Improved oncolytic and immunostimulatory activity of the spontaneous jin-3 reovirus mutant in preclinical bladder cancer models.

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Introduction

Urothelial carcinoma of the bladder (UCB) is the seventh most prevalent cancer globally, with a 5-year prevalence surpassing 5 million cases. 1 Approximately 75% of newly diagnosed cases encompass non-muscle invasive bladder cancer (NMIBC), which are usually removed by transurethral resection. 2 Patients are then categorized as low, intermediate, or high risk for recurrence and progression according to the guidelines of the European Urological Association. 3 Current therapy for patients with intermediate- and high-risk NMIBC includes immunotherapy with Bacillus Calmette-Guérin (BCG) or chemotherapy. To date, BCG instillations have proven to be the most successful adjuvant therapy. 3 , 4 However, 30%–50% of the patients fail to respond to BCG therapy, and 15% of patients show progression to the muscle invasive disease, MIBC. 2 , 5 , 6 These MIBC patients have a relatively poor prognosis (5-year survival of 40%–60%). UCB recurs frequently after initial treatment and requires long-term clinical monitoring, causing substantial morbidity and diminished quality of life. 4 , 7 , 8 Immunotherapy (including immune checkpoint inhibition [ICI], cell-based therapy, and cancer vaccines) is most effective in tumors with relatively high somatic mutation rates as is the case for UCB. 9 , 10 , 11 , 12 , 13 , 14 , 15

ICIs, including pembrolizumab, nivolumab, atezolizumab, durvalumab, and avelumab, have improved the treatment landscape for bladder cancer, particularly for metastatic urothelial carcinoma. 16

These ICIs have shown impressive and durable responses in bladder cancer patients. 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 In high-risk NMIBC patients who are unresponsive to BCG, 41% of patients display a clinically desirable response to pembrolizumab (i.e., absence of high-risk NMIBC or progressive disease after 3 months). 29 About 30% of patients with metastatic MIBC will respond to ICI treatment (objective response rate and overall survival), 30 although these benefits appear to be limited to patients with pre-existing anti-tumor responses. 25 , 31 , 32 , 33

Currently, ICIs are standard-of-care options in multiple clinical scenarios: as first-line therapy for cisplatin-ineligible patients, second-line therapy after platinum-based chemotherapy failure, and in novel combination regimens.

Despite these encouraging results in a subgroup of UCB patients, treatment modalities that can further improve immunotherapy are urgently required for unresponsive bladder cancer patients.

Oncolytic viruses represent a possible new approach since these viruses can infect, replicate, and lyse malignant tumor cells, while minimizing harm to normal cells. 34 Furthermore, oncolytic viruses can activate the adaptive and innate immune system 35 , 36 , 37 , 38 via the induction of immunogenic cell death (ICD), a type of cell death that triggers immunity against tumor antigens of dying cells, resulting in the exposure of damage-associated molecular patterns (DAMPs), e.g., HMGB1 release. 39 , 40 DAMPs can activate antigen-presenting cells, which subsequently can stimulate the expansion and activation of effector T cells, including cytotoxic CD8+, helper CD4+ T cells, and natural killer (NK) cells. 41 , 42

Reoviruses are double-stranded RNA viruses and have not been associated with severe disease in humans. 43 Mutant reovirus jin-3 harbors a mutation in the spike protein Sigma-1 and can infect cells via the high-affinity receptor junction adhesion molecule A (JAM-A) but also independently of JAM-A on the tumor cell surface by high-affinity binding to negatively charged sialic acids. We previously demonstrated that jin-3 reoviruses display extended tumor tropism compared to the wild-type reovirus. 44 , 45 Interestingly, this JAM-A protein is involved in various biological processes including tight junctions involved in epithelial barrier function. 46 Expression levels of JAM-A in various cancers have been analyzed using multiple databases, including TIMER2.0, GEPIA2, and the UALCAN portal and was shown to be upregulated in multiple cancers. 47 JAM-A was significantly upregulated in urothelial carcinoma compared to normal bladder. However, bladder cancer patients with low JAM-A expression showed decreased overall survival. 47 Potentially, this reduced overall survival could be due to the acquisition of a more mesenchymal invasive phenotype (EMT) by the cancer cells due to the shedding of epithelial tight junctions. 47 Mutational profile analysis (cBioportal based on TCGA datasets) revealed that JAM-A was altered in more than 15% of bladder cancer patients experiencing either amplification or mutation of JAM-A. 47

In this study, the direct oncolytic and indirect immunomodulatory effects of reovirus mutant jin-3 were determined and compared to those of the wild-type T3D reovirus R124 in state-of-the-art preclinical bladder cancer models, including monolayer cultures and three-dimensional cell (co-) cultures and ex vivo cultured bladder tumor tissue slices derived from patients with disease ranging from low-grade NMIBC to MIBC.

Results

Direct cytolytic effects of jin-3 and R124 reoviruses

A panel of human bladder cancer cell lines of different molecular subtypes was exposed to a dose range of either R124 or jin-3 , and viral expression levels were detected by RT-PCR after 24 h. In the UM-UC-3, HT-1197, and RT-4 cell lines, more viral transcripts were detected in the cells exposed to jin-3 , whereas in the T24 and RT-112 cell lines either no difference or less viral transcripts were found ( Figures 1 A and S1 A–S1C; Table S1 ). In addition, viral protein levels of Sigma-3 were determined in various tumor cells by flow cytometry ( Figure 1 B) or immunofluorescence ( Figure 1 C) after 72 h of treatment. A similar Sigma-3 pattern was observed, i.e., increased level of expression in jin-3 -treated UM-UC3, HT-1197, and RT-4 and a similar expression level of Sigma-3 in T24 and RT-112 cell lines. Figure 1 Comparison of the oncolytic effects of mutant jin-3 versus R124 reovirus in bladder cancer cell lines in vitro (A) Mean viral S4Q copy number (log fold change S4Q mRNA expression vs. mock treated cells) in bladder cancer cell lines exposed to a range of MOI (0.01–0.1–1–10) of either R124 or jin-3 reoviruses after 24 h. In red, the highest infection with virus is shown. Two-way ANOVA followed by Tukey’s post hoc test. n = 3 (2 replicates). UM-UC-3 cells R124 reovirus MOI1 vs. mock p = 0.0184; ∗∗∗ p < .001, $$$ p < .001, asterisks indicate mock versus reovirus infection, and dollar signs indicate R124 versus jin-3. (B) Viral load (% of viable, single cells expressing Sigma-3 protein) in bladder cancer cell lines exposed to MOI10 of either R124 or jin-3 after 72 h mean (SD) ( N = 3). Two-way ANOVA followed by Tukey’s post hoc test. ∗∗∗ p < .001, $$$ p < .001, asterisks indicate mock versus reovirus infection, and dollar signs indicate R124 versus jin-3. (C) Confocal images of viral protein (Sigma-3, green) and DAPI (blue)-stained bladder cancer cells treated with MOI10 of R124 or jin-3 for 72h. Scale bars, 25 μm. (D) Mean percentage of viable cells after exposure to a range of MOI of either R124 or jin-3 for 6 days. n = 3 (6 replicates). p values are depicted (vs. mock; when 2 depicted upper p value is R124 vs. jin-3 ); ∗∗∗ p < .001, $$$ p < .001, asterisks indicate mock versus reovirus infection, and dollar signs indicate R124 versus jin-3. (E) Percentage of single, viable, JAM-A protein expressing cells. Mean (SD), n = 3. MOI = multiplicity of infection.

Both jin-3 and R124 effectively killed the majority of bladder cancer cells in a dose- and time-dependent manner ( Figures 1 D and S1 D). In cell lines of different type (MIBC or NMIBC; see Table S1 ) and molecular cancer subtypes, jin-3 and R124 displayed comparable anti-tumor effects in 5637 (mixed phenotype), RT-4 (luminal phenotype), and SW780 (basal phenotype). In the HT-1197, HT-1376 (both mixed phenotype), and RT-112 cells (luminal phenotype), jin-3 was found to be more effective. 48 In the basal molecular subtype bladder cancer cell panel, T24 cells were significantly more effectively killed by jin-3 compared with wild-type reovirus R124, while both viruses were equally effective in TCCSUP cells. Strikingly, while R124 viruses did not affect the viability of the basal type UM-UC-3 cells, jin-3 reduced their viability. Moreover, in J82 cells, R124 viruses resulted in decreased viability only after exposure to high MOI, whereas exposure to jin-3 resulted in reduced viability at lower MOI.

R124 reoviruses enter cancer cells via its high-affinity receptor junction adhesion molecule A (JAM-A), 44 , 49 , 50 while the spontaneous mutant jin-3 virus has a broader tropism due to its ability to also infect cancer cells independently of JAM-A, i.e., by binding negatively charged cell surface sialic acids. 44

As expected, the expression levels of the canonical viral entry receptor JAM-A in tested cancer cell lines were in line with the ability of the reovirus variants to infect and/or replicate in the tumor cells ( Figures 1 E and S1 G).

