Mouse Bladder Tumor Research Articles

Assessment of Therapeutic Effects of Combined Treatment With Cisplatin and Anti-mouse Programmed Death (PD)-1 Antibody in a Mouse Urothelial Cancer Model.

The therapeutic impact of combination treatment with an immune checkpoint inhibitor (ICI) and chemotherapeutic agent on patients with urothelial cancer (UC) remains controversial. Therefore, the present study investigated differences in the therapeutic effects of combination therapy with cisplatin plus anti-mouse programmed death (PD)-1 antibody according to the dose of cisplatin using the mouse bladder tumor model MBT2. The effects of treatment with two different doses cisplatin and/or anti-mouse PD-1 antibody on tumor growth after the subcutaneous injection of MBT2 cells were compared. Infiltrating patterns of lymphocytes into tumors after treatment were assessed using immunohistochemical staining. MBT2 tumor volumes were significantly larger in mice receiving high-dose cisplatin alone than in those receiving low-dose cisplatin alone. Combination treatment with cisplatin plus anti-mouse PD-1 antibody exerted significantly stronger growth inhibitory effects on MBT2 tumors than treatment with either agent alone, irrespective of cisplatin doses; however, no significant differences were observed in MBT2 tumor volumes between mice receiving anti-mouse PD-1 antibody plus high-dose cisplatin and those receiving anti-mouse PD-1 antibody plus low-dose cisplatin. Furthermore, CD8+ to CD3+ and CD8+ to CD11b+ T-lymphocyte ratios in MBT2 tumors were both significantly higher in the low-dose cisplatin alone group than in the high-dose cisplatin alone group, whereas no significant differences were noted in either ratio between the two different combination treatment regimens. When combined with ICI, a lower dose of cisplatin may achieve favorable antitumor effects in UC patients by preventing lymphocyte exhaustion.

Abstract 1552: Tumorigenesis of basal muscle invasive bladder cancer was mediated by PTEN protein degradation resulting from SNHG1 upregulation

Abstract hosphatase and tensin homolog deleted on chromosome ten (PTEN) serves as a powerful tumor suppressor, and has been found to be downregulated in human bladder cancer (BC) tissues. Despite this observation, the mechanisms contributing to PTEN’s downregulation have remained elusive. In this study, we discovered that mice exposed to N-butyl-N-(4-hydroxybu-tyl)nitrosamine (BBN), a specific bladder chemical carcinogen, exhibited primary basal muscle invasive BC (BMIBC) accompanied by a pronounced reduction in PTEN protein expression in vivo. Utilizing a lncRNA deep sequencing high-throughput platform, along with gain- and loss-of-function analyses, we identified small nucleolar RNA host gene 1 (SNHG1) as a critical lncRNA that might drive the formation of primary BMIBCs in BBN-treated mice. Cell culture results further demonstrated that BBN exposure significantly induced SNHG1 in normal human bladder urothelial cell UROtsa. Notably, the ectopic expression of SNHG1 alone was sufficient to induce malignant transformation in human urothelial cells, while SNHG1 knockdown effectively inhibited anchorage-independent growth of human BMIBCs. Our detailed investigation revealed that SNHG1 overexpression led to PTEN protein degradation through its direct interaction with HUR. This interaction reduced HUR binding to ubiquitin-specific peptidase 8 (USP8) mRNA, causing degradation of USP8 mRNA and a subsequent decrease in USP8 protein expression. The downregulation of USP8, in turn, increased PTEN poly-ubiquitination and degradation, culminating in cell malignant transformation and BMIBC anchorage-independent growth. In vivo studies confirmed the downregulation of PTEN and USP8, as well as their positive correlations in both BBN-treated mouse bladder urothelium and tumor tissues of bladder cancer in nude mice.Our findings, for the first time, demonstrate that overexpressed SNHG1 competes with USP8 for binding to HUR. This competition attenuates USP8 mRNA stability and protein expression, leading to PTEN protein degradation, consequently, this process drives urothelial cell malignant transformation and fosters BMIBC growth and primary BMIBC formation. Citation Format: Chuanshu Huang, Tengda Li, Honglei Jin. Tumorigenesis of basal muscle invasive bladder cancer was mediated by PTEN protein degradation resulting from SNHG1 upregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1552.

