Murine Bladder Cancer Cell Research Articles

PD-1/PD-L1 Immune Checkpoint Inhibition with Radiation in Bladder Cancer: In Situ and Abscopal Effects.

The combination of radiation with immune checkpoint inhibitors was reported in some cancers to have synergic effects both locally and distally. Our aim was to assess this combined therapy on both radiated and nonradiated bladder tumors and to characterize the immune landscape within the tumor microenvironment. Murine bladder cancer cells (MB49) were injected subcutaneously in both flanks of C57BL/6 mice. Mice were randomly assigned to the following treatments: placebo, anti-PD-L1 (four intraperitoneal injections over 2 weeks), radiation to right flank (10 Gy in two fractions), or radiation+anti-PD-L1. Tumor digestion, flow cytometry, and qPCR were performed. Log-rank analysis was used for statistical significance. Radiation+anti-PD-L1 group demonstrated statistically significant slower tumor growth rate both in the radiated and nonirradiated tumors (P < 0.001). Survival curves demonstrated superior survival in the combination group compared with each treatment alone (P = 0.02). Flow cytometry showed increased infiltration of immunosuppressive cells as well as CTL in the radiation and combination groups (P = 0.04). Ratio of immunosuppressive cells to CTL shifted in favor of cytotoxic activity in the combination arm (P < 0.001). The qPCR analysis revealed downregulation of immunosuppressive genes (CCL22, IL22, and IL13), as well as upregulation of markers of CTL activation (CXCL9, GZMA, and GZMB) within both the radiated and distant tumors within the combination group. Combining radiation with immune checkpoint inhibitor provided better response in the radiated tumors and also the distant tumors along with a shift within the tumor microenvironment favoring cytotoxic activity. These findings demonstrate a possible abscopal effect in urothelial carcinoma with combination therapy.

Development And In Vitro Characterization Of Bladder Tumor Cell Targeted Lipid-Coated Polyplex For Dual Delivery Of Plasmids And Small Molecules.

BackgroundBladder cancer is the fourth most common cancer in men and eleventh most common in women. Combination therapy using a gene and chemotherapeutic drug is a potentially useful strategy for treating bladder cancer in cases where a synergistic benefit can be achieved successfully. This approach relies on developing drug combinations using carrier systems that can load both hydrophilic genes and hydrophobic drugs. Ideally, the formulation for carrier system should be free of traditional high shear techniques such as sonication and extrusion to reduce shear-induced nucleic acid strand breakage. Moreover, the system should be able to protect the nucleic acid from enzymatic attack and deliver it specifically to the tumor site.Materials and methodsA dual payload carrier system that was formulated using a simple flow mixing technique to complex anionic plasmid (EGFP-NLS) using a cationic polymer (CD-PEI2.5kD) followed by coating of the polyplex using lipid membranes. The resulting lipid-coated polyplex (LCP) formulations are targeted to bladder cancer cells by employing a bacterial adhesive peptide sequence, RWFV, that targets the LCP to the tumor stroma for efficiently delivering reporter plasmid, EGFP-NLS and a model small molecule drug, pyrene, to the cancer cells.ResultsEncapsulation efficiency of the peptide targeted carrier for the plasmid was 50% ± 0.4% and for pyrene it was 16% ± 0.4%. The ability of the targeted LCP to transfect murine bladder cancer cells was 4-fold higher than LCP bearing a scrambled peptide sequence. Fluorescence of cells due to pyrene delivery was highest after 4 hrs using targeted LCP. Finally, we loaded the peptide targeted LCP with anti-cancer agent, curcumin. The targeted formulation of curcumin resulted in only 45% viable cancer cells at a concentration of 5 µg/mL, whereas the empty and non-targeted formulations did not result any significant cell death.ConclusionThese results demonstrate the specificity of the targeting peptide sequence in engaging tumor cells and the utility of the developed carrier platform to deliver a dual payload to bladder tumor cells.

