Reovirus mutant jin-3 exhibits lytic and immune-stimulatory effects in preclinical human prostate cancer models

Treatment of castration-resistant prostate cancer remains a challenging clinical problem. Despite the promising effects of immunotherapy in other solid cancers, prostate cancer has remained largely unresponsive. Oncolytic viruses represent a promising therapeutic avenue, as oncolytic virus treatment combines tumour cell lysis with activation of the immune system and mounting of effective anti-tumour responses. Mammalian Orthoreoviruses are non-pathogenic human viruses with a preference of lytic replication in human tumour cells. In this study, we evaluated the oncolytic efficacy of the bioselected oncolytic reovirus mutant jin-3 in multiple human prostate cancer models. The jin-3 reovirus displayed efficient infection, replication, and anti-cancer responses in 2D and 3D prostate cancer models, as well as in ex vivo cultured human tumour slices. In addition, the jin-3 reovirus markedly reduced the viability and growth of human cancer cell lines and patient-derived xenografts. The infection induced the expression of mediators of immunogenic cell death, interferon-stimulated genes, and inflammatory cytokines. Taken together, our data demonstrate that the reovirus mutant jin-3 displays tumour tropism, and induces potent oncolytic and immunomodulatory responses in human prostate cancer models. Therefore, jin-3 reovirus represents an attractive candidate for further development as oncolytic agent for treatment of patients with aggressive localised or advanced prostate cancer.Experimental cancer immunology and therap

Generation and exploitation of stem cell derived organoid cultures

Following the identification of the intestinal epithelium stem cell (ISC) Barker et al. 2007 by Lgr5, the transcriptome of the ISC was defined using microarray technology. One of the genes specifically expressed by ISC’s is Nr2e3. Originally Nr2e3 was identified as a retinal specific gene, where it fulfills a crucial function in cone and rod photoreceptor progenitor cells. We postulated that Nr2e3 might mark novel stem cell populations and has a function in stem cell maintenance. Therefore we generated a mouse model were eGFP and the recombinase CreERT2 are controlled by endogenous DNA sequences of Nr2e3 (Nr2e3KI). The Nr2e3KI mouse model allows detection and isolation of Nr2e3+ve cells by eGFP expression, and enabled us to perform lineage-tracing studies when crossed with a Cre reporter line (Chapter 2). Analysis of eGFP expression revealed restricted Nr2e3+ve cell populations in a variety of organs, including the Small intestine, lung, eye and stomach. Lineage tracing revealed that none of the novel Nr2e3+ve populations represent stem cells. Moreover we show Nr2e3 is indispensible for retinal progenitor cells. How disturbances in the intestinal milieu i.e. commensal bacteria influence Lgr5+ve stem cells function is not well understood. In chapter 3 we describe the generation of a mouse model where the whole regenerating gene family is knocked-out. The regenerating (Reg) gene family is composed of highly homologous, secreted proteins containing a c-type lectin-binding domain and are known to have a bactericidal function. Several members of the regenerating gene family are expressed in the small intestine. Future studies will elucidate the role of these proteins in the intestine and their influence of homeostasis and stem cell function. Based on the requirements of Lgr5+ve ISC, a novel culture method for intestinal epithelium was developed by Sato et al. called organoid culture. In organoid culture the small intestinal epithelium is composed of a defined crypt and villus region and contains all intestinal epithelial celltypes that exist in vivo (Stem cells, Paneth cells, Goblet cells, Enteroendocrine cells and tuft cells). This system allows for the study of the epithelium in an isolated fashion, which could lead to novel insights into diseases with a complex etiology like inflammatory bowel disease. In chapter 4 we study the effects of a key inflammatory factor interferon gamma (Ifnγ) on the intestinal epithelium using the organoid culture. We found that Ifnγ uniquely acts as a degranulation agent for Paneth cells in the intestine. In contrast to prior findings we find that pathogen associated molecular patterns (PAMPs) do not induce Paneth cell degranulation. We find that PAMP activated immune cells act as a critical mediator in Ifnγ induced degranulation of Paneth cells. Based on the small intestinal organoid system, similar culture methods were developed for other gastrointestinal organs. In chapter 5 we describe the development of the first non-gastrointestinal organoid culture, one of the prostate. Organoids derived from the murine prostate can be cultured in the presence or androgens and are composed of the two distinct epithelial lineages, luminal and basal, that are also present in vivo. Moreover murine organoids mimicked in vivo phenotypes of murine models of prostate cancer (PCa) and are easily manipulated with chemical compounds and lentivirusses, enabling future studies for novel PCa genes. Next we developed a culture system that allows for the growth for human prostate epithelium. Human prostate organoids are also composed of distinct luminal and basal epithelial cells. We show that prostate organoids are responsive to clinically approved anti-androgens, Casodex and Enzalutamide. Next we tried to identify stem cell populations in the prostate (Chapter 6). From the transcriptome of Lgr5+ve ISC, Tnfrsf19 was found to be an ISC specific gene. Using the Tnfrsf19KI mouse generated by Stange et al., were eGFP and CreERT2 are controlled by endogenous Tnfrsf19 DNA sequences, we defined the expression pattern of Tnfrsf19 in the murine prostate. Tnfrsf19 is expressed in distinct luminal and basal cell populations. By crossing the Tnfrsf19KI mouse with a Cre reporter line, we show that in vivo luminal Tnfrsf19+ve serve as unipotent progenitor cells for the luminal lineage. But when single luminal Tnfrsf19+ve cells are bipotent (i.e. capable of generating both luminal and basal lineage) when placed in organoid culture conditions