Upon challenge with R124 and jin-3, a dose-dependent increase in viral copy number was detected in 3D cultures of RT-112 cells ( Figure S1 H), coinciding with reduced viability of the treated tumoroids after 3 days ( Figure S1 I).

In organoids cultured from the patient-derived muscle-invasive bladder cancer xenograft model PDX TM00024, R124 and jin-3 showed dose-dependent infection and replication ( Figure 2 A). 51 Furthermore, a significant reduction in cancer cell viability was observed after reovirus exposure for 7 days ( Figure 2 B). Viral protein (Sigma-3) was detected in the outer cell layers of the PDXOs, indicating active viral infection and replication after 3 days ( Figures 2 D and S2 A). 52 , 53 Moreover, an increased number of c-CASP-3-positive cells was measured ( Figures 2 E and S2 A). Further increase of the Sigma-3- and c-CASP-3-positive cells was observed after 7 days ( Figures 2 C–2E and S2 B for dose range), coinciding with a decrease in cell proliferation (nuclear PCNA; Figures 2 C, 2F, and S2 B) and loss of structural integrity of the PDXOs ( Figures 2 C and S2 B). No significant differences were observed between the viruses. Figure 2 Comparison of the oncolytic effects of reovirus mutant jin-3 versus wild-type reovirus R124 in 3D cultures in vitro TM00024 bladder PDXOs were exposed to the indicated plaque-forming units (PFUs) of reoviruses for 3 or 7 days. (A) Mean (SD) viral copy number of TM00024 PDXOs treated for 3 days with the indicated PFUs of the reoviruses. Two-way ANOVA followed by Tukey’s post hoc comparison ( n = 3) ∗∗∗ p < .001. (B–F) Viability (mean [SD] was measured using cell titer glo 3D after days 3 and 7 of reovirus exposure. N = 3 (3 replicates). Two-way ANOVA followed by Tukey’s post hoc comparison. ∗∗∗ p < .001. (C) Confocal images of PDXOs (day 7) stained for respectively H&E, panKRT (red); Sigma-3, c-CASP3, or PCNA (green); and DAPI (blue). Scale bars, 20 μm. Cells expressing the respective proteins Sigma-3 (D), c-CASP-3 (E), and nuclear PCNA (F) were counted with ImageJ and divided by the amount of panKRT+_DAPI+ cells. At least five fields were scored per technical replicate. Mean (SD) of N = 3 (3 replicates). ∗∗∗ p < .001, asterisks indicate reovirus infection versus mock same day. Two-way ANOVA followed by Tukey’s post hoc comparison.

Subsequently, viral infection and replication was determined in tumor tissue slices derived from cell-line-derived xenograft (CDX) and patient-derived xenograft (PDX) models after reovirus exposure. Upon exposure of tumor tissue slices of RT-112 bladder cancer cells 54 to R124 and jin-3 ex vivo for 3 days, the viruses were capable of infecting the cancer cells and replicated in viral factories in the cell ( Figures 3 A and 3B). Furthermore, apoptosis was stimulated upon reovirus exposure as assessed by a relative increase of c-CASP-3-positive cells coinciding with a decline in tumor cell proliferation ( Figures 3 C–3E and S3 A for dose range). In another CDX model J82 ( Figure S4 ), similar results were obtained. Moreover, in ex vivo cultured tumor slices obtained from the PDX model TM00024, which displayed cell membranous JAM-A protein expression ( Figure S3 C), reovirus exposure also induced increased apoptosis and impaired tumor cell proliferation ( Figures 3 F–3J and S3 B). No significant changes were observed between the two viruses. Figure 3 Comparison of jin-3 and R124 reovirus infection in ex vivo cultured tumor tissue Explanted tissue slices from either RT-112 CDX (A–E) or TM00024 PDX (F–J) were exposed to the indicated PFU of R124 or jin-3 reovirus for 3 days. Tissues were stained for H&E, panKRT (red); Sigma-3, c-CASP3 or PCNA (green), type I collagen (white), and DAPI (blue). Representative confocal images are shown. Scale bars, 20 μm. Cells expressing the respective proteins were counted with ImageJ and divided by the number of panKRT+_DAPI+ cells. At least four fields were scored per technical replicate. Mean (SD) of N = 2 (4 replicates). The ratio fragmented tumor cells was measured by the number of fragmented cells/total tumor cells (E and J). ∗∗∗ p < .001, reovirus infection versus mock. Mean (SD), n = 2 (4 replicates). Two-way ANOVA followed by Tukey’s post hoc comparison.

Consistent with earlier findings, 47 JAM-A expression was upregulated in bladder cancer compared to normal bladder (TCGA-BLCA database), although low levels of JAM-A are observed in 103/412 samples ( Figures S1 E and S1F). Next, tumor tissue slices were generated from freshly obtained tissues from patients diagnosed with various stages of bladder cancer ( n = 15; Table 1 ). Tissue slices were exposed to mock, R124, or jin-3 reoviruses for 3 days ( Figures 4 and S5 ). Immunolocalization of JAM-A was determined in the patient tissues, and membranous localization was observed in all the patients, with one patient displaying lower levels of staining (pt C1-005; Figure S5 B). Explanted tumor tissue was stained for c-CASP-3, PCNA (proliferation) and integrity of the cells (H&E staining and panKRT/DAPI staining). In the reovirus-treated patient-derived ex vivo cultured tumor explants, interpatient heterogeneity in Sigma-3 protein levels was observed, with Sigma-3 expression found in regions in multiple tissue slices per patient ( Figures 4 A and 4B). Exposure to reoviruses resulted in increased number of c-CASP-3-positive cells ( Figures 4 C and S5 C), whereas the number of proliferating tumor cells was reduced ( Figures 4 D and S5 C) as well as the integrity of the cells as indicated by the H&E staining and the increased ratio of fragmented cells ( Figures 4 E and S5 A–S5C). Table 1 Patient information: clinical histopathological characteristics of the patients included. Disease stage and grade at time of sampling of the tumor material Patient identifier Disease Risk group 55 Disease stage Disease grade Treatment history Age Gender CIS C1-005 MIBC – pT2 high-grade CIS BCG bladder instillations 70 male yes 01–032 MIBC – pT2 G3 unknown 69 male no C1-015 NMIBC very high pT1 focal G3 none 61 male no 01–037 NMIBC high pT1 G2 none 82 female no C1-020 NMIBC high pTa/pT1 G3 none 76 male no C1-013 NMIBC intermediate pT1 low grade none 87 male no C1-012 NMIBC intermediate pTa G3 none 77 male no 01–034 NMIBC intermediate pTa G2 none 80 male no C1-008 NMIBC intermediate pT1 G1 none 62 male no C1-022 NMIBC intermediate pTa G1 none 79 male no C1-047 NMIBC intermediate pTa G1 none 64 male no C1-086 NMIBC low pTa G1 none 67 male no C1-088 NMIBC low pTa G1 none 73 male no C1-018 NMIBC low pTa G1 none 67 male no C1-014 NMIBC low pTa low grade none 70 female No pTa, p(pathological) non-invasive papillary carcinoma; pT1, tumor invades subepithelial connective tissue; pT2, tumor invades muscle. Grading was scored using the WHO grading 1973 (G1: well differentiated; G2: moderately differentiated; G3: poorly differentiated) or WHO 2004 (low/high grade) MIBC, muscle-invasive bladder carcinoma; NMIBC, non-muscle invasive bladder carcinoma; CIS, carcinoma in situ ; BCG, Bacillus Calmette-Guerin. Figure 4 Comparison of jin-3 and R124 reovirus infection in ex vivo cultured tumor tissue slices from human bladder cancer patients Explanted tissue slices from patients ( n = 15) diagnosed with bladder cancer were exposed to either mock, R124, or jin-3 reovirus for 3 days and stained for viral protein, c-CASP-3, PCNA (proliferation), and integrity of the cell (nuclear DAPI and panKRT tumor marker). (A–E) Representative confocal images of the mock and 10 7 PFU condition of both viruses from five patients ranging from low-risk NMIBC to MIBC (viral protein [green, Sigma-3], tumor markers [red, panKRT], and nuclei [DAPI, blue]. Scale bars, 20 μm). Cells expressing the respective proteins [(B) Sigma-3, (C) c-CASP3, or (D) nuclear PCNA] were counted with ImageJ and divided by the number of panKRT+_DAPI+ cells. At least four fields were scored per technical replicate. Mean (SD) (3–4 replicates) with each dot representing one bladder cancer patient. The ratio of fragmented tumor cells was measured by the number of fragmented cells/total tumor cells (E). ∗∗∗ p < 0.001, reovirus infection versus mock, one-way ANOVA followed by Tukey’ post hoc comparison.

