Tumor microenvironment-modulating oncolytic adenovirus combined with GSK-3β inhibitor enhances antitumor immune response against bladder cancer

Bladder cancer is a common type of cancer around the world, and the majority of patients are diagnosed with non-muscle-invasive bladder cancer (NMIBC). Although low-risk NMIBC has a good prognosis, the disease recurrence rate and development of treatment-refractory disease remain high in intermediate- to high-risk NMIBC patients. To address these challenges for the treatment of NMIBC, a novel combination therapy composed of an oncolytic adenovirus (oAd) co-expressing interleukin (IL)-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), and relaxin (RLX; HY-oAd) and a clinical-stage glycogen synthase kinase (GSK)-3β inhibitor (9-ING-41; elraglusib) was investigated in the present report. Our findings demonstrate that HY-oAd and 9-ING-41 combination therapy (HY-oAd+9-ING-41) exerted superior inhibition of tumor growth compared with respective monotherapy in a syngeneic NMIBC tumor model. HY-oAd+9-ING-41 induced high-level tumor extracellular matrix (ECM) degradation and a more potent antitumor immune response than the respective monotherapy. In detail, HY-oAd+9-ING-41 induced superior accumulation of intratumoral T cells, prevention of immune cell exhaustion, and induction of tumor-specific adaptive immune response compared to either monotherapy. Collectively, these results demonstrate that the combination of HY-oAd and 9-ING-41 may be a promising approach to elicit a potent antitumor immune response against bladder cancer.