Tumorigenesis of basal muscle invasive bladder cancer was mediated by PTEN protein degradation resulting from SNHG1 upregulation

BackgroundPhosphatase and tensin homolog deleted on chromosome ten (PTEN) serves as a powerful tumor suppressor, and has been found to be downregulated in human bladder cancer (BC) tissues. Despite this observation, the mechanisms contributing to PTEN’s downregulation have remained elusive.MethodsWe established targeted genes’ knockdown or overexpressed cell lines to explore the mechanism how it drove the malignant transformation of urothelial cells or promoted anchorageindependent growth of human basal muscle invasive BC (BMIBC) cells. The mice model was used to validate the conclusion in vivo. The important findings were also extended to human studies.ResultsIn this study, we discovered that mice exposed to N-butyl-N-(4-hydroxybu-tyl)nitrosamine (BBN), a specific bladder chemical carcinogen, exhibited primary BMIBC accompanied by a pronounced reduction in PTEN protein expression in vivo. Utilizing a lncRNA deep sequencing high-throughput platform, along with gain- and loss-of-function analyses, we identified small nucleolar RNA host gene 1 (SNHG1) as a critical lncRNA that might drive the formation of primary BMIBCs in BBN-treated mice. Cell culture results further demonstrated that BBN exposure significantly induced SNHG1 in normal human bladder urothelial cell UROtsa. Notably, the ectopic expression of SNHG1 alone was sufficient to induce malignant transformation in human urothelial cells, while SNHG1 knockdown effectively inhibited anchorage-independent growth of human BMIBCs. Our detailed investigation revealed that SNHG1 overexpression led to PTEN protein degradation through its direct interaction with HUR. This interaction reduced HUR binding to ubiquitin-specific peptidase 8 (USP8) mRNA, causing degradation of USP8 mRNA and a subsequent decrease in USP8 protein expression. The downregulation of USP8, in turn, increased PTEN polyubiquitination and degradation, culminating in cell malignant transformation and BMIBC anchorageindependent growth. In vivo studies confirmed the downregulation of PTEN and USP8, as well as their positive correlations in both BBN-treated mouse bladder urothelium and tumor tissues of bladder cancer in nude mice.ConclusionsOur findings, for the first time, demonstrate that overexpressed SNHG1 competes with USP8 for binding to HUR. This competition attenuates USP8 mRNA stability and protein expression, leading to PTEN protein degradation, consequently, this process drives urothelial cell malignant transformation and fosters BMIBC growth and primary BMIBC formation.

Type 2 innate lymphoid cells are not involved in mouse bladder tumor development.

Therapies for bladder cancer patients are limited by side effects and failures, highlighting the need for novel targets to improve disease management. Given the emerging evidence highlighting the key role of innate lymphoid cell subsets, especially type 2 innate lymphoid cells (ILC2s), in shaping the tumor microenvironment and immune responses, we investigated the contribution of ILC2s in bladder tumor development. Using the orthotopic murine MB49 bladder tumor model, we found a strong enrichment of ILC2s in the bladder under steady-state conditions, comparable to that in the lung. However, as tumors grew, we observed an increase in ILC1s but no changes in ILC2s. Targeting ILC2s by blocking IL-4/IL-13 signaling pathways, IL-5, or IL-33 receptor, or using IL-33-deficient or ILC2-deficient mice, did not affect mice survival following bladder tumor implantation. Overall, these results suggest that ILC2s do not contribute significantly to bladder tumor development, yet further investigations are required to confirm these results in bladder cancer patients.

Orthotopic Mouse Models of Urinary Bladder Cancer.

Urinary bladder cancer has various etiologies and tends to recur and then progress to a higher grade. When muscles are invaded, the response to conventional therapy is poor and the quality of life deteriorates rapidly. Here, we summarize and compare two representative methods used to create the syngeneic mouse models required for immunological research. In this study, we utilized six-week-old female C3H/HeNCrl mice and the mouse bladder tumor cell line MBT-2. The first method involved transurethral catheterization with poly-L-lysine pretreatment (catheter group), while the second method involved transperitoneal incision and direct injection of tumor cells into the bladder wall (open group). Mouse postoperative status was monitored on a weekly basis using magnetic resonance imaging (MRI). The catheter group had a tumor development rate of 47% (7 out of 15 mice), with only 1 mouse developing an intravesical tumor. In contrast, the open group had a higher tumor formation rate of 69% (47 out of 68 mice), with 27 mice showing intravesical tumor formation. Notably, with a lower cell count, urinary obstruction events were observed 2 weeks post-inoculation, which is one week later than the higher cell count group. In this study, we conducted a comparative analysis between the transurethral catheterization method and the transperitoneal incision and direct injection method in animal bladder tumor models. Our findings provide evidence of the consistent effectiveness in constructing a stable model within the open group. Well-designed orthotopic animal models are essential.