Reovirus-induced immune activation

Inflammatory cytokines, type I interferons (IFNs), and IFN-stimulated genes (ISGs) are of key significance in oncolytic virotherapy that largely relies on the stimulation of anti-tumor and anti-viral immune cell activation. 55 , 56 , 57 Exposure to R124 and jin-3 reovirus resulted in a significant and dose-dependent upregulation of IFN-β gene expression ( IFNB1 ) after 24 h in multiple bladder cancer cell lines ( Figures 5 A–5G). jin-3 treatment resulted in a significantly stronger upregulation in IFNB1 (6/7) compared to R124 (4/7) in all the tested cell lines ( Figures 5 C–5G), except for UM-UC-3 and T24 ( Figures 5 A and 5B). Moreover, exposure to jin-3 led to significantly stronger induction of ISGs (i.e., ISG15 , IFIT1 , IFIT2 , RSAD2 , and MX1 ) in most of the tested cell lines compared to R124 ( Figure 5 ). Exposure to jin-3 led to a superior and significant expression of inflammatory cytokines CXCL10 (in 7/7 cell lines), TNF (4/7), and IL1B (2/7) compared with R124 ( Figure 5 ). Moreover, the cytosolic RNA sensor RIG-I was significantly upregulated after jin-3 exposure compared to R124 in 6/7 cell lines ( Figure 5 ). In the TM00024 PDXOs, exposure to jin-3 reovirus resulted in significant upregulation of TNF, CXCL10 , IFNB1 , and RSAD2 gene expression, whereas R124 reovirus only induced TNF gene expression ( Figure 5 H). Figure 5 Comparison of immune modulation induced by jin-3 and R124 reovirus treatment of bladder cancer cell lines Heat maps of various inflammatory cytokines and interferon-stimulated genes (log fold change mRNA expression [2 −ΔΔCt ] vs. mock treated cells) in bladder cancer cell lines UM-UC-3 (A), T24 (B), HT-1197 (C), RT-112 (D), RT-4 (E), TCCSUP (F), J82 (G), and PDXOs (H) exposed to a range of either R124 or jin-3 reoviruses after respectively 24 h (cell lines) and 3 days (PDXOs). p values are depicted (vs. mock; when 2 depicted upper p value is R124 vs. jin-3 ) ∗∗∗ p < .001, $$$ p < .001, asterisks indicate mock versus reovirus infection, and dollar signs indicate R124 versus jin-3.

Cell surface expression of the DAMPs ecto-calreticulin and ecto-HSP90 was significantly increased upon exposure to reoviruses ( Figures 6 A and 6B). Strikingly, jin-3 caused a more pronounced increase in HMGB1 release, a well-established marker of immunogenic cell death 42 ( Figure 6 C). Correlation between these markers at an MOI of 10 is depicted in Figure 6 D. Figure 6 Comparison of immunogenic cell death induced by jin-3 and R124 reovirus treatment of bladder cancer cell lines (A–C) Cell lines were treated with oncolytic viruses for 48 h at an MOI of 10, and the DAMPs ecto-calreticulin (A) and ecto-HSP90 (B) were measured using flow cytometry. Secreted HMGB1 protein was measured with an ELISA after 48 h of treatment with a dose range of oncolytic viruses (C). ∗∗∗ p < 0.001, $$$ p < 0.001, asterisks indicate reovirus infection versus mock same day, and dollar signs indicate R124 vs. jin-3. Mean (SD), N = 3 (2 replicates). Two-way ANOVA followed by Tukey’s posthoc comparison. (D) Correlation graph of the HMGB1 release ( x axis), percentage of viable cells (size of the dots), and fold change of ecto-CRT ( y axis)-positive cells at MOI 10 of the indicated virus.

Subsequently, co-culturing of 3D tumoroids of human RT-112 cells with partially HLA-matched peripheral blood mononuclear cells (PBMCs) led to increased tumoroid killing ( Figure S7 A). This was accompanied with significantly increased release of fragmented cytokeratin-18 (KRT18; Figure S7 B), CXCL10 ( Figure S7 C), and IFN-γ ( Figure S7 D). Addition of reoviruses to the 3D cultures resulted in expression of viral transcripts in the reovirus-treated RT-112 cells, with a lower copy number when the 3D cultures were co-cultured with PBMCs ( Figure S7 E). Reovirus treatment in the co-cultures resulted in augmented tumoroid killing compared to the mock-treated conditions, with jin-3 displaying significantly more pronounced effects ( Figures 7 A and 7B). Moreover, chemo- and cytokine production (CXCL10 and IFN-γ) and mRNA expression of type I IFN response genes (e.g., IFNB1 , RSAD2 , MX1 , IFIT2 , and ISG15 mRNA levels) was significantly enhanced upon reovirus treatment in these cultures ( Figures 7 C–7E and S7 F–S7P). Figure 7 Activation of PBMCs in RT-112 co-cultures (A) Brightfield images of RT-112 bladder cancer cells that were cultured in 60% Matrigel and allowed to form 3D structures for approximately 3 days. Subsequently, PBMCs were added at an effector: target ratio of 20:1 in absence or presence of either R124 or jin-3 reovirus at the indicated PFU for an additional 3 days. (B) Fragmented KRT18 was determined as an outcome measure for tumor cell killing 3 days after OV exposure. (C–E) CXCL10 levels and (D) IFN-γ levels were measured 3 days after OV exposure. ( n = 3, 2 replicates). Mean (SD). Two-way ANOVA with Tukey’s post hoc comparison. (E) Heatmap of various inflammatory cytokines and interferon-stimulated genes (log fold change mRNA expression [2 −ΔΔCt ] vs. mock treated cells) in 3D cultured RT-112 cells or RT-112 cells co-cultured with PBMCs exposed to either R124 or jin-3 reoviruses. Mean (SD). p values are depicted (vs. mock; when two depicted upper p value is R124 vs. jin-3 ) ∗∗∗ p < .001, $$$ p < .001, asterisks indicate mock versus reovirus infection and dollar signs R124 versus jin-3. , n = 2 (2 replicates). Two-way ANOVA followed by Tukey’s post hoc comparison.

Discussion

In this study, we evaluated the direct oncolytic and immunomodulatory effects of reoviruses R124 and jin-3 in preclinical human bladder cancer models.

We observed that reoviruses induce potent oncolytic effects in flatbottom 2D and 3D cultures of human bladder cancer. Despite the differences in oncolytic responses, which are likely to be due to well-documented clinical inter-tumoral heterogeneity in bladder cancer, 58 , 59 we observed viral infection upon reovirus administration in most human bladder cancer cells. Moreover, we detected viral infection and replication in near-patient ex vivo cultured bladder cancer tissue slices derived from CDX and PDX models and primary bladder cancer patient material.

Besides oncolytic properties, oncolytic viruses can induce both anti-viral immunity and anti-tumor immune responses, which is required for long-lasting and systemic tumor control. 60 Effective oncolytic virotherapy relies on the production of pro-inflammatory cytokines and type I IFNs and the induction of interferon-stimulated genes (ISGs). Pro-inflammatory chemokines and cytokines are released and attract lymphocytes toward the tumor. 60 , 61 , 62 These anti-viral immunological events could turn immunological “cold” tumors into “hot” tumors. Our study demonstrated that jin-3 reovirus led to elevated expression of ISG and inflammatory cytokine production by infected bladder cancer cells compared to R124 wild-type reovirus. This is further substantiated by the augmented release of immunogenic cell death markers HMGB1 and DAMPs (i.e., ectopic cell surface expression of calreticulin and HSP90 after exposure to jin-3 reovirus). Co-culturing of bladder tumoroids with PBMCs resulted in a significant, dose-dependent increase in tumor cell death and elevated production of CXCL10 and IFN-γ after reovirus exposure. This is consistent with findings in a colorectal cancer co-culture system in which autologous PBMCs acquire tumor-targeting capabilities, which correlated with clinical responses to checkpoint inhibitors. 63

Overall, our preclinical results support the notion that infection of the human bladder cancer cells with reoviruses, especially jin-3 , induces oncolysis and may contribute to a switch from an immunologically “cold’’ toward an immunological “hot’’ inflammatory phenotype. Additionally, jin-3 has an extended tropism compared to the wild-type reovirus because these viruses can infect cells independently of JAM-A expression on the tumor cell surface. These results are in line with previous reports in prostate cancer cells showing that intra-tumoral injection of wild-type reovirus induced pro-inflammatory cytokines production. 45 , 64

Intravesical administration of reovirus in an orthotopic model of superficial bladder cancer revealed minor side effects of therapy with significantly higher tumor-free survival compared to BCG treatment. 65 Moreover, in a syngeneic mouse model, intravesical therapy of reovirus in combination with an anti-programmed cell death protein 1 (PD-1) antibody improved survival and induced a more immune-active (hot) tumor microenvironment. 66

