MB49 Cells Research Articles

Single-cell RNA sequencing analysis identifies acute changes in the tumor microenvironment induced by interferon α gene therapy in a murine bladder cancer model

IntroductionNadofaragene firadenovec (Ad-IFNα/Syn3) is now approved for BCG-unresponsive bladder cancer (BLCA). IFNα is a pleiotropic cytokine that causes direct tumor cell killing via TRAIL-mediated apoptosis, angiogenesis inhibition, and activation of the innate and adaptive immune system. We established an immunocompetent murine BLCA model to study the effects of murine adenoviral IFNα (muAd-Ifnα) gene therapy on cancer cells and the tumor microenvironment using a novel murine equivalent of Nadofaragene firadenovec (muAd-Ifnα).MethodsTumors were induced by instilling MB49 cells into the bladders of mice; luciferase imaging confirmed tumor development. Mice were treated with adenovirus control (Ad-Ctrl; empty vector), or muAd-Ifnα (3x1011 VP/mL), and survival analysis was performed. For single-cell sequencing (scRNAseq) analysis (72h), bladders were harvested and treated with collagenase/hyaluronidase and TrypLE for cell dissociation. Single cells were suspended in PBS/1% FBS buffer; viability was assessed with Vicell cell counter. scRNAseq analysis was performed using 10X genomics 3’ sequencing. Raw RNAseq data were pre-processed using Cell Ranger single-cell software. Seurat (R package) was used to normalize and cluster the scRNA data. Pooled differential gene expression analysis in specific cell clusters was performed with DESeq2.ResultsWe identified 16 cell clusters based on marker expression which were grouped into epithelial (tumor), uroplakin-enriched, endothelial, T-cells, neutrophils, and macrophage clusters. Top differentially expressed genes between muAd-Ifnα and Ad-Ctrl were identified. Within the specific cell clusters, IPA analysis revealed significant differences between muAd-Ifnα and control. IFNα signaling and hypercytokinemia/chemokinemia were upregulated in all clusters. Cell death pathways were upregulated in tumor and endothelial clusters. T-cells demonstrated upregulation of the immunogenic cell death signaling pathway and a decrease in the Th2 pathway genes. Macrophages showed upregulation of PD1/PD-L1 pathways along with downregulation of macrophage activation pathways (alternate and classical). Multiplex immunofluorescence confirmed increased infiltration with macrophages in muAd-Ifnα treated tumors compared to controls. PD1/PD-L1 expression was reduced at 72h.DiscussionThis single-cell analysis builds upon our understanding of the impact of Ad-IFNα on tumor cells and other compartments of the microenvironment. These data will help identify mechanisms to improve patient selection and therapeutic efficacy of Nadofaragene firadenovec.

Abstract LB437: Development of a bladder cancer-on-a-chip assay to assess the invasive potential of bladder cancer cells

Abstract Goals: The primary goal of this study was to develop a 3D functional bioassay to assess the relative invasiveness of bladder cancer cells. This model will support the testing of migrastatic drugs and investigation of the molecular mechanisms which allow for bladder cancer cell invasion to occur. Methods: Our model uses an organ-on-a-chip platform to assess bladder cancer cell invasiveness. This platform supports 3D culture of cells as well as pharmacokinetic studies. Our chips were designed to have a center channel to hold cells supported in Matrigel®, 2 adjacent side channels filled with Matrigel® only, and 2 outer media channels. Total cell and Matrigel® side channel length is 3 cm, channel height is 150 µm, and each channel width is 1 mm. Four bladder cancer cell lines (MB49, T24, J82, RT4) and cells isolated from a mouse bladder were used to develop our model. Cells were mixed with 1:1 media and Matrigel® and a total of 5,000 cells/µl introduced into the center channel. The 2 adjacent side channels were then filled with Matrigel®. The upper media channel was filled with 10% FBS cell culture media while the lower media channel was filled with 1% charcoal stripped cell culture media to thereby create a nutrient and growth factor gradient. Invasion of cells into the Matrigel®-containing side channels was assessed every 24 hours. Using the ImageXpress machine (Molecular Dynamics), we measured the furthest distance travelled by the cells. The chips were then fixed in 4% paraformaldehyde and stained using FITC-phalloidin and DAPI. Results: J82 and T24 cell lines, derived from patients with muscle invasive bladder cancer, and the MB49 cell line, a highly invasive mouse bladder cancer cell line, started invading into the Matrigel® side channels within 24 hours. T24, J82, and MB49 cells moved a maximum of 110, 87, and 170 µm by 48 hours, respectively. In contrast, RT4 cells, derived from a patient with non-invasive bladder cancer, took 48 hours to invade the Matrigel® channels and only invaded a maximum of 3 µm. Cells isolated from a mouse bladder did not invade into the Matrigel® side channels but survived within the chips for over 20 days. Staining of chips with DAPI and FITC-phalloidin allowed for clear visualization of cells and the actin cytoskeleton and demonstrated that immunostaining of cells within chips is possible. Conclusions: Our chip model allows for studying the invasiveness of bladder cancer cells and immunostaining experiments which can be used to assess the molecular mechanisms which drive invasion. It can also support the growth and assessment of primary cells. This model has the potential to improve bladder cancer patient treatments and outcomes by providing a physiologically relevant system to test new and existing drug combinations and to better understand the molecular mechanisms which drive bladder cancer cell invasion. Citation Format: Desiree Ewell, Nita Vue, Sakib Moinuddin, Tanoy Sarkar, Fakhrul Ahsan, Ruth L. Vinall. Development of a bladder cancer-on-a-chip assay to assess the invasive potential of bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB437.