Introduction
Non-muscle-invasive bladder cancer (NMIBC) is the most common genitourinary malignancy and comprises more than 70% of all bladder cancer cases (1).Currently, transurethral resection of bladder tumors and adjuvant intravesical treatments, like chemotherapy or Bacillus Calmette-Gueŕin (BCG), are widely utilized and demonstrated favorable outcomes for the majority of the patients (2)(3)(4).Nevertheless, approximately 40% of these NMIBC patients eventually experience tumor recurrence within a span of 2 years, while 10% of the cases progress to muscle-invasive bladder cancer (MIBC) with a worse prognosis (5).
To address the limitations of conventional therapeutic options, various immunotherapeutics, like cancer vaccines, gene therapies, and immune checkpoint inhibitors (ICIs), have been extensively investigated in both preclinical and clinical environments (6).Recently, gene therapies utilizing adenovirus (Ad) have shown promising clinical efficacy against BCG-unresponsive or advanced NMIBC cases (7).In detail, Adstiladrin, a non-replicating Ad expressing interferon (IFN)-a2b, was approved by the US Food and Drug Administration (FDA) for the treatment of BCGunresponsive NMIBC patients in 2022 after achieving 51% complete response rate (8).Similarly, CG0070, which is an oncolytic Ad (oAd) expressing granulocyte-macrophage colonystimulating factor (GM-CSF), has shown promising clinical activity for the treatment of BCG-refractory NMIBC patients in a phase II clinical trial (9).Currently, CG0070 is being evaluated in a phase III trial as monotherapy and in combination with pembrolizumab in a phase II trial for the treatment of BCG-unresponsive high-grade NMIBC patients (10), highlighting the promising nature of Adbased gene therapy for disease management of NMIBC.
oAds, which can preferentially replicate in and lyse tumor cells (11-15), are more actively evaluated in the clinical environment than replication-incompetent counterparts for the treatment of cancer owing to several advantageous attributes like prolonged biological persistence, higher level of therapeutic gene expression, and superior induction of antitumor immune response (16,17).Briefly, cancer-specific viral replication and subsequent cytolysis of tumor cells by oAd release tumor-specific antigens and dangerassociated signaling molecules that promote tumor-specific immune response.Arming the oAd with immune-stimulatory therapeutic genes, like cytokines and chemokines, further potentiates the antitumor immune response mediated by the virus via exponential amplification of transgene products in a tumorspecific manner (18,19).Indeed, the majority of the oncolytic viruses under clinical investigations express at least one immunestimulatory therapeutic gene, with interleukin (IL)-12 and GM-CSF being the most frequently utilized antitumor immune transgene candidates to date (17,20).
Of the two cytokines, GM-CSF has been more extensively investigated with several different types of oncolytic viruses, including Ad, herpes simplex virus (HSV), and vaccinia virus (VV), expressing GM-CSF (CG0070, Imlygic, and Pexa-vec, respectively) either completing or under ongoing investigation in phase III clinical trials.Although GM-CSF has been extensively investigated in clinical trials as a therapeutic transgene for oncolytic viruses or as a recombinant cytokine for cancer immunotherapy, there are increasing number of evidences that demonstrate the potentially pro-tumorigenic role of GM-CSF and inadequate therapeutic benefit of GM-CSF monotherapy in clinical environment (21)(22)(23)(24), suggesting that GM-CSF as sole therapeutic gene may exert suboptimal antitumor immune response.Indeed, there are evidences that suggest that there are better antitumor cytokine candidates, like IL-12, that can exert a superior therapeutic effect compared with GM-CSF for oncolytic virotherapy (25-28).Thus, more recently developed oncolytic viruses in clinical trials have been armed with immune transgenes, like IL-12, IL-15, and IL-21, instead of GM-CSF (17).Alternatively, co-expression of GM-CSF with other immune-stimulatory transgenes, like IL-12, by an oncolytic virus has been shown to exert a superior antitumor effect over the expression of respective immune transgene alone in preclinical setting (26,27,29), suggesting that GM-CSF as an adjuvant transgene may warrant further investigations.
One of the major obstacles to effective disease management of NMIBC cases remains innate or acquired resistance to BCG therapy.There are several purported mechanisms, like differential tumor mutation burden or mutation signatures, to patients becoming unresponsive to BCG treatment (30).Recent lines of evidence also suggest that T-cell exhaustion, evidenced by elevated immune checkpoint molecules like PD-1, LAG3, CTLA-4, TIGIT, or TIM-3, is correlated with BCG unresponsiveness of NMIBC patients (30,31).In line with these findings, PD-1-targeted ICI, pembrolizumab, was shown to exert promising therapeutic efficacy against BCG-unresponsive NMIBC cases, ultimately leading to US FDA approval in 2020 (32,33), showing that reversal of T-cell exhaustion via checkpoint blockade can be a promising strategy for the treatment of BCG-unresponsive NMIBC.
Based on these backgrounds, the present report investigated a novel combination immunotherapy regimen utilizing oAd coexpressing IL-12, GM-CSF, and relaxin (RLX; HY-oAd) and glycogen synthase kinase (GSK)-3b inhibitor (iGSK3b), 35), for the treatment of NMIBC.Clinically evaluated iGSK3b, 9-ING-41, was chosen for combination therapy based on its previously reported function to inhibit the expression of various checkpoint molecules, like PD-1, TIM-3, and TIGIT, on CD8 + T cells (35).As oAd-mediated antitumor immune response has been reported to induce T-cell exhaustion and T-cell exhaustion is associated with BCG unresponsiveness of NMIBC, the present report sought to overcome T-cell exhaustion by combining HY-oAd with iGSK3b to induce synergistic antitumor immune response.To this end, HY-oAd as a single agent induced potent bladder cancer-specific killing efficacy and inhibited tumor growth by inducing a potent antitumor immune response in the syngeneic murine bladder tumor model.The combination of HY-oAd and 9-ING-41 (HY-oAd+9-ING-41) exerted superior anticancer efficacy both in vitro and in vivo over the respective monotherapy.The superior efficacy of HY-oAd+9-ING-41 was achieved via superior induction of CD4 + and CD8 + T-cell infiltration into tumor tissues, as well as tumor-specific immune response over the respective monotherapy, indicating that combining both oAd and iGSK3b inhibitor can be a promising strategy to overcome T-cell exhaustion and exert potent antitumor immunity against NMIBC.