Previous clinical studies have also demonstrated the potential of oncolytic viruses in cancer therapy, highlighting their ability to target and destroy cancer cells selectively. For example, T-VEC is a Food and Drug Administration (FDA)-approved herpes-simplex-virus-based therapy for advanced melanoma. 67 Other oncolytic viruses including vesicular stomatitis virus, adenovirus, and vaccinia virus have been studied in multiple cancer types, including bladder cancer. 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76

Some oncolytic viruses, for example, ICAM-1 targeted Coxsackievirus A21 (CVA21) and vaccinia virus, have shown acceptable safety profiles and proof of viral targeting and replication in the bladder tumor cells in clinical trials. 68 , 71 , 77 In addition, in early stage bladder cancer, oncolytic adenoviruses have been efficacious in BCG-refractory patients. 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85

Importantly, clinical trials have revealed that pelareorep, also known as Reolysin, an isolate of wild-type T3 Dearing reovirus, can be safely administered to cancer patients, including patients with advanced cancer such as multiple melanoma, prostate, and esophagus cancer, with relatively few adverse effects. 43 , 49 , 86 , 87 , 88 , 89 However, the clinical benefit of wild-type reovirus administration is limited, and phase II studies have revealed no survival benefit. 90 No clinical trials with reovirus have been reported for bladder cancer yet. Clinical benefit may be enhanced by using our reovirus mutant jin-3 with an expanded tropism, beyond the canonical reovirus entry receptor JAM-A. 44

Due to the significant variability between and within tumors—including genetic mutations and differences in the tumor microenvironment—it is essential to develop diverse and improved oncolytic virotherapy strategies using multiple types of oncolytic viruses.

Matching the most effective oncolytic virus(es) to an individual patient is key for virotherapy to become more effective. Stratification of patients who are more likely to respond to a specific oncolytic virus is required but—to date—not yet accomplished. If proven, this personalized approach will enhance treatment outcomes by matching the best candidate virus to an individual tumor based on its unique biological characteristics.

Accumulating evidence shows that oncolytic virotherapy, when used as a monotherapy, is unlikely to fully overcome the immunosuppressive tumor microenvironment in a clinical setting. Several clinical studies are with oncolytic reovirus in combination with immune checkpoint inhibition in various cancers including pancreatic adenocarcinoma, metastatic non-small lung cancer, multiple myeloma, and metastatic colorectal cancer. 91 , 92 , 93 , 94 , 95 , 96 , 97 For example, in the GOBLET multi-cohort phase 1/2 study in pancreatic and anal cancer, intravenous pelareorep, in combination with the PD-L1 inhibitor atezolizumab, displayed promising efficacy (respectively 62% and 37.5% objective response rate for metastatic pancreatic and anal cancer). 98 Furthermore, combination of pelareorep with the PD-1 inhibitor pembrolizumab modestly promoted anti-tumor activity in advanced pancreatic cancer. 99

To better understand the interactions between oncolytic viruses and the immune system, tumor-immune cell co-culture systems—as the ones described here—can be exploited. By incorporating both tumor cells and relevant immune cell populations (e.g., T cell populations and dendritic cells), these systems can be used to assess how different OVs modulate immune cell activation, suppression, or recruitment, to monitor cytokine and chemokine production in response to viral infection and to evaluate the potential for synergy or antagonism when combining oncolytic viruses with current immunotherapy (immune checkpoint inhibition or cell-based immunotherapy). Correct timing, optimal oncolytic virus (hence the repertoire of OVs), optimal dosing, and treatment combinations are key.

Collectively, our findings underscore the oncolytic potential of reoviruses in diverse preclinical models of human bladder cancer. Notably, the mutant reovirus jin-3 exhibits a more robust immunomodulatory response in preclinical bladder cancer models compared to the wild-type reovirus R124. This promising result positions jin-3 as a compelling candidate for future clinical translation.

Material and methods

Reovirus production

Wild-type T3D reovirus strain R124 was plaque-purified from the wild-type reovirus T3D (ATCC) on HER911 cells. 44 , 100 Reovirus mutant jin-3 was isolated from JAM-A-deficient U118MG cells after passaging of the wild-type T3D strain R124. 44 Both R124 and jin-3 reoviruses were propagated, purified, and titrated on human HER911 cells as previously described. 44

Preclinical models and culture conditions

Cell lines as monolayers : a panel of human bladder cancer cell lines was cultured in monolayers with the respective culture medium ( Table S1 ).

Three-dimensional bladder cancer tumoroids or patient-derived xenograft (PDX) organoids (PDXOs) were cultured from cells isolated from subcutaneously growing PDX TM00024 tumors 51 (Jackson Laboratory; ethical permit AVD16605 ) or from the RT-112 and J82 cell lines as described previously. 52

Three-dimensional tumor-immune cell co-cultures : 10,000 RT-112 cells were seeded in 40 μL domes in 60% Matrigel. After 3 days, partially HLA-matched PBMCs (Sanquin NVT0631.01) were added at a 20:1 effector target ratio to the 3D cultures. After 3 days, conditioned medium was collected, and RNA was isolated as described below.

Ex vivo cultured tissue explants were sliced and cultured as previously described. 54 Bladder cancer tissue was derived from either CDX, PDX, or directly from patient tumor material (Biobank Urology protocol B20.060, animal ethical protocol AVD16605 ; Table 1 ). Multiple tissue slices were used per condition and cultured for 4 days.

Viability assays

Monolayer experiments: 1,500 cells per well were seeded in a 96-well plates. After 24 h, these cells were exposed to a range of either R124 or jin-3 (MOI 0–100 plaque-forming units [PFUs]/cell). Cells were incubated for 72 or 144 h, after which a CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega) was performed according to manufacturer’s protocol. After 2 h of incubation time, optical density was measured at 490 nm using SpectraMax iD3 plate reader to assess cell viability. 45

The 3D tumoroids or PDXOs were exposed to a range of R124 and jin-3 reoviruses. After 3–7 days, viability was determined using the Cell Titer Glo assay, according to the manufacturer’s protocol (Promega). 45

Flow cytometry

Four hundred thousand cells/well were seeded in a 6-well plate. Each cell line was exposed to either R124 or jin-3 (mock solution only, MOI 10) and incubated for 48 h. Subsequently, levels of viral protein expression (Sigma-3), apoptosis (cleaved caspase-3 [c-CASP-3]), and protein expression of several DAMPs (cell surface expression of calreticulin and HSP90) for each cell line/virus combination were acquired using LSR Fortessa X-20 Cell Analyzer and analyzed using FlowJo software ( Table S1 ).

Histology and immunofluorescence

H&E and immunofluorescent stainings were executed as previously described. 54 H&E and immunofluorescent stainings were visualized using the Pannoramic MIDI slide scanner (3DHISTECH). All fluorescent stainings were visualized by confocal microscopy (63× magnification, resolution 1,024 × 1,024) (Leica SP8). At least four fields of the fluorescent staining were scored per TS by two independent reviewers. The average of these 4 fields was shown for each of the technical replicates. Cells expressing the respective proteins were counted with ImageJ and divided by the number of pancytokeratin-positive and DAPI-positive (panKRT+_DAPI+) cells (i.e., intact tumor cells with intact nuclei). The ratio of fragmented tumor cells was measured by the number of fragmented cells divided by the number of total tumor cells.

Real-time quantitative polymerase chain reaction

Cells were infected with the viruses and after the indicated time RNA was isolated according to the manufacturer’s protocol (Nucleospin RNA kit Macherey-Nagel). cDNA was generated by using random primers (Promega), and real-time qPCR was performed with GoTaq Mastermix (Promega) according to the manufacturer’s protocol in technical duplicates and biological triplicates (Promega). Gene expression was normalized to GAPDH. Primer sequences can be found in Table S1 .

ELISAs

Cells were exposed to R124 or jin-3 with an MOI of 10. At the indicated time post exposure, HMGB1 (high mobility group box 1) release was measured by performing the Lumit HMGB1 Human/Mouse Immunoassay (Promega).

Three-dimensional (co-) cultures were exposed to R124 or jin-3 . At the indicated time points post exposure, conditioned medium was collected, and either fragmented KRT18 (Abcam), IFN-γ (Abcam), or CXCL10 (Invitrogen) levels were measured as indicated by the manufacturers protocol.

Statistical analysis

Statistical analyses were performed by using GraphPad Prism 8.0. Two-way ANOVA was performed for viability and ELISA experiments followed by Tukey post hoc test for multiple comparison. For ex vivo cultures, sample size was calculated using proportional odds models as described. 101 Multiple slices were scored per condition per patient (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001).

