The Potential Antitumor Effects of Combining Intravesical Therapy with Recombinant Bacillus Calmette-Guérin and an Immune Checkpoint Inhibitor in Bladder Cancer

The Potential Antitumor Effects of Combining Intravesical Therapy with Recombinant Bacillus Calmette-Guérin and an Immune Checkpoint Inhibitor in Bladder Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Potential Antitumor Effects of Combining Intravesical Therapy with Recombinant Bacillus Calmette-Guérin and an Immune Checkpoint Inhibitor in Bladder Cancer Jung Hoon Kim, Sejung Maeng, Joongwon Choi, Chung Un Lee, Yong Seong Lee, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6798208/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Mar, 2026 Read the published version in BMC Cancer → Version 1 posted 14 You are reading this latest preprint version Abstract Background Recombinant Bacillus Calmette-Guérin (rBCG) is a genetically modified BCG that enhances therapeutic efficacy and safety in patients with bladder cancer. Pembrolizumab is an immune checkpoint inhibitor that is used to treat bladder cancer. We aimed to evaluate the effectiveness of the combination of rBCG and pembrolizumab in in vitro and in vivo bladder cancer models. Methods We investigated the antitumor effects of rBCG- dltA and pembrolizumab in a bladder cancer-on-a-chip (BCOC) model, an organoid three-dimensional cell culture tool. An orthotopic mouse model was employed to assess the in vivo effects of the combination therapy. To overcome errors caused by differences between species, an in vivo study was conducted using a mouse PD1 inhibitor instead of pembrolizumab. Results The T24 bladder cancer cell line declined independently when rBCG- dltA and pembrolizumab were used in the BCOC model. Monocyte migration was also significantly greater than that in the other groups. In the orthotopic bladder cancer model, the combination treatment inhibited the growth of bladder cancer. Conclusions Intravesical therapy with rBCG- dltA and pembrolizumab is effective in treating bladder cancer. This study suggests that the combination of rBCG and immune checkpoint inhibitors is a novel strategy for treating patients with BCG-unresponsive bladder cancer. Bacillus carmette-guérin Bladder cancer Pembrolizumab 3-dimensional cell culture Organ-on-a-chip devices Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background According to Global Cancer Statistics 2020, bladder cancer is the 10th most commonly diagnosed malignant tumor in the world ( 1 ). It is the second most common type of urinary tract cancer after prostate cancer. Although surgical management of bladder cancer has remained relatively unchanged, the field has witnessed significant advancements in therapeutic agents over recent years. Recently, traditional and innovative therapeutic agents, such as chemotherapy or immune checkpoint inhibitors, have been integrated to manage both non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). Intravesical immunotherapy with Bacillus Calmette-Guérin (BCG) is a common therapeutic modality for patients with bladder cancer. The European Association of Urology (EAU) guidelines strongly recommend intravesical BCG after transurethral tumor resection in patients with intermediate- and high-risk bladder cancer ( 2 ). BCG prevents disease progression in NMIBC by up to 27% and reduces the recurrence rate by 32% ( 3 ). However, BCG therapy has several side effects, such as hematuria, dysuria, and urinary tract infections. Severe adverse events, such as BCG sepsis, are rarely reported ( 4 ). Owing to the collapse of the global supply chain, BCG shortages are an emerging issue. Various studies have been conducted to overcome the unresponsiveness of BCG, increase its effectiveness, and decrease its side effects ( 5 , 6 ). Recombinant BCG (rBCG) is a modified version of BCG. Compared with conventional BCG treatment, genetically modified BCG products aim to increase the efficiency of immunotherapy and reduce complication rates. rBCGs have been developed to induce specific immune responses or include foreign antigens ( 7 ). rBCG induced the overexpression of cytokines more than did conventional BCG treatment. Bioengineered 3D tumor models are used to construct tumor microenvironments (TMEs) that play key roles in tumor growth, drug resistance, and metastasis ( 8 ). The cancer-on-a-chip model better emulates the TME, cancer architecture, and flow dynamics ( 9 ). Bladder cancer-on-a-chip (BCOC) is a form of organ-on-chip. It is a layered structure of cell blocks printed via 3D bioprinting ( 10 ). Pembrolizumab is a highly selective monoclonal antibody against PD-1 that disrupts the linkage between PD-1 ligands. Pembrolizumab is an effective immune checkpoint inhibitor (ICI) that has antitumor activity in patients with bladder cancer ( 11 ). Compared with systemic chemotherapy, pembrolizumab has shown significant overall survival gains for 3 months ( 12 ). The antitumor effects of pembrolizumab are achieved not only through systemic administration but also through intravesical instillation. A phase I clinical trial of intravesical pembrolizumab and BCG treatment reported safe and feasible results in patients with BCG-unresponsive NMIBC ( 13 ). We hypothesized that the combination of rBCG and pembrolizumab would enhance immunological reactions more than the conventional BCG immunotherapy. In this study, we used a high-throughput BCOC model to evaluate the immunotherapeutic effects of rBCG and pembrolizumab. Methods 1. Cell lines and reagents of the BCOC Model To evaluate the combined antitumor effect of rBCG and pembrolizumab, we employed a high-throughput BCOC system, composed of four parallel modules driven by syringe pump ( 10 , 14 ). The 3D bioprinted BCOC blocks consisted of the T24 bladder cancer cells, MRC-5 fibroblast, Jurkat T lymphocyte, THP-1 monocytes, and human umbilical vein endothelial cell line (HUVEC), cultured using standard protocols provided by the suppliers; Korean Cell Line Bank (Seoul, Republic of Korea) and Lonza (Basel, Switzerland). MBT2-luc murine bladder cancer cells were generously provided by Dr. Sang-Jin Lee (National Cancer Center, Republic of Korea). Commercially available Mycobacterium bovis BCG was obtained from OncoTice (Merck Sharp, Kenilworth, NJ, USA). BCG was prepared at a multiplicity of infection (MOI) of 30 (1.8×10 6 cells/ml) and stored at -80°C until use. The GelMA prepolymer mixture used was Gel4Cell (Innoregen, Daegu, Republic of Korea). The rBCG strain was engineered to express dltA gene, cloned into a pMV306 vector using Hind Ⅲ and Sal I restriction enzymes, following previously describe protocols ( 15 ). 2. Live/dead cell viability and THP-1 cell migration assay Cell viability in the 3D structures was assessed on the first and third days following the administration of rBCG- dltA and pembrolizumab (Keytruda; Merck Sharp and Dohme Ireland, Carlow, Ireland). Prior to fluorescent live and dead staining solutions (Thermo Fisher, Waltham, MA, USA), each 3D cell construct was washed three times with Dulbecco’s phosphate-buffered saline. A fluorescence microscope (Leica DMi8; Leica, Germany) was used to observe the cell morphology, and three independent samples were analyzed. THP-1 monocytes were transformed into macrophages by incubation with 25 nM phorbol 12-myristate 13-acetate (PMA; Sigma‒Aldrich, St. Louis, MO, USA) for 24 h. The differentiated cells were seeded into the bottom layer of the chip at a density of 2×10 4 cells/20 µL, and the THP-1 cells were visualized via an Olympus CKX41 inverted microscope (Olympus, Tokyo, Japan) and counted in three randomly selected fields, and the average was calculated. 