Abstract 2657: Immune checkpoint therapy as a treatment option for non-muscle invasive bladder cancer

Abstract Bladder cancer (BCa) is one of the most common malignancies with more than 500,000 new cases diagnosed each year. Intravesical instillations of Bacillus Calmette-Guérin (BCG) is the gold-standard treatment for non-muscle invasive BCa (NMIBC) patients despite significant side effects and high rates of refractory disease and/or recurrence. Indeed, treatment failure occurs in ~40% of cases emphasizing the urgent need to evaluate alternative and/or complementary new therapies including immunotherapies. Recently, the use of immune checkpoint inhibitor (ICI) anti-PD-1 therapy has been approved for the treatment of BCG-unresponsive NMIBC patients. Here, we investigated whether targeting PD-1 or CTLA-4 can boost anti-tumor immunity in the orthotopic murine MB49 BCa model. To this purpose, 2.5 × 105 luciferase-expressing MB49 cells (MB49-Luc) have been instilled into the preconditioned bladder of female C57Bl/6 mice by intra-urethral catheterization. In vivo tumor growth has been monitored three times a week using the IVIS spectrum in vivo imaging system. All the inoculated mice showed a luciferase signal at day 3 post-bladder tumor inoculation, demonstrating 100% of tumor uptake. Based on the in vivo bioluminescence, a randomization has been performed before ICI treatment consisting of anti-CTLA-4 (10 μg/mouse; q3dx4, intraperitoneal) or anti-PD-1 (100 μg/mouse, q3dx4, intraperitoneal). Our data indicate that ICI treatments significantly decreased the in vivo bioluminescence, indicating reduced tumor growth. Furthermore, anti-CTLA-4 and anti-PD-1 administration resulted in a significant long term anti-tumor activity, with ~90% and ~70% overall survival of anti-CTLA-4 and anti-PD-1 treated mice, respectively, while isotype control mice displayed ~10% survival by day 74 post-MB49-Luc cells inoculation. To investigate the potential development of systemic adaptive anti-tumor immunity elicited by ICI treatment, remaining ICI-treated mice and newly onboarded non-treated control mice have been (re)-challenged with MB49 tumor cells subcutaneously (s.c.). While all the control mice showed tumor outgrowth and needed to be sacrificed due to humane endpoint by the day 32 post-s.c. challenge, rechallenged ICI-treated mice showed potent abscopal effect and displayed no tumor engraftment with 100% overall survival. Altogether, these findings demonstrate that systemic ICI immunotherapies such as PD-1 and CTLA-4 elicit potent anti-tumor activity and induce protective anti-tumor immune memory. ICI might present an alternative or a complementary treatment to BCG therapy for NMIBC patients. Citation Format: Sulayman Benmerzoug, Katie Louche, Thibault Angles, Nizar Serhan, Frederique Dol-Gleizes, Pascale Lejeune, Sophie Chabot. Immune checkpoint therapy as a treatment option for non-muscle invasive bladder cancer [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 2657.

Abstract 506: TRIP13 is a promising therapeutic target for bladder cancer

Abstract Bladder cancer (BCa) is the second most common genitourinary cancer. High recurrence rate, progression to MIBC and poor prognosis in metastatic disease are major challenges for this disease management. Therapeutic options for advanced stage BCa are very limited, hence developing agents to treat this disease progression are highly needed. Thyroid receptor-interacting protein 13 (TRIP13), a member of the AAA-ATPase family, regulates various cellular processes such as DNA repair and spindle assembly. TCGA data analysis showed significant overexpression of TRIP13 that correlates with disease stage and mortality. Recent studies have shown that TRIP13 amplification occurs in several types of human cancers, including BCa, and it regulates cell proliferation, tumor growth, and drug resistance. However, the role of TRIP13 in BCa and its potential as a therapeutic target remain elusive. Therefore, in the present study we performed a preclinical investigation of the TRIP13 role in BCa progression, underlying molecular mechanisms, and develop promising therapeutics against BCa. Initially we profiled TRIP13 expression in 10 different bladder cancer cell lines from a panel of human and rodent origin and found elevated protein expression in multiple BCa cell lines. TRIP13 levels were also significantly elevated in the BBN induced rat- and SV40T driven mouse- bladder tumor tissues compared to normal bladder tissues. Pharmacological inhibition of TRIP13 with a potent drug DCZ0415 significantly suppressed cell proliferation (IC50 ~7μM) in J82, NBT-II and MB49 cell lines and induced cell death. DCZ0415 treatment led to cell cycle arrest at G1 phase (P<0.001) in BCa cells. In mechanistic investigations on its effect on cell cycle progression by DCZ0415 treatment decreased the expression of CDK4, CDK6, Cyclin D1, and PCNA protein expression. TRIP13 inhibition also suppressed the migration and invasion potential of BCa cells. Annexin V and γ-H2AX staining suggested that DCZ0415 induced apoptosis in BCa cells and increased DNA damage. These findings were also complemented by an increase in cleaved caspase-3 and PARP proteins. We also observed modulation of cGAS-STING signaling pathway in BCa cells with DCZ0415 treatment, indicating potential for its combination with cGAS-STING stimulators. Importantly, we found that TRIP13 inhibition with DCZ0415 significantly suppressed the growth of 3D BCa models as well BBN-induced rat BCa tumoroids and spheroids. Overall, these results suggest that TRIP13 could be a potential therapeutic target for prevention of bladder cancer and warrants further validation in in vivo models (Funding supported by P30CA225520 and ACS-IRG grant). Citation Format: Surya Pratap Singh, Krishnendu Goswami, Adam S. Asch, Chinthalapally V. Rao, Venkateshwar Madka. TRIP13 is a promising therapeutic target for bladder cancer [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 506.

Mucoadhesive Thiolated Hyaluronic Acid/Pluronic F127 Nanogel Formation via Thiol-Maleimide Click Reaction for Intravesical Drug Delivery.