Data and code availability

Data supporting the findings of this study are available within the paper and its supplemental information.

Acknowledgments

This study was supported by the OAK Foundation ( www.supportcasper.nl;STRATIVIR and SOAK18.01 ), the 10.13039/501100004622 KWF Dutch Cancer Society ( KWF15932 ), and a kind gift from the Franje Foundation .

Author contributions

Conceptualization, R.H., G.P., and G.H.; data curation, A.F., M.M., and G.H.; formal analysis, A.F., M.M., L.H., D.W., and G.H.; funding acquisition, R.H., G.P., and G.H.; investigation, A.F., M.M., L.H., D.W., and G.H.; methodology, A.F., M.M., D.W., and G.H.; project administration, G.P. and G.H.; resources, R.H., D.W., R.F., and W.Z.; supervision, R.H., G.P., and G.H.; validation, A.F., M.M., G.P., and G.H.; visualization, A.F., M.M., and G.H.; writing—original draft, A.F. and G.H.; writing—review and editing, A.F., R.H., M.K., G.P., and G.H.

Declaration of interests

The authors declare no competing interests.

References

1 Wéber A. Vignat J. Shah R. Morgan E. Laversanne M. Nagy P. Kenessey I. Znaor A. Global burden of bladder cancer mortality in 2020 and 2040 according to GLOBOCAN estimates World J. Urol. 42 2024 237 38625417 10.1007/s00345-024-04949-8 PMC11021283

2 Babjuk M. Burger M. Compérat E.M. Gontero P. Mostafid A.H. Palou J. van Rhijn B.W.G. Rouprêt M. Shariat S.F. Sylvester R. European Association of Urology Guidelines on Non-muscle-invasive Bladder Cancer (TaT1 and Carcinoma In Situ) - 2019 Update Eur. Urol. 76 2019 639 657 31443960 10.1016/j.eururo.2019.08.016

3 Zhang Y. Zhang X. Huang X. Tang X. Zhang M. Li Z. Hu X. Zhang M. Wang X. Yan Y. Tumor stemness score to estimate epithelial-to-mesenchymal transition (EMT) and cancer stem cells (CSCs) characterization and to predict the prognosis and immunotherapy response in bladder urothelial carcinoma Stem Cell Res. Ther. 14 2023 15 36721217 10.1186/s13287-023-03239-1 PMC9890713

4 Schmidt S. Kunath F. Coles B. Draeger D.L. Krabbe L.M. Dersch R. Kilian S. Jensen K. Dahm P. Meerpohl J.J. Intravesical Bacillus Calmette-Guérin versus mitomycin C for Ta and T1 bladder cancer: Abridged summary of the Cochrane Review Investig. Clin. Urol. 61 2020 349 354 10.4111/icu.2020.61.4.349 PMC7329645 32665991

5 Chan K.S. Volkmer J.P. Weissman I. Cancer stem cells in bladder cancer: a revisited and evolving concept Curr. Opin. Urol. 20 2010 393 397 20657288 10.1097/MOU.0b013e32833cc9df PMC2997612

6 Chang J.T. Mani S.A. Sheep, wolf, or werewolf: cancer stem cells and the epithelial-to-mesenchymal transition Cancer Lett. 341 2013 16 23 23499890 10.1016/j.canlet.2013.03.004 PMC3762892

7 Ferlay J. Steliarova-Foucher E. Lortet-Tieulent J. Rosso S. Coebergh J.W.W. Comber H. Forman D. Bray F. Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012 Eur. J. Cancer 49 2013 1374 1403 23485231 10.1016/j.ejca.2012.12.027

8 Dyba T. Randi G. Bray F. Martos C. Giusti F. Nicholson N. Gavin A. Flego M. Neamtiu L. Dimitrova N. The European cancer burden in 2020: Incidence and mortality estimates for 40 countries and 25 major cancers Eur. J. Cancer 157 2021 308 347 34560371 10.1016/j.ejca.2021.07.039 PMC8568058

9 Chalmers Z.R. Connelly C.F. Fabrizio D. Gay L. Ali S.M. Ennis R. Schrock A. Campbell B. Shlien A. Chmielecki J. Analysis of 100,000 human cancer genomes reveals the landscape of tumor mutational burden Genome Med. 9 2017 34 28420421 10.1186/s13073-017-0424-2 PMC5395719

10 Yarchoan M. Hopkins A. Jaffee E.M. Tumor Mutational Burden and Response Rate to PD-1 Inhibition N. Engl. J. Med. 377 2017 2500 2501 29262275 10.1056/NEJMc1713444 PMC6549688

11 Hussain S.A. Birtle A. Crabb S. Huddart R. Small D. Summerhayes M. Jones R. Protheroe A. From Clinical Trials to Real-life Clinical Practice: The Role of Immunotherapy with PD-1/PD-L1 Inhibitors in Advanced Urothelial Carcinoma Eur. Urol. Oncol. 1 2018 486 500 31158093 10.1016/j.euo.2018.05.011

12 Hsu M.M. Balar A.V. PD-1/PD-L1 Combinations in Advanced Urothelial Cancer: Rationale and Current Clinical Trials Clin. Genitourin. Cancer 17 2019 e618 e626 31005473 10.1016/j.clgc.2019.03.009

13 Gupta M. Kates M. Bivalacqua T.J. Immunotherapy in nonmuscle invasive bladder cancer: current and emerging treatments Curr. Opin. Oncol. 31 2019 183 187 30893148 10.1097/CCO.0000000000000533

14 van Dijk N. Funt S.A. Blank C.U. Powles T. Rosenberg J.E. van der Heijden M.S. The Cancer Immunogram as a Framework for Personalized Immunotherapy in Urothelial Cancer Eur. Urol. 75 2019 435 444 30274701 10.1016/j.eururo.2018.09.022 PMC6697269

15 Hurwitz M.E. Sokhn J. Petrylak D.P. Cancer immunotherapy: new applications in urologic oncology Curr. Opin. Urol. 26 2016 535 542 27584025 10.1097/MOU.0000000000000345

16 Yang R. Yu Y. Patient-derived organoids in translational oncology and drug screening Cancer Lett. 562 2023 216180 10.1016/j.canlet.2023.216180 37061121

17 Balar A.V. Immune Checkpoint Blockade in Metastatic Urothelial Cancer J. Clin. Oncol. 35 2017 2109 2112 28481707 10.1200/JCO.2017.72.8444

18 Balar A.V. Castellano D. O'Donnell P.H. Grivas P. Vuky J. Powles T. Plimack E.R. Hahn N.M. de Wit R. Pang L. First-line pembrolizumab in cisplatin-ineligible patients with locally advanced and unresectable or metastatic urothelial cancer (KEYNOTE-052): a multicentre, single-arm, phase 2 study Lancet Oncol. 18 2017 1483 1492 28967485 10.1016/S1470-2045(17)30616-2

19 Balar A.V. Galsky M.D. Rosenberg J.E. Powles T. Petrylak D.P. Bellmunt J. Loriot Y. Necchi A. Hoffman-Censits J. Perez-Gracia J.L. Atezolizumab as first-line treatment in cisplatin-ineligible patients with locally advanced and metastatic urothelial carcinoma: a single-arm, multicentre, phase 2 trial Lancet 389 2017 67 76 27939400 10.1016/S0140-6736(16)32455-2 PMC5568632

20 Balar A.V. Weber J.S. PD-1 and PD-L1 antibodies in cancer: current status and future directions Cancer Immunol. Immunother. 66 2017 551 564 28213726 10.1007/s00262-017-1954-6 PMC11028560

21 Bellmunt J. Bajorin D.F. Pembrolizumab for Advanced Urothelial Carcinoma N. Engl. J. Med. 376 2017 2304 28591526 10.1056/NEJMc1704612

22 Bellmunt J. de Wit R. Vaughn D.J. Fradet Y. Lee J.L. Fong L. Vogelzang N.J. Climent M.A. Petrylak D.P. Choueiri T.K. Pembrolizumab as Second-Line Therapy for Advanced Urothelial Carcinoma N. Engl. J. Med. 376 2017 1015 1026 28212060 10.1056/NEJMoa1613683 PMC5635424

23 Bellmunt J. Kerst J.M. Vazquez F. Morales-Barrera R. Grande E. Medina A. Gonzalez Graguera M.B. Rubio G. Anido U. Fernandez Calvo O. A randomized phase II/III study of cabazitaxel versus vinflunine in metastatic or locally advanced transitional cell carcinoma of the urothelium (SECAVIN) Ann. Oncol. 28 2017 1517 1522 28419193 10.1093/annonc/mdx186

24 Bellmunt J. Nadal R. Urothelial cancer in 2017: Changes in expectations for metastatic urothelial carcinoma Nat. Rev. Clin. Oncol. 15 2018 73 74 29203837 10.1038/nrclinonc.2017.184

