Orthotopic Bladder Cancer Mouse Model Research Articles

Targeting DAD1 gene with CRISPR-Cas9 system transmucosally delivered by fluorinated polylysine nanoparticles for bladder cancer intravesical gene therapy.

Background: Intravesical chemotherapy is highly recommended after transurethral resection of bladder tumor for patients with bladder cancer (BCa). However, this localized adjuvant therapy has drawbacks of causing indiscriminate damage and inability to penetrate bladder mucosal. Methods: Fluorinated polylysine micelles (PLLF) were synthesized by reacting polylysine (PLL) with heptafluorobutyrate anhydride. Anti-apoptotic gene defender against cell death 1 (DAD1) was selected by different gene expression analysis between BCa patients and healthy individuals and identified by several biological function assays. The gene transfection ability of PLLF was verified by multiple in vitro and in vivo assays. The therapeutic efficiency of PLLF nanoparticles (NPs) targeting DAD1 were confirmed by intravesical administration using an orthotopic BCa mouse model. Results: Decorated with fluorinated chains, PLL can self-assemble to form NPs and condense plasmids with excellent gene transfection efficiency in vitro. Loading with the CRISPR-Cas9 system designed to target DAD1 (Cas9-sgDAD1), PLLF/Cas9-sgDAD1 NPs strongly inhibited the expression of DAD1 in BCa cells and induced BCa cell apoptosis through the MAPK signaling pathway. Furthermore, intravesical administration of PLLF/Cas9-sgDAD1 NPs resulted in significant therapeutic outcomes without systemic toxicity in vivo. Conclusion: The synthetized PLLF can transmucosally deliver the CRISPR-Cas9 system into orthotopic BCa tissues to improve intravesical instillation therapy for BCa. This work presents a new strategy for targeting DAD1 gene in the intravesical therapy for BCa with high potential for clinical applications.

Immunotherapeutic effects of recombinant Bacillus Calmette-Guérin containing sic gene in ex vivo and in vivo bladder cancer models.

PurposeThe recombinant Bacillus Calmette–Guérin (BCG) containing the streptococcal inhibitor of the complement gene (rBCG-sic) may be more resistant to antimicrobial peptides and improve internalization; therefore, it can enhance the immunotherapeutic effect of the BCG. Here we determined the optimal dose of rBCG-sic and compared its effectiveness with that of BCG.Materials and MethodsWe fabricated a high-throughput 3D-bioprinted bladder cancer-on-a-chip (BCOC) and used it to evaluate the effectiveness of the rBCG-sic in terms of cell viability, cell migration, and cytokine concentrations. Using an orthotopic mouse model, we evaluated its anticancer effect and toxicity via bioluminescence imaging.ResultsT24 cell viability was decreased after treatment with rBCG-sic 30 multiplicities of infection (MOI) versus the same dosage of mock BCG (42.8%±6.4% vs. 75.7%±6.6%, p<0.05). THP-1 cell migration was positively correlated with rBCG-sic concentration (2.42-fold at 30MOI, p<0.01). The interleukin-6 concentration of rBCG-sic 30MOI was significantly higher than that of mock BCG 30MOI (11.2±1.3 pg/mL vs. 6.7±0.6 pg/mL, p<0.05). In the orthotopic bladder cancer mouse model, lower tumor volume was observed in the rBCG-sic 30MOI group than in the BCG 30MOI group after 10 days of treatment (p<0.05).ConclusionsWe concluded that rBCG-sic is a useful tool for overcoming BCG unresponsiveness in non-muscle invasive bladder cancer. Additionally, high-throughput BCOC with a microfluidic system can successfully reflect the bladder cancer microenvironment.

PD42-02 THE EFFECTS OF RECOMBINANT BACILLUS CALMETTE-GUERIN RESISTANT TO ANTIMICROBIAL PEPTIDES ON ORTHOTOPIC BLADDER CANCER MOUSE MODEL