The development of nanocarriers to prolong the residence time and enhance the permeability of chemotherapeutic drugs on bladder mucosa is important in the postsurgery treatment of superficial bladder cancers (BCs). Here, the mucoadhesive HA-SH/PF127 nanogels composed of a temperature-sensitive Pluronic F127 (PF127) core and thiolated hyaluronic acid (HA-SH) shell were prepared by the emulsification/solvent evaporation method. The nanogels were constructed through the thiol-maleimide click reaction in the HA-SH aqueous side of the oil-water interface and self-oxidized cross-linking thiols between HA-SH. The HA-SH/PF127 nanogels prepared at different thiol-to-maleimide group molar ratios, water-to-oil volume ratios, and cross-linking reaction times were characterized regarding hydrodynamic diameter (Dh) and zeta potential (ζ), and the optimal formulation was obtained. The excellent mucoadhesive properties of the HA-SH/PF127 nanogels were evaluated by using the mucin particle method. Doxorubicin (DOX) was encapsulated in the PF127 core of DOX@HA-SH/PF127 nanogels with a high loading efficiency (87.5%) and sustained release from the nanogels in artificial urine. Ex vivo studies on porcine bladder mucosa showed that the DOX@HA-SH/PF127 nanogels enhanced the penetration of the DOX into the bladder mucosa without disrupting the mucus structure or the bladder tissue. A significant dose-dependent cytotoxic effect of DOX@HA-SH/PF127 nanogels on both T24 and MB49 cells was observed. The present study demonstrates that the mucoadhesive HA-SH/PF127 nanogels are a promising intravesical drug delivery system for superficial BC therapy.

Homogeneous Polyporus polysaccharide exerts anti-bladder cancer effects via autophagy induction

Context Polyporus polysaccharide (PPS), the leading bioactive ingredient extracted from Polyporus umbellatus (Pers.) Fr. (Polyporaceae), has been demonstrated to exert anti-bladder cancer and immunomodulatory functions in macrophages. Objective To explore the effects of homogeneous Polyporus polysaccharide (HPP) on the proliferation and autophagy of bladder cancer cells co‐cultured with macrophages. Materials and methods MB49 bladder cancer cells and RAW264.7 macrophages were co-cultured with or without HPP intervention (50, 100, or 200 μg/mL) for 24 h. The cell counting kit-8 (CCK-8) assay and 5-ethynyl-2″-deoxyuridine (EdU) staining evaluated MB49 cell proliferation. Monodansylcadaverine (MDC) staining and transmission electron microscopy (TEM) observed autophagosomes. Western blotting detected the expression levels of autophagy-related proteins and PI3K/Akt/mTOR pathway proteins. Results HPP inhibited the proliferation of MB49 cells co-cultured with RAW264.7 cells but not MB49 cells alone. HPP altered the expression of autophagy-related proteins and promoted the formation of autophagosomes in MB49 cells in the co-culture system. Autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) not only antagonized HPP-induced autophagy but also attenuated the inhibitory effects of HPP on MB49 cell proliferation in the co-culture system. HPP or RAW264.7 alone was not sufficient to induce autophagy in MB49 cells. In addition, HPP suppressed the protein expression of the PI3K/Akt/mTOR pathway in MB49 cells in the co-culture system. Discussion and conclusions HPP induced bladder cancer cell autophagy by regulating macrophages in the co-culture system, resulting in the inhibition of cancer cell proliferation. The PI3K/Akt/mTOR pathway was involved in HPP-induced autophagy in the co-culture system.

6]-Shogaol Induces Apoptosis of Murine Bladder Cancer Cells.

Bladder cancer is considered one of the most aggressive neoplasms due to its recurrence and progression profile, and even with the improvement in diagnosis and treatment methods, the mortality rate has not shown a declining trend in recent decades. From this perspective, the search and development of more effective and safer therapeutic alternatives are necessary. Phytochemicals are excellent sources of active principles with therapeutic potential. [6]-Shogaol is a phenolic compound extracted from the ginger rhizomes that has shown antitumor effects in a wide variety of cancer models. However, there is no record in the literature of studies reporting these effects in models of bladder cancer. Thus, this study aimed to investigate the in vitro cytotoxic and pro-apoptotic potential of [6]-Shogaol against murine bladder cancer urothelial cells (MB49). The cytotoxic effects of [6]-Shogaol on cell viability (MTT method), cell morphology (light microscopy), alteration of proliferative processes (clonogenic assay), oxidative stress pathway (levels of reactive oxygen species) and the induction of apoptotic events (flow cytometry and high-resolution epifluorescence imaging) were evaluated in murine urothelial bladder cancer cell lines (MB49), relative to non-tumor murine fibroblasts (L929). The results showed that [6]-Shogaol was able to induce concentration-dependent cytotoxic effects, which compromised cell viability, exhibiting an inhibitory concentration of 50% of cells (IC50) of 146.8 µM for MB49 tumor cells and 236.0 µM for L929 non-tumor fibroblasts. In addition to inhibiting and altering the proliferative processes if colony formation, it presented pro-apoptotic activity identified through a quantitative analysis and the observation of apoptotic phenotypes, events apparently mediated by the induction of nuclear fragmentation. The data presented suggest that [6]-Shogaol has a higher concentration-dependent cytotoxic and apoptosis-inducing potential in MB49 cells than in L929 fibroblasts. These results may contribute to the development of therapeutic alternatives for bladder cancer.

Exosomes derived from bladder epithelial cells infected with uropathogenic Escherichia coli increase the severity of urinary tract infections (UTIs) by impairing macrophage function.