25 Bellmunt J. Powles T. Vogelzang N.J. A review on the evolution of PD-1/PD-L1 immunotherapy for bladder cancer: The future is now Cancer Treat Rev. 54 2017 58 67 28214651 10.1016/j.ctrv.2017.01.007

26 Massard C. Gordon M.S. Sharma S. Rafii S. Wainberg Z.A. Luke J. Curiel T.J. Colon-Otero G. Hamid O. Sanborn R.E. Safety and Efficacy of Durvalumab (MEDI4736), an Anti-Programmed Cell Death Ligand-1 Immune Checkpoint Inhibitor, in Patients With Advanced Urothelial Bladder J. Clin. Oncol. 34 2016 3119 3125 27269937 10.1200/JCO.2016.67.9761 PMC5569690

27 Sharma P. Callahan M.K. Bono P. Kim J. Spiliopoulou P. Calvo E. Pillai R.N. Ott P.A. de Braud F. Morse M. Nivolumab monotherapy in recurrent metastatic urothelial carcinoma (CheckMate 032): a multicentre, open-label, two-stage, multi-arm, phase 1/2 trial Lancet Oncol. 17 2016 1590 1598 27733243 10.1016/S1470-2045(16)30496-X PMC5648054

28 Sari Motlagh R. Pradere B. Mori K. Miura N. Abufaraj M. Shariat S.F. Bladder-preserving strategies for Bacillus Calmette-Guerin unresponsive non-muscle invasive bladder cancer; where are we and what will be expected? Curr. Opin. Urol. 30 2020 584 593 32304379 10.1097/MOU.0000000000000792

29 Balar A.V. Kamat A.M. Kulkarni G.S. Uchio E.M. Boormans J.L. Roumiguié M. Krieger L.E.M. Singer E.A. Bajorin D.F. Grivas P. Pembrolizumab monotherapy for the treatment of high-risk non-muscle-invasive bladder cancer unresponsive to BCG (KEYNOTE-057): an open-label, single-arm, multicentre, phase 2 study Lancet Oncol. 22 2021 919 930 34051177 10.1016/S1470-2045(21)00147-9

30 Lopez-Beltran A. Cimadamore A. Blanca A. Massari F. Vau N. Scarpelli M. Cheng L. Montironi R. Immune Checkpoint Inhibitors for the Treatment of Bladder Cancer Cancers (Basel) 13 2021 131 10.3390/cancers13010131 PMC7795541 33401585

31 Roumiguié M. Compérat E. Chaltiel L. Nouhaud F.X. Verhoest G. Masson-Lecomte A. Colin P. Audenet F. Houédé N. Larré S. PD-L1 expression and pattern of immune cells in pre-treatment specimens are associated with disease-free survival for HR-NMIBC undergoing BCG treatment World J. Urol. 39 2021 4055 4065 32666225 10.1007/s00345-020-03329-2

32 Ghate K. Amir E. Kuksis M. Hernandez-Barajas D. Rodriguez-Romo L. Booth C.M. Vera-Badillo F.E. PD-L1 expression and clinical outcomes in patients with advanced urothelial carcinoma treated with checkpoint inhibitors: A meta-analysis Cancer Treat Rev. 76 2019 51 56 31125908 10.1016/j.ctrv.2019.05.002

33 Maiorano B.A. Di Maio M. Cerbone L. Maiello E. Procopio G. Roviello G. MeetURO Group Significance of PD-L1 in Metastatic Urothelial Carcinoma Treated With Immune Checkpoint Inhibitors: A Systematic Review and Meta-Analysis JAMA Netw. Open 7 2024 e241215 10.1001/jamanetworkopen.2024.1215 PMC10918499 38446479

34 Davola M.E. Mossman K.L. Oncolytic viruses: how “lytic” must they be for therapeutic efficacy? OncoImmunology 8 2019 e1581528 10.1080/2162402X.2019.1596006 PMC6492965 31069150

35 Errington F. Steele L. Prestwich R. Harrington K.J. Pandha H.S. Vidal L. de Bono J. Selby P. Coffey M. Vile R. Reovirus activates human dendritic cells to promote innate antitumor immunity J. Immunol. 180 2008 6018 6026 18424722 10.4049/jimmunol.180.9.6018

36 Lee P. Gujar S. Potentiating prostate cancer immunotherapy with oncolytic viruses Nat. Rev. Urol. 15 2018 235 250 29434366 10.1038/nrurol.2018.10

37 Prestwich R.J. Errington F. Harrington K.J. Pandha H.S. Selby P. Melcher A. Oncolytic viruses: do they have a role in anti-cancer therapy? Clin. Med. Oncol. 2 2008 83 96 21892269 10.4137/cmo.s416 PMC3161683

38 Prestwich R.J. Ilett E.J. Errington F. Diaz R.M. Steele L.P. Kottke T. Thompson J. Galivo F. Harrington K.J. Pandha H.S. Immune-mediated antitumor activity of reovirus is required for therapy and is independent of direct viral oncolysis and replication Clin. Cancer Res. 15 2009 4374 4381 19509134 10.1158/1078-0432.CCR-09-0334 PMC4821072

39 Tang D. Kang R. Berghe T.V. Vandenabeele P. Kroemer G. The molecular machinery of regulated cell death Cell Res. 29 2019 347 364 30948788 10.1038/s41422-019-0164-5 PMC6796845

40 Galluzzi L. Vitale I. Warren S. Adjemian S. Agostinis P. Martinez A.B. Chan T.A. Coukos G. Demaria S. Deutsch E. Consensus guidelines for the definition, detection and interpretation of immunogenic cell death J. Immunother. Cancer 8 2020 e000337 10.1136/jitc-2019-000337 PMC7064135 32209603

41 Tesniere A. Panaretakis T. Kepp O. Apetoh L. Ghiringhelli F. Zitvogel L. Kroemer G. Molecular characteristics of immunogenic cancer cell death Cell Death Differ. 15 2008 3 12 18007663 10.1038/sj.cdd.4402269

42 Kepp O. Senovilla L. Vitale I. Vacchelli E. Adjemian S. Agostinis P. Apetoh L. Aranda F. Barnaba V. Bloy N. Consensus guidelines for the detection of immunogenic cell death OncoImmunology 3 2014 e955691 10.4161/21624011.2014.955691 PMC4292729 25941621

43 Vidal L. Pandha H.S. Yap T.A. White C.L. Twigger K. Vile R.G. Melcher A. Coffey M. Harrington K.J. DeBono J.S. A phase I study of intravenous oncolytic reovirus type 3 Dearing in patients with advanced cancer Clin. Cancer Res. 14 2008 7127 7137 18981012 10.1158/1078-0432.CCR-08-0524

44 van den Wollenberg D.J.M. Dautzenberg I.J.C. van den Hengel S.K. Cramer S.J. de Groot R.J. Hoeben R.C. Isolation of reovirus T3D mutants capable of infecting human tumor cells independent of junction adhesion molecule-A PLoS One 7 2012 e48064 10.1371/journal.pone.0048064 PMC3480499 23110175

45 van de Merbel A.F. van der Horst G. van der Mark M.H. Bots S.T.F. van den Wollenberg D.J.M. de Ridder C.M.A. Stuurman D. Aalders T. Erkens-Schulz S. van Montfoort N. Reovirus mutant jin-3 exhibits lytic and immune-stimulatory effects in preclinical human prostate cancer models Cancer Gene Ther. 29 2021 1 10 34135475 10.1038/s41417-021-00360-2 PMC9209329

46 Mitchell L.A. Ward C. Kwon M. Mitchell P.O. Quintero D.A. Nusrat A. Parkos C.A. Koval M. Junctional adhesion molecule A promotes epithelial tight junction assembly to augment lung barrier function Am. J. Pathol. 185 2015 372 386 25438062 10.1016/j.ajpath.2014.10.010 PMC4305184

47 Ren T. Zheng Y. Liu F. Liu C. Zhang B. Ren H. Gao X. Wei Y. Sun Q. Huang H. Identification and Validation of JAM-A as a Novel Prognostic and Immune Factor in Human Tumors Biomedicines 12 2024 1423 10.3390/biomedicines12071423 PMC11275048 39061997

48 Zuiverloon T.C.M. de Jong F.C. Costello J.C. Theodorescu D. Systematic Review: Characteristics and Preclinical Uses of Bladder Cancer Cell Lines Bladder Cancer 4 2018 169 183 29732388 10.3233/BLC-180167 PMC5929350

49 Thirukkumaran C.M. Nodwell M.J. Hirasawa K. Shi Z.Q. Diaz R. Luider J. Johnston R.N. Forsyth P.A. Magliocco A.M. Lee P. Oncolytic viral therapy for prostate cancer: efficacy of reovirus as a biological therapeutic Cancer Res. 70 2010 2435 2444 20215509 10.1158/0008-5472.CAN-09-2408