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II (PD42)1 Sep 2021PD42-02 THE EFFECTS OF RECOMBINANT BACILLUS CALMETTE-GUERIN RESISTANT TO ANTIMICROBIAL PEPTIDES ON ORTHOTOPIC BLADDER CANCER MOUSE MODEL Jae Hun Shim, Joonhee Gook, Se Young Choi, Byung Hoon Chi, Jin Wook Kim, Tae-Hyoung Kim, Soon Chul Myung, and In Ho Chang Jae Hun ShimJae Hun Shim More articles by this author , Joonhee GookJoonhee Gook More articles by this author , Se Young ChoiSe Young Choi More articles by this author , Byung Hoon ChiByung Hoon Chi More articles by this author , Jin Wook KimJin Wook Kim More articles by this author , Tae-Hyoung KimTae-Hyoung Kim More articles by this author , Soon Chul MyungSoon Chul Myung More articles by this author , and In Ho ChangIn Ho Chang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002056.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Although Mycobacterium bovis Bacillus Calmette-Guerin (BCG) is the most widely used bladder cancer immunotherapy, innate immune responses involving antimicrobial peptides (AMPs) cause BCG failure. Here, we developed genetically modified recombinant BCG (rBCG) strains which escape AMPs and evaluate the efficacy and effects of rBCG. METHODS: We constructed rBCG strains expressing Streptococcal inhibitor of complement (Sic), which confers resistance to human α-defensin-1 and cathelicidin, and d-alanyl carrier protein ligase (dltA), which confers resistance to cationic AMPs. Sic and dltA were separately cloned into the pMV306 plasmid and introduced into BCG via electroporation. The efficacy of the Sic and dltA gene electroporation into BCG was evaluated by quantitative real-time polymerase chane reaction (qRT-PCR). The internalization rates and anti-cancer effects of the rBCG strains containing Sic (rBCG-Sic) and dltA (rBCG-dltA) was evaluated by the orthotopic bladder cancer mouse model. RESULTS: The cycle quantification (Cq) values of rBCG-Sic (y=-4.8823x+13.645, R2=0.9996) and rBCG-dltA (y=-5.438x+11.641, R2=0.9995) were inverse correlations to the amount of Sic and dltA genes dose dependently. The mean introduction proportions of Sic and dltA genes into BCG by electroporation were 22.2%, 27.5% and showed constant efficacy. In the orthotopic bladder cancer mouse model, the relative internalization number of rBCG-Sic, and rBCG-dltA into bladder cell in mouse bladder were higher than that of BCG and the tumor volume at rBCG-Sic were lower than at BCG and rBCG-dltA at 11, 14 m and 18 days. CONCLUSIONS: Our results showed that constructed rBCG-Sic and rBCG-dltA by electroporation and the rBCG-Sic and rBCG-dltA can effectively escape BCG-stimulated AMPs, and significantly improved immunotherapeutic tools to treat bladder cancer in orthotopic bladder cancer mouse model. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e724-e724 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jae Hun Shim More articles by this author Joonhee Gook More articles by this author Se Young Choi More articles by this author Byung Hoon Chi More articles by this author Jin Wook Kim More articles by this author Tae-Hyoung Kim More articles by this author Soon Chul Myung More articles by this author In Ho Chang More articles by this author Expand All Advertisement Loading ...

Intravesical delivery of rapamycin via folate-modified liposomes dispersed in thermo-reversible hydrogel.

PurposeTo develop an intravesical instillation system for the treatment of bladder cancer, rapamycin (Rap) was encapsulated into liposomes and then homogeneously dispersed throughout a poloxamer 407 (P407)-based hydrogel.MethodsRap-loaded conventional liposomes (R-CL) and folate-modified liposomes (R-FL) were prepared using a film hydration method and pre-loading technique, and characterized by particle size, drug entrapment efficiency, and drug loading. The cellular uptake behavior in folate receptor-expressing bladder cancer cells was observed by flow cytometry and confocal laser scanning microscopy using a fluorescent probe. In vitro cytotoxic effects were evaluated using MTT assay, colony forming assay, and Western blot. For in vivo intravesical instillation, Rap-loaded liposomes were dispersed in P407-gel, generating R-CL/P407 and R-FL/P407. Gel-forming capacities and drug release were evaluated. Using the MBT2/Luc orthotopic bladder cancer mouse model, in vivo antitumor efficacy was evaluated according to regions of interest (ROI) measurement.ResultsR-CL and R-FL were successfully prepared, at approximately <160 nm, 42% entrapment efficiency, and 57 μg/mg drug loading. FL cellular uptake was enhanced over 2-fold than that of CL; folate receptor-mediated endocytosis was confirmed using a competitive assay with folic acid pretreatment. In vitro cytotoxic effects increased dose-dependently. Rap-loaded liposomes inhibited mTOR signaling and induced autophagy in urothelial carcinoma cells. With gelation time of <30 seconds and gel duration of >12 hrs, both R-CL/P407 and R-FL/P407 preparations transformed into gel immediately after instillation into the mouse bladder. Drug release from the liposomal gel was erosion controlled. In orthotopic bladder cancer mouse model, statistically significant differences in ROI values were found between R-CL/P407 and R-FL/P407 groups at day 11 (P=0.0273) and day 14 (P=0.0088), indicating the highest tumor growth inhibition by R-FL/P407.ConclusionIntravesical instillation of R-FL/P407 might represent a good candidate for bladder cancer treatment, owing to its enhanced retention and FR-targeting.