Uncovering the Inhibitory Molecular Mechanism of Pomegranate Peel to Urinary Bladder Urothelial Carcinoma Using Proteomics Techniques.

Pomegranate (Punica granatum L.) fruit demonstrates the repressive effectiveness of many tumors. Our previous studies showed that the PEP (pomegranate peel extract) E2 fraction obtained from the ethyl acetate layer of the pomegranate peel's ethanol extract exhibited the highest inhibitory activities to induce Urinary bladder urothelial carcinoma (UBUC) cell apoptosis. The ethyl acetate layer could lower the volume and weight of T24 tumors and initiate apoptosis in nude mice xenografted bladder tumors. In this study, we intended to clarify the inhibitory molecular process of Taiwanese local pomegranate peel to urinary bladder urothelial carcinoma using a proteomics strategy. Gel-based proteomics (two-dimensional gel electrophoresis coupled with tandem mass spectrometry) was used to get an insight into the molecular mechanisms initiated by PEPE2 to evoke bladder cancer cell apoptosis. We found eleven down-regulated and eight up-regulated proteins in PEPE2-treated T24 cells. Our results implied that these PEPE2-dysregulated proteins belong to cell apoptosis, cell proliferation, death receptor signaling, JAK/STAT signaling, the PPAR pathway, the PPARα/RXR α pathway, Rho family GTPase signaling, and RhoGDI signaling. In addition, HSP90 and PTP1B proteins, associated with apoptosis, were de-regulated in xenografted bladder tumors in nude mice fed with an ethyl acetate layer of ethanol extract. The findings above implied that pomegranate might be a potential chemopreventive resource for UBUC carcinogenesis.

Detection of PD-L1-Expressing Myeloid Cell Clusters in the Hyaluronan-Enriched Stroma in Tumor Tissue and Tumor-Draining Lymph Nodes.

Expression of the transmembrane protein PD-L1 is frequently upregulated in cancer. Because PD-L1-expressing cells can induce apoptosis or anergy of T lymphocytes through binding to the PD1 receptor, the PD-L1-mediated inhibition of activated PD1+ T cells is considered a major pathway for tumor immune escape. However, the mechanisms that regulate the expression of PD-L1 in the tumor microenvironment are not fully understood. Analysis of organotypic tumor tissue slice cultures, obtained from mice with implanted syngeneic tumors (MBT2 bladder tumors in C3H mice, Renca kidney, and CT26 colon tumors in BALB/c mice), as well as from patients with cancer, revealed that tumor-associated hyaluronan (HA) supports the development of immunosuppressive PD-L1+ macrophages. Using genetically modified tumor cells, we identified epithelial tumor cells and cancer-associated mesenchymal fibroblast-like cells as a major source of HA in the tumor microenvironment. These HA-producing tumor cells, and particularly the vimentin-positive fibroblast-like cells of bone marrow origin, directly interact with tumor-recruited myeloid cells to form large stromal congregates/clusters that are highly enriched for both HA and PD-L1. Furthermore, similar cell clusters composed of HA-producing fibroblast-like cells and PD-L1+ macrophages were detected in tumor-draining, but not in distant, lymph nodes. Collectively, our findings indicate that the formation of multiple large HA-enriched stromal clusters that support the development of PD-L1-expressing APCs in the tumor microenvironment and draining lymph nodes could contribute to the immune escape and resistance to immunotherapy in cancer.

Magnetic-Powered Janus Cell Robots Loaded with Oncolytic Adenovirus for Active and Targeted Virotherapy of Bladder Cancer.

A unique robotic medical platform is designed by utilizing cell robots as the active "Trojan horse" of oncolytic adenovirus (OA), capable of tumor-selective binding and killing. The OA-loaded cell robots are fabricated by entirely modifying OA-infected 293T cells with cyclic arginine-glycine-aspartic acid tripeptide (cRGD) to specifically bind with bladder cancer cells, followed by asymmetric immobilization of Fe3 O4 nanoparticles (NPs) on the cell surface. OA can replicate in host cells and induce cytolysis to release the virus progeny to the surrounding tumor sites for sustainable infection and oncolysis. The asymmetric coating of magnetic NPs bestows the cell robots with effective movement in various media and wireless manipulation with directional migration in a microfluidic device and bladder mold under magnetic control, further enabling steerable movement and prolonged retention of cell robots in the mouse bladder. The biorecognition of cRGD and robust, controllable propulsion of cell robots work synergistically to greatly enhance their tissue penetration and anticancer efficacy in the 3D cancer spheroid and orthotopic mouse bladder tumor model. Overall, this study integrates cell-based microrobots with virotherapy to generate an attractive robotic system with tumor specificity, expanding the operation scope of cell robots in biomedical community.