50 Barton E.S. Forrest J.C. Connolly J.L. Chappell J.D. Liu Y. Schnell F.J. Nusrat A. Parkos C.A. Dermody T.S. Junction adhesion molecule is a receptor for reovirus Cell 104 2001 441 451 11239401 10.1016/s0092-8674(01)00231-8

51 Pan C.X. Zhang H. Tepper C.G. Lin T.Y. Davis R.R. Keck J. Ghosh P.M. Gill P. Airhart S. Bult C. Development and Characterization of Bladder Cancer Patient-Derived Xenografts for Molecularly Guided Targeted Therapy PLoS One 10 2015 e0134346 10.1371/journal.pone.0134346 PMC4535951 26270481

52 Mullenders J. de Jongh E. Brousali A. Roosen M. Blom J.P.A. Begthel H. Korving J. Jonges T. Kranenburg O. Meijer R. Mouse and human urothelial cancer organoids: A tool for bladder cancer research Proc. Natl. Acad. Sci. USA 116 2019 4567 4574 30787188 10.1073/pnas.1803595116 PMC6410883

53 Lee S.H. Hu W. Matulay J.T. Silva M.V. Owczarek T.B. Kim K. Chua C.W. Barlow L.J. Kandoth C. Williams A.B. Tumor Evolution and Drug Response in Patient-Derived Organoid Models of Bladder Cancer Cell 173 2018 515 528.e17 29625057 10.1016/j.cell.2018.03.017 PMC5890941

54 van de Merbel A.F. van der Horst G. van der Mark M.H. van Uhm J.I.M. van Gennep E.J. Kloen P. Beimers L. Pelger R.C.M. van der Pluijm G. An ex vivo Tissue Culture Model for the Assessment of Individualized Drug Responses in Prostate and Bladder Cancer Front. Oncol. 8 2018 400 30333957 10.3389/fonc.2018.00400 PMC6176278

55 Vitiello G.A.F. Ferreira W.A.S. Cordeiro de Lima V.C. Medina T.D.S. Antiviral Responses in Cancer: Boosting Antitumor Immunity Through Activation of Interferon Pathway in the Tumor Microenvironment Front. Immunol. 12 2021 782852 10.3389/fimmu.2021.782852 PMC8674309 34925363

56 Matveeva O.V. Chumakov P.M. Defects in interferon pathways as potential biomarkers of sensitivity to oncolytic viruses Rev. Med. Virol. 28 2018 e2008 10.1002/rmv.2008 PMC6906582 30209859

57 Kurokawa C. Iankov I.D. Anderson S.K. Aderca I. Leontovich A.A. Maurer M.J. Oberg A.L. Schroeder M.A. Giannini C. Greiner S.M. Constitutive Interferon Pathway Activation in Tumors as an Efficacy Determinant Following Oncolytic Virotherapy J. Natl. Cancer Inst. 110 2018 1123 1132 29788332 10.1093/jnci/djy033 PMC6186520

58 Knowles M.A. Hurst C.D. Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity Nat. Rev. Cancer 15 2015 25 41 25533674 10.1038/nrc3817

59 Lawson A.R.J. Abascal F. Coorens T.H.H. Hooks Y. O'Neill L. Latimer C. Raine K. Sanders M.A. Warren A.Y. Mahbubani K.T.A. Extensive heterogeneity in somatic mutation and selection in the human bladder Science 370 2020 75 82 33004514 10.1126/science.aba8347

60 Lemos de Matos A. Franco L.S. McFadden G. Oncolytic Viruses and the Immune System: The Dynamic Duo Mol. Ther. Methods Clin. Dev. 17 2020 349 358 32071927 10.1016/j.omtm.2020.01.001 PMC7015832

61 Li R. Zhang J. Gilbert S.M. Conejo-Garcia J. Mulé J.J. Using oncolytic viruses to ignite the tumour immune microenvironment in bladder cancer Nat. Rev. Urol. 18 2021 543 555 34183833 10.1038/s41585-021-00483-z

62 Kaufman H.L. Kohlhapp F.J. Zloza A. Oncolytic viruses: a new class of immunotherapy drugs Nat. Rev. Drug Discov. 14 2015 642 662 26323545 10.1038/nrd4663 PMC7097180

63 Jeong S.R. Kang M. Exploring Tumor-Immune Interactions in Co-Culture Models of T Cells and Tumor Organoids Derived from Patients Int. J. Mol. Sci. 24 2023 14609 10.3390/ijms241914609 PMC10572813 37834057

64 Gujar S.A. Pan D.A. Marcato P. Garant K.A. Lee P.W.K. Oncolytic virus-initiated protective immunity against prostate cancer Mol. Ther. 19 2011 797 804 21245852 10.1038/mt.2010.297 PMC3070098

65 Hanel E.G. Xiao Z. Wong K.K. Lee P.W.K. Britten R.A. Moore R.B. A novel intravesical therapy for superficial bladder cancer in an orthotopic model: oncolytic reovirus therapy J. Urol. 172 2004 2018 2022 15540781 10.1097/01.ju.0000142657.62689.f6

66 Smelser W.W. Wang J. Ogden K.M. Chang S.S. Kirschner A.N. Intravesical oncolytic virotherapy and immunotherapy for non-muscle-invasive bladder cancer mouse model BJU Int. 132 2023 298 306 36961272 10.1111/bju.16012 PMC10518025

67 Zhang T. Jou T.H.T. Hsin J. Wang Z. Huang K. Ye J. Yin H. Xing Y. Talimogene Laherparepvec (T-VEC): A Review of the Recent Advances in Cancer Therapy J. Clin. Med. 12 2023 1098 10.3390/jcm12031098 PMC9917711 36769745

68 Taguchi S. Fukuhara H. Homma Y. Todo T. Current status of clinical trials assessing oncolytic virus therapy for urological cancers Int. J. Urol. 24 2017 342 351 28326624 10.1111/iju.13325

69 Cozzi P.J. Malhotra S. McAuliffe P. Kooby D.A. Federoff H.J. Huryk B. Johnson P. Scardino P.T. Heston W.D. Fong Y. Intravesical oncolytic viral therapy using attenuated, replication-competent herpes simplex viruses G207 and Nv1020 is effective in the treatment of bladder cancer in an orthotopic syngeneic model FASEB J. 15 2001 1306 1308 11344122 10.1096/fj.00-0533fje

70 Hadaschik B.A. Zhang K. So A.I. Fazli L. Jia W. Bell J.C. Gleave M.E. Rennie P.S. Oncolytic vesicular stomatitis viruses are potent agents for intravesical treatment of high-risk bladder cancer Cancer Res. 68 2008 4506 4510 18559493 10.1158/0008-5472.CAN-08-0238

71 Annels N.E. Mansfield D. Arif M. Ballesteros-Merino C. Simpson G.R. Denyer M. Sandhu S.S. Melcher A.A. Harrington K.J. Davies B. Phase I Trial of an ICAM-1-Targeted Immunotherapeutic-Coxsackievirus A21 (CVA21) as an Oncolytic Agent Against Non Muscle-Invasive Bladder Cancer Clin. Cancer Res. 25 2019 5818 5831 31273010 10.1158/1078-0432.CCR-18-4022

72 Shalhout S.Z. Miller D.M. Emerick K.S. Kaufman H.L. Therapy with oncolytic viruses: progress and challenges Nat. Rev. Clin. Oncol. 20 2023 160 177 36631681 10.1038/s41571-022-00719-w

73 Engeland C.E. Bell J.C. Introduction to Oncolytic Virotherapy Methods Mol. Biol. 2058 2020 1 6 31486028 10.1007/978-1-4939-9794-7_1

74 Li M. Zhang M. Ye Q. Liu Y. Qian W. Preclinical and clinical trials of oncolytic vaccinia virus in cancer immunotherapy: a comprehensive review Cancer Biol. Med. 20 2023 646 661 37615308 10.20892/j.issn.2095-3941.2023.0202 PMC10546091

75 Muhlebach M.D. Measles virus in cancer therapy Curr. Opin. Virol. 41 2020 85 97 32861945 10.1016/j.coviro.2020.07.016

76 Javanbakht M. Tahmasebzadeh S. Cegolon L. Gholami N. Kashaki M. Nikoueinejad H. Mozafari M. Mozaffari M. Zhao S. Khafaei M. Oncolytic viruses: A novel treatment strategy for breast cancer Genes Dis. 10 2023 430 446 37223527 10.1016/j.gendis.2021.11.011 PMC10201557

77 Annels N.E. Arif M. Simpson G.R. Denyer M. Moller-Levet C. Mansfield D. Butler R. Shafren D. Au G. Knowles M. Oncolytic Immunotherapy for Bladder Cancer Using Coxsackie A21 Virus Mol. Ther. Oncolytics 9 2018 1 12 29989024 10.1016/j.omto.2018.02.001 PMC6035483