Targeted Elastin-like Fusion Proteins for Enhanced Imaging and Treatment of Non-Muscle Invasive Bladder Cancer

Background and Hypothesis: A recent study estimates 17,670 individuals will die from bladder cancer in the United States in 2019. With current imaging and treatment technology, bladder cancer is associated with high recurrence rates making it the highest lifetime treatment cost per patient. High expense is primarily due to the profound heterogeneity associated with bladder cancer necessitating repeated intervention. Traditional treatment of non-muscle invasive bladder (NMIBC) cancer following intravesical tumor resection often includes chemotherapy or immunostimulatory therapy via Bacillus Calmette-Guerin (BCG) infusions. Both lack specificity for cancerous tissue, leading to sub-optimal prognosis and unnecessary discomfort for patients. Furthermore, bladder cancer detection is limited in its precision. Methods such as CT/MRI urography and cystoscopies are unable to accurately detect tumors under 2 centimeters in diameter. Blue light cystoscopy, a gold standard detection method, is unable to accurately differentiate tumors from inflamed tissue. This leads to issues detecting aggressive carcinoma in situ. The goal of this project is to enhance tumor detection by fusing various targeting ligands to elastin-like peptides (ELP).&#x0D; Experimental Design or Project Methods: Using synthetic biology approaches, a library of ELP fusions were made and purified using a simplified organic extraction method. (Biomaterials Science 2018, 6 (4), 863-876) This resulted in exceptionally pure protein within a matter of hours. Contrast agents were covalently attached, and performance testing was done in murine bladder cancer cells.&#x0D; Results: Novel ELP fusions were successfully produced and sequences verified. Protein expression and purification yielded active targeted constructs which showed enhanced binding efficiency over non-targeted constructs. These ELP fusion proteins are effective transporters for rapid and preferential cargo entry into bladder cancer tissue.&#x0D; Conclusion and Potential Impact: Chemotherapeutic, immunostimulatory, and tumor imaging cargo are all bright candidates for targeted ELP fusion technology. Targeted ELP delivery may be a superior tool key to improving tumor visualization and treatment, leading to improved comfort and prognosis in patients with NMIBC.&#x0D;

Abstract 3743: Neutrophil extracellular traps and their implication with radioresistance in muscle invasive bladder cancer

Abstract PURPOSE: Radiotherapy modifies diverse components of the tumor microenvironment and inflammation plays a pivotal role in modulating radiation responsiveness of tumors. Neutrophils are one of the first-line responders during the acute phase of inflammation and are increasingly being recognized as drivers of tumor progression. One mechanism by which neutrophils play a role in tumor progression is through the formation of neutrophil extracellular traps (NETs). NETs are web-like structures expelled by the neutrophil composed of DNA studded with various proteins. Initially, this was described as a mechanism of antimicrobial defense but lately NETs have been associated with a variety of adverse effects, such as pathogenesis of autoimmune diseases, surgical stress, tumor progression, and metastasis. Recent studies show that the protein High Mobility Group Box-1 (HMGB1), a key player in radioresistance can in fact promote NETs. Importantly, the impact of NETs has not yet been explored in the context of radiation, so we sought to explore this further. METHODS: In vitro: a) Human neutrophils isolated from healthy donors were stimulated with 50ng of rHMGB1 for 4 hours. NETs were quantified through Sytox green fluorescence. b) Neutrophils were co-cultured with irradiated or non-irradiated conditioned media from UM-UC3 human bladder cancer cell line in combination with glycyrrhizin (GLZ), a natural inhibitor of HMGB1. In vivo: Murine bladder cancer cell line (MB49) was subcutaneously implanted into flanks of C57BL/6 and NETosis deficient PAD4-/- mice. Tumors were irradiated (2x5Gy) using the XRAD Smart Irradiator. Intraperitoneal injections of GLZ were used to modulate HMGB1 and intramuscular injections of DNAse were used to deplete NETs. Tumor volumes were measured using a digital caliper till endpoint. RESULTS: Our in vitro results demonstrate incubation of neutrophils with 50ng rHMGB1 significantly induced NETs formation compared to controls (p&amp;lt;0.0001) and this was reversed through addition of GLZ (p&amp;lt;0.0001). Similarly, co-culture of neutrophils with irradiated MB49 conditioned media induced NETs formation (p=0.01) and this effect was reversed with GLZ (p=0.009). Our in vivo results demonstrate that NETosis deficient mice treated with an HMGB1 inhibitor significantly improves response to radiation therapy. PAD4-/- mice treated with GLZ showed delayed tumor growth kinetics (p=0.023) and increased overall survival post radiation (p=0.0231) compared to all other irradiated arms: C57BL/6, PAD4-/-, C57BL/6 + DNAse and C57BL/6 + GLZ. Similarly, C57BL/6 mice treated with DNAse and GLZ also showed a delay in tumor growth kinetics post radiation (p&amp;lt;0.0001). CONCLUSION: NETs may induce radioresistance through interactions with HMGB1. Highlighting the role of HMGB1 in NET formation will provide valuable information on the responses that occur in the tumor microenvironment post radiation therapy. Citation Format: Surashri Shinde-Jadhav, Jose Joao Mansure, Roni Rayes, Mina Ayoub, Jonathan Spicer, Wassim Kassouf. Neutrophil extracellular traps and their implication with radioresistance in muscle invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3743.

