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

Abstract

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.

Fig. 1. Infection, replication, and oncolytic effects of reovirus jin-3 mutant versus wild-type R124 parental reovirus in prostate cancer cell lines in vitro.

A Detection of viral transcripts (capsid protein S4) by RT-qPCR indicated dose- and time-dependent infection and replication of R124 reovirus (upper row) and jin-3 reovirus (bottom row) in human prostate cancer cells PC-3M-Pro4luc2, DU145, and 22Rv1. Gene expression is represented as 2^−ddCt ± standard error of the mean (SEM), N = 3. Two-way ANOVA. MOI = multiplicity of infection. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. * mock versus reovirus infection. B Confocal microscopy for Sigma-3 viral capsid protein (green) in PC-3M-Pro4luc2 cells indicated dose-dependent and time-dependent immunofluorescent localisation of Sigma-3 viral capsid protein (green) in PC-3M-Pro4luc2 prostate cancer cells that were exposed to R124 wild-type and jin-3 reoviruses. Green Sigma-3 viral capsid protein, blue DAPI (nuclei). Magnification is 63×, scale bar = 25 μm. C Dose-dependent killing of human PC-3M-Pro4luc2, DU145, and 22Rv1 prostate cancer cell lines (cell viability) upon exposure of these cells with R124 wild-type and jin-3 reoviruses for 6 days. *p < 0.05, ***p < 0.001, ****p < 0.0001, ^$p < 0.05,^$$$$p < 0.0001. * mock versus reovirus infection, ^$ R124 versus jin-3. Mean ± standard error of the mean (SEM), N = 3. Two-way ANOVA. MOI multiplicity of infection.

Fig. 2. Reovirus infection and replication in three-dimensional cultures of human prostate cancer.

A Three-dimensional cultures from MSK-PCa1 prostate cancer cells, derived from a bone metastasis [17] were exposed to 10⁷ and 10⁸ plaque forming units (p.f.u.) of R124 or jin-3 reovirus. After 3 days, staining for reovirus (Sigma-3) was observed in the outer cell layers of the 3D cultures, indicating active viral infection and replication. Green Sigma-3 (viral protein), red pan-cytokeratin (tumour cells), blue DAPI (nuclei). Magnification is 63×, scale bar = 25 μm. B-D Three-dimensional cultures of metastatic human prostate cancer were generated and exposed to jin-3 reovirus for 7 and 10 days. Viability assays indicated a significant reduction of cellular viability after exposure to jin-3. Mean ± standard error of the mean (SEM), *p < 0.05, **p < 0.01, ****p < 0.0001. One-way ANOVA.

Fig. 3. jin-3 reovirus infection in ex vivo cultured tumour tissue slices from human prostate cancer cell line-derived xenografts.

A Prostate cancer tissue slices from PC-3M-Pro4luc2 tumours were exposed to 10⁸ p.f.u. jin-3 reovirus for 2, 3, 4, or 7 days. Scoring of Sigma-3 viral protein indicated a time-dependent increase in Sigma-3 score. B Viral infection and replication in ex vivo cultured tissue slices with reovirus. Green Sigma-3 (viral protein), red pan-cytokeratin (tumour cells), blue DAPI (nuclei). Magnification is 63×, scale bar = 25 μM.

Fig. 4. jin-3 reovirus infection in prostate cancer tissue slices derived from patient-derived xenografts (PDX) and primary prostate cancer.

A Scoring of Sigma-3 viral protein in ex vivo infected human prostate cancer tissue slices indicated heterogeneous response to exposure to 10⁸ p.f.u. jin-3 reovirus. B Scoring of Sigma-3 viral protein in ex vivo infected primary prostate cancer material with 10⁸ jin-3 reovirus for 4 days. Green viral capsid protein Sigma-3, red pan-cytokeratin or AMACR (tumour cell markers), blue DAPI (nuclei). Magnification is 63×, scale bar = 25 μm .

Fig. 5. Direct oncolytic effects of jin-3 reovirus in xenograft models of human prostate cancer models.

The effect of intra-tumoural administration of jin-3 in subcutaneously growing human prostate tumours from cell line-derived xenograft (CDX) PC-3M-Pro4luc2 (A–F) and patient-derived xenograft (PDX) model PCa-15.01 (G–J). A Effect of jin-3 reovirus administration on total tumour burden was measured by whole-body bioluminescent reporter imaging (BLI) of firefly-luciferase2 expressing PC-3M-Pro4luc2 cells (n = 6 per group). B Whole-body bioluminescent optical imaging (BLI) at the start and end of the experiment. C Change of tumour burden (BLI) was significantly reduced in jin-3-treated tumours (p < 0.05). D Tumour volume (calliper measurements) was reduced after jin-3 administration. E S4 RNA expression, indicative of viral Sigma-3 gene expression, was observed in PC-3M-Pro4luc2 tumours treated with jin-3 reovirus at day 21 (p < 0.0001). F Histological evaluation depicted a strong oncolytic response, the presence of viral proteins (Sigma-3), a reduction in tumour cell proliferation (PCNA), and a loss of cytokeratins in PC-3M-Pro4luc2 tumours treated with jin-3 reovirus. G Treatment with jin-3 reovirus significantly reduced tumour volume in the PCa-15.01 PDX model (n = 10 per group; p < 0.01). H Significant tumour shrinkage upon intra-tumoural Jin 3 administration (tumour weight; p < 0.05). I In tumours treated with jin-3 reovirus, viral S4 RNA was detected (p < 0.0001). J Histological evaluation of jin-3 reovirus-mediated anti-tumour effects indicated a loss of tissue architecture, the presence of viral proteins (Sigma-3), an induction of apoptosis (cleaved caspase-3), a reduction in proliferation (PCNA), and a loss of tumour-associated cytokeratins (PANKRT). Magnification is 63×, scale bar = 25 μm. Error bars indicated ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA and t tests.

Fig. 6. Immune modulatory response of R124 parental and jin-3 mutant reovirus in prostate cancer cultures.

A The role of the STING pathway and IFN signalling in recognising viral RNA and induction of anti-viral and anti-tumour immune responses. B Induction of IFNβ mRNA in prostate cancer cells after treatment with R124 or jin-3 reovirus for 48 h. C Induction of interferon-stimulated genes (ISGs) in human prostate cancer cells after treatment with MOI10 of R124 or jin-3 reovirus for 48 h (log-transformed). White boxes indicated that the gene was not expressed. D Induction of inflammatory cytokines gene expression of CXCL10, TNFα, and IL-1β and cytosolic RNA sensor RIG-I after treatment of human prostate cancer cells with reovirus. E HMGB1 protein release (danger-associated molecular pattern) by human prostate cancer cells after 48 h treatment with oncolytic reovirus. *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001, ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001, ^$$ p < 0.0001. Mean ± standard error of the mean (SEM), N = 2. Two-way ANOVA.