Ancient role of vasopressin/oxytocin-type neuropeptides as regulators of feeding revealed in an echinoderm.

BACKGROUND: Vasopressin/oxytocin (VP/OT)-type neuropeptides are well known for their roles as regulators of diuresis, reproductive physiology and social behaviour. However, our knowledge of their functions is largely based on findings from studies on vertebrates and selected protostomian invertebrates. Little is known about the roles of VP/OT-type neuropeptides in deuterostomian invertebrates, which are more closely related to vertebrates than protostomes. RESULTS: Here, we have identified and functionally characterised a VP/OT-type signalling system comprising the neuropeptide asterotocin and its cognate G-protein coupled receptor in the starfish (sea star) Asterias rubens, a deuterostomian invertebrate belonging to the phylum Echinodermata. Analysis of the distribution of asterotocin and the asterotocin receptor in A. rubens using mRNA in situ hybridisation and immunohistochemistry revealed expression in the central nervous system (radial nerve cords and circumoral nerve ring), the digestive system (including the cardiac stomach) and the body wall and associated appendages. Informed by the anatomy of asterotocin signalling, in vitro pharmacological experiments revealed that asterotocin acts as a muscle relaxant in starfish, contrasting with the myotropic actions of VP/OT-type neuropeptides in vertebrates. Furthermore, in vivo injection of asterotocin had a striking effect on starfish behaviour-triggering fictive feeding where eversion of the cardiac stomach and changes in body posture resemble the unusual extra-oral feeding behaviour of starfish. CONCLUSIONS: We provide a comprehensive characterisation of VP/OT-type signalling in an echinoderm, including a detailed anatomical analysis of the expression of both the VP/OT-type neuropeptide asterotocin and its cognate receptor. Our discovery that asterotocin triggers fictive feeding in starfish provides important new evidence of an evolutionarily ancient role of VP/OT-type neuropeptides as regulators of feeding in animals.

PD-1 Blockade Following Isolated Limb Perfusion with Vaccinia Virus Prevents Local and Distant Relapse of Soft-tissue Sarcoma.

PURPOSE: The prevention and treatment of metastatic sarcoma are areas of significant unmet need. Immune checkpoint inhibitor monotherapy has shown little activity in sarcoma and there is great interest in identifying novel treatment combinations that may augment responses. In vitro and in vivo, we investigated the potential for an oncolytic vaccinia virus (GLV-1h68) delivered using isolated limb perfusion (ILP) to promote antitumor immune responses and augment response to PD-1 blockade in sarcoma.Experimental Design: In an established animal model of extremity sarcoma, we evaluated the potential of locoregional delivery of a vaccinia virus (GLV-1h68) alongside biochemotherapy (melphalan/TNFα) in ILP. Complementary in vitro assays for markers of immunogenic cell death were performed in sarcoma cell lines. RESULTS: PD-1 monotherapy had minimal efficacy in vivo, mimicking the clinical scenario. Pretreatment with GLV-1h68 delivered by ILP (viral ILP) significantly improved responses. Furthermore, when performed prior to surgery and radiotherapy, viral ILP and PD-1 blockade prevented both local and distant relapse, curing a previously treatment-refractory model. Enhanced therapy was associated with marked modulation of the tumor microenvironment, with an increase in the number and penetrance of intratumoral CD8+ T cells and expansion and activation of dendritic cells. GLV-1h68 was capable of inducing markers of immunogenic cell death in human sarcoma cell lines. CONCLUSIONS: Viral ILP augments the response to PD-1 blockade, transforming this locoregional therapy into a potentially effective systemic treatment for sarcoma and warrants translational evaluation.

ATR Inhibition Potentiates the Radiation-induced Inflammatory Tumor Microenvironment.

PURPOSE: ATR inhibitors (ATRi) are in early phase clinical trials and have been shown to sensitize to chemotherapy and radiotherapy preclinically. Limited data have been published about the effect of these drugs on the tumor microenvironment.Experimental Design: We used an immunocompetent mouse model of HPV-driven malignancies to investigate the ATR inhibitor AZD6738 in combination with fractionated radiation (RT). Gene expression analysis and flow cytometry were performed posttherapy. RESULTS: Significant radiosensitization to RT by ATRi was observed alongside a marked increase in immune cell infiltration. We identified increased numbers of CD3+ and NK cells, but most of this infiltrate was composed of myeloid cells. ATRi plus radiation produced a gene expression signature matching a type I/II IFN response, with upregulation of genes playing a role in nucleic acid sensing. Increased MHC I levels were observed on tumor cells, with transcript-level data indicating increased antigen processing and presentation within the tumor. Significant modulation of cytokine gene expression (particularly CCL2, CCL5, and CXCL10) was found in vivo, with in vitro data indicating CCL3, CCL5, and CXCL10 are produced from tumor cells after ATRi + RT. CONCLUSIONS: We show that DNA damage by ATRi and RT leads to an IFN response through activation of nucleic acid-sensing pathways. This triggers increased antigen presentation and innate immune cell infiltration. Further understanding of the effect of this combination on the immune response may allow modulation of these effects to maximize tumor control through antitumor immunity.