Radiotherapy-induced immunogenic effects in bladder cancer.

418 Background: Many patients with bladder cancer (BC) undergo radiotherapy (RT) during the course of their treatment. There is emerging evidence that RT can cause immune stimulatory changes within the tumour microenvironment (TME), potentially contributing to its efficacy. We aimed to determine if RT induces immunogenic changes in murine BC cell lines, and develop a pre-clinical model of BC with a TME reflective of de novo tumours in order to test this premise in vivo. Methods: Immunogenic effects of RT were determined using murine vaccination studies with irradiated tumour cells. RT-induced immuno-phenotypic changes in surface antigen expression on tumour cells were ascertained using flow cytometry. An orthotopic BC model was established using MBT2 cells instilled intravesically in C3H/Hen mice, and resulting tumours monitored with ultrasound (US). We used immunohistochemical (IHC) staining to determine the immune contexture of the TME within developing orthotopic tumours. Results: C57BL/6 mice inoculated with irradiated MB49 cells demonstrated improved survival compared to control mice after subsequent rechallenge with viable tumour cells. This effect was not seen in C3H mice implanted with irradiated MBT2 cells. RT led to upregulation of immune stimulatory molecules CD80, MHC I, and Fas on MB49 but not MBT2 cells. Tumours developed in 80% of mice following catheter implant, and visible on US 3-4 weeks after instillation. Profiling of the TME with IHC demonstrated that tumours contained few CD8+ T-cells, but high numbers of myeloid cells. Conclusions: RT induces immune stimulatory effects on murine BC cells, including upregulation of several surface proteins. In future work, we will determine the effects of RT on the TME in the orthotopic model, and correlate these with the expression of various immunogenic cell surface proteins. This may lead to the discovery of a biomarker to predict which patients with BC would benefit from combination of an immunomodulatory agent with RT.

Lentiviral interferon: A novel method for gene therapy in bladder cancer.

456 Background: Gene therapy for bladder cancer (BLCA) is rapidly evolving. We reported that intravesical adenoviral interferon-alpha (Ad-IFNα) produced a complete response in 35% of patients with BCG-unresponsive BLCA enrolled in a Phase II trial. Lentivirus (LV) is another potential vector for intravesical delivery of IFNα. Unlike the adenovirus, LV can infect non-dividing cells and integrate into the host’s genome, making it one of the most efficient gene delivery vectors. The objective of this study was to investigate lentiviral interferon-alpha (LV-IFNα) BLCA gene therapy in preclinical models. Methods: Murine BLCA cell lines were transduced in-vitro with LV-IFNα using a multiplicity of infection (MOI) of 2:1. IFNα levels were measured by ELISA. Cell viability was assessed using Trypan blue dye exclusion. qPCR was used to identify expression of IFNα target genes. A LV-βGalactosidase reporter construct was delivered intravesically, and urinary IFNα levels were measured in mice treated with LV-IFNα or control virus to assess gene transfer. To assess survival benefit, p53+/- C57/B6 mice were exposed to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) to induce CIS and then treated with LV-IFNα or control virus, and sacrificed when moribund. Results: Efficient LV-IFNα transduction of BLCA cells was observed at an MOI of 2:1, resulting in increased expression of IFNα and its target genes PDL-1, TRAIL, and IRF7 (p&lt;0.001), and reduced cell viability vs. controls (p&lt;0.001). Mechanistically, TRAIL dependent cytotoxicity in the LV-IFNα cells was rescued by Caspase 8 inhibition. βGal expression confirmed efficient transduction of murine urothelium. Urinary IFNα levels were elevated in mice receiving LV-IFNα compared with control virus. BBN mice treated with LV-IFNα had longer overall survival than mice treated with control virus (p=0.04). LV-IFNα induced intratumoral CD8+ T cell infiltration, high expression of PD-L1, and inhibited angiogenesis. Conclusions: LV-IFNα effectively upregulated IFNα target genes, was cytotoxic to murine BLCA cells, and improved the survival of BBN tumor-bearing mice. LV appears to be a promising vector for intravesical gene delivery.