3. Animal studies A syngeneic orthotopic bladder cancer mouse model was established as described previously ( 10 , 16 ). After a mouse bladder cancer model was established, the mice were divided into four groups. Four groups of mice, consisting of five mice each, received either PBS, rBCG- dltA , anti-mPD1, or a combination of both directly into the bladder through a catheter twice weekly. Bladder cancer progression was monitored via bioluminescence imaging (BLI), and body weight measurements were taken twice weekly for 2 weeks. The BLI data were obtained and analyzed via the Living Image software CleVue version 3.1.3.2054 (Vieworks, Anyang, Republic of Korea). A schematic of the treatment protocol is shown in Fig. 1 . Prior to tissue collection, all animals underwent anesthesia utilizing an inhalation anesthesia system. The anesthetic agent was Ifran Liquid for Inhalation (Isoflurane; Hana Pharm, Seoul, Korea). Anesthesia was initiated within a chamber utilizing a gas mixture of nitrogen and oxygen in a 1:1 ratio, with isoflurane administered at a concentration of 2–3%. After approximately 5 min of induction, the animals were transitioned to a nose cone connected to the anesthesia system to sustain isoflurane anesthesia during the tissue harvesting process. Following the collection of samples, the animals were euthanized via cervical dislocation while still under deep anesthesia. This approach was chosen to minimize animal suffering and to ensure a humane endpoint. 4. Hematoxylin and eosin (H&E) staining Bladder cancer tissues were harvested from an orthotopic bladder cancer mouse model and fixed in 4% paraformaldehyde solution (Biosesang, Yongin, Republic of Korea) at 4°C for 24 h. Following standard processing, tissues were dehydrated, embedded in paraffin, and sectioned at 4 µm thickness. Sections were deparaffinized, rehydrated, and stained with H&E using conventional protocols. The stained slides were imaged via a image scanner (Pannoramic MIDI; 3DHISTECH, Budapest, Hungary) for histological evaluation. 5. Statistical analysis Experimental data were obtained from a minimum of three independent experiments. The Statistical Package for Social Sciences (SPSS) version 25.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Student’s t test was performed to compare the means between two different groups, and statistical significance was defined as p < 0.05. 6. Ethics approval The procedures and animal care were approved by the Institutional Animal Care and Use Committee of Chung-Ang University (Approval No. A2021020, date: Jan 09, 2023, Seoul, Republic of Korea) and performed in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals. In addition, this study was conducted in accordance with the ARRIVE guidelines. Results 1. Cellular viability after treatment with rBCG- dltA with or without pembrolizumab in BCOCs Cell viability was assessed at two different time intervals, on the first and third days, for each treatment group. In the live and dead staining assay, the T24 cancer cell line in the combination group of rBCG- dltA and pembrolizumab exhibited predominantly decreased viability, whereas MRC-5 cells and HUVECs maintained a normal level of viability (Fig. 2 A). The merged image represents the relative proportions of living (green) and dead (red) cells. The images clearly indicate a higher rate of T24 cell death in the combination group. The fluorescence intensity of the T24 cells (mean ± standard error [SE], %) was significantly lower in the combination group (22.24 ± 5.01) than in the control group (Fig. 2 B). The other MRC-5 cells and HUVECs did not significantly differ between the groups. Compared with both the rBCG-dltA and pembrolizumab groups, the combination treatment group demonstrated superior effectiveness in inhibiting tumor growth ( p < 0.05). 2. Effects of chemotaxis following the administration of rBCG- dltA and/or pembrolizumab The chemotactic response of monocytic THP-1 cells within the membrane was verified in the control, rBCG- dltA and pembrolizumab groups (Fig. 3 A). The average migration rate (mean ± SE, %) of THP-1 cells was significantly greater in the combination group (10,540 ± 1,551, p < 0.01) than in the control group (554.7 ± 354.6) (Fig. 3 B). 3. Evaluation of combination treatment in an orthotopic mouse model of bladder cancer ( in vivo study) To assess the inhibition of tumor growth in the combination treatment group, we compared the BLI intensities in an orthotopic mouse model of bladder cancer (Fig. 4 A). Following the implantation of bladder cancer cells on day 0, all experimental mice were randomly allocated to the three groups and subjected to BLI. The imaging signal intensities were assessed by calculating the average of each measurement (Fig. 4 B). At the time of randomization, there was no statistically significant difference in signal intensity between the groups. The mean ± standard error (SE) of the signal intensity, as measured by BLI, in the combination group (9.44 ± 1.36 × 10 10 ) was significantly lower than that in both the control group (1.69 ± 1.47 × 10 11 , p < 0.05) and the anti-mPD1 group (1.88 ± 3.46 × 10 11 , p < 0.05) on day 10. Compared with the individual treatment groups, the combined treatment group presented a significantly decreased BLI of the tumor (p < 0.05). Following the completion of the in vivo experiment, the bladders were extracted from the mice. The presence of the tumors within the bladder was visually verified via H&E staining (Fig. 4 C). 4. Evaluation of drug toxicity and safety Weight measurements were performed on the mice in each experimental group to assess the potential toxicity of the treatment agent prior to BLI (Fig. 5 ). No notable differences in weight changes were observed between the groups. Discussion The primary focus of future cell culture techniques in cancer research is the effective implementation of the TME. The combination of 3D bioprinted cell culture with a microfluidic system has been demonstrated to represent a dynamic TME. We previously demonstrated the effectiveness of BCOC and rBCG in in vivo and ex vivo bladder cancer models ( 10 , 14 ). In this study, we investigated the synergistic antitumor effects of rBCG and ICIs. Although 3D cell culture studies involving ICIs have been carried out in a range of carcinomas, such as hepatomas, there is limited research on the combination of ICIs with rBCG in bladder cancer models ( 17 ). Intravenous therapeutic approaches using ICIs have been studied for the treatment of patients with BCG-unresponsive bladder cancer. Various ICIs, such as nivolumab, atezolizumab, durvalumab, and avelumab, have been evaluated for diverse treatment strategies ( 18 – 20 ). Conversely, our study focused on two specific features. First, the study was conducted using rBCG rather than conventional BCG. We developed an rBCG strain expressing dltA derived from bacterial proteins that are resistant to antimicrobial peptides (AMPs) ( 21 ). rBCG- dltA can prevent immune responses related to AMP. Second, rBCG- dltA and anti-mPD1 were administered intravesically to