HY-oAd expresses all three therapeutic genes and elicits bladder cancer-specific cell-killing effect
To overcome the limitations of oncolytic virus expressing GM-CSF as a single therapeutic gene for the treatment of NMIBC, HY-oAd co-expressing single-chain murine IL-12 (scIL-12), murine GM-CSF, and human RLX has been utilized (Figure 1A).As shown in Figure 1B, infection of murine NMIBC cell line MB49 cells with HY-oAd led to a dose-dependent expression of all therapeutic genes, showing that HY-oAd has been properly generated.
Infection of human or murine bladder cancer cell lines with HY-oAd led to a dose-dependent cancer cell-killing effect that was significantly more potent than those observed using control oAd lacking therapeutic transgene (control oAd) (Figure 1C; **p < 0.01, ***p < 0.001).Neither control oAd nor HY-oAd exerted off-target cytopathic effects in murine or human fibroblast cell lines up to 500 or 50 multiplicity of infection (MOI), respectively.Together, these results demonstrate that the three therapeutic genes expressed by HY-oAd enhanced bladder cancer-specific cell-killing effect with no observable off-target activity in normal cell lines.

HY-oAd induces potent antitumor effect against subcutaneous bladder tumor model
To evaluate the antitumor efficacy of HY-oAd, mice bearing subcutaneous MB49 tumors were intratumorally treated with 2 × 10 10 viral particles (VPs) of HY-oAd or an oAd expressing GM-CSF alone on days 0, 2, and 4 along with phosphate-buffered saline (PBS) as negative control (Figure 2A).As shown in Figures 2B, C, the PBStreated group exhibited rapid tumor growth and reached an average tumor volume of 3,464.2 ± 295.2 mm 3 at day 30 after the initial treatment.In contrast, both oAd expressing GM-CSF alone and HY-oAd induced significant tumor growth inhibition compared to the PBS-treated group (***p < 0.001).Importantly, HY-oAd exerted a significantly more potent antitumor effect compared with oAd expressing GM-CSF alone, as evidenced by complete regression of all tumors in six out of six mice compared to three out of six regression, respectively.Together, these findings demonstrated that the expression of multiple therapeutic genes by HY-oAd led to a more potent anticancer effect against NMIBC compared to the expression of GM-CSF as the only therapeutic gene.compared with either HY-oAd or 9-ING-41 monotherapy (*p < 0.05), showing 72.6% and 67.2% superior tumor burden reduction, respectively.Importantly, both HY-oAd and HY-oAd+9-ING-41 treatments exerted robust anti-metastatic effects compared to PBS or 9-ING-41 groups where extensive lung metastasis (>20 metastatic nodules per lung) was observed (Supplementary Figure S3).In detail, 6/6 and 8/8 mice in PBS and 9-ING-41 groups, respectively, showed extensive lung metastasis, whereas 5/9 mice were either free of metastatic nodules or with less than 10 nodules for both HY-oAd monotherapy and HY-oAd+9-ING-41 treatment groups.Notably, HY-oAd+9-ING-41 treatment led to the highest percentage of mice that were nearly absent of metastatic nodules (5/ 9 for combination therapy versus 3/9 for HY-oAd monotherapy for mice with 0-2 metastatic nodules).None of the treatments induced any significant body weight loss and observable toxicity up to 28 days after the initial treatment (Supplementary Figure S4), indicating negligible systemic toxicity.Taken together, these results suggest that the combination of HY-oAd and 9-ING-41 can effectively control the growth of both in a safe manner.