MP06-14 ROLE OF LATROPHILINS AS KEY DOWNSTREAM EFFECTORS OF THE ANDROGEN RECEPTOR IN MODULATING UROTHELIAL TUMORIGENESIS

You have accessJournal of UrologyCME1 May 2022MP06-14 ROLE OF LATROPHILINS AS KEY DOWNSTREAM EFFECTORS OF THE ANDROGEN RECEPTOR IN MODULATING UROTHELIAL TUMORIGENESIS Takuro Goto, Yuki Teramoto, Masato Yasui, Guiyang Jiang, Yujiro Nagata, George Netto, and Hiroshi Miyamoto Takuro GotoTakuro Goto More articles by this author , Yuki TeramotoYuki Teramoto More articles by this author , Masato YasuiMasato Yasui More articles by this author , Guiyang JiangGuiyang Jiang More articles by this author , Yujiro NagataYujiro Nagata More articles by this author , George NettoGeorge Netto More articles by this author , and Hiroshi MiyamotoHiroshi Miyamoto More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.14AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: We and others have indicated that androgen receptor (AR) signaling plays a critical role in urothelial cancer outgrowth. Meanwhile, our DNA microarray data suggest that latrophilin-3 (LPHN3), one of latrophilins (LPHNs) that are a group of the G-protein-coupled receptor where a spider venom latrotoxin (LTX) is known to bind, represents a downstream target of AR in bladder cancer cells. The present study aimed to determine the functional role of LPHNs, including LPHN1, LPHN2, and LPHN3, in the development of urothelial cancer. METHODS: We compared the levels of LPHN expression in 83 bladder tumors vs. paired normal bladder tissues (by immunohistochemistry) as well as in human normal urothelial SVHUC sublines stably expressing wild-type AR vs. vector only (by western blot). We then assessed the effects of ligand (e.g. LTX, FLRT3) treatment, as well as silencing of each LPHN via shRNA virus infection, on the neoplastic transformation of SVHUC-derived cells induced by a chemical carcinogen 3-methylcholanthrene (MCA). Bladder tumor development was also compared in C57BL/6 mice treated with a carcinogen BBN and LTX/FLRT3. RESULTS: The rates of immunoreactivity for LPHN1 (87% vs. 69%, P=0.012), LPHN2 (55% vs. 31%, P=0.002), and LPHN3 (51% vs. 37%, P=0.087) were significantly higher in bladder tumors than in non-neoplastic urothelial tissues. Moreover, LPHN3 positivity in non-muscle-invasive tumors was associated with the risk of disease recurrence after transurethral surgery (P=0.051). The expression levels of each LPHN were also higher in SVHUC-AR cells than in AR-negative SVHUC-vector cells, and androgen treatment in SVHUC-AR further increased them. Chromatin immunoprecipitation assay then revealed the binding of AR to the promoter region of each LPHN in SVHUC-derived cells. Meanwhile, induction in LPHN expression by LTX or FLRT3 treatment was seen in SVHUC cells. The in vitro transformation assay showed that LTX and FLRT3 induced the MCA-mediated oncogenic activity particularly in SVHUC-vector cells, while knockdown of LPHN1, LPHN2, or LPHN3 resulted in similar inhibition in that of MCA-SVHUC-AR cells. LTX (P=0.002) and FLRT3 (P=0.001) also accelerated the development of bladder tumors in BBN-treated female mice. CONCLUSIONS: These findings suggest that LPHNs, which are activated in bladder tumors as key downstream effectors of the AR, promote urothelial tumorigenesis. Accordingly, LPHN inhibition, along with AR inactivation, has the potential of being an effective chemopreventive approach for urothelial cancer. Source of Funding: Ferring Research Institute © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e82 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takuro Goto More articles by this author Yuki Teramoto More articles by this author Masato Yasui More articles by this author Guiyang Jiang More articles by this author Yujiro Nagata More articles by this author George Netto More articles by this author Hiroshi Miyamoto More articles by this author Expand All Advertisement PDF DownloadLoading ...