Cationized liposomal keto-mycolic acids isolated from Mycobacterium bovis bacillus Calmette-Guérin induce antitumor immunity in a syngeneic murine bladder cancer model.

Intravesical therapy using Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the most established cancer immunotherapy for bladder cancer. However, its underlying mechanisms are unknown. Mycolic acid (MA), the most abundant lipid of the BCG cell wall, is suspected to be one of the essential active components of this immunogenicity. Here, we developed cationic liposomes incorporating three subclasses (α, keto, and methoxy) of MA purified separately from BCG, using the dendron-bearing lipid D22. The cationic liposomes using D22 were efficiently taken up by the murine bladder cancer cell line MB49 in vitro, but the non-cationic liposomes were not. Lip-kMA, a cationic liposome containing keto-MA, presented strong antitumor activity in two murine syngeneic graft models using the murine bladder cancer cell lines MB49 and MBT-2 in comparison to both Lip-aMA and Lip-mMA, which contained α-MA and methoxy-MA, respectively. Interestingly, Lip-kMA(D12), which was made of D12 instead of D22, did not exhibit antitumor activity in the murine syngeneic graft model using MB49 cells, although it was successfully taken up by MB49 cells in vitro. Histologically, compared to the number of infiltrating CD4 lymphocytes, the number of CD8 lymphocytes was higher in the tumors treated with Lip-kMA. Antitumor effects of Lip-kMA were not observed in nude mice, whereas weak but significant effects were observed in beige mice with natural killer activity deficiency. Thus, a cationized liposome containing keto-MA derived from BCG induced in vivo antitumor immunity. These findings will provide new insights into lipid immunogenicity and the underlying mechanisms of BCG immunotherapy.

Animal model of local tumor recurrence after resection using the murine MBT2 bladder cancer cell line in syngeneic C3H mice.

Animal model of local tumor recurrence after resection using the murine MBT2 bladder cancer cell line in syngeneic C3H mice.

Abstract 709: Anti-PD-1 therapy reduces bone lesion growth in a novel syngeneic bladder cancer bone metastasis model

Abstract Bladder cancer is a common and aggressive type of cancer occurring both in women and in men. At diagnosis, lymph node involvement is frequently observed and in some cases metastatic spread can already be seen at early stage. With advanced stages, the metastatic incidence increases and the most common metastatic sites are bones, lung, liver and peritoneum. Bone metastases count about 50% of all metastases and the 5-year survival rate in metastatic disease is only 15%. Bone metastases are incurable but can be treated to reduce tumor burden in bone, and new therapies such as immunotherapies hold the potential to treat patients with bone metastatic bladder cancer. The aim of the study was to establish a novel syngeneic model for bladder cancer bone metastasis that could be used to study efficacy of new immunotherapies, and to test efficacy of anti-PD-1 therapy in the model. In the model establishment study, an intratibial injection of varying number of murine MBT-2 bladder cancer cells was given to 5-6 weeks old female C3H/HeN mice. Tumor-induced bone changes were followed by X-ray imaging once a week. At sacrifice, tumor-bearing tibias were collected and evaluated by histology. In the efficacy study, 5x105 MBT-2 cells were used. The mice were stratified to treatment groups based on similar bone lesion areas at 10 days after cancer cell inoculations and treated with anti-PD-1 therapy (RMP1-14) or isotype control (rat IgG2a, 200 µg per dose) at Q3D schedule. Bone lesion growth was followed by X-ray imaging one week after start of treatment and at sacrifice after three weeks on treatment. In the model establishment study, tumor take rate of 50% was observed. MBT-2 cells induced an osteolytic bone reaction resulting in substantial bone loss in the mice. The osteolytic bone lesions started to be visible between 7-14 days and the maximum duration of the study was 4 weeks from the cancer cell inoculations. Histology confirmed a large tumor and decreased bone mass in the inoculated tibia. In the efficacy study, treatment with anti-PD-1 reduced bone lesion growth. A trend towards decreased bone lesion area was seen already at one week on treatment and a significant reduction of bone lesion area was seen at sacrifice. A novel syngeneic bladder cancer bone metastasis model was established. According to the observed significant effects on bone lesion growth by anti-PD-1 treatment this model can be used in preclinical efficacy assessment of new immunotherapies as monotherapy or in combination with other therapies. Citation Format: Tiina E. Kähkönen, Mari I. Suominen, Jussi M. Halleen, Jenni Bernoulli, Arne Scholz. Anti-PD-1 therapy reduces bone lesion growth in a novel syngeneic bladder cancer bone metastasis model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 709.