Uropathogenic Escherichia coli (UPEC) is the primary causative agent of urinary tract infections (UTIs) in humans. Moreover, as one of the most common bacterial pathogens, UPEC imposes a substantial burden on healthcare systems worldwide. Epithelial cells and macrophages are two major components of the innate immune system, which play critical roles in defending the bladder against UPEC invasion. Yet, the routes of communication between these cells during UTI pathogenesis are still not fully understood. In the present study, we investigated the role of membrane-bound nanovesicles (exosomes) in the communication between bladder epithelial cells and macrophages during UPEC infection, using an array of techniques such as flow cytometry, miRNA profiling, RNA sequencing, and western blotting. Moreover, our in vitro findings were validated in a mouse model of UPEC-induced cystitis. We found that UPEC infection induced the bladder epithelial MB49 cell line to secrete large numbers of exosomes (MB49-U-Exo), which were efficiently absorbed by macrophages both in vivo and in vitro. Assimilation of MB49-U-Exo induced macrophages to produce proinflammatory cytokines, including tumor necrosis factor (TNF)α. Exposure of macrophages to MB49-U-Exo reduced their phagocytic activity (by downregulating the expression of phagocytosis-related genes) and increased their rate of apoptosis. Mechanistically, we showed that MB49-U-Exo were enriched in miR-18a-5p, which induced TNFα expression in macrophages by targeting PTEN and activating the MAPK/JNK signaling pathway. Moreover, administration of the exosome secretion inhibitor GW4869 or a TNFα-neutralizing antibody alleviated UPEC-mediated tissue damage in mice with UPEC-induced cystitis by reducing the bacterial burden of the bladder and dampening the associated inflammatory response. Collectively, these findings suggest that MB49-U-Exo regulate macrophage function in a way that exacerbates UPEC-mediated tissue impairment. Thus, targeting exosomal -release or TNFα signaling during UPEC infection may represent promising non-antibiotic strategies for treating UTIs.

The transcription factor sex-determining region Y-box 2 (SOX2) in bladder cancer.

Sex-determining region Y-box 2 (SOX2) is a transcription factor with a central role in embryologic development. SOX2 is also an oncogene in several cancer types. Prior work by our group has shown SOX2 activity associates with cell cycle dysregulation in early-stage bladder cancer. The present study was thus undertaken to broadly investigate SOX2 in bladder cancer, with emphasis on associations with tumor stage, clinical outcomes, and tumorigenicity. Gene expression was quantified by immunohistochemistry in an established tissue microarray (n=303 cystectomy specimens, all stages) and whole tissue sections of noninvasive papillary urothelial carcinoma (n=25). Gene expression by RNA sequencing was evaluated in non-muscle invasive and muscle-invasive cohorts from publicly available repositories. By immunohistochemistry, SOX2 was expressed in 40% of whole tissue sections of noninvasive papillary carcinoma, which correlated with SOX2 expression by RNA sequencing (r=0.6, P=0.001, Spearman correlation). Expression tended to be focal (median H-score =6). SOX2 was expressed in only 9% of TMA cases, consistent with focal expression. SOX2 expression was substantially higher in muscle-invasive compared with noninvasive papillary urothelial carcinoma by RNA sequencing (P<0.001, Wilcoxon rank sum test). SOX2 expression associated with stage progression in lamina-propria invasive cancers (hazard ratio =2, P=0.05, Cox model, binary, RNA sequencing) but not noninvasive papillary cancers (P=0.5, Cox model, binary, RNA sequencing). SOX2 expression did not associate with overall survival in muscle-invasive carcinoma. Activity of SOX2 in bladder cancer was tested in vivo using murine allografts created with MB49 cells that express human SOX2 (MB49-SOX). MB49-SOX allografts expressed this protein focally by immunohistochemistry, much like human tumors. Compared with controls, MB49 allografts demonstrated larger tumor size (P=0.03, Wilcoxon rank sum test) and higher tumor burden in mesenteric metastases (P=0.009, Wilcoxon rank sum test). Though SOX2 expression is focal within tumors, it may drive tumorigenesis, increase growth rate, and promote aggressive features of bladder cancer, particularly stage progression of early-stage disease.

Abstract B051: GSTT2 modulates the bladder tumor environment and response to BCG Immunotherapy

Abstract The purpose of this study was to elucidate the impact of Glutathione -S-transferase Theta 2 (GSTT2) loss on the response of bladder tumors to Mycobacterium Bovis, Bacillus Calmette Guerin (BCG) immunotherapy. GSTT2 gene expression is controlled by a promoter deletion that occurs frequently in 25-29% of Asians and 41% of Caucasians. The GSTT2 promoter deletion correlated with better response to BCG therapy in a retrospective analysis of patients who received less BCG therapy. To understand this response, we generated GSTT2 knockout mice to elucidate the role of GSTT2 in BCG immunotherapy. Female C57BL/6J mice from GSTT2 wildtype (WT) and KO backgrounds were orthotopically implanted with MB49-PSA cells (murine MB49 cells secreting human PSA). To view the impact of GSTT2 loss on BCG immunotherapy, mice were treated with only 4 instillations of BCG Tokyo. This therapeutic schedule would not result in a high cure rate and but was chosen as we expected it to provide insights into the anti-tumor response. Mice were treated weekly with intravesical BCG Tokyo instillations and tumor growth was monitored by urinary PSA secretion. Final tumor presence was confirmed by real-time PCR analysis of PSA gene expression. One day after the last instillation the bladders (n=7) were harvested for single cell RNA sequencing (Parse Biosciences). Cell clusters were identified by their gene expression profiles. The response to BCG in terms of the cure rate was similar in KO and WT mice (cure rate of 18-20%) but several genes were differentially expressed. Genes with a Log2FC value of +1.5 in KO mice (Peak1, Rpph1, Gphn and Gm19551) and -1.5 in KO (Rora, Tshz2, Sh3gl2, Ankfn1, Rbms3, Naaladl2, Prkn, Mecom, Fmo5, Saa3, Tmprss2, C3, Gm10800 and Gm10801) relative to WT mice were examined. Based on the literature, these genes either block tumor progression (Rora, Tshz2, Sh3gl2 and Gphn); promote tumor progression (Ankfn1, Rbms3, Naaladl2, Prkn, Mecom, Fmo5, Tmprss2, Saa3, C3, Peak1, Rpph1) or modulate immune cells (C3, Saa3 and Rpph1). The majority of these genes were not previously associated with bladder cancer. Interrogation of the TCGA database showed that high expression of Gphn, Peak1 and GSTT2 correlated with poorer outcomes in bladder cancer patients in terms of disease-free survival (HR=1.9 log rank p=0.00038). While expression of the genes downregulated in KO mice and GSTT2 resulted in better outcomes with lower expression of all the genes (HR=1.4, log rank p=0.042). This data indicates that the loss of GSTT2 expression impacts the response of tumors to BCG immunotherapy, primarily by modulating the expression of genes associated with cancer progression and the expression of genes in multiple cell types. Given the common loss of GSTT2 expression in man this gene may be a parameter that should be evaluated in further detail in patients receiving BCG immunotherapy. Citation Format: Ratha Mahendran, Mugdha Vijay Patwardhan, Qin Kane Toh, Edmund Chiong. GSTT2 modulates the bladder tumor environment and response to BCG Immunotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B051.