Targeting an Autocrine Regulatory Loop in Cancer Stem-like Cells Impairs the Progression and Chemotherapy Resistance of Bladder Cancer.

Cancer stem-like cells (CSCs) contribute to bladder cancer chemotherapy resistance and progression, but the associated mechanisms have not been elucidated. This study determined whether blocking an autocrine signaling loop in CSCs improves the therapeutic effects of cis-platinum on bladder cancer. The expression of the epithelial marker OV6 and other markers in human bladder cancer specimens was examined by IHC. The CSC properties of magnetic-activated cell sorting (MACS)-isolated OV6+ and OV6- bladder cancer cells were examined. Molecular mechanisms were assessed through RNA-Seq, cytokine antibody arrays, co-immunoprecipitation (co-IP), chromatin immunoprecipitation (ChIP) and other assays. An orthotopic bladder cancer mouse model was established to evaluate the in vivo effects of a YAP inhibitor (verteporfin) and a PDGFR inhibitor (CP-673451) on the cis-platinum resistance of OV6+ CSCs in bladder cancer. Upregulated OV6 expression positively associated with disease progression and poor prognosis for bladder cancer patients. Compared with OV6- cells, OV6+ bladder cancer cells exhibited strong CSC characteristics, including self-renewal, tumor initiation in NOD/SCID mice, and chemotherapy resistance. YAP, which maintains the stemness of OV6+ CSCs, triggered PDGFB transcription by recruiting TEAD1. Autocrine PDGF-BB signaling through its receptor PDGFR stabilized YAP and facilitated YAP nuclear translocation. Furthermore, blocking the YAP/TEAD1/PDGF-BB/PDGFR loop with verteporfin or CP-673451 inhibited the cis-platinum resistance of OV6+ bladder cancer CSCs in an orthotopic bladder cancer model. OV6 could be a helpful indicator of disease progression and prognosis for patients with bladder cancer, and targeting the autocrine YAP/TEAD1/PDGF-BB/PDGFR loop might serve as a remedy for cis-platinum resistance in patients with advanced bladder cancer.

Effectiveness of two different dose administration regimens of an IL-15 superagonist complex (ALT-803) in an orthotopic bladder cancer mouse model

BackgroundWe set out to determine if the administration of subcutaneous (SQ) ALT-803 was non-inferior to standard intravesical BCG treatment in a carcinogen induced mouse (C57BL/6J) bladder cancer model.MethodsUsing this well-established carcinogen induced mouse model, we studied the effects of various dosing schemas of ALT-803 (SQ alone, SQ with intravesical BCG, intravesical alone, intravesical with intravesical BCG) compared to intravesical BCG alone (positive control) and PBS (negative control). The non-inferiority margin for the difference in bladder weight, as a surrogate for tumor mass, was defined as 7%.ResultsAll treatment groups (i.e., ALT-803 SQ alone, ALT-803 SQ with intravesical BCG, ALT-803 intravesical alone, ALT-803 intravesical with intravesical BCG and intravesical BCG alone) demonstrated a significant reduction in tumor burden as evident by bladder weights and H&E stain (p < 0.005). Non-inferiority tests between the intravesical BCG alone group and the additional treatment groups showed that SQ ALT-803 alone (p = 0.04) and BCG plus SQ ALT-803 (p = 0.009) were non-inferior to intravesical BCG alone. In this model, we did not see an appreciable infiltration of CD4+ T, CD8+ T or CD161/KLRB1+ natural killer (NK) cells in the bladder/tumor. When assessing peripheral blood mononuclear cells, SQ ALT-803 alone resulted in a robust induction of CD8+ T cells (p < 0.01), NKG2D+ NK cells (p < 0.005) and CD3+/NKG2D+ NKT cells (p < 0.005) compared to other groups, while in splenic tissue, SQ ALT-803 alone resulted in a robust induction of CD3+/NKG2D+ NKT cells (p < 0.005) compared to other groups.ConclusionSubcutaneous ALT-803 treatment alone or in combination with intravesical BCG was well tolerated and was not inferior to intravesical BCG alone. CD8+ T, NKG2D+ NK and CD3+/NKG2D+ NKT cell induction along with induction of key cytokines remain steadfast mechanisms behind ALT-803. The enhanced therapeutic index seen with BCG and ALT-803, administered SQ or intravesically, provides a powerful justification for the further development of these regimens.