induce an antitumor response in an orthotopic mouse bladder cancer model. This study selected rBCG- dltA as a combination drug with anti-mPD1 therapy and demonstrated that this combination was superior to rBCG- dltA alone or anti-mPD1 therapy alone. Ultimately, our study revealed real-time alterations in the dimensions of bladder cancer within living mice. During this process, the efficacy and potential toxicity of the administered agent can be assessed concurrently. In the latter part of the study, it was demonstrated that the actual volume of bladder cancer decreased when the bladder was extracted from the sacrificed mice. Several emerging therapeutic strategies combined with ICIs have been developed to treat bladder cancer. Among several types of ICIs, it is widely known that PD-1 and PD-L1 inhibitors are strongly associated with bladder cancer. In addition, PD-L1 expression in bladder cancer may facilitate cancer progression and BCG unresponsiveness ( 22 ). PD-1 and PD-L1 inhibitors have been collaborated with other antitumor drugs, such as chemotherapeutic agents or cancer vaccines, to improve immunotherapeutic performance. In an animal model experiment, the use of a dendritic cell vaccine with PD-1 antibodies enhanced immunologic responses ( 23 ). Various combination therapies have been assessed to enhance the effectiveness of PD-1 agents, including anti-CTLA-4 drugs, chemotherapy, radiotherapy, targeted therapies, and other immunomodulatory agents ( 24 ). In recent years, there has been interest in combining BCG therapy with other treatments, such as immunotherapy with pembrolizumab. This approach is based on the idea that BCG and pembrolizumab work in different ways to stimulate the immune system and target cancer cells and that combining the two may lead to better outcomes for patients. Several clinical trials have investigated the use of combination therapy with BCG and pembrolizumab in patients with bladder cancer. The use of this regimen is restricted to patients with NMIBC who have previously failed BCG therapy and are either unwilling or unable to undergo radical cystectomy. This study offers a distinct advantage in exploring a combinatorial approach that incorporates rBCG- dltA and extends beyond the combination of conventional BCG and pembrolizumab. Furthermore, this research is noteworthy because it examined the administration of therapeutic agents via the intravesical route in a murine model. This study acknowledges several limitations. While 3D-bioprinted BCOC platform offers a promising method for mimicking the complexity of human tumors and microenvironments, organoid-based systems are still in the nascent stage of development. There are ongoing challenges in accurately reproducing the full heterogeneity and dynamic evolution of patient-derived tumors in a personalized context. Additionally, although the utilization of commercial cell lines provides convenience and standardization, these lines may not adequately represent the genomic and phenotypic diversity of primary bladder tumors due to their immortalization and the accumulation of genetic mutations. Furthermore, the current model is deficient in possessing a fully functional immune system and vasculature, which constrains the assessment of long-term immunological interactions, systemic pharmacokinetics, and metastatic behavior. Future research should aim to incorporate patient-derived cells and autologous immune components into the BCOC platform to more closely mimic in vivo tumor biology Conclusions This research illustrates the therapeutic efficacy of the combination of rBCG- dltA and pembrolizumab in the treatment of bladder cancer. Utilizing a BCOC platform alongside an in vivo murine model, we identified synergistic antitumor effects resulting from this combinatorial strategy. Although additional preclinical and clinical validation is necessary, our results indicate that this approach may represent a promising direction for the advancement of immunotherapy in bladder cancer. Abbreviations Recombinant Bacillus Calmette-Guérin (rBCG); bladder cancer-on-a-chip (BCOC); non-muscle invasive bladder cancer (NMIBC); muscle invasive bladder cancer (MIBC); Bacillus Calmette-Guérin (BCG); three-dimensional (3D); tumor microenvironments (TMEs); immune checkpoint inhibitor (ICI) Declarations Conflicts of interest There are no conflicts to declare. Funding This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (RS-2021-NR056999 and RS-2021-NR058108). Author Contribution J.H.K: conceptualization, funding acquisition, writing-original draft, and writing-review & editing. S.M.: Data curation, formal analysis, visualization, and writing-original draft. J.C.: Data curation. C.U.L: investigation. Y.S.L.: supervision and investigation. M.K.: data curation, validation, and methodology. S.Y.C.: supervision and validation. I.H.C.: conceptualization, supervision, funding acquisition, and writing-review & editing. Availability of data and materials All the data needed to evaluate the conclusions are presented in the paper. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209-49. 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Cite Share Download PDF Status: Published Journal Publication published 29 Mar, 2026 Read the published version in BMC Cancer → Version 1 posted Editorial decision: Revision requested 06 Nov, 2025 Reviews received at journal 23 Oct, 2025 Reviews received at journal 22 Oct, 2025 Reviews received at journal 13 Oct, 2025 Reviews received at journal 12 Oct, 2025 Reviewers agreed at journal 11 Oct, 2025 Reviewers agreed at journal 08 Oct, 2025 Reviewers agreed at journal 05 Oct, 2025 Reviewers agreed at journal 30 Sep, 2025 Reviewers invited by journal 11 Jul, 2025 Editor assigned by journal 09 Jun, 2025 Editor invited by journal 09 Jun, 2025 Submission checks completed at journal 09 Jun, 2025 First submitted to journal 09 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6798208","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":483990316,"identity":"c9b57322-4a23-46ff-932e-213614bcac35","order_by":0,"name":"Jung Hoon Kim","email":"","orcid":"","institution":"Chung-Ang University Gwangmyeong Hospital, Chung-Ang University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jung","middleName":"Hoon","lastName":"Kim","suffix":""},{"id":483990317,"identity":"cf4d66c2-fa5c-47b4-92ef-975a7c725d26","order_by":1,"name":"Sejung Maeng","email":"","orcid":"","institution":"Chung-Ang University Hospital, Chung-Ang University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Sejung","middleName":"","lastName":"Maeng","suffix":""},{"id":483990318,"identity":"0b8789cf-24c3-4d04-a3fc-0d7579f4aea8","order_by":2,"name":"Joongwon Choi","email":"","orcid":"","institution":"Chung-Ang University Gwangmyeong Hospital, Chung-Ang University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Joongwon","middleName":"","lastName":"Choi","suffix":""},{"id":483990319,"identity":"2e57f523-e44a-4bbb-b888-0949fd43f4ed","order_by":3,"name":"Chung Un Lee","email":"","orcid":"","institution":"Chung-Ang University Gwangmyeong Hospital, Chung-Ang University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chung","middleName":"Un","lastName":"Lee","suffix":""},{"id":483990320,"identity":"9ad29278-959e-4b23-81b0-d987212eac87","order_by":4,"name":"Yong Seong Lee","email":"","orcid":"","institution":"Chung-Ang