Dysfunction of sinus macrophages in tumor-bearing host induces resistance to immunotherapy.

Sinus macrophages in draining lymph nodes (DLNs) are involved in anti-tumor immune reactions. CD169 (Sialoadhesin, Siglec-1) is expressed on sinus macrophages and is considered a surrogate marker for the immunostimulatory phenotype of macrophages. In this study, the significance of sinus macrophages in immunotherapy was evaluated using mouse models. Treatment with anti-programmed death-ligand 1 (PD-L1) antibody suppressed the subcutaneous tumor growth of MC38 and E0771 cells but was not effective against MB49 and LLC tumors. Decreased cytotoxic T-lymphocyte (CTL) infiltration in tumor tissues and CD169 expression in sinus macrophages were observed in MB49 and LLC cells compared to corresponding parameters in MC38 and E0771 cells. The anti-tumor effects of the anti-PD-L1 antibody on MC38 and E0771 cells were abolished when sinus macrophages in DLNs were depleted, suggesting that sinus macrophages are involved in the therapeutic effect of the anti-PD-L1 antibody. Naringin activated sinus macrophages. Naringin inhibited tumor growth in MB49- and LLC-bearing mice but did not affect that in MC38- and E0771-bearing mice. The infiltration of CTLs in tumor tissues and their activation were increased by naringin, and this effect was impaired when sinus macrophages were depleted. Combination therapy with naringin and anti-PD-L1 antibody suppressed MB49 tumor growth. In conclusion, CD169-positive sinus macrophages in DLNs are critical for anti-tumor immune responses, and naringin suppresses tumor growth by activating CD169-positive sinus macrophages and anti-tumor CTL responses. The activation status of sinus macrophages has been suggested to differ among tumor models, and this should be investigated in future studies.

Ultrasound Molecular Imaging of Bladder Cancer via Extradomain B Fibronectin-Targeted Biosynthetic GVs.

Ultrasound molecular imaging (UMI) has proven promising to diagnose the onset and progression of diseases such as angiogenesis, inflammation, and thrombosis. However, microbubble-based acoustic probes are confined to intravascular targets due to their relatively large particle size, greatly reducing the application value of UMI, especially for extravascular targets. Extradomain B fibronectin (ED-B FN) is an important glycoprotein associated with tumor genesis and development and highly expressed in many types of tumors. Here, we developed a gas vesicles (GVs)-based nanoscale acoustic probe (ZD2-GVs) through conjugating ZD2 peptides which can specially target to ED-B FN to the biosynthetic GVs. ED-B FN expression was evaluated in normal liver and tumor tissues with immunofluorescence and Western blot. ZD2-GVs were prepared by conjugating ZD2 to the surface of GVs by amide reaction. The inverted microscope was used to analyze the targeted binding capacity of ZD2-GVs to MB49 cells (bladder cancer cell line). The contrast-enhanced imaging features of GVs, non-targeted control GVs (CTR-GVs), and targeted GVs (ZD2-GVs) were compared in three MB49 tumor mice. The penetration ability of ZD2-GVs in tumor tissues was assessed by fluorescence immunohistochemistry. The biosafety of GVs was evaluated by CCK8, blood biochemistry, and HE staining. Strong ED-B FN expression was observed in tumor tissues while little expression in normal liver tissues. The resulting ZD2-GVs had only 267.73 ± 2.86 nm particle size and exhibited excellent binding capability to the MB49 tumor cells. The in vivo UMI experiments showed that ZD2-GVs produced stronger and longer retention in the BC tumors than that of the non-targeted CTR-GVs and GVs. Fluorescence immunohistochemistry confirmed that ZD2-GVs could penetrate the tumor vascular into the interstitial space of the tumors. Biosafety analysis revealed there was no significant cytotoxicity to these tested mice. Thus, ZD2-GVs can function as a potential UMI probe for the early diagnosis of bladder cancer.

MiR-3960 inhibits bladder cancer progression via targeting of DEXI

PurposeMicroRNAs (miRNAs) are dominant cargo in exosomes and act as master regulators of cell function, inhibiting mRNA translation and affecting gene silencing. Some aspects of tissue-specific miRNA transport in bladder cancer (BC) and its role in cancer progression are not fully understood. Materials and methodsA microarray was used to identify miRNAs in mouse bladder carcinoma cell line MB49 exosomes. Real-time reverse transcription polymerase chain reaction was used to examine the expression of miRNAs in BC and healthy donor serum. Western blotting and immunohistochemical staining were used to examine the expression of dexamethasone-induced protein (DEXI) in patients with BC. CRISPR-Cas 9 was used to knock out Dexi in MB49, and flow cytometry was performed to test cell proliferation ability and apoptosis under chemotherapy. Human BC organoid culture, miR-3960 transfection, and 293T-exosome-loaded miR-3960 delivery were used to analyze the effect of miR-3960 on BC progression. ResultsThe results showed that miR-3960 levels in BC tissue were positively correlated with patient survival time. Dexi was a major target of miR-3960. Dexi knockout inhibited MB49 cell proliferation and promoted cisplatin- and gemcitabine-induced apoptosis. Transfection of miR-3960 mimic inhibited DEXI expression and organoid growth. In parallel, 293T-exosome-loaded miR-3960 delivery and Dexi knockout significantly inhibited subcutaneous growth of MB49 cells in vivo. ConclusionOur results demonstrate the potential role of miR-3960-mediated inhibition of DEXI as a therapeutic strategy against BC.

