreated control group (1.9μM, P< 0.0001). Relative dosimetry of Western blotting showed that HSP70 and HMGB1 in the supernatant was significantly higher than the untreated control group (13-fold for HSP70; P< 0.05 and 1.8-fold for HMGB1; P< 0.05, respectively). There was no significant difference in macrophage survival and proliferation compared with the untreated control group. Transwell migration assay showed significant migration per field compared with no treatment group (38.0 vs 11.6 cells/field; P< 0.05). After adding supernatant, M2 phenotype skewing is observed over time. There was significantly more M2 macrophages at 24 hours time point compared with no treatment group (49.3 vs 23.4%; P< 0.01). DAMPs released by tumor cells after IRE results in M2 phenotype skewing in macrophages.