University Gwangmyeong Hospital, Chung-Ang University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"Seong","lastName":"Lee","suffix":""},{"id":483990321,"identity":"0ec1af30-6f79-40df-96fa-0fe77bb0bd38","order_by":5,"name":"Mirinae Kim","email":"","orcid":"","institution":"Chung-Ang University Hospital, Chung-Ang University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Mirinae","middleName":"","lastName":"Kim","suffix":""},{"id":483990322,"identity":"87de846a-1e52-450b-b247-d8a92edda66b","order_by":6,"name":"Se Young Choi","email":"","orcid":"","institution":"Chung-Ang University Hospital, Chung-Ang University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Se","middleName":"Young","lastName":"Choi","suffix":""},{"id":483990323,"identity":"2450155c-cc43-491c-adb2-2ab263a29036","order_by":7,"name":"In Ho Chang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYLCCxAYbGDOBaC1ppGphbDhMghb+9jOGHx7uOC/PPyOB8cMPhrR8glokzuQYSySeuW0440YCs2QPQ45lAyEtBgw5ZgyJbbcTGG4kMEgzMFQYELTFgP8NSMu5BHmgLb+J0yIBtuVAgsGNBDagLTmEtUjceFYskdiWbLjxzMM2yx6DNMJa+PuTN3782WYnL3c8+fCNHxXJhLUgAcYGUGiMglEwCkbBKKAGAABqjDfillkjFgAAAABJRU5ErkJggg==","orcid":"","institution":"Chung-Ang University Hospital, Chung-Ang University College of Medicine","correspondingAuthor":true,"prefix":"","firstName":"In","middleName":"Ho","lastName":"Chang","suffix":""}],"badges":[],"createdAt":"2025-06-02 02:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6798208/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6798208/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12885-026-15890-x","type":"published","date":"2026-03-29T16:09:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86777327,"identity":"5c49f7c0-a15d-4f67-883f-81010e3a111d","added_by":"auto","created_at":"2025-07-15 12:49:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23646,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of the drug treatment protocol for anorthotopic mouse model of bladder cancer. After 10 to 14 days of intravesical instillation of MBT2-luc cells, the mice were divided into four groups: the control, rBCG-\u003cem\u003edltA\u003c/em\u003e, pembrolizumab, and combination groups. The target drugs were administereda total of 4 times every 3–4days, and the experiments were terminated on the 10th day of administration.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6798208/v1/2b65330dd9b9c31309222d12.png"},{"id":86778591,"identity":"e6177d92-d705-4747-9b2d-4091a56f9062","added_by":"auto","created_at":"2025-07-15 12:57:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":669950,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of 3D bioprintedbladder cancer on a chip model.(A) Live/dead staining on the3rd day after drug treatment. T24 bladder cancer cell blocks in the rBCG-\u003cem\u003edltA\u003c/em\u003eand pembrolizumab combination group had lower cell survival rates than those in the other groups did. Noncancerouscell lines, such as HUVECsand MRC-5 cells, survived well in all groups. (B) According to the results of the quantitative cell density test, T24 cancer cells wereaffected by rBCG-\u003cem\u003edltA\u003c/em\u003e and pembrolizumab. The group with the lowest cell density was the combination-treated group. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. control.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6798208/v1/6d28693fa301b667a10b02e3.png"},{"id":86777330,"identity":"c7ffe22c-6a68-478c-90e1-0320b50753d2","added_by":"auto","created_at":"2025-07-15 12:49:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":529851,"visible":true,"origin":"","legend":"\u003cp\u003eIllustrates the chemotactic behavior of monocytic THP-1 cells within a chip model featuring a permeable membrane of bladder cancer cells. (A) Migration and distribution patterns of THP-1 cells in response to the treated agents. (B) Quantitative analysis of the migrationrates of THP-1 cells.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6798208/v1/299a96863bc470a41f82e173.png"},{"id":86777332,"identity":"c8f1d274-9de9-43f2-9f4b-7a3bfb22d19d","added_by":"auto","created_at":"2025-07-15 12:49:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":372893,"visible":true,"origin":"","legend":"\u003cp\u003eTherapeutic efficacy of the combination of rBCG-\u003cem\u003edltA\u003c/em\u003e and pembrolizumab in an orthotopic mouse model of bladder cancer. (A) Illustration of bioluminescence intensity demonstrating that the volume of bladder cancer decreased in the combination therapy group compared with the other treatment groups. (B) On the 10\u003csup\u003eth\u003c/sup\u003e day, quantitative analysis of signal intensity revealed a significant decrease in the rBCG-\u003cem\u003edltA\u003c/em\u003e and pembrolizumab combination group. (C) Bladder images from each group were stained with hematoxylin and eosin (H\u0026amp;E) staining following the sacrifice of the mice.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6798208/v1/c65af454826033ccb42d40b4.png"},{"id":86778593,"identity":"1cc09e8f-80ec-4c07-98cd-7ca4e224c818","added_by":"auto","created_at":"2025-07-15 12:57:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":31768,"visible":true,"origin":"","legend":"\u003cp\u003eEstimation of body weights in each treatment group. A small amount of weight loss was observed in all four groups; however, there was no significant weight loss compared with that in the control group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6798208/v1/3c63449c6159cd2b35806b64.png"},{"id":105755069,"identity":"c85cd65e-f7c7-4fdd-b363-ef0d3d2c3089","added_by":"auto","created_at":"2026-03-30 16:24:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1767386,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6798208/v1/e438d4b0-2e1c-4568-9965-c50e5742f89c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Potential Antitumor Effects of Combining Intravesical Therapy with Recombinant Bacillus Calmette-Guérin and an Immune Checkpoint Inhibitor in Bladder Cancer","fulltext":[{"header":"Background","content":"\u003cp\u003eAccording to Global Cancer Statistics 2020, bladder cancer is the 10th most commonly diagnosed malignant tumor in the world (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). It is the second most common type of urinary tract cancer after prostate cancer. Although surgical management of bladder cancer has remained relatively unchanged, the field has witnessed significant advancements in therapeutic agents over recent years. Recently, traditional and innovative therapeutic agents, such as chemotherapy or immune checkpoint inhibitors, have been integrated to manage both non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). Intravesical immunotherapy with Bacillus Calmette-Guérin (BCG) is a common therapeutic modality for patients with bladder cancer. The European Association of Urology (EAU) guidelines strongly recommend intravesical BCG after transurethral tumor resection in patients with intermediate- and high-risk bladder cancer (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). BCG prevents disease progression in NMIBC by up to 27% and reduces the recurrence rate by 32% (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). However, BCG therapy has several side effects, such as hematuria, dysuria, and urinary tract infections. Severe adverse events, such as BCG sepsis, are rarely reported (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Owing to the collapse of the global supply chain, BCG shortages are an emerging issue. Various studies have been conducted to overcome the unresponsiveness of BCG, increase its effectiveness, and decrease its side effects (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRecombinant BCG (rBCG) is a modified version of BCG. Compared with conventional BCG treatment, genetically modified BCG products aim to increase the efficiency of immunotherapy and reduce complication rates. rBCGs have been developed to induce specific immune responses or include foreign antigens (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). rBCG induced the overexpression of cytokines more than did conventional BCG treatment. Bioengineered 3D tumor models are used to construct tumor microenvironments (TMEs) that play key roles in tumor growth, drug resistance, and metastasis (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The cancer-on-a-chip model better emulates the TME, cancer architecture, and flow dynamics (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Bladder cancer-on-a-chip (BCOC) is a form of organ-on-chip. It is a layered structure of cell blocks printed via 3D bioprinting (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePembrolizumab is a highly selective monoclonal antibody against PD-1 that disrupts the linkage between PD-1 ligands. Pembrolizumab is an effective immune checkpoint inhibitor (ICI) that has antitumor activity in patients with bladder cancer (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Compared with systemic chemotherapy, pembrolizumab has shown significant overall survival gains for 3 months (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The antitumor effects of pembrolizumab are achieved not only through systemic administration but also through intravesical instillation. A phase I clinical trial of intravesical pembrolizumab and BCG treatment reported safe and feasible results in patients with BCG-unresponsive NMIBC (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe hypothesized that the combination of rBCG and pembrolizumab would enhance immunological reactions more than the conventional BCG immunotherapy. In this study, we used a high-throughput BCOC model to evaluate the immunotherapeutic effects of rBCG and pembrolizumab.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e1. Cell lines and reagents of the BCOC Model\u003c/p\u003e\u003cp\u003eTo evaluate the combined antitumor effect of rBCG and pembrolizumab, we employed a high-throughput BCOC system, composed of four parallel modules driven by syringe pump (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The 3D bioprinted BCOC blocks consisted of the T24 bladder cancer cells, MRC-5 fibroblast, Jurkat T lymphocyte, THP-1 monocytes, and human umbilical vein endothelial cell line (HUVEC), cultured using standard protocols provided by the suppliers; Korean Cell Line Bank (Seoul, Republic of Korea) and Lonza (Basel, Switzerland). MBT2-luc murine bladder cancer cells were generously provided by Dr. Sang-Jin Lee (National Cancer Center, Republic of Korea). Commercially available \u003cem\u003eMycobacterium bovis\u003c/em\u003e BCG was obtained from OncoTice (Merck Sharp, Kenilworth, NJ, USA). BCG was prepared at a multiplicity of infection (MOI) of 30 (1.8×10\u003csup\u003e6\u003c/sup\u003e cells/ml) and stored at -80°C until use. The GelMA prepolymer mixture used was Gel4Cell (Innoregen, Daegu, Republic of Korea). The rBCG strain was engineered to express \u003cem\u003edltA\u003c/em\u003e gene, cloned into a pMV306 vector using \u003cem\u003eHind\u003c/em\u003eⅢ and \u003cem\u003eSal\u003c/em\u003eI restriction enzymes, following previously describe protocols (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e2. Live/dead cell viability and THP-1 cell migration assay\u003c/p\u003e\u003cp\u003eCell viability in the 3D structures was assessed on the first and third days following the administration of rBCG-\u003cem\u003edltA\u003c/em\u003e and pembrolizumab (Keytruda; Merck Sharp and Dohme Ireland, Carlow, Ireland). Prior to fluorescent live and dead staining solutions (Thermo Fisher, Waltham, MA, USA), each 3D cell construct was washed three times with Dulbecco’s phosphate-buffered saline. A fluorescence microscope (Leica DMi8; Leica, Germany) was used to observe the cell morphology, and three independent samples were analyzed.\u003c/p\u003e\u003cp\u003eTHP-1 monocytes were transformed into macrophages by incubation with 25 nM phorbol 12-myristate 13-acetate (PMA; Sigma‒Aldrich, St. Louis, MO, USA) for 24 h. The differentiated cells were seeded into the bottom layer of the chip at a density of 2×10\u003csup\u003e4\u003c/sup\u003e cells/20 µL, and the THP-1 cells were visualized via an Olympus CKX41 inverted microscope (Olympus, Tokyo, Japan) and counted in three randomly selected fields, and the average was calculated.\u003c/p\u003e\u003cp\u003e3. Animal studies\u003c/p\u003e\u003cp\u003eA syngeneic orthotopic bladder cancer mouse model was established as described previously (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). After a mouse bladder cancer model was established, the mice were divided into four groups. Four groups of mice, consisting of five mice each, received either PBS, rBCG-\u003cem\u003edltA\u003c/em\u003e, anti-mPD1, or a combination of both directly into the bladder through a catheter twice weekly. Bladder cancer progression was monitored via bioluminescence imaging (BLI), and body weight measurements were taken twice weekly for 2 weeks. The BLI data were obtained and analyzed via the Living Image software CleVue version 3.1.3.2054 (Vieworks, Anyang, Republic of Korea). A schematic of the treatment protocol is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003ePrior to tissue collection, all animals underwent anesthesia utilizing an inhalation anesthesia system. The anesthetic agent was Ifran Liquid for Inhalation (Isoflurane; Hana Pharm, Seoul, Korea). Anesthesia was initiated within a chamber utilizing a gas mixture of nitrogen and oxygen in a 1:1 ratio, with isoflurane administered at a concentration of 2–3%. After approximately 5 min of induction, the animals were transitioned to a nose cone connected to the anesthesia system to sustain isoflurane anesthesia during the tissue harvesting process. Following the collection of samples, the animals were euthanized via cervical dislocation while still under deep anesthesia. This approach was chosen to minimize animal suffering and to ensure a humane endpoint.\u003c/p\u003e\u003cp\u003e4. Hematoxylin and eosin (H\u0026amp;E) staining\u003c/p\u003e\u003cp\u003eBladder cancer tissues were harvested from an orthotopic bladder cancer mouse model and fixed in 4% paraformaldehyde solution (Biosesang, Yongin, Republic of Korea) at 4°C for 24 h. Following standard processing, tissues were dehydrated, embedded in paraffin, and sectioned at 4 µm thickness. Sections were deparaffinized, rehydrated, and stained with H\u0026amp;E using conventional protocols. The stained slides were imaged via a image scanner (Pannoramic MIDI; 3DHISTECH, Budapest, Hungary) for histological evaluation.\u003c/p\u003e\u003cp\u003e5. Statistical analysis\u003c/p\u003e\u003cp\u003eExperimental data were obtained from a minimum of three independent experiments. The Statistical Package for Social Sciences (SPSS) version 25.