Irradiation plus myeloid-derived suppressor cell-targeted therapy for overcoming treatment resistance in immunologically cold urothelial carcinoma

BackgroundRadiotherapy (RT) has recently been highlighted as a partner of immune checkpoint inhibitors. The advantages of RT include activation of lymphocytes while it potentially recruits immunosuppressive cells, such as myeloid-derived suppressor cells (MDSCs). This study aimed to investigate the mechanism of overcoming treatment resistance in immunologically cold tumours by combining RT and MDSC-targeted therapy.MethodsThe abscopal effects of irradiation were evaluated using MB49 and cisplatin-resistant MB49R mouse bladder cancer cells, with a focus on the frequency of immune cells and programmed cell death-ligand 1 (PD-L1) expression in a xenograft model.ResultsMB49R was immunologically cold compared to parental MB49 as indicated by the fewer CD8+ T cells and lower PD-L1 expression. Polymorphonuclear MDSCs increased in both MB49 and MB49R abscopal tumours, whereas the infiltration of CD8+ T cells increased only in MB49 but not in MB49R tumours. Interestingly, PD-L1 expression was not elevated in abscopal tumours. Finally, blocking MDSC in combination with RT remarkably reduced the growth of both MB49 and MB49R abscopal tumours regardless of the changes in the frequency of infiltrating CD8+ T cells.ConclusionsThe combination of RT and MDSC-targeted therapy could overcome treatment resistance in immunologically cold tumours.

Abstract 3282: Regorafenib enhance PD-L1 expression via ATM activation and evaluate the therapeutic efficacy of combining PD-L1 antibody on bladder cancer

Abstract Bladder cancer is one of the most common cancer in urinary system in Taiwan. According to Ministry of Health and Welfare’s research, bladder cancer plays 15th leading causes of death in Taiwan in 2020. Recently immune checkpoint inhibitor (ICI) therapy show several benefits on preventing T cell exhaustion, accumulating evidences showed ICI therapy still remain some limitation, including unable to regulated immunosuppressor cells and tumor microenvironment (TME) related elements which might lead to ICI therapy dysfunction. Recently, study report that DNA damage may activate the innate immune response and improve ICI efficacy. Thus, regulating DNA damage or illustrate the role of DNA damage related factor may bring the twilight to immunotherapy. To identify the possible combination methods with ICIs, we used MB49 cells and animal model to test the combination property of regorafenib (a multiple receptor tyrosine kinase inhibitor) and 10F.9G2 (a PD-L1 monoclonal antibody). Our research illustrated the mechanism between regorafenib and PD-L1, we proved that regorafenib-induced regulated PD-L1 is associated with ATM activation. TUENL assay showed regorafenib are able to activate ATM by causing DNA break and thus enhance the expression of PD-L1 on tumor surface. This effect could be an important step to open the interaction potential of PD-L1 antibodies and thus enhance its efficacy. Through the interaction analysis of miRNA targeting gene (RNA22), we found that IDO1 mRNA could specifically interact with endogenous miR-124. Here, the propose mechanism in our research is that regorafenib may downregulate IDO1 expression via increasing of endogenous miR-124 expression. In animal model, the combination therapy showed extraordinary therapeutic effect, not only dramatically decreased tumor volume, but also upregulating the positive TME components, such as CD8+T cells and M1 macrophage. Though regorafenib may induce PD-L1 expression, regorafenib still showed several therapeutic benefits, including downregulating immunosuppressive factors, such as IDO1, VEGF, and suppressing the accumulation of regulatory T cells, myeloid-derived suppressor cells and also repolarizing macrophage from M2-to-M1⋯etc. In conclusion, this study points out the combination therapy of regorafenib and anti-PD-L1 has present several benefits to bladder cancer patients and is associated with miR-124/ATM signaling. Citation Format: Kai-Jen Pai, I-Tsang Chiang, Fei-Ting Hsu, Jai-Jen Tsai. Regorafenib enhance PD-L1 expression via ATM activation and evaluate the therapeutic efficacy of combining PD-L1 antibody on bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3282.

PD25-10 A NEWLY DISCOVERED COLLECTIVE MIGRATION OF UROTHELIAL CELLS: A POTENTIAL DEFENSE MECHANISM AGAINST BLADDER CANCER DEVELOPMENT

You have accessJournal of UrologyCME1 Apr 2023PD25-10 A NEWLY DISCOVERED COLLECTIVE MIGRATION OF UROTHELIAL CELLS: A POTENTIAL DEFENSE MECHANISM AGAINST BLADDER CANCER DEVELOPMENT Zhang Ning, Takeshi Sano, Ryosuke Ikeuchi, Hideaki Takada, Kenji Nakamura, Toru Sakatani, Akihiro Hamada, Yuki Kita, Michiyuki Matsuda, and Takashi Kobayashi Zhang NingZhang Ning More articles by this author , Takeshi SanoTakeshi Sano More articles by this author , Ryosuke IkeuchiRyosuke Ikeuchi More articles by this author , Hideaki TakadaHideaki Takada More articles by this author , Kenji NakamuraKenji Nakamura More articles by this author , Toru SakataniToru Sakatani More articles by this author , Akihiro HamadaAkihiro Hamada More articles by this author , Yuki KitaYuki Kita More articles by this author , Michiyuki MatsudaMichiyuki Matsuda More articles by this author , and Takashi KobayashiTakashi Kobayashi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003303.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Imaging of live animals at microscopic resolution (intravital imaging) found that bladder tumor (BT) development caused a unique collective migration of urothelial cells in a mouse orthotopic bladder cancer model. This study aimed to investigate the relationship between this newly discovered migration and BT growth. METHODS: MB49 cells (a mouse bladder cancer cell line) were labeled with a red fluorescent protein to distinguish them from urothelial cells of C57BL/6 mice expressing a cyan fluorescent protein. Labeled MB49 cells were then injected into mouse bladders pretreated with poly-l-lysine (PLL) to initiate BTs. While mice underwent general anesthesia using isoflurane, two-photon excitation microscopy, which enables deep tissue imaging, was performed to visualize the urothelium and the attached MB49 cells. Dasatinib, a Src/focal adhesion kinase signaling inhibitor, was intravenously or orally administered. RESULTS: A collective migration of urothelial cells was observed four days after intravesical injection of 1×106 MB49 cells (Figure 1). Pretreatment with PLL alone did not cause this migration. Intravenous and oral administration of dasatinib each remarkably retarded MB49-induced migration (Figure 2a). Surprisingly, inhibition of this migration by daily oral administration of dasatinib starting 24 hours after MB49 injection significantly enhanced BT growth despite dasatinib generally inhibiting cell-growth (Figure 2b). In a subcutaneous xenograft mouse model using MB49 cells, dasatinib slightly suppressed tumor growth. Intriguingly, transfer of urine from which cells had been removed by filtration from an MB49-injected bladder to another C57BL/6 mouse bladder caused the urothelial cell migration (Figure 2c). CONCLUSIONS: Cancer cell injection into the bladder caused a unique collective migration of urothelial cells. Inhibition of this migration enhances BT growth, suggesting that this migration may play a defensive role against BT development. We are currently working to identify the molecule(s) that trigger the migration. Enhancement of this migration may be a novel strategy for preventing BT recurrence after transurethral resection of BTs. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e733 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Zhang Ning More articles by this author Takeshi Sano More articles by this author Ryosuke Ikeuchi More articles by this author Hideaki Takada More articles by this author Kenji Nakamura More articles by this author Toru Sakatani More articles by this author Akihiro Hamada More articles by this author Yuki Kita More articles by this author Michiyuki Matsuda More articles by this author Takashi Kobayashi More articles by this author Expand All Advertisement PDF downloadLoading ...