HY-oAd+9-ING-41 promotes ECM degradation, apoptosis, and viral dispersion in bladder tumor tissue
To further evaluate the therapeutic effect of the combination treatment regimen, histological and immunohistochemical analyses of tumor tissues were performed.As shown in Figure 5A

HY-oAd+9-ING-41 promotes T-cell accumulation in tumor and spleen tissues
To investigate the mechanism behind the antitumor immune response mediated by each treatment, the splenic and intratumoral CD4 + and CD8 + T-cell populations were analyzed.As shown in Figure 6A, the number of CD4 + and CD8 + T-cell populations was unaffected by 9-ING-41 monotherapy in comparison with the PBS control group.In contrast, both HY-oAd monotherapy and its combination with 9-ING-41 led to significantly elevated CD4 + and CD8 + T-cell population in comparison with the PBS or 9-ING-41 group (*p < 0.05, **p < 0.01, or ***p < 0.001), suggesting that HY-oAd was integral to induction of T cell-mediated antitumor immune response.Of note, HY-oAd+9-ING-41 combination therapy induced a higher level of CD8 + T-cell accumulation in the spleen with respect to HY-oAd (*p < 0.05), whereas the CD4 + Tcell accumulation was similarly high between the two groups, showing that the synergistic effect induced by the combination therapy was more reliant on CD8 + T-cell response.In line with these findings, the analysis of tumor-infiltrating lymphocyte (TIL) population also revealed that both CD4 + and CD8 + T-cell accumulation was significantly elevated in tumor tissues after treatment with HY-oAd monotherapy or its combination with 9-ING-41 in comparison with the PBS control group with extremely low-level T-cell infiltration (less than 5% of total tumor cell population was either CD4+ or CD8+ T cells; ***p < 0.001; Figure 6B).One notable difference between spleen and tumor tissues was that 9-ING-41 significantly elevated CD4 + and CD8 + T-cell infiltration into the tumor tissues in comparison with PBS control, whereas the therapy failed to elevate T-cell recruitment in the spleen tissues, suggesting that systemically administered 9-ING-41 may induce a more pronounced T-cell response in immunosuppressive tumor microenvironment.Importantly, HY-oAd plus 9-ING-41 combination therapy led to the highest level of both CD4 + and CD8 + T-cell infiltration among all treatment groups (**p < 0.01 or ***p < 0.001 versus 9-ING-41 monotherapy).Similar results were also obtained by immunohistochemical analysis, as HY-oAd+9-ING-41 induced the highest level of CD4 + and CD8 + Tcell recruitment in the tumor (Figure 6C; *p < 0.05 or ***p < 0.001 versus HY-oAd or 9-ING-41 monotherapy).Together, these results suggested that the combination therapy induced a potent antitumor effect through robust initiation of T cell-mediated antitumor immune response.

Tumor-specific immune response and prevention of T-cell exhaustion by combination of HY-oAd and 9-ING-41
To further characterize the antitumor immune response induced by HY-oAd+9-ING-41 combination therapy, the splenocytes from tumor-bearing mice treated with PBS, 9-ING-41, 1 × 10 9 VPs of HY-oAd, and HY-oAd+9-ING-41 were harvested and co-cultured with irradiated MB49 cells to determine IFN-g-secreting lymphocyte population by IFN-g ELISpot assay.As shown in Figure 7A, the HY-oAd+9-ING-41 combination group showed a significantly higher number of IFNg-secreting lymphocytes over the respective monotherapy (*p < 0.05, **p < 0.01), suggesting that it could induce a potent tumorspecific adaptive immune response.
Next, we evaluated whether the HY-oAd+9-ING-41 combination therapy abates cytotoxic T-cell exhaustion in the spleen and tumor tissues.As shown in Figure 7B, all treatments (9-ING-41, HY-oAd, or HY-oAd+9-ING-41) elevated frequency of CD8 + PD1 − T-cell subsets in spleen tissues in comparison with the PBS control group (*p < 0.05, **p < 0.01), suggesting that all three treatments can enhance the accumulation of functional cytotoxic T cells that were not exhausted in the secondary lymphoid organ.In contrast, only HY-oAd+9-ING-41 combination therapy significantly elevated the number of unexhausted CD8 + PD1 − T-cell population in the tumor tissues (***p < 0.01), whereas the respective monotherapy could not.In support, immunofluorescence detection of PD-1 expression in MB49 tumor tissues also revealed that the periphery of tumor tissues of mice in the PBS group was enriched with PD-1 + cells (Figure 7C).Both 9-ING-41 and HY-oAd monotherapy markedly attenuated intratumoral infiltration of PD-1 + cells in comparison with the PBS control group.Of the two monotherapies, 9-ING-41 exerted superior inhibition of PD-1 + cell infiltration.Importantly, HY-oAd+9-ING-41 combination therapy showed a markedly lower level of PD-1 + cell infiltration compared to the HY-oAd monotherapy or PBS control group, suggesting that the potent PD-1 inhibitory effect of 9-ING-41 enabled a synergistic increase in intratumoral infiltration of unexhausted CD8 + PD1 − T cells.Together, these results demonstrate that the HY-oAd+9-ING-41 combination therapy could effectively induce a tumor-specific immune response and enhance the recruitment of unexhausted cytotoxic T cells in the immunosuppressive tumor milieu.