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Student’s \u003cem\u003et\u003c/em\u003e test was performed to compare the means between two different groups, and statistical significance was defined as \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e6. Ethics approval\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e The procedures and animal care were approved by the Institutional Animal Care and Use Committee of Chung-Ang University (Approval No. A2021020, date: Jan 09, 2023, Seoul, Republic of Korea) and performed in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals. In addition, this study was conducted in accordance with the ARRIVE guidelines.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e1. Cellular viability after treatment with rBCG-\u003cem\u003edltA\u003c/em\u003e with or without pembrolizumab in BCOCs\u003c/p\u003e\u003cp\u003eCell viability was assessed at two different time intervals, on the first and third days, for each treatment group. In the live and dead staining assay, the T24 cancer cell line in the combination group of rBCG-\u003cem\u003edltA\u003c/em\u003e and pembrolizumab exhibited predominantly decreased viability, whereas MRC-5 cells and HUVECs maintained a normal level of viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The merged image represents the relative proportions of living (green) and dead (red) cells. The images clearly indicate a higher rate of T24 cell death in the combination group. The fluorescence intensity of the T24 cells (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error [SE], %) was significantly lower in the combination group (22.24\u0026thinsp;\u0026plusmn;\u0026thinsp;5.01) than in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The other MRC-5 cells and HUVECs did not significantly differ between the groups. Compared with both the rBCG-dltA and pembrolizumab groups, the combination treatment group demonstrated superior effectiveness in inhibiting tumor growth (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e2. Effects of chemotaxis following the administration of rBCG-\u003cem\u003edltA\u003c/em\u003e and/or pembrolizumab\u003c/p\u003e\u003cp\u003eThe chemotactic response of monocytic THP-1 cells within the membrane was verified in the control, rBCG-\u003cem\u003edltA\u003c/em\u003e and pembrolizumab groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The average migration rate (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE, %) of THP-1 cells was significantly greater in the combination group (10,540\u0026thinsp;\u0026plusmn;\u0026thinsp;1,551, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) than in the control group (554.7\u0026thinsp;\u0026plusmn;\u0026thinsp;354.6) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e3. Evaluation of combination treatment in an orthotopic mouse model of bladder cancer (\u003cem\u003ein vivo\u003c/em\u003e study)\u003c/p\u003e\u003cp\u003eTo assess the inhibition of tumor growth in the combination treatment group, we compared the BLI intensities in an orthotopic mouse model of bladder cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Following the implantation of bladder cancer cells on day 0, all experimental mice were randomly allocated to the three groups and subjected to BLI. The imaging signal intensities were assessed by calculating the average of each measurement (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). At the time of randomization, there was no statistically significant difference in signal intensity between the groups. The mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE) of the signal intensity, as measured by BLI, in the combination group (9.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36 \u0026times; 10\u003csup\u003e10\u003c/sup\u003e) was significantly lower than that in both the control group (1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and the anti-mPD1 group (1.88\u0026thinsp;\u0026plusmn;\u0026thinsp;3.46 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) on day 10. Compared with the individual treatment groups, the combined treatment group presented a significantly decreased BLI of the tumor (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Following the completion of the \u003cem\u003ein vivo\u003c/em\u003e experiment, the bladders were extracted from the mice. The presence of the tumors within the bladder was visually verified via H\u0026amp;E staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e4. Evaluation of drug toxicity and safety\u003c/p\u003e\u003cp\u003eWeight measurements were performed on the mice in each experimental group to assess the potential toxicity of the treatment agent prior to BLI (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). No notable differences in weight changes were observed between the groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe primary focus of future cell culture techniques in cancer research is the effective implementation of the TME. The combination of 3D bioprinted cell culture with a microfluidic system has been demonstrated to represent a dynamic TME. We previously demonstrated the effectiveness of BCOC and rBCG in \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e bladder cancer models (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). In this study, we investigated the synergistic antitumor effects of rBCG and ICIs. Although 3D cell culture studies involving ICIs have been carried out in a range of carcinomas, such as hepatomas, there is limited research on the combination of ICIs with rBCG in bladder cancer models (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Intravenous therapeutic approaches using ICIs have been studied for the treatment of patients with BCG-unresponsive bladder cancer. Various ICIs, such as nivolumab, atezolizumab, durvalumab, and avelumab, have been evaluated for diverse treatment strategies (\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Conversely, our study focused on two specific features. First, the study was conducted using rBCG rather than conventional BCG. We developed an rBCG strain expressing \u003cem\u003edltA\u003c/em\u003e derived from bacterial proteins that are resistant to antimicrobial peptides (AMPs) (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). rBCG-\u003cem\u003edltA\u003c/em\u003e can prevent immune responses related to AMP. Second, rBCG-\u003cem\u003edltA\u003c/em\u003e and anti-mPD1 were administered intravesically to induce an antitumor response in an orthotopic mouse bladder cancer model. This study selected rBCG-\u003cem\u003edltA\u003c/em\u003e as a combination drug with anti-mPD1 therapy and demonstrated that this combination was superior to rBCG-\u003cem\u003edltA\u003c/em\u003e alone or anti-mPD1 therapy alone. Ultimately, our study revealed real-time alterations in the dimensions of bladder cancer within living mice. During this process, the efficacy and potential toxicity of the administered agent can be assessed concurrently. In the latter part of the study, it was demonstrated that the actual volume of bladder cancer decreased when the bladder was extracted from the sacrificed mice.