Abstract 3582: Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors

Abstract Adoptive transfer of tumor-reactive T cells is a promising anti-tumor therapy for many cancers. To enhance tumor recognition by T cells, chimeric antigen receptors (CAR) consisting of signaling domains fused to receptors that recognize tumor antigens can be created and expressed in T cells. One receptor that is a prospective target for a new chimeric antigen receptor is PD1 because the ligands for the PD1 receptor are expressed on many cancer types. Therefore, we developed a murine chimeric PD1 receptor (chPD1) consisting of the PD1 receptor extracellular domain and the activation domain of CD3 zeta. In addition, current chimeric antigen receptor therapies utilize various costimulatory domains to enhance anti-tumor efficacy. Therefore, we also compared the inclusion of CD28, Dap10, 41BB, GITR, ICOS, or OX40 costimulatory domains in our chPD1 receptor to determine which costimulatory domain induced optimal anti-tumor immunity. To determine if this novel CAR could potentially target a wide variety of tumors, the anti-tumor efficacy of chPD1 T cells against murine lymphoma, melanoma, kidney, pancreatic, liver, colon, breast, ovarian, prostate, and bladder cancer cell lines was measured. Of the eighteen cell lines tested, all expressed PD1 ligands on their cell surface, making them potential targets for chPD1 T cells. Regardless of the costimulatory domain in the CAR, all of the chPD1 T cells induced similar levels of T cell proliferation and tumor cell lysis. However, differences were observed in the cytokine secretion profiles depending on which costimulatory receptor was included in the CAR. While most of the chPD1 T cell receptor combinations secreted both pro-inflammatory (IFNγ, TNFα, IL-2, GM-CSF, IL-17, and IL-21) and anti-inflammatory cytokines (IL-10 and IL-5) as determined by ELISA and LegendPlex analysis, chPD1 T cells containing a Dap10 costimulatory domain secreted high levels of proinflammatory cytokines, but did not secrete a significant amount of anti-inflammatory cytokines. Furthermore, T cells expressing chPD1 receptors with a Dap10 domain also had the strongest anti-tumor efficacy in vivo. ChPD1 T cells did not survive for longer than 14 days in vivo, however treatment with chPD1 T cells induced long-lived protective host-anti-tumor immune responses in tumor-bearing mice. Therefore, adoptive transfer of chPD1 T cells could be a novel therapeutic strategy to treat multiple types of cancer and inclusion of the Dap10 costimulatory domain in chimeric antigen receptors may induce a preferential cytokine profile for anti-tumor therapies. Citation Format: Amorette E. Barber, Geoffrey Parriott, Shane Crean, Hailey Kintz. Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3582.

Cell-intrinsic PD-L1 and PD-1 signal effects in bladder cancer

Abstract PD-1/PD-L1 provides a mechanism of immune escape, the blockage of which has reinvigorated interest in the treatment of urothelial cancer. We recently reported on cell-intrinsic PD-L1 effects in melanoma and ovarian cancer, and considered that effects in bladder cancer cells could differ based on distinct mutational landscapes. Using CRISPER/Cas9 methodology, we knockout PD-L1 in the murine bladder cancer cell line MB49 and the human bladder caner cell line RT. We show here that cell-intrinsic MB49 and RT4 cells express PD-L1 and PD-1 that each mediate cell-intrinsic signals. PD-L1 knockout has little effect on cells proliferation in vitro, but significant difference could be found in tumor size when we used MB49 Ctrl or MB49 PD-L1KO cells to challenge C57B16 mice. αPD-L1 and αPD-1 antibodies reduced bladder cancer cell proliferation in vitro demonstrating direct signaling effects. Bladder cancer cell-intrinsic PD-L1 promoted mTORC1 and tumor initiating cell generation similar to melanoma and ovarian cancer cells. By contrast to melanoma and ovarian cancer cells, bladder cancer cell PD-L1 promoted autophagy and had little effect on in vivo immune-independent growth. Base one the functional role, PD-L1 knockout increased proliferation inhibition induced by Rapamycin in both mouse and human bladder cancer cells. Furthermore, we found intrinsic PD-L1 suppress cytokine production, including CXCL10. Corresponding with increased CXCL10 secretion, more CXCR3+ NK cells could be attracted by PD-L1KO cancer cells. Overall, our findings further illustrated the intrinsic role of PD-L1 in bladder cancer development and found PD-1 expression on human bladder cancer cells for the first time.