PD25-06 COMBINATION TREATMENT WITH INTRAVESICAL INTERFERON GENE THERAPY AND PAN-ERBB RECEPTOR FAMILY BLOCKER IMPROVES SURVIVAL IN MICE WITH BLADDER CANCER

You have accessJournal of UrologyCME1 Apr 2023PD25-06 COMBINATION TREATMENT WITH INTRAVESICAL INTERFERON GENE THERAPY AND PAN-ERBB RECEPTOR FAMILY BLOCKER IMPROVES SURVIVAL IN MICE WITH BLADDER CANCER Akshay Sood, Jan Rudzinski, Alberto Martini, Come Tholomier, Sharada Mokkapati, and Colin Dinney Akshay SoodAkshay Sood More articles by this author , Jan RudzinskiJan Rudzinski More articles by this author , Alberto MartiniAlberto Martini More articles by this author , Come TholomierCome Tholomier More articles by this author , Sharada MokkapatiSharada Mokkapati More articles by this author , and Colin DinneyColin Dinney More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003303.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Recently, intravesical interferon (IFNα) gene therapy has demonstrated success in treatment of BCG-unresponsive NMIBC in a large, randomized, phase-III trial. While the results have been promising, there is room for improvement. One strategy to improve treatment efficacy and durability may be to identify actionable resistance mechanisms and employ targeted combination therapies. METHODS: We performed end-tumor RNA-seq analysis of MB49 mice tumors treated with IFNα gene therapy to identify potential mechanisms of therapy resistance – ErbB pathway was discovered as a potential target (Figure 1a). We therefore tested combination treatment with ErbB pathway blocker and IFNα in MB49 cells using lentiviral IFNα (LV-IFNα) with/without Afatinib in vitro. Next, in vivo studies were conducted in the MB49 orthotopic mice bladder cancer model: mice were randomized in to 5 treatment groups (n=10 each), saline (Ctrl), LV-Ctrl, Afatinib monotherapy, LV-IFNα monotherapy, and the experimental LV-IFNα + Afatinib combination therapy. Overall survival was assessed. RESULTS: Combination therapy significantly reduced the viability of MB49 cells in vitro compared to any of the other treatment conditions (mean relative ATPase activity for combination treatment at 72 h 4%, compared to 100%, 26%, and 28% for Ctrl, LV-IFNα, and Afatinib, p<0.001; Figure 1b). This effect on cell viability appeared to be driven by a combination of additive cytostatic and cytolytic effects (Figure 1c-d). The combination treatment also decreased cell migration (mean migrated cells/10x field on Boyden chamber assay: 92.3 for combination therapy and 631.0, 600.4, and 270.3 for Ctrl, LV-IFNα, and Afatinib, p<0.001; Figure 1e). Finally, in vivo studies demonstrated improved OS with combination therapy (median OS 49 d in the combination group vs 15, 29, and 26 d in the Ctrl, LV-IFNα, and Afatinib groups, Log-rank p<0.001; Figure 1f). No mice in the combination group died of drug toxicity. CONCLUSIONS: Our preliminary investigations suggest that ErbB pathway may be a clinically meaningful resistance mechanism, which when targeted alongside IFNα gene therapy may improve overall treatment efficacy. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e731 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akshay Sood More articles by this author Jan Rudzinski More articles by this author Alberto Martini More articles by this author Come Tholomier More articles by this author Sharada Mokkapati More articles by this author Colin Dinney More articles by this author Expand All Advertisement PDF downloadLoading ...