\u003c/p\u003e\u003cp\u003eSeveral emerging therapeutic strategies combined with ICIs have been developed to treat bladder cancer. Among several types of ICIs, it is widely known that PD-1 and PD-L1 inhibitors are strongly associated with bladder cancer. In addition, PD-L1 expression in bladder cancer may facilitate cancer progression and BCG unresponsiveness (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). PD-1 and PD-L1 inhibitors have been collaborated with other antitumor drugs, such as chemotherapeutic agents or cancer vaccines, to improve immunotherapeutic performance. In an animal model experiment, the use of a dendritic cell vaccine with PD-1 antibodies enhanced immunologic responses (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Various combination therapies have been assessed to enhance the effectiveness of PD-1 agents, including anti-CTLA-4 drugs, chemotherapy, radiotherapy, targeted therapies, and other immunomodulatory agents (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn recent years, there has been interest in combining BCG therapy with other treatments, such as immunotherapy with pembrolizumab. This approach is based on the idea that BCG and pembrolizumab work in different ways to stimulate the immune system and target cancer cells and that combining the two may lead to better outcomes for patients. Several clinical trials have investigated the use of combination therapy with BCG and pembrolizumab in patients with bladder cancer. The use of this regimen is restricted to patients with NMIBC who have previously failed BCG therapy and are either unwilling or unable to undergo radical cystectomy. This study offers a distinct advantage in exploring a combinatorial approach that incorporates rBCG-\u003cem\u003edltA\u003c/em\u003e and extends beyond the combination of conventional BCG and pembrolizumab. Furthermore, this research is noteworthy because it examined the administration of therapeutic agents via the intravesical route in a murine model.\u003c/p\u003e\u003cp\u003eThis study acknowledges several limitations. While 3D-bioprinted BCOC platform offers a promising method for mimicking the complexity of human tumors and microenvironments, organoid-based systems are still in the nascent stage of development. There are ongoing challenges in accurately reproducing the full heterogeneity and dynamic evolution of patient-derived tumors in a personalized context. Additionally, although the utilization of commercial cell lines provides convenience and standardization, these lines may not adequately represent the genomic and phenotypic diversity of primary bladder tumors due to their immortalization and the accumulation of genetic mutations. Furthermore, the current model is deficient in possessing a fully functional immune system and vasculature, which constrains the assessment of long-term immunological interactions, systemic pharmacokinetics, and metastatic behavior. Future research should aim to incorporate patient-derived cells and autologous immune components into the BCOC platform to more closely mimic in vivo tumor biology\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis research illustrates the therapeutic efficacy of the combination of rBCG-\u003cem\u003edltA\u003c/em\u003e and pembrolizumab in the treatment of bladder cancer. Utilizing a BCOC platform alongside an \u003cem\u003ein vivo\u003c/em\u003e murine model, we identified synergistic antitumor effects resulting from this combinatorial strategy. Although additional preclinical and clinical validation is necessary, our results indicate that this approach may represent a promising direction for the advancement of immunotherapy in bladder cancer.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eRecombinant Bacillus Calmette-Gu\u0026eacute;rin (rBCG); bladder cancer-on-a-chip (BCOC); non-muscle invasive bladder cancer (NMIBC); muscle invasive bladder cancer (MIBC); Bacillus Calmette-Gu\u0026eacute;rin (BCG); three-dimensional (3D); tumor microenvironments (TMEs); immune checkpoint inhibitor (ICI)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflicts of interest\u003c/h2\u003e\u003cp\u003eThere are no conflicts to declare.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT \u0026amp; Future Planning (RS-2021-NR056999 and RS-2021-NR058108).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.H.K: conceptualization, funding acquisition, writing-original draft, and writing-review \u0026amp; editing. S.M.: Data curation, formal analysis, visualization, and writing-original draft. J.C.: Data curation. C.U.L: investigation. Y.S.L.: supervision and investigation. M.K.: data curation, validation, and methodology. S.Y.C.: supervision and validation. I.H.C.: conceptualization, supervision, funding acquisition, and writing-review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\u003cp\u003eAll the data needed to evaluate the conclusions are presented in the paper. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209-49.\u003c/li\u003e\n\u003cli\u003eSylvester RJ, Rodriguez O, Hernandez V, Turturica D, Bauerova L, Bruins HM, et al. European Association of Urology (EAU) Prognostic Factor Risk Groups for Non-muscle-invasive Bladder Cancer (NMIBC) Incorporating the WHO 2004/2016 and WHO 1973 Classification Systems for Grade: An Update from the EAU NMIBC Guidelines Panel. Eur Urol. 2021;79(4):480-8.\u003c/li\u003e\n\u003cli\u003eSylvester RJ, van der MA, Lamm DL. Intravesical bacillus Calmette-Guerin reduces the risk of progression in patients with superficial bladder cancer: a meta-analysis of the published results of randomized clinical trials. J Urol. 2002;168(5):1964-70.\u003c/li\u003e\n\u003cli\u003evan der Meijden AP, Sylvester RJ, Oosterlinck W, Hoeltl W, Bono AV, Group EG-UTC. Maintenance Bacillus Calmette-Guerin for Ta T1 bladder tumors is not associated with increased toxicity: results from a European Organization for Research and Treatment of Cancer Genito-Urinary Group Phase III Trial. Eur Urol. 2003;44(4):429-34.\u003c/li\u003e\n\u003cli\u003eChoi SY, Ha MS, Kim JH, Chi BH, Kim JW, Chang IH, et al. Low-dose versus standard-dose bacille Calmette-Guerin for non-muscle-invasive bladder cancer: Systematic review and meta-analysis of randomized controlled trials. Investig Clin Urol. 2022;63(2):140-50.\u003c/li\u003e\n\u003cli\u003eAghamir SMK, Khatami F, Farrokhpour H, Oliveira Reis L, Ahmadi Pishkuhi M, Mohammadi A. Oncologic outcomes of Bacillus Calmette-Guerin therapy in elderly patients with non-muscle-invasive bladder cancer: A meta-analysis. PLoS One. 2022;17(5):e0267934.\u003c/li\u003e\n\u003cli\u003eBegnini KR, Buss JH, Collares T, Seixas FK. Recombinant \u003cem\u003eMycobacterium bovis\u003c/em\u003e BCG for immunotherapy in nonmuscle invasive bladder cancer. Appl Microbiol Biotechnol. 2015;99(9):3741-54.\u003c/li\u003e\n\u003cli\u003eEmon B, Bauer J, Jain Y, Jung B, Saif T. Biophysics of Tumor Microenvironment and Cancer Metastasis - A Mini Review. Comput Struct Biotechnol J. 2018;16:279-87.\u003c/li\u003e\n\u003cli\u003eMonteiro MV, Zhang YS, Gaspar VM, Mano JF. 3D-bioprinted cancer-on-a-chip: level-up organotypic \u003cem\u003ein vitro\u003c/em\u003e models. Trends Biotechnol. 2022;40(4):432-47.\u003c/li\u003e\n\u003cli\u003eKim JH, Choi J, Kim M, Kang SJ, Choi YW, Choi SY, et al. Immunotherapeutic effects of recombinant Bacillus Calmette-Guerin containing sic gene in ex vivo and \u003cem\u003ein vivo\u003c/em\u003e bladder cancer models. Investig Clin Urol. 2022;63(2):228-37.