Flavonoid silybin improves the response to radiotherapy in invasive bladder cancer

Conservative treatment for invasive bladder cancer (BC) involves a complete transurethral tumor resection combined with chemotherapy (CT) and radiotherapy (RT). The major obstacles of chemo-radiotherapy are the addition of the toxicities of RT and CT, and the recurrence due to RT and CT resistances. The flavonoid Silybin (Sb) inhibits pathways involved in cell survival and resistance mechanisms, therefore the purpose of this paper was to study in vitro and in vivo, the ability of Sb to improve the response to RT, in two murine BC cell lines, with different levels of invasiveness, placing emphasis on radio-sensitivity, and pathways involved in radio-resistance and survival. In vitro, Sb radio-sensitized murine invasive cells through the inhibition of RT-induced NF-κB and PI3K pathways, and the increase of oxidative stress, while non-invasive cells did not show to be sensitized. In vivo, Sb improved RT-response and overall survival in invasive murine tumors. As Sb is already being tested in clinical trials for other urological cancers and it improves RT-response in invasive BC, these results could have translational relevance, supporting further research.

Down-regulation of PKM2 enhances anticancer efficiency of THP on bladder cancer.

Pyruvate kinase M2 (PKM2) regulates the final step of glycolysis levels that are correlated with the sensitivity of anticancer chemotherapeutic drugs. THP is one of the major drugs used in non‐muscle‐invasive bladder cancer instillation chemotherapy. However, low response ratio of THP (19.7%) treatment to human genitourinary tumours using collagen gel matrix has been observed. This study aims to investigate the effect of down‐regulation of PKM2 on THP efficiency. Via inhibitor or siRNA, the effects of reduced PKM2 on the efficiency of THP were determined in 2 human and 1 murine bladder cancer cell lines, using MTT, cologenic and fluorescence approaches. Molecular mechanisms of PKM2 on THP sensitization were explored by probing p‐AMPK and p‐STAT3 levels via WB. Syngeneic orthotopic bladder tumour model was applied to evaluate this efficiency in vivo, analysed by Kaplan‐Meier survival curves, body and bladder weights plus immunohistochemistric tumour biomarkers. PKM2 was overexpressed in bladder cancer cells and tissues, and down‐regulation of PKM2 enhanced the sensitivity of THP in vitro. Activation of AMPK is essential for THP to exert anti‐bladder cancer activities. On the other hand, down‐regulating PKM2 activates AMPK and inhibits STAT3, correlated with THP sensitivity. Compared with THP alone (400 μmol L−1, 50 μL), the combination with metformin (60 mmol L−1, 50 μL) stopped growth of bladder cancer completely in vivo (combination group VS normal group P = .078). Down‐regulating the expression of PKM2 enhances the anticancer efficiency of THP. This study provides a new insight for improving the chemotherapeutic effect of THP.

Enhanced Mortality to Metastatic Bladder Cancer Cell Line MB49 in Vasoactive Intestinal Peptide Gene Knockout Mice.

To identify if the absence of the vasoactive intestinal peptide (VIP) gene enhances susceptibility to death from metastatic bladder cancer, two strains of mice were injected with MB49 murine bladder cancer cells. The growth and spread of the cancer was measured over a period of 4 weeks in C57BL/6 mice and 5 weeks in VIP knockout (KO) mice. A Kaplan–Meier plot was constructed to compare control C57BL/6 mice and C57BL/6 mice with MB49 vs. VIP KO controls and VIP KO mice with MB49. The wild-type (WT) strain (C57BL/6) contained the VIP gene, while the other strain, VIP knockout backcrossed to C57BL/6 (VIP KO) did not and was thus unable to endogenously produce VIP. VIP KO mice had increased mortality compared to C57BL/6 mice at 4 weeks. The number of ulcers between both groups was not statistically significant. In vitro studies indicated that the presence VIP in high doses reduced MB49 cell growth, as well as macrophage inhibitory factor (MIF), a growth factor in bladder cancer cells. These findings support the concept that VIP may attenuate susceptibility to death from bladder cancer, and that it exerts its effect via downregulation of MIF.