Discussion
BCG has been used as the first-line immunotherapeutic treatment for NMIBC.Despite BCG having long been the standard therapy, its failure occurred in 30% to 50% of patients (36).The response to BCG is often limited by several factors, like activation of the PD-1/PD-L1 signaling axis that induces immune anergy and Tcell exhaustion, which abrogate the efficacy of BCG therapy over the treatment course (31).Currently, oncolytic virus expressing proinflammatory cytokine can boost the induction of antitumor immune response and reprogram the immunosuppressive tumor milieu (10,(37)(38)(39).Still, the oncolytic viruses' effect on T-cell exhaustion and immune checkpoint regulation within the tumor microenvironment have not been conclusively elucidated: some suggested that T-cell exhaustion can be prevented or reversed by oncolytic virotherapy, while others demonstrated that the virus infection can upregulate PD-L1 expression on the surface of tumor cells and simultaneously increase PD-1 expression on T cells that are suggestive of exhaustion (40)(41)(42).
Although the complexity of PD-1 regulation patterns for T cells makes it difficult for PD-1 expression status alone to discriminate between exhausted and activated T cells within the tumor microenvironment (43), our present findings demonstrated that clinical-stage GSK-3b inhibitor, 9-ING-41, which has been reported to activate and transcriptionally attenuate expression of various immune checkpoint molecules of T cells in either preclinical or clinical environment (35,44,45), in combination with oAd armed with immune-stimulatory and ECM-degrading therapeutic genes could effectively diminish PD-1 + cell infiltration into the periphery of the bladder tumor and synergistically enhanced the infiltration and recruitment of CD8 + PD-1 − T cells in both tumor and spleen (Figures 7B, C).These findings suggested that the potential risk of T-cell exhaustion via activation of PD-1/PD-L1 signaling axis could be effectively mitigated by the rational combination of HY-oAd and 9-ING-41.The combination therapy was also shown to induce the highest level of tumor-specific immune response (Figure 7A), which likely contributed to it inducing the most robust inhibition of lung metastasis (Supplementary Figure S3).These findings are in line with previous publications that highlight the importance of tumor-specific immunity in the initiation of strong abscopal effect and systemic immune response by the therapeutic (46)(47)(48).Although these findings showed that PD-1 downregulation by combination may exert an anti-metastatic effect via abscopal effect and systemic immune activation, more in-depth preclinical characterization of the combination therapy regimen across different types of tumors, especially those that are refractory to PD-1/PD-L1 checkpoint inhibitors, is still needed to translate these findings into clinical stages.
Additionally, GM-CSF therapy has been reported to exert conflicting biological functions across different types of tumors, as both pro-tumorigenic and anticancer activity of the cytokine have been reported (22,49).The inhibition of the GSK-3 signaling pathway has also been reported to exert potentially protumorigenic function, as it can induce anti-inflammatory reactions by suppressing the NF-kB signaling axis (34, 50, 51).
These findings demonstrate that further evaluations of HY-oAd and 9-ING-41 in other types of cancers will be necessary in the future to better identify patient demographics that would benefit from this treatment regimen.
Another important clinical feature of both HY-oAd and 9-ING-41 for cancer therapy is that they are capable of degrading aberrant tumor ECM (Figure 5A), which is a well-known barrier against intratumoral penetration and dispersion of various cancer    S2).These findings highlighted that degradation of ECM had a critical role in positively reshaping TIL composition within bladder tumors.
There are several studies showing the role of GM-CSF in immune cell homeostasis.These studies illustrate that a small amount of GM-CSF hampers the proper generation of innate immune cells and subsequent activation of the adaptive anticancer immune response, whereas excessive GM-CSF can deplete immune cells and foster cancer growth (22, 49).Additionally, the impact of GM-CSF signaling on cancer progression varied based on cancer type and the tumor microenvironment as well as GM-CSF levels.Even though GM-CSF has a double-edged sword mechanism in cancer immunotherapy, our results show that the strategy of combining 9-ING-41 with HY-oAd-expressing GM-CSF does not negatively affect the eradication of bladder tumors.Nevertheless, for the combination dosing strategy proposed in this study to be applied in the clinic with expanded indications, a serious approach to the effect of GM-CSF on the anticancer immune response is required.
Despite significant advancements made with Ad-based immunotherapy for the treatment of bladder cancer, no oAd treatment has reached clinical approval for the treatment of NMIBC by the US FDA or European Medicines Agency to date.Additionally, there are many patients who still suffer from NMIBC and do not respond to either BCG or ICI treatments, with more recent lines of evidence suggesting that T-cell exhaustion is correlated with BCG unresponsiveness of NMIBC patients (30,31).To address these challenges, the present report investigated GSK-3b inhibitor, 9-ING-41, rather than PD-1/PD-L1-targeted ICI to pharmacologically inhibit checkpoint upregulation within the tumor milieu and combat T-cell exhaustion in combination with multifunctional HY-oAd.Collectively, our findings demonstrate that oAd armed with multiple immune-stimulatory and ECMdegrading therapeutic genes can outperform those observed using oAd expressing GM-CSF as a single therapeutic gene and that it could synergize with 9-ING-41 to induce robust degradation of tumor ECM and tumor-specific immune response and prevent immune cell exhaustion, ultimately enabling effective control of primary and metastatic bladder tumor growth.