\u003c/li\u003e\n\u003cli\u003eBalar AV, Kamat AM, Kulkarni GS, Uchio EM, Boormans JL, Roumiguie M, et al. 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. 2021;22(7):919-30.\u003c/li\u003e\n\u003cli\u003eBellmunt J, de Wit R, Vaughn DJ, Fradet Y, Lee JL, Fong L, et al. Pembrolizumab as Second-Line Therapy for Advanced Urothelial Carcinoma. N Engl J Med. 2017;376(11):1015-26.\u003c/li\u003e\n\u003cli\u003eMeghani K, Cooley LF, Choy B, Kocherginsky M, Swaminathan S, Munir SS, et al. First-in-human Intravesical Delivery of Pembrolizumab Identifies Immune Activation in Bladder Cancer Unresponsive to Bacillus Calmette-Guerin. Eur Urol. 2022;82(6):602-10.\u003c/li\u003e\n\u003cli\u003eKim JH, Lee S, Kang SJ, Choi YW, Choi SY, Park JY, et al. Establishment of Three-Dimensional Bioprinted Bladder Cancer-on-a-Chip with a Microfluidic System Using Bacillus Calmette-Guerin. Int J Mol Sci. 2021;22(16).\u003c/li\u003e\n\u003cli\u003eCho MJ, Kim MJ, Kim K, Choi YW, Lee SJ, Whang YM, et al. The immunotherapeutic effects of recombinant Bacillus Calmette-Guerin resistant to antimicrobial peptides on bladder cancer cells. Biochem Biophys Res Commun. 2019;509(1):167-74.\u003c/li\u003e\n\u003cli\u003eSeo HK, Shin SP, Jung NR, Kwon WA, Jeong KC, Lee SJ. The establishment of a growth-controllable orthotopic bladder cancer model through the down-regulation of c-myc expression. Oncotarget. 2017;8(31):50500-9.\u003c/li\u003e\n\u003cli\u003eLi Y, Zhang T, Pang Y, Li L, Chen ZN, Sun W. 3D bioprinting of hepatoma cells and application with microfluidics for pharmacodynamic test of Metuzumab. Biofabrication. 2019;11(3):034102.\u003c/li\u003e\n\u003cli\u003eMassard C, Gordon MS, Sharma S, Rafii S, Wainberg ZA, Luke J, et al. Safety and Efficacy of Durvalumab (MEDI4736), an Anti-Programmed Cell Death Ligand-1 Immune Checkpoint Inhibitor, in Patients With Advanced Urothelial Bladder Cancer. J Clin Oncol. 2016;34(26):3119-25.\u003c/li\u003e\n\u003cli\u003eInman BA, Longo TA, Ramalingam S, Harrison MR. Atezolizumab: A PD-L1-Blocking Antibody for Bladder Cancer. Clin Cancer Res. 2017;23(8):1886-90.\u003c/li\u003e\n\u003cli\u003ePowles T, Park SH, Voog E, Caserta C, Valderrama BP, Gurney H, et al. Avelumab Maintenance Therapy for Advanced or Metastatic Urothelial Carcinoma. N Engl J Med. 2020;383(13):1218-30.\u003c/li\u003e\n\u003cli\u003eHong SJ, Hwang GY, Kang SJ, Choi SY, Chi BH, Chang IH. The Effects of Recombinant Bacillus Calmette-Gu\u0026eacute;rin Resistant to Antimicrobial Peptides on Orthotopic Bladder Cancer Mouse Model. Korean J Urol Oncol. 2021;19(1):40-7.\u003c/li\u003e\n\u003cli\u003eInman BA, Sebo TJ, Frigola X, Dong H, Bergstralh EJ, Frank I, et al. PD-L1 (B7-H1) expression by urothelial carcinoma of the bladder and BCG-induced granulomata: associations with localized stage progression. Cancer. 2007;109(8):1499-505.\u003c/li\u003e\n\u003cli\u003eLim S, Park JH, Chang H. Enhanced anti-tumor immunity of vaccine combined with anti-PD-1 antibody in a murine bladder cancer model. Investig Clin Urol. 2023;64(1):74-81.\u003c/li\u003e\n\u003cli\u003eBalar AV, Weber JS. PD-1 and PD-L1 antibodies in cancer: current status and future directions. Cancer Immunol Immunother. 2017;66(5):551-64.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bacillus carmette-guérin, Bladder cancer, Pembrolizumab, 3-dimensional cell culture, Organ-on-a-chip devices","lastPublishedDoi":"10.21203/rs.3.rs-6798208/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6798208/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eRecombinant Bacillus Calmette-Gu\u0026eacute;rin (rBCG) is a genetically modified BCG that enhances therapeutic efficacy and safety in patients with bladder cancer. Pembrolizumab is an immune checkpoint inhibitor that is used to treat bladder cancer. We aimed to evaluate the effectiveness of the combination of rBCG and pembrolizumab in \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e bladder cancer models.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe investigated the antitumor effects of rBCG-\u003cem\u003edltA\u003c/em\u003e and pembrolizumab in a bladder cancer-on-a-chip (BCOC) model, an organoid three-dimensional cell culture tool. An orthotopic mouse model was employed to assess the \u003cem\u003ein vivo\u003c/em\u003e effects of the combination therapy. To overcome errors caused by differences between species, an \u003cem\u003ein vivo\u003c/em\u003e study was conducted using a mouse PD1 inhibitor instead of pembrolizumab.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe T24 bladder cancer cell line declined independently when rBCG-\u003cem\u003edltA\u003c/em\u003e and pembrolizumab were used in the BCOC model. Monocyte migration was also significantly greater than that in the other groups. In the orthotopic bladder cancer model, the combination treatment inhibited the growth of bladder cancer.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eIntravesical therapy with rBCG-\u003cem\u003edltA\u003c/em\u003e and pembrolizumab is effective in treating bladder cancer. This study suggests that the combination of rBCG and immune checkpoint inhibitors is a novel strategy for treating patients with BCG-unresponsive bladder cancer.\u003c/p\u003e","manuscriptTitle":"The Potential Antitumor Effects of Combining Intravesical Therapy with Recombinant Bacillus Calmette-Guérin and an Immune Checkpoint Inhibitor in Bladder Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-15 12:49:33","doi":"10.21203/rs.3.rs-6798208/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-06T10:35:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-23T13:49:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-22T16:51:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-13T13:01:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-12T08:59:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"217103945883920415506543882804333033893","date":"2025-10-11T12:13:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"229440646816303492717630113983999114283","date":"2025-10-08T09:00:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"287867786041912809139595955992813067440","date":"2025-10-05T23:09:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"14225811816372792219597652519612214767","date":"2025-09-30T13:25:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-11T08:24:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-10T01:59:42+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-09T16:15:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-09T14:36:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2025-06-09T14:33:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6260cc91-568c-4192-9797-3c463a6a4fc8","owner":[],"postedDate":"July 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:20:36+00:00","versionOfRecord":{"articleIdentity":"rs-6798208","link":"https://doi.org/10.1186/s12885-026-15890-x","journal":{"identity":"bmc-cancer","isVorOnly":false,"title":"BMC Cancer"},"publishedOn":"2026-03-29 16:09:06","publishedOnDateReadable":"March 29th, 2026"},"versionCreatedAt":"2025-07-15 12:49:33","video":"","vorDoi":"10.1186/s12885-026-15890-x","vorDoiUrl":"https://doi.org/10.1186/s12885-026-15890-x","workflowStages":[]},"version":"v1","identity":"rs-6798208","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6798208","identity":"rs-6798208","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}