Abstract 2037: Oral nitroxoline in combination with intravesical bacille calmette-guerin (BCG) shows synergistic antitumor efficacy in mouse bladder cancer orthotopic xenograft

Abstract Nitroxoline, an antibiotic used to treat urinary tract infection, had previously been identified as a MetAP2 inhibitor and found to show antitumor efficacy in mouse models of human breast cancer xenograft and bladder cancer orthotopic xenograft [1]. Our purpose in this study was to evaluate the feasibility of combining oral nitroxoline with intravesical BCG, the first-line therapy for intermediate- and high-risk non-muscle invasive bladder cancer (NMIBC), in mouse bladder cancer orthotopic xenograft. The luciferase-expressing murine bladder cancer cell line MBT-2-Luc cells was transurethrally implanted in the bladder of syngeneic female C3H/He mice. The animals were randomly divided into groups and treated with phosphate-buffered saline (PBS), oral nitroxoline, intravesical BCG alone, and a combination as described in methods. The IVIS spectrum images analyses of luciferin-stained tumor at 21 days after inoculation revealed that combining oral nitroxoline with intravesical BCG showed synergistically enhanced antitumor efficacy and prolonged long-term survival, implicating the potential of combining bladder cancer treatment using oral nitroxoline with intravesical BCG in the clinical setting. The mechanism behind this observation is currently under investigated. Citation Format: Qiang Li, Kevin Pan, Xiezhao Li, Naijin Xu, Peng Huang. Oral nitroxoline in combination with intravesical bacille calmette-guerin (BCG) shows synergistic antitumor efficacy in mouse bladder cancer orthotopic xenograft [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2037. doi:10.1158/1538-7445.AM2017-2037

Myeloid-specific genetic ablation of ATP-binding cassette transporter ABCA1 is protective against cancer.

Increased circulating levels of apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), by genetic manipulation or infusion, protects against melanoma growth and metastasis. Herein, we explored potential roles in melanoma tumorigenesis for host scavenger receptor class B, type 1 (SR-B1), and ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), all mediators of apoA-I and HDL sterol and lipid transport function. In a syngeneic murine melanoma tumor model, B16F10, mice with global deletion of SR-B1 expression exhibited increased plasma HDL cholesterol (HDLc) levels and decreased tumor volume, indicating host SR-B1 does not directly contribute to HDL-associated anti-tumor activity. In mice with myeloid-specific loss of ABCA1 (Abca1−M/−M; A1−M/−M), tumor growth was inhibited by ∼4.8-fold relative to wild type (WT) animals. Abcg1−M/−M (G1−M/−M) animals were also protected by 2.5-fold relative to WT, with no further inhibition of tumor growth in Abca1/Abcg1 myeloid-specific double knockout animals (DKO). Analyses of tumor-infiltrating immune cells revealed a correlation between tumor protection and decreased presence of the immune suppressive myeloid-derived suppressor cell (MDSC) subsets, Ly-6G+Ly-6CLo and Ly-6GnegLy-6CHi cells. The growth of the syngeneic MB49 murine bladder cancer cells was also inhibited in A1−M/−M mice. Collectively, our studies provide further evidence for an immune modulatory role for cholesterol homeostasis pathways in cancer.

A novel synthetic derivative of quercetin, 8-trifluoromethyl-3,5,7,3',4'-O-pentamethyl-quercetin, inhibits bladder cancer growth by targeting the AMPK/mTOR signaling pathway.

Quercetin is a naturally existing compound and shows attractive anticancer properties for a variety of solid tumors including glioma, bladder cancer, hepatocellular carcinoma, breast cancer, hematological malignancies and prostate carcinoma. However, these anticancer properties have not been clinically approved due to unclear mechanistic information and its low bioactivity. In our previous study, we elucidated that quercetin activates AMPK pathway which is the major mechanism for its unique anticancer effect in bladder cancer. In the present study, we are trying to enhance its bioactivity by chemical modification using fluorination approach to prepare novel chemical entities, based on the principle of intermediate derivative method (IDM). The compound we obtained is named 8-trifluoromethyl-3,5,7,3′,4′-O-pentamethyl- quercetin (TFQ), characterized by NMR spectra and mass spectrum (MS). The results from MTT and cologenic assay in two human and one murine bladder cancer cell lines showed that TFQ exhibits more potent inhibition on the three bladder cancer cell lines than quercetin (Que) although this enhanced effects is not very dramatic. Furthermore, we found that the survival of normal bladder cells PEBC was not significantly suppressed by TFQ compared with Que. Western blot analysis showed that TFQ possess more potent AMPK activation than Que. The downstream of AMPK was further examined by western blot. TFQ treatment is able to inactivate mTOR signaling pathway with the regulation of mTOR, 4EBP1 and P70S6K. These results demonstrated that the fluorinated quercetin derivative TFQ inhibits bladder cancer cell growth through the AMPK/mTOR pathway. Altogether, our findings suggest that TFQ could serve as a new potential therapeutic agent for bladder cancer more effective than Que.