PD37-09 INTERMITTENT WST-11 VASCULAR TARGET PHOTODYNAMIC THERAPY IN A MOUSE ORTHOTOPIC BLADDER MODEL

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (PD37)1 Sep 2021PD37-09 INTERMITTENT WST-11 VASCULAR TARGET PHOTODYNAMIC THERAPY IN A MOUSE ORTHOTOPIC BLADDER MODEL Jie Chen, Jasmine Thomas, Karan Nagar, Lucas Nogueira, Peter Reisz, Kwanghee Kim, and Jonathan Coleman Jie ChenJie Chen More articles by this author , Jasmine ThomasJasmine Thomas More articles by this author , Karan NagarKaran Nagar More articles by this author , Lucas NogueiraLucas Nogueira More articles by this author , Peter ReiszPeter Reisz More articles by this author , Kwanghee KimKwanghee Kim More articles by this author , and Jonathan ColemanJonathan Coleman More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002047.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Vascular targeted photodynamic therapy (VTP) is a non-surgical tumor ablation approach used to treat early-stage prostate cancer and may also be effective for urothelial cancer based on preclinical data in heterotopic models. While orthotopic models are valuable as they reproduce factors such as local tissue environment and cancer progression pathogenesis compared to ectopic implantation, its implementation is technically difficult. Herein we describe our work in developing an orthotopic murine model for bladder urothelial cancer using intermittent laser pulses to ensure mouse survival and local tumor control. METHODS: The MB-49 murine urothelial cancer line was used for all experiments after establishing stable luciferase expression. Tumors were implanted in 16 1C57BL-6 mice by surgical approach by direct injection of 1x105 MB49-Luc cells into the bladder. Successful tumor implantation was assessed by IVIS images 72 hours after implantation. Mice were divided in two groups: control and VTP treated. VTP was performed 96 hours after cell injection at a power of 100mW in 30 seconds pulses, with 30 seconds interval. Mouse bladders were harvested 13 days post implantation. Safety, tumor control, and histology parameters were assessed. RESULTS: Adequate tumor growth was observed in all mice, and no major toxicity was observed in both groups. Final tumor size was 71% smaller, and histological viability was decreased in VTP treated tumors when compared to controls (p <0.001, figure 1). CD31, Ki67 expression were decreased 2.75 and 1.87-fold respectively, and tunnel staining was increased 3.2-fold in control tumors when compared to VTP treated. (p <0.05, figure 2). CONCLUSIONS: A mouse orthotopic bladder urothelial model via injection of MB-49 cell line is feasible and displays a very high tumor take rates. Intermittent VTP treatment has an effect on mouse bladder tumors as shown by the decrease in tumor size, weight and change in histology. Additional refinement to the model may provide a means to establish an appropriate timeline and utility for future studies. Source of Funding: NIH/NCI (P30 CA008748) and the Thompson Family Foundation © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e658-e659 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jie Chen More articles by this author Jasmine Thomas More articles by this author Karan Nagar More articles by this author Lucas Nogueira More articles by this author Peter Reisz More articles by this author Kwanghee Kim More articles by this author Jonathan Coleman More articles by this author Expand All Advertisement Loading ...