78 Burke J. Virus therapy for bladder cancer Cytokine Growth Factor Rev. 21 2010 99 102 20382064 10.1016/j.cytogfr.2010.02.003

79 Fodor I. Timiryasova T. Denes B. Yoshida J. Ruckle H. Lilly M. Vaccinia virus mediated p53 gene therapy for bladder cancer in an orthotopic murine model J. Urol. 173 2005 604 609 15643273 10.1097/01.ju.0000143196.37008.2c

80 Kuball J. Wen S.F. Leissner J. Atkins D. Meinhardt P. Quijano E. Engler H. Hutchins B. Maneval D.C. Grace M.J. Successful adenovirus-mediated wild-type p53 gene transfer in patients with bladder cancer by intravesical vector instillation J. Clin. Oncol. 20 2002 957 965 11844817 10.1200/JCO.2002.20.4.957

81 Freund C.T. Tong X.W. Block A. Contant C.F. Kieback D.G. Rowley D.R. Lerner S.P. Adenovirus-mediated suicide gene therapy for bladder cancer: comparison of the cytomegalovirus- and Rous sarcoma virus-promoter Anticancer Res. 20 2000 2811 2816 11062688

82 Gomella L.G. Mastrangelo M.J. McCue P.A. Maguire HC J.R. Mulholland S.G. Lattime E.C. Phase i study of intravesical vaccinia virus as a vector for gene therapy of bladder cancer J. Urol. 166 2001 1291 1295 11547060

83 Hu C. Liu Y. Lin Y. Liang J.K. Zhong W.W. Li K. Huang W.T. Wang D.J. Yan G.M. Zhu W.B. Intravenous injections of the oncolytic virus M1 as a novel therapy for muscle-invasive bladder cancer Cell Death Dis. 9 2018 274 29449555 10.1038/s41419-018-0325-3 PMC5833719

84 Kohno S.i. Luo C. Goshima F. Nishiyama Y. Sata T. Ono Y. Herpes simplex virus type 1 mutant HF10 oncolytic viral therapy for bladder cancer Urology 66 2005 1116 1121 16286150 10.1016/j.urology.2005.05.041

85 Shiau A.L. Lin Y.P. Shieh G.S. Su C.H. Wu W.L. Tsai Y.S. Cheng C.W. Lai M.D. Wu C.L. Development of a conditionally replicating pseudorabies virus for HER-2/neu-overexpressing bladder cancer therapy Mol. Ther. 15 2007 131 138 17164784 10.1038/sj.mt.6300001

86 Kemp V. Lamfers M.L.M. van der Pluijm G. van den Hoogen B.G. Hoeben R.C. Developing oncolytic viruses for clinical use: A consortium approach Cytokine Growth Factor Rev. 56 2020 133 140 32553482 10.1016/j.cytogfr.2020.06.010

87 Gujar S. Bell J. Diallo J.S. SnapShot: Cancer Immunotherapy with Oncolytic Viruses Cell 176 2019 1240 1240.e1 30794777 10.1016/j.cell.2019.01.051

88 Comins C. Spicer J. Protheroe A. Roulstone V. Twigger K. White C.M. Vile R. Melcher A. Coffey M.C. Mettinger K.L. REO-10: a phase I study of intravenous reovirus and docetaxel in patients with advanced cancer Clin. Cancer Res. 16 2010 5564 5572 20926400 10.1158/1078-0432.CCR-10-1233 PMC3934355

89 Kicielinski K.P. Chiocca E.A. Yu J.S. Gill G.M. Coffey M. Markert J.M. Phase 1 clinical trial of intratumoral reovirus infusion for the treatment of recurrent malignant gliomas in adults Mol. Ther. 22 2014 1056 1062 24553100 10.1038/mt.2014.21 PMC4015229

90 Eigl B.J. Chi K. Tu D. Hotte S.J. Winquist E. Booth C.M. Canil C. Potvin K. Gregg R. North S. A randomized phase II study of pelareorep and docetaxel or docetaxel alone in men with metastatic castration resistant prostate cancer: CCTG study IND 209 Oncotarget 9 2018 8155 8164 29487723 10.18632/oncotarget.24263 PMC5814290

91 Bradbury P.A. Morris D.G. Nicholas G. Tu D. Tehfe M. Goffin J.R. Shepherd F.A. Gregg R.W. Rothenstein J. Lee C. Canadian Cancer Trials Group (CCTG) IND211: A randomized trial of pelareorep (Reolysin) in patients with previously treated advanced or metastatic non-small cell lung cancer receiving standard salvage therapy Lung Cancer 120 2018 142 148 29748010 10.1016/j.lungcan.2018.03.005

92 Clark A.S. Zhao F. Klein P. Montero A.J. Falkson C. Krill-Jackson E. Rowland K. Sardesai S. Incorvati J. Dillon P. A Phase II Randomized Study of Paclitaxel Alone or Combined with Pelareorep with or without Avelumab in Metastatic Hormone Receptor-Positive Breast Cancer: The BRACELET-01/PrE0113 Study Clin. Cancer Res. 31 2025 2655 2662 40300087 10.1158/1078-0432.CCR-24-2701 PMC12209820

93 Goel S. Ocean A.J. Parakrama R.Y. Ghalib M.H. Chaudhary I. Shah U. Viswanathan S. Kharkwal H. Coffey M. Maitra R. Elucidation of Pelareorep Pharmacodynamics in A Phase I Trial in Patients with KRAS-Mutated Colorectal Cancer Mol. Cancer Ther. 19 2020 1148 1156 32156785 10.1158/1535-7163.MCT-19-1117 PMC7207225

94 Mahalingam D. Fountzilas C. Moseley J. Noronha N. Tran H. Chakrabarty R. Selvaggi G. Coffey M. Thompson B. Sarantopoulos J. A phase II study of REOLYSIN(®) (pelareorep) in combination with carboplatin and paclitaxel for patients with advanced malignant melanoma Cancer Chemother. Pharmacol. 79 2017 697 703 28289863 10.1007/s00280-017-3260-6

95 Mahalingam D. Wilkinson G.A. Eng K.H. Fields P. Raber P. Moseley J.L. Cheetham K. Coffey M. Nuovo G. Kalinski P. Pembrolizumab in Combination with the Oncolytic Virus Pelareorep and Chemotherapy in Patients with Advanced Pancreatic Adenocarcinoma: A Phase Ib Study Clin. Cancer Res. 26 2020 71 81 31694832 10.1158/1078-0432.CCR-19-2078 PMC6942612

96 Nawrocki S.T. Olea J. Villa Celi C. Dadrastoussi H. Wu K. Tsao-Wei D. Colombo A. Coffey M. Fernandez Hernandez E. Chen X. Comprehensive Single-Cell Immune Profiling Defines the Patient Multiple Myeloma Microenvironment Following Oncolytic Virus Therapy in a Phase Ib Trial Clin. Cancer Res. 29 2023 5087 5103 37812476 10.1158/1078-0432.CCR-23-0229 PMC10722139

97 Parakrama R. Fogel E. Chandy C. Augustine T. Coffey M. Tesfa L. Goel S. Maitra R. Immune characterization of metastatic colorectal cancer patients post reovirus administration BMC Cancer 20 2020 569 32552875 10.1186/s12885-020-07038-2 PMC7301987

98 Collienne M. Loghmani H. Heineman T.C. Arnold D. GOBLET: a phase I/II study of pelareorep and atezolizumab +/- chemo in advanced or metastatic gastrointestinal cancers Future Oncol. 18 2022 2871 2878 35796248 10.2217/fon-2022-0453

99 Mahalingam D. Chen S. Xie P. Loghmani H. Heineman T. Kalyan A. Kircher S. Helenowski I.B. Mi X. Maurer V. Combination of pembrolizumab and pelareorep promotes anti-tumour immunity in advanced pancreatic adenocarcinoma (PDAC) Br. J. Cancer 129 2023 782 790 37443348 10.1038/s41416-023-02344-5 PMC10449917

100 Dautzenberg I.J.C. van den Wollenberg D.J.M. van den Hengel S.K. Limpens R.W.A. Bárcena M. Koster A.J. Hoeben R.C. Mammalian orthoreovirus T3D infects U-118 MG cell spheroids independent of junction adhesion molecule-A Gene Ther. 21 2014 609 617 24739522 10.1038/gt.2014.34

101 Walters S.J. Sample size and power estimation for studies with health related quality of life outcomes: a comparison of four methods using the SF-36 Health Qual. Life Outcomes 2 2004 26 15161494 10.1186/1477-7525-2-26 PMC421748

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Document S1. Figures S1–S7 and Table S1 Table S1. Key resources, critical equipment and software Document S2. Article plus supplemental information

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