MP98-09 SYNERGISTIC IMMUNO-PHOTOTHERMAL NANOTHERAPY (SYMPHONY): A NOVEL TREATMENT FOR LOCALIZED AND METASTATIC BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology V1 Apr 2017MP98-09 SYNERGISTIC IMMUNO-PHOTOTHERMAL NANOTHERAPY (SYMPHONY): A NOVEL TREATMENT FOR LOCALIZED AND METASTATIC BLADDER CANCER Steven C. Brousell, Yang Liu, Paolo F. Maccarini, Gregory M. Palmer, Wiguins Etienne, Yulin Zhao, Chen-Ting Lee, Tuan Vo-Dinh, and Brant A. Inman Steven C. BrousellSteven C. Brousell More articles by this author , Yang LiuYang Liu More articles by this author , Paolo F. MaccariniPaolo F. Maccarini More articles by this author , Gregory M. PalmerGregory M. Palmer More articles by this author , Wiguins EtienneWiguins Etienne More articles by this author , Yulin ZhaoYulin Zhao More articles by this author , Chen-Ting LeeChen-Ting Lee More articles by this author , Tuan Vo-DinhTuan Vo-Dinh More articles by this author , and Brant A. InmanBrant A. Inman More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.3074AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES We developed a novel treatment for localized and metastatic bladder cancer comprised of gold nanoparticle-based photothermal therapy and immunotherapy (SYMPHONY). We demonstrate that it effectively ablates primary tumors, destroys metastases abscopally, and induces potent anti-tumor immunity. METHODS MB49 murine bladder cancer cells were injected into the bilateral flanks of C57BL/6 mice and grown until 100 mm3 in size. PEG-functionalized gold nanostars, developed and manufactured by our team, were administered intravenously. A 808-nm laser (0.6 W/cm2) was used to trigger plasmonic heat production from the gold nanostars in the left flank 24 hours after injection, while the contralateral flank was left untreated. Anti-PD-L1 antibody immunotherapy was co-administered intraperitoneally and repeated q3days. Mice were assessed for ipsilateral and contralateral tumor response and survival. Flow cytometry, multiplex cytokine profiling, and T cell receptor sequencing were used to characterize the immune response. Mice achieving a complete response were rechallenged with an additional injection of MB49 tumor cells 90 days later. RESULTS Gold nanostar-mediated phototherapy alone completely ablated ipsilateral tumors in 4/5 of mice (pT0 at necropsy) but contralateral tumors grew and all 5 mice required sacrifice within 14 days. Anti-PD-L1 therapy alone slowed tumor growth in 3/5 mice, but tumors rapidly began growing again and 5/5 mice required sacrifice by 45 days. Combined treatment (i.e. SYMPHONY) ablated 5/5 ipsilateral tumors and resulted in partial (3/5) and complete responses (2/5) of untreated contralateral tumors, demonstrating a strong abscopal effect. After 90 days of follow-up, the two mice achieving a complete response with SYMPHONY were rechallenged with MB49 and neither developed a tumor over the ensuing 4 weeks indicating strong and effective immune memory. Flow cytometry showed CD4 and CD8 T cell proliferation, decreased myeloid derived suppressor cells, and increased IL2 with SYMPHONY. CONCLUSIONS SYMPHONY treatment resulted not only in effective ablation of primary tumors but also in immune-mediated abscopal destruction of untreated distant tumors. Strong and permanent anti-tumor immunity developed in some mice, indicating that with further optimization, SYMPHONY may be able to cure more advanced bladder cancers. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1315 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Steven C. Brousell More articles by this author Yang Liu More articles by this author Paolo F. Maccarini More articles by this author Gregory M. Palmer More articles by this author Wiguins Etienne More articles by this author Yulin Zhao More articles by this author Chen-Ting Lee More articles by this author Tuan Vo-Dinh More articles by this author Brant A. Inman More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 µL of poly-L-lysine (PLL) is slowly instilled at a rate of 10 µL/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 µL) at a rate of 10 µL/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation. This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 µL of poly-L-lysine (PLL) is slowly instilled at a rate of 10 µL/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 µL) at a rate of 10 µL/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation. This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.