Topical and systemic immunoreaction triggered by intravesical chemotherapy in an N-butyl-N-(4-hydroxybutyl) nitorosamine induced bladder cancer mouse model.

Intravesical bacillus Calmette-Guerin (BCG) treatment is the most common therapy to prevent progression and recurrence of non-muscle invasive bladder cancer (NMIBC). Although the immunoreaction elicited by BCG treatment is well documented, those induced by intravesical treatment with chemotherapeutic agents are much less known. We investigated the immunological profiles caused by mitomycin C, gemcitabine, adriamycin and docetaxel in the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced orthotopic bladder cancer mouse model. Ninety mice bearing orthotopic bladder cancer induced by BBN were randomly divided into six groups and treated with chemotherapeutic agents once a week for four weeks. After last treatment, bladder and serum samples were analyzed for cell surface and immunological markers (CD4, CD8, CD56, CD204, Foxp3, and PD-L1) using immunohistochemistry staining. Serum and urine cytokine levels were evaluated by ELISA. All chemotherapeutic agents presented anti-tumor properties similar to those of BCG. These included changes in immune cells that resulted in fewer M2 macrophages and regulatory T cells around tumors. This result was compatible with those in human samples. Intravesical chemotherapy also induced systemic changes in cytokines, especially urinary interleukin (IL)-17A and granulocyte colony stimulating factor (G-CSF), as well as in the distribution of blood neutrophils, lymphocytes, and monocytes. Our findings suggest that intravesical treatment with mitomycin C and adriamycin suppresses protumoral immunity while enhancing anti-tumor immunity, possibly through the action of specific cytokines. A better understanding of the immunoreaction induced by chemotherapeutic agents can lead to improved outcomes and fewer side effects in intravesical chemotherapy against NMIBC.

Guizhi Fuling Wan as a Novel Agent for Intravesical Treatment for Bladder Cancer in a Mouse Model.

Alternative intravesical agents are required to overcome the side effects currently associated with the treatment of bladder cancer. This study used an orthotopic bladder cancer mouse model to evaluate Guizhi Fuling Wan (GFW) as an intravesical agent. The effects of GFW were compared with those of mitomycin-C (Mito-C) and bacille Calmette-Guérin (BCG). We began by evaluating the response of the mouse bladder cancer cell line MB49 to GFW treatment, with regard to cell viability, cell cycle progression and apoptosis. MB49 cells were subsequently implanted into the urothelial walls of the bladder in female C57BL/6 mice. The success of the model was confirmed by the appearance of hematuria and tumor growth in the bladder. Intravesical chemotherapy was administered in accordance with a published protocol. In vitro data revealed that GFW arrested MB49 cell cycle in the G0/G1 phase, resulting in the suppression of cell proliferation and induced apoptosis. One possible mechanism underlying these effects is an increase in intracellular reactive oxygen species (ROS) levels leading to the activation of ataxia telangiectasia-mutated (ATM)/checkpoint kinase 2 (CHK2) and ATM/P53 pathways, thereby mediating cell cycle progression and apoptosis, respectively. This mouse model demonstrates the effectiveness of GFW in the tumor growth, with results comparable to those achieved by using BCG and Mito-C. Furthermore, GFW was shown to cause only mild hematuria. The low toxicity of the compound was confirmed by a complete lack of lesions on bladder tissue, even after 10 consecutive treatments using high concentrations of GFW. These results demonstrate the potential of GFW for the intravesical therapy of bladder cancer.

Abstract 3127: The effect of Guizhi Fuling Wan (GFW) on bladder tumor growth in a mouse model