PD37-06 LATROPHILIN-3 AS A KEY DOWNSTREAM EFFECTOR OF THE ANDROGEN RECEPTOR INDUCES UROTHELIAL TUMORIGENESIS

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (PD37)1 Sep 2021PD37-06 LATROPHILIN-3 AS A KEY DOWNSTREAM EFFECTOR OF THE ANDROGEN RECEPTOR INDUCES UROTHELIAL TUMORIGENESIS Takuro Goto, Yuki Teramoto, Yujiro Nagata, Guiyang Jiang, Taichi Mizushima, Satoshi Inoue, Hiroki Ide, George Netto, and Hiroshi Miyamoto Takuro GotoTakuro Goto More articles by this author , Yuki TeramotoYuki Teramoto More articles by this author , Yujiro NagataYujiro Nagata More articles by this author , Guiyang JiangGuiyang Jiang More articles by this author , Taichi MizushimaTaichi Mizushima More articles by this author , Satoshi InoueSatoshi Inoue More articles by this author , Hiroki IdeHiroki Ide More articles by this author , George NettoGeorge Netto More articles by this author , and Hiroshi MiyamotoHiroshi Miyamoto More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002047.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: We and others have indicated that androgen receptor (AR) signaling plays a critical role in urothelial cancer outgrowth. Meanwhile, our DNA microarray data suggest that latrophilin-3 (LPHN3), one of latrophilins that are a group of the G-protein-coupled receptor where a spider venom latrotoxin (LTX) is known to bind, represents a downstream target of AR in bladder cancer cells. The present study aims to determine the functional role of LPHN3 in urothelial carcinogenesis. METHODS: We determined the expression of LPHN3 in 145 bladder tumors and paired normal bladder tissues (by immunohistochemistry) as well as in human normal urothelial SVHUC sublines stably expressing wild-type AR or vector only (by RT-PCR and western blot). We then assessed the effects of ligand (e.g. LTX, FLRT3) treatment on neoplastic/malignant transformation of SVHUC-derived cells, as well as bladder tumor development in C57BL/6 mice, induced by chemical carcinogens (e.g. MCA, BBN). RESULTS: Immunohistochemistry showed that LPHN3 was positive in 52% (47% 1+, 5% 2+) of tumors, which was significantly (P=0.049) higher than in non-neoplastic urothelial tissues [37% (all 1+)], and that LPHN3 positivity in non-muscle-invasive tumors was associated with the risk of tumor recurrence after transurethral surgery (P=0.051). The level of LPHN3 expression was higher in SVHUC-AR cells than in AR-negative SVHUC-vector cells. In addition, significant induction in the LPHN3 expression by LTX or FLRT3 treatment was seen in SVHUC, whereas LPHN3 knockdown via shRNA virus infection in SVHUC-AR resulted in considerable induction or reduction of the expression of the proteins positively (e.g. cleaved caspase-3) or negatively (e.g. Bcl-2, Bcl-xL), respectively, associated with apoptosis. Chromatin immunoprecipitation assay further revealed the binding of AR to the promoter region of LPHN3 in SVHUC-derived cells. The in vitro transformation assay showed that LTX and FLRT3 induced the MCA-mediated oncogenic activity in both SVHUC-AR and SVHUC-vector cells, while the inhibitory effect of LPHN3 knockdown on the neoplastic transformation was observed in MCA-SVHUC-AR cells. LTX/FLRT3 also significantly accelerated the development of bladder tumors in BBN-treated female mice. CONCLUSIONS: These findings suggest that LPHN3, which is activated in bladder tumors as a key downstream effector of the AR, promotes urothelial tumorigenesis. Accordingly, LPHN3 inhibition, along with AR inactivation, has the potential of being an effective chemopreventive approach for urothelial cancer. Source of Funding: Ferring Research Institute © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e657-e657 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takuro Goto More articles by this author Yuki Teramoto More articles by this author Yujiro Nagata More articles by this author Guiyang Jiang More articles by this author Taichi Mizushima More articles by this author Satoshi Inoue More articles by this author Hiroki Ide More articles by this author George Netto More articles by this author Hiroshi Miyamoto More articles by this author Expand All Advertisement Loading ...

Abstract LB173: APVO603: A dual 4-1BB and OX40 bispecific approach utilizing ADAPTIRTMtechnology designed to deliver a conditional T cell/NK response against solid tumors

Abstract APVO603 is Aptevo's bispecific candidate targeting 4-1BB and OX40. It was designed to have unique properties with the potential to overcome some of the clinical challenges observed with monoclonal antibody targeting these receptors. APVO603 is engineered as an FcγR-signaling deficient bispecific antibody that utilizes Aptevo's ADAPTIR technology for a distinct approach for dual targeting of 4-1BB and OX40 in the absence of additional effector activity. The distinct characteristics of APVO603 may enable conditional activation of 4-1BB and OX40 via agonism of these receptors only when cross-linked via engagement of the other receptor via cis and/or trans cellular interactions. Thus, APVO603 is designed with the potential to overcome both the on-target toxicity and limited efficacy observed with 4-1BB and OX40 monoclonal antibody treatment in the clinic. Anti-4-1BB and OX40 binding domains were optimized to increase affinity, function and stability, then incorporated into the ADAPTIR bispecific antibody platform to produce the APVO603 lead candidate. APVO603 was found to augment 4-1BB and OX40 activity in a dose-dependent manner and is strictly dependent on engagement of the reciprocal receptor to elicit 4-1BB or OX40 signaling in vitro. In preclinical assays using PBMCs sub-optimally stimulated with anti-CD3, APVO603 induces synergistic proliferation of CD4+, CD8+ T and NK cells when compared to anti-OX40 or 4-1BB antibodies with a wt Fc, included either individually or in combination. Additionally, APVO603 enhances proinflammatory cytokine production, granzyme B expression, and reduces the T cell exhaustion phenotype. The mechanistic activity of APVO603 resulted in dose-dependent control of in vivo tumor growth in a preclinical humanized murine xenograft model using established murine MB49 bladder tumors in human 4-1BB and OX40 double knock-in mice. APVO603 is a dual-agonistic bispecific antibody that augments the effector function of activated CD4+ and CD8+ T cells and NK cells in a dose-dependent manner and reduces growth of established tumors in vivo. This preclinical data demonstrates conditional dual stimulation of 4-1BB and OX40 and supports further development of APVO603, a promising immuno-oncology therapeutic with potential for benefit in solid tumors. This program is progressing into IND-enabling studies later this year. Citation Format: Michelle H. Nelson, Robert E. Miller, Secil Franke-Welch, Ruth Chenault, Hang Fang, Allison Chunyk, Gabriela Hernandez-Hoyos, Hilario J. Ramos, David Bienvenue, Catherine McMahan. APVO603: A dual 4-1BB and OX40 bispecific approach utilizing ADAPTIRTMtechnology designed to deliver a conditional T cell/NK response against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB173.