Preparation of HY-oAd
To produce HY-oAd, the HY-oAd genome containing plasmid was linearized with PacI and then transfected into 293A cells using JetPrime transfection reagent (Polyplus, Illkirch, France) according to the manufacturer's instruction.The construction and generation of hypoxia-and telomerase-responsive control oAd lacking therapeutic transgene (control oAd) have been described previously (50).All Ads were propagated in A549 cells and purified by CsCl gradient centrifugation.The number of VPs was determined by measuring the optical density at 260 nm, for which an absorbance value of 1 is equivalent to 1.1 × 10 12 VPs/mL.Purified viruses were stored at −80°C until use.

Quantification of IL-12, GM-CSF, and RLX expression level
MB49 cells were plated onto 6-well plates at 2 × 10 5 cells per well overnight and then infected with HY-oAd at a MOI of 20, 50, or 100.At 48 h after infection, the supernatants were harvested.The expression levels of mouse IL-12 and mouse GM-CSF secreted into the supernatants were determined using conventional mouse IL-12p70 (R&D Systems, Minneapolis, MN, USA) and mouse GM-CSF Duo Set ELISA kit (R&D Systems) according to the manufacturer's instructions.For the assessment of the mRNA expression level of RLX, cell lysates were obtained from the same plate as those utilized for ELISA by treating these cells with RNA iso Plus kit (Takara, Otsu, Japan) according to the manufacturer's protocol.
Total RNA was purified from the cell lysates, and cDNA was obtained using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) under the following conditions: 25°C for 10 min, 37°C for 120 min, and 85°C for 5 min.RLX mRNA was amplified by PCR with the following primer set: 5′-CCTGGAGCAAAAGGTCTCTG-3′ as the sense primer and 5′-TCTCAGATAGGGCTGCCTTC-3′ as the antisense primer.PCR products were analyzed by gel electrophoresis using 1% agarose gels.carried out as described previously (57).The spots were measured using a computer-based immunospot system (AID ELISpot Reader System version 3.4; Autoimmun Diagnostika GmbH, Strassberg, Germany).

