Pulse mode irradiation regimen of PDT results in high progression free and overall survival in mice with model tumour

Abstract

Focused Ultrasound-induced Mechanical Ablation (boiling histotripsy) in the treatment of solid tumors; (b) examine the relationship between the degree of mechanical damage induced by boiling histotripsy and the level of immune activity, and (c) finally to provide a better understanding of the effects of boiling histotripsy on immune response. Methods: In vitro 3D model system was used to investigate the effects of histotripsy on tumor immune microenvironment. MDA-MB-231 tumor spheroids were made with 10,000 cells for 72hrs and embedded in collagen gels (6mg/ml). Cell Death Pathway array was performed to determine the mechanism of tumor death. Proteome cytokine array was performed to identify antigenic factors that were secreted by damaged solid tumors. Macrophage polarization induced by immunogenic cell death was examined using qPCR array. Results: Our results showed that cancer cells (MDA-MB-231) underwent immuno-genic cell death via TNF-mediated necrosis signaling pathway after the boiling histo-tripsy exposure. Significant increases in secretions of damage-associated molecular patterns (CRT, HSP70, HMGB-1), pro-inflammatory cytokines (IFN-c, IL-1a, IL-1b, IL-18) and chemokines (IL-8) which were related to M1 macrophage activation were also observed. Furthermore, the levels of these signaling proteins increased with the degree of mechanical damage induced by the boiling histotripsy. Conclusions: We demonstrate that histotripsy causes the mechanical destruction of cancer cells and promotes immunogenic cell death, ultimately leading to an anticancer immune response. Background: The NOD2 agonist muramyl-dipeptide (MDP), a peptidoglycan motif common to all bacteria, supports LGR5þ intestinal stem cells (ISCs) survival through NOD2 activation upon an otherwise lethal oxidative-stress-mediated signal. Yet the underlying protective mechanisms remains unknown. Methods: Irradiation was used as stressor and primarily murine-derived intestinal organoids as a model system. Results: MDP induced a strong reduction of total and mitochondrial reactive oxygen species (ROS) within ISCs, which was associated with mitophagy induction. ATG16L1 KO and NOD2 KO organoids did not benefit from the MDP-induced cytoprotection. We showed the MDP-dependent induction of ISC mitophagy upon stress in vivo. Conclusions: In LGR5þ ISCs, NOD2 allows irradiation-generated ROS clearance through ATG16L1-activated mitophagy. Legal entity responsible for the study: The authors. Background: Photodynamic therapy (PDT) is one of actively developing modalities of cancer treatment. During PDT procedure systemically injected photosensitizer molecules are activated by laser irradiation in tumor location leading to generation of singlet oxygen which induces cytotoxic effect. In modern clinical practice PDT uses continuous wave irradiation. In such mode fast depletion of molecular (triplet) oxygen in tissue occurs leading to reduction of PDT efficiency. Previously we have shown that photody-namic treatment using pulse mode irradiation regimens could be more efficient in damaging tumor cells than standard PDT due to reoxygenation (Klimenko et. al., Photodiagnosis Photodyn Ther., 2016). In present study we have evaluated specially designed pulse mode PDT regimes on tumor model in vivo. Methods: PDT pulse mode irradiation regimens were designed using physics and mathematical modelling. BALB/c male mice were subcutaneous injected into right flanks with CT26 murine colon carcinoma cells. At day ten after inoculation mice were randomized into 3 groups with 10 animals in each (control group, PDT at continuous wave irradiation mode-CW group, PDT at pulse irradiation mode-PM group) and PDT was made. The Radachlorins R photosensitizer and «Lahta-Milon» semiconductor laser (662 nm) were used. After treatment the tumor size was estimated every two days. All experiments were performed in accordance with the animal ethics guidelines. Results: We have designed new pulse mode irradiation regimens of PDT. We have shown that antitumor efficiency of PDT at pulse irradiation mode is much better than one at standard continuous wave irradiation mode. Median survival after day of treatment was 14 days in control group, 16 days in CW group and 28 days in PM group. In PM group 5 animals had total regress of tumor and progression free survival till the end of experiment (100 days after treatment) compared with only 1 animal in CW group. Thus, we can suggest that overall survival should be much higher in PM group. Conclusions: We have demonstrated that PDT using pulse mode irradiation regimens is much more effective that PDT using standard regimens and leads to higher progression free and overall survival in mice with CT26 colon carcinoma. Background: Histone acetylation plays an important role in regulating the chromatin structure and is tightly regulated by histone acetyltransferases (HAT) and histone deacetylases (HDAC). Recent studies have demonstrated that several chromatin-regulating proteins can modulate cellular responses to other cytotoxic modalities including ionizing radiation and chemotherapeutic drugs. Even though histone acetylation is one of the key mechanisms of epigenetic regulation, relatively little is known about the cancer therapeutic potential of HAT inhibitors. This is in stark contrast to the well-studied effects of HDAC inhibitors. In this study, we investigated the proton beam-sensitizing effect of C646, a selective small molecule inhibitor of p300 histone acetyltransferase in human pancreatic cancer cells. Methods: Cell viability assay (CCK-8; Dojindo), Clonogenic survival assay, Cell cycle analysis (PI staining). Annexin V-FITC and PI staining, c-H2AX foci, Western blot, BxPC-3 Xenograft tumor model, TUNEL assay. Results: AsPC-1, BxPC-3 and Mia-paca2 cells exhibited increase in radiosensitivity when exposed to C646 at all doses of proton beam tested. C646 pre-treatment induce led to increase of sub-G1 population and abolishment of G2/M arrest. Flow cytometry analysis with annexin V staining and Western blot analysis showed that The pre-treatment with C646 significantly enhanced proton-induced apoptotic cell death through the down-regulation of anti-apoptotic molecules. Our in vivo results demonstrate syn-ergistic effects of combination therapy with C646 and proton beam. In BxPC-3 xeno-graft tumor model, C646 pre-treatment increased proton-induced tumor growth inhibition and apoptotic cell death in tumor tissues. In addition, the combination treatment of C646 with proton beam increased DNA damage and decreased activation of DNA repair pathway compared with proton alone. Conclusions: Our results suggest that the C646 may increase proton-induced apopto-sis through the modulation of various pro-and anti-apoptotic molecules in human pancreatic cancer cells. These results provide proof of principle that the inhibition of histone acetylation by HATis can be exploited to develop new proton-sensitizer.