How Cancer Cells Invade Bladder Epithelium and Form Tumors: The Mouse Bladder Tumor Model as a Model of Tumor Recurrence in Patients.

Non-muscle-invasive bladder cancer is the most common form of bladder cancer. The main problem in managing bladder tumors is the high recurrence after the transurethral resection of bladder tumors (TURBT). Our study aimed to examine the fate of intravesically applied cancer cells as the implantation of cancer cells after TURBT is thought to be a cause of tumor recurrence. We established an orthotopic mouse bladder tumor model with MB49-GFP cancer cells and traced them during the first three days to define their location and contacts with normal urothelial cells. Data were obtained by Western blot, immunolabeling, and light and electron microscopy. We showed that within the first two hours, applied cancer cells adhered to the traumatized epithelium by cell projections containing α3β1 integrin on their tips. Cancer cells then migrated through the epithelium and on day 3, they reached the basal lamina or even penetrated it. In established bladder tumors, E-cadherin and desmoplakin 1/2 were shown as feasible immunohistochemical markers of tumor margins based on the immunolabeling of various junctional proteins. Altogether, these results for the first time illustrate cancer cell implantation in vivo mimicking cellular events of tumor recurrence in bladder cancer patients.

Oral Recombinant Methioninase Sensitizes a Bladder Cancer Orthotopic Xenograft Mouse Model to Low-dose Cisplatinum and Prevents Metastasis.

The aim of the study was to determine if oral recombinant methioninase (o-rMETase) can sensitize an orthotopic bladder tumor in nude mice to low-dose cisplatinum (CDDP). The green fluorescent protein (GFP)-expressing UM-UC-3-GFP bladder cancer was surgically orthotopically implanted (SOI) to the bladder in nude mice. The treatment was initiated when the primary tumor volume reached 100 mm3 Mice were assigned to 3 groups: G1: Saline vehicle (0.1 ml per mouse, oral, twice per day); G2: low-dose CDDP (0.5 mg/kg, intraperitoneal twice per week); G3: o-rMETase + low-dose CDDP (100 units per mouse, oral, twice per day + 0.5 mg/kg, intraperitoneal twice per week, respectively). Tumor volume and body weight were measured twice per week. The expression of Ki-67 was detected by immunohistochemistry to evaluate cell proliferation. The combination of o-rMETase and low-dose CDDP increased inhibition efficacy compared to low-dose CDDP monotherapy, on primary-tumor growth (p=0.032) and metastasis (p=0.002). The combination of o-rMETase with low-dose CDDP has future clinical potential for bladder cancer.

PD-L1 targeting high-affinity NK (t-haNK) cells induce direct antitumor effects and target suppressive MDSC populations

BackgroundAlthough immune checkpoint inhibitors have revolutionized cancer treatment, clinical benefit with this class of agents has been limited to a subset of patients. Hence, more effective means to target tumor...

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids.