Animal studies
Five-week-old male C57BL/6 mice (DBL Inc., Eumseong, South Korea) were maintained in a laminar airflow cabinet with specific pathogen-free conditions.All facilities have been approved by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).Animal studies were conducted according to the institutional guidelines established by the Hanyang University Institutional Animal Care and Use Committee.

Statistical analysis
No statistical methods were used to predetermine sample sizes for in vitro or in vivo experiments.All results are expressed as the mean ± SEM unless indicated otherwise.Comparisons between groups were made using the two-tailed Student's t-test or one-way ANOVA and Tukey's post-hoc tests for multiple groups.Statistical significance was denoted as *p < 0.05, **p < 0.01, and ***p < 0.001.Statistical analysis was performed in GraphPad Prism 5 (GraphPad, San Diego, CA, USA).

1
FIGURE 1 Characterization of HY-oAd.(A) Schematic representation for the genomic structure of the HY-oAd.(B) MB49, a murine bladder cancer cell, was infected with control oAd or HY-oAd at 0 to 100 MOI for 48 h The supernatants were harvested, and then murine IL-12 and GM-CSF expression levels were analyzed by ELISA.Total RNA was extracted from MB49 cell lysates, and then mRNA expression levels of RLX and b-actin were analyzed by conventional reverse-transcriptase PCR.Each cell line was tested at least three times, and the data shown are representative of experiments performed in triplicate.Bars represent mean ± SD. ***p < 0.001 (n = 3).p-Values were determined using Student's t-test.(C) Murine bladder cancer cell line (MB49), human bladder cancer cell lines (RT-4 and UM-UC-3), murine normal fibroblast (NIH3T3), and human normal fibroblast (BJ) were infected with 0-500 MOI of control oAd or HY-oAd, and then the cell viability was determined by MTT assay.Each cell line was tested at least three times, and the data shown are representative of experiments performed in triplicate.Bars represent mean ± SD. ***p < 0.001, **p < 0.01 (n = 3).p-Values were determined using Student's t-test.MOI, multiplicity of infection; GM-CSF, granulocyte-macrophage colony-stimulating factor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Prior to the evaluation of HY-oAd+9-ING-41 combination therapy, we first sought to investigate the anticancer effect of 9-ING-41 monotherapy against bladder cancer.As shown in Figures3A and Supplementary
FIGURE 4 Antitumor effect of HY-oAd with 9-ING-41 combination therapy against subcutaneous MB49 tumors.(A) Graphical outlay of the treatment schedule and dosing for the combination therapy efficacy evaluation study.When the mean tumor volume of MB49 reached 200 mm 3 , mice were intratumorally treated three times with 2 × 10 10 VPs of HY-oAd and/or intraperitoneally administered with 10 mg/kg of 9-ING-41 (n = 8-9 per group), along with PBS as negative control.(B) The mean tumor volume throughout the course of the study and individual tumor volume of all mice are provided.Data are presented as mean ± SEM (n = 8-9).*p < 0.05, ***p < 0.001.p-Values were determined using the one-way ANOVA.Individual tumor growth curves in MB49 tumor model treated with PBS, 9-ING-41, HY-oAd, or HY-oAd+ 9-ING-41 were plotted.VPs, viral particles; PBS, phosphate-buffered saline.
therapeutics and immune cells (52-54).In line with this literature, effective degradation of tumor ECM by HY-oAd and 9-ING-41 was shown to facilitate CD4 + and CD8 + T-cell infiltration into the tumor tissues (Figures 6B, C, 7B), as well as simultaneously improving HY-oAd dispersion and accumulation throughout the bladder tumor tissues in a viral replication-independent manner (Figures 5B, Supplementary