PD25-09 TUMOR ASSOCIATED ANTIGEN VACCINE ENHANCES ANTI-TUMOR EFFECTS OF IMMUNE CHECKPOINT INHIBITORS AGAINST REFRACTORY CANCERS

You have accessJournal of UrologyCME1 Apr 2023PD25-09 TUMOR ASSOCIATED ANTIGEN VACCINE ENHANCES ANTI-TUMOR EFFECTS OF IMMUNE CHECKPOINT INHIBITORS AGAINST REFRACTORY CANCERS Shohei Ueda, Miho Ushijima, Atsushi Irie, Yasuharu Nishimura, and Masatoshi Eto Shohei UedaShohei Ueda More articles by this author , Miho UshijimaMiho Ushijima More articles by this author , Atsushi IrieAtsushi Irie More articles by this author , Yasuharu NishimuraYasuharu Nishimura More articles by this author , and Masatoshi EtoMasatoshi Eto More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003303.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Progressive and chemoresistant bladder cancer is problematic because of the poor prognosis. Immune checkpoint blockade (ICB) has exhibited drastic anti-tumor efficacy in some patients, but the response rates were limited. Therefore, to improve efficacy of immunotherapy for refractory cancer, we have tried to develop a combination immunotherapy of tumor antigen vaccine and ICB in tumor-bearing mouse models. METHODS: We identified genes highly expressed in tumors but not in normal tissues by comparing RNA-seq data of mouse bladder cancer MB49, cisplatin-resistant MB49-CR, and lymphoma RMA cell lines, all of which are refractory to ICB, with datasets of 26 normal mouse tissues. Subsequently tumor-associated antigens-derived peptides predicted to have high binding affinities to MHC-I and -II molecules were identified by prediction algorithms. We also selected immunogenic and cancer-specific mutated Neo-antigenic peptides. MB49, MB49-CR, or RMA bearing mice were treated with these peptides and ICB. Finally, we investigated tumor infiltrating immune cells by flow cytometry, T cell receptor (TCR) repertoire analysis and RNA-seq of inoculated tumors. RESULTS: In 50-80% of mice pre-immunized with identified peptides, growths of MB49 and RMA inoculated s.c. were prevented and these immunized mice rejected re-transplanted tumors. Furthermore, combination therapy of these peptide vaccines and ICB synergistically inhibited tumor-growth of MB49-CR, MB49 and RMA indicating that the combination immunotherapy enhances therapeutic efficacy to treat refractory cancers. We also observed combination therapy with MHC-I antigen vaccination and ICB synergistically increased limited CD8+ TCR-alpha and beta genes in tumors, suggesting increased antigen-specific tumor-infiltrating CD8+ T cells. Furthermore, combination therapy with MHC-I/II antigen vaccine and ICB synergistically induced drastic changes of various types of tumor-infiltrating immune cells such as CD8+ T cells, CD4+ type 1 helper T cells, regulatory CD4+ T cells and natural killer cells. CONCLUSIONS: These results suggest that the combination immunotherapy described above shapes tumor-infiltrating immune cells and enhances therapeutic efficacy to treat refractory cancer. Source of Funding: This work was supported by KAKENHI. © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e732 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shohei Ueda More articles by this author Miho Ushijima More articles by this author Atsushi Irie More articles by this author Yasuharu Nishimura More articles by this author Masatoshi Eto More articles by this author Expand All Advertisement PDF downloadLoading ...

Bioengineered bacterial outer membrane vesicles encapsulated Polybia-mastoparan I fusion peptide as a promising nanoplatform for bladder cancer immune-modulatory chemotherapy.

Nanosized bacterial outer membrane vesicles (OMVs) secreted by Gram-negative bacteria have emerged as a novel antitumor nanomedicine reagent due to their immunostimulatory properties. The encapsulated bacterial composition in OMVs can be edited via manipulating bioengineering technology on paternal bacteria, allowing us to design an ingenious antitumor platform by loading the Polybia-mastoparan I (MPI) fusion peptide into OMVs. OMVs containing the MPI fusion peptide were obtained from bioengineered Escherichia coli transformed with recombinant plasmid. The antitumor efficacy of bioengineered OMVs in vitro was verified by performing cell viability and wound-healing and apoptosis assays using MB49 and UMUC3 cells, respectively. Subcutaneous MB49 tumor-bearing mice were involved to investigate the tumor inhibition ability of bioengineered OMVs. Moreover, the activated immune response in tumor and the biosafety were also evaluated in detail. The resulting OMVs had the successful encapsulation of MPI fusion peptides and were subjected to physical characterization for morphology, size, and zeta potential. Cell viabilities of bladder cancer cells including MB49 and UMUC3 rather than a non-carcinomatous cell line (bEnd.3) were decreased when incubated with bioengineered OMVs. In addition, bioengineered OMVs restrained migration and induced apoptosis of bladder cancer cells. With intratumor injection of bioengineered OMVs, growths of subcutaneous MB49 tumors were significantly restricted. The inherent immunostimulation of OMVs was demonstrated to trigger maturation of dendritic cells (DCs), recruitment of macrophages, and infiltration of cytotoxic T lymphocytes (CTLs), resulting in the increased secretion of pro-inflammatory cytokines (IL-6, TNF-α, and IFN-γ). Meanwhile, several lines of evidence also indicated that bioengineered OMVs had satisfactory biosafety. Bioengineered OMVs fabricated in the present study were characterized by strong bladder cancer suppression and great biocompatibility, providing a new avenue for clinical bladder cancer therapy.

Injectable Thermosensitive Nanocomposites Based on Poly(N-vinylcaprolactam) and Silica Particles for Localized Release of Hydrophilic and Hydrophobic Drugs.

The systemic delivery of drugs employed by conventional methods has shown to be less effective than a localized delivery system. Many drugs have the effectiveness reduced by fast clearance, increasing the amount required for an efficient treatment. One way to overcome this drawback is through the use of thermoresponsive polymers that undergo a sol-gel transition at physiological temperature, allowing their injection directly in the desired site. In this work, thermosensitive nanocomposites based on poly(N-vinylcaprolactam) and silica particles with 80 and 330 nm were synthesized to be employed as delivery systems for hydrophobic (naringin) and hydrophilic (doxorubicin hydrochloride) drugs. The insertion of SiO2 increased the rheological properties of the nanocomposite at 37 °C, which helps to prevent its diffusion away from the site of injection. The synthesized materials were also able to control the drug release for a period of 7 days under physiological conditions. Due to its higher hydrophobicity and better interaction with the PNVCL matrix, naringin presented a more controlled release. The Korsmeyer-Peppas model indicated different release mechanisms for each drug. At last, a preliminary in vitro study of DOX-loaded nanocomposites cultured with L929 and MB49 cells showed negligible toxic effects on healthy cells and better efficient inhibition of carcinoma cells.