Tigilanol tiglate is an oncolytic small molecule that induces immunogenic cell death and enhances the response of both target and non-injected tumors to immune checkpoint blockade.

BACKGROUND: Tigilanol tiglate (TT) is a protein kinase C (PKC)/C1 domain activator currently being developed as an intralesional agent for the treatment of various (sub)cutaneous malignancies. Previous work has shown that intratumoral (I.T.) injection of TT causes vascular disruption with concomitant tumor ablation in several preclinical models of cancer, in addition to various (sub)cutaneous tumors presenting in the veterinary clinic. TT has completed Phase I dose escalation trials, with some patients showing signs of abscopal effects. However, the exact molecular details underpinning its mechanism of action (MoA), together with its immunotherapeutic potential in oncology remain unclear. METHODS: A combination of microscopy, luciferase assays, immunofluorescence, immunoblotting, subcellular fractionation, intracellular ATP assays, phagocytosis assays and mixed lymphocyte reactions were used to probe the MoA of TT in vitro. In vivo studies with TT used MM649 xenograft, CT-26 and immune checkpoint inhibitor refractory B16-F10-OVA tumor bearing mice, the latter with or without anti-programmed cell death 1 (PD-1)/anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) mAb treatment. The effect of TT at injected and non-injected tumors was also assessed. RESULTS: Here, we show that TT induces the death of endothelial and cancer cells at therapeutically relevant concentrations via a caspase/gasdermin E-dependent pyroptopic pathway. At therapeutic doses, our data demonstrate that TT acts as a lipotoxin, binding to and promoting mitochondrial/endoplasmic reticulum (ER) dysfunction (leading to unfolded protein responsemt/ER upregulation) with subsequent ATP depletion, organelle swelling, caspase activation, gasdermin E cleavage and induction of terminal necrosis. Consistent with binding to ER membranes, we found that TT treatment promoted activation of the integrated stress response together with the release/externalization of damage-associated molecular patterns (HMGB1, ATP, calreticulin) from cancer cells in vitro and in vivo, characteristics indicative of immunogenic cell death (ICD). Confirmation of ICD in vivo was obtained through vaccination and rechallenge experiments using CT-26 colon carcinoma tumor bearing mice. Furthermore, TT also reduced tumor volume, induced immune cell infiltration, as well as improved survival in B16-F10-OVA tumor bearing mice when combined with immune checkpoint blockade. CONCLUSIONS: These data demonstrate that TT is an oncolytic small molecule with multiple targets and confirms that cell death induced by this compound has the potential to augment antitumor responses to immunotherapy.

Use of Bacteriophages to Control Vibrio Contamination of Microalgae Used as a Food Source for Oyster Larvae During Hatchery Culture.

Cultured microalgae are the primary food source for oyster larvae during hatchery culture and are a potential vector for Vibrio spp. infection of larval cultures. Bacteriophages have shown potential for controlling contamination of Vibrio spp. in aquaculture systems and their application could be an effective biological control method to eliminate such bacterial contamination of microalgae. This study investigated whether Vibrio-free microalgae sources could be ensured via the application of Vibrio specific phages. As a first step, four different Vibrio bacteriophages (belonging to the Myoviridae viral family) were isolated from marine waters in Queensland, Australia and used in challenge tests against a Vibrio host species, previously isolated from New South Wales oyster hatchery and found to be closely related to V. alginolyticus (ATCC 17749). The genome sequence of one of the four isolated bacteriophages, Vibrio Φ-2, that displayed strongest virulence against the host was determined. The 242446 bp genome of this bacteriophage was predicted to encode 217 proteins with an average GC content of 43.91%, containing putative thymidine kinases and a lysin enzyme. Application of these bacteriophages to pathogenic Vibrio spp. contaminating microalgae suspensions resulted in significant decreases in their numbers within 2 h. Findings indicated that direct application of bacteriophages to microalgae suspensions could be an effective method of reducing the occurrence of vibriosis in oyster hatcheries.

In vitro antibacterial phenolic extracts from "sugarbag" pot-honeys of Australian stingless bees (Tetragonula carbonaria).

Australian stingless bee honeys have been shown to exert antioxidant and in vitro antimicrobial properties; however their bioactive factors remained unidentified. This study investigated the antibacterial properties of phenolic extracts from Tetragonula carbonaria honeys. Honeys were harvested from beehives in three sites of South East Australia. Liquid-liquid extractions yielded the phenolic concentrates, for analyses by liquid and gas chromatography mass spectrometry. Antibacterial assays were conducted against Staphylococcus aureus and Klebsiella pneumoniae by in vitro agar diffusion and broth dilution assays. The phenolic extracts averaged to 5.87 mg/100 g of raw honeys, and constituents were 3-phenyllactic acid, lumichrome, diglycosylflavonoids, norisoprenoids. The honeys did not contain methylglyoxal, dihydroxyacetone or phenolics characteristic of Leptospermum nectars. Hydrogen peroxide content amounted up to 155.8 µM in honeys. Beside the bactericidal effects of hydrogen peroxide at 760 µM, other antibacterial factors were the phenolic extracts of "sugarbag" honeys that were active at minimum bactericidal concentrations of 1.2-1.8 mg/mL.