Abstract Bladder cancer is the second most common malignancy of the genitourinary tumors in the world and tenth most common cancer in Taiwan. The most common type of bladder cancer is urothelial carcinoma (UC, formerly known as transitional cell carcinoma, TCC). The incidence rate is particularly high in southwestern coast of Taiwan. Treatment for superficial non-invasive bladder carcinoma is currently using intravesical chemotherapy with mitomycin C and BCG after transurethral resection of bladder tumor (TUR-BT). However, this therapy usually causes adverse effects including urinary frequency, urinary urgency, hematuria and tuberculosis infection. Therefore, we intend to find a complementary and alternative medicine for bladder cancer treatment which can bring a good result with less side effects. In our previous study, we found that a traditional Chinese Medicine (TCM), Guizhi Fuling Wan (GFW) had an effect on growth inhibition of human bladder cancer cell lines as same as mitomycin-C, doxorubicin and cisplatin, but had mild toxicicity on normal human bladder epithelial cells. In this study we investigated whether GFW could be used as an intravesical chemotherapy agent in our orthotopic bladder cancer mouse model and compared its adverse effect with mitomycin-C and BCG. A mouse bladder cancer cell line, MB49, was selected and evaluated its response to GFW treatment. Cell Counting Kit-8 (CCK-8) assay was employed to analyze cell viability and flow cytometry was used to determine cell cycle and apoptosis. MB49 cells were implanted into female C57BL/6 mice to generate our orthotopic bladder tumor model which was confirmed under a microscope in histology by hematoxylin and eosin staining. Our data showed that GFW was a potent inhibitor of the proliferation of mouse bladder cancer cells in vitro as well as inhibited the tumor growth on MB49 bladder cancer orthotopic model. In addition, bladder hemorrhage which was observed in bladder cancer mice had completely disappeared 17 days after the intravesical GFW-treatment. These results support GFW as a strong candidate for intravesicle chemotherapy against bladder cancer. Citation Format: Chi-Chen Lu, Ling-Huei Tseng, Syue-Yi Chen, Jiann-Der Wu, Shu-Fen Wu, Michael W.Y. Chan, Yu-Wei Leu, Cheng-Da Hsu. The effect of Guizhi Fuling Wan (GFW) on bladder tumor growth in a mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3127. doi:10.1158/1538-7445.AM2014-3127

RNAi-based Therapeutics Targeting Survivin and PLK1 for Treatment of Bladder Cancer

Harnessing RNA interference (RNAi) to silence aberrant gene expression is an emerging approach in cancer therapy. Selective inhibition of an overexpressed gene via RNAi requires a highly efficacious, target-specific short interfering RNA (siRNA) and a safe and efficient delivery system. We have developed siRNA constructs (UsiRNA) that contain unlocked nucleobase analogs (UNA) targeting survivin and polo-like kinase-1 (PLK1) genes. UsiRNAs were encapsulated into dialkylated amino acid-based liposomes (DiLA(2)) containing a nor-arginine head group, cholesteryl hemisuccinate (CHEMS), cholesterol and 1, 2-dimyristoyl-phosphatidylethanolamine-polyethyleneglycol 2000 (DMPE-PEG2000). In an orthotopic bladder cancer mouse model, intravesical treatment with survivin or PLK1 UsiRNA in DiLA(2) liposomes at 1.0 and 0.5 mg/kg resulted in 90% and 70% inhibition of survivin or PLK1 mRNA, respectively. This correlated with a dose-dependent decrease in tumor volumes which was sustained over a 3-week period. Silencing of survivin and PLK1 mRNA was confirmed to be RNA-induced silencing complex mediated as specific cleavage products were detected in bladder tumors over the duration of the study. This report suggests that intravesical instillation of survivin or PLK1 UsiRNA can serve as a potential therapeutic modality for treatment of bladder cancer.

Antitumor Effects of Imidazoquinolines in Urothelial Cell Carcinoma of the Bladder

Imidazoquinolines (Toll-like receptor-7 agonists) are a class of synthetic immune modulating agents. Imiquimod, a member of this drug family, is currently used as first line topical therapy for genital condyloma. It recently showed clinical efficacy against several benign and malignant skin lesions, including actinic keratosis and basal cell carcinoma. Working primarily through the stimulation of a proinflammatory immune response, the mechanism of action of imiquimod may be similar to that through which bacillus Calmette-Guerin is thought to act. We hypothesized that imidazoquinolines have therapeutic potential against bladder cancer. We determined the in vitro and in vivo effects of imidazoquinolines against bladder cancer cells. The human and murine J82, T24, TCC-SUP (American Tissue Culture Collection, Manassas, Virginia) and MBT-2 bladder cancer cell lines were cultured in normal culture medium or medium supplemented with imidazoquinoline. Effects on cell viability, apoptosis induction and cytokine production were evaluated. In addition, the effects of imidazoquinoline on in vivo bladder tumor growth were determined via intravesical instillation in an orthotopic bladder tumor model in the mouse. A dose dependent decrease in cell viability was observed in all tumor cell lines treated with imidazoquinoline. In addition, imidazoquinoline significantly induced apoptosis and cytokine production. In in vivo experiments most mice treated with imidazoquinoline showed only an intense inflammatory response with no evidence of tumor, while control mice showed tumor growth. Imidazoquinolines have potent direct activity against bladder cancer cells by decreasing cell viability and inducing apoptosis and cytokine production. In addition, in vivo data suggest antitumor effects in an orthotopic bladder cancer mouse model. Therefore, imidazoquinolines may have therapeutic potential as a synthetic intravesical agent against bladder cancer.