| Popis: |
Introduction: Extracellular ATP (eATP) is a damage-associated molecular pattern (DAMP) which plays a critical role in the activation of the NLRP3 inflammasome, an important mediator of the innate immune response. Ultrasound (US) and microbubbles (MB) have been shown to release ATP in muscle locally [1]. Our hypothesis is that US+MB could release ATP in tumor microenvironment following by the activation of pro-inflammatory immune response. The eATP release following US+MB and its quantification in muscle and 4T1 model will be presented. Methods: eATP quantification in vitro: 4T1 cells were incubated with D-luciferin (250 µM), luciferase (0.7 µM) and MB (5x106MB/ml) and the eATP signal was measured by bioluminescence (BLI) using Optix MX2. eATP quantification in vivo : We quantified eATP in mice hindlimbs ( $\mathrm{n}=6$ ) and 4T1 subcutaneous tumors ( $\mathrm{n}=8$ ). After IP injection of D-luciferin (3 mg) and IV infusion of luciferase (270 µg) and MB (Definity, 4 µL/min), the mice received US treatment (1 MHz, 5000 cycles, 800 kPa) using a therapeutic probe for 10 min. The therapy was guided by US contrast imaging and the eATP signal was measured by BLI. A positive control was performed by IM administration of ATP (250 µM) in the hindlimb ( $\mathrm{n}=3$ ). Results and conclusion: US+MB treatment released ATP in vitro and the signal increased with the number of cycle and pressure. Following the IM injection of ATP (250 µM), we detected a BLI signal at 4 min that was higher than baseline but was back to baseline level at 8 min. In muscle, After US+MB, the signal was 5.5 folds greater in the treated side compared to the non-treated side at 15 min and persisted until 75 min post-treatment. In tumors, we observed a significant increase in eATP signal post-US+MB in the treated side at 15 min that persisted up to 45 min post-treatment compared to the control side. In conclusion, US+MB treatment releases ATP in muscle and in 4T1 tumors which persisted longer in time compared to a direct intramuscular injection. The immune response characterization in 4T1 tumors after US+MB is in progress. |