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

Halofuginone: a novel oral and intravesical agent for the treatment of non-muscle invasive bladder cancer

Background: Non-muscle invasive form (NMIBC) is a chronic disease with a high recurrence rate and requires lifelong surveillance. Various intravesical agents were shown to reduce tumor recurrence but unfortunately, none of these agents proved to be of benefit in long-term prevention of local recurrence or disease progression. Aim of Research: Previous studies have shown that Halofuginone (HF), an antiprotozoal agent, exerts anti-neoplastic activity in various cancer models. Our aim was to evaluate the in vivo activity of oral and intravesical HF treatment in an experimental mouse model harboring NMIBC. Methods: Initially, 60 mice were divided into six treatment groups to evaluate the toxicity of this anti-parasitic agent on the bladder mucosa. The second stage included 126 mice which underwent intravesical implantation with Mouse Bladder Tumor cells (MBT-2): Group 1 (n = 30) received no treatment, group 2 (n = 32) received 6 intravesical instillations of PBS, group 3 (n = 32) received 6 doses of 250 µg oral HF, whereas group 4 (n = 32) received 6 intravesical instillations of 250 µg HF. Results: The average weight of bladders, which reflects the anti-neoplastic activity, differed significantly between the control and treated groups: 88.8 mg ± 15.58 SEM and 81.2 mg ± 13.79 SEM for untreated and PBS-treated mice, respectively, versus 38.0 mg ± 4.02 SEM and 39.6 mg ± 5.97 SEM for animals treated with oral and intravesical HF, respectively. Conclusions: HF exerted a significant anti-neoplastic activity in mice bearing NMIBC upon oral as well as intravesical administration. These results may constitute the basis for the maintenance of oral treatment with HF in patients with NMIBC.

How cancer cells attach to urinary bladder epithelium in vivo: study of the early stages of tumorigenesis in an orthotopic mouse bladder tumor model.

The majority of bladder cancers in humans are non-muscle-invasive cancers that recur frequently after standard treatment procedures. Mouse models are widely used to develop anti-tumor treatments. The purpose of our work was to establish an orthotopic mouse bladder tumor model and to explore early stages of implantation of cancerous MB49 cells in vivo using various labeling and microscopic techniques. To distinguish cancer cells from normal urothelial cells in mouse urinary bladders, we performed molecular characterization of MB49 cells before intravesical injection experiments. In this new approach we applied internalized metal nanoparticles to unequivocally discriminate cancer cells from normal cells. This method revealed that cancer cells attached to the urothelium or basal lamina within just 1hour of intravesical injection, whereas small tumors and localized hyperplastic urothelial regions developed within two days. We found that cancer cells initially adhere to normal urothelial cells through filopodia and by focal contacts with basal lamina. This is the first in vivo characterization of intercellular contacts between cancerous and normal urothelial cells in the bladder. Our study yields new data about poorly known early events of tumorigenesis in vivo, which could be helpful for the translation into clinic.

Lapatinib, a Dual Inhibitor of Epidermal Growth Factor Receptor (EGFR) and HER-2, Enhances Radiosensitivity in Mouse Bladder Tumor Line-2 (MBT-2) Cells In Vitro and In Vivo

BackgroundThe aim of this study was to evaluate the effect of lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR) and HER-2, on the radiosensitivity of murine bladder tumor line-2 (MBT-2) cells in vitro and in vivo.Material/MethodsMBT-2 cells were pretreated with lapatinib at doses ranging from 200–1,000 nM for 30 min followed by radiation at doses ranging from 2.5–10 Gy for 30 min. A clonogenic assay (colony formation assay) assessed cell survival. Western blot measured phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated AKT (p-AKT), and phosphorylated HER-2 (p-HER2) and the apoptosis marker, PARP. The C3H/HeN mouse tumor xenograft model underwent subcutaneous injection of MBT-2 cells; mice were divided into four groups, treated with lapatinib (200 mg/kg), radiation (15 Gy), a combination of both, and with vehicle (control).ResultsLapatinib pretreatment, combined with radiation, decreased MBT-2 cell survival, and suppressed radiation-activated levels of p-EGFR and p-HER-2. MBT-2 cells treated with a 10 Gy dose of radiation and 1000 nM of lapatinib showed combination index (CI) values of <1 indicating synergy. Increased expression of γ-H2AX, indicated increased apoptosis. In mice with tumor xenografts, a daily dose of lapatinib (200 mg/kg/day) for seven days combined with radiation on the fourth day suppressed tumor growth to a greater degree than radiation alone.ConclusionsLapatinib treatment enhanced the radiation sensitivity in an in vitro and in vivo murine bladder cancer model by decreasing radiation-mediated EGFR and HER-2 activation, and by causing DNA damage leading to cell apoptosis.