Murine Bladder Cancer Cell Line Research Articles

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.

Abstract 2060: Treatment combination strategies to improve radiation efficacy in immunologically cold tumors in vivo

Abstract Introduction: Radiation therapy (RT) is a promising bladder-sparing therapy in muscle-invasive bladder cancer (MIBC). However, 30% of patients exhibit radioresistant tumors requiring salvage surgery. Combining RT with immune checkpoint inhibitor was reported to have synergic effects on anti-tumor immunity. Yet, it is also met with treatment resistance. In addition, activation of the STING pathway was shown to induce cell death, cancer cell antigens release and presentation, and promote the priming, activation and trafficking of T cells into tumors. Previous in vivo results from our team using the MB49 murine bladder cancer cell line - and immunologically ‘hot’ tumor model - demonstrated significant improvement of survival and immune cell infiltration upon STING agonist treatment, when combined with RT and anti-PDL1 treatment. Oppositely, treating the ‘cold’ tumor model UPPL with a combination of RT and anti-PDL1 treatment did not improve survival compared to RT alone. Consistently, UPPL tumors present low T cell and high neutrophil infiltration in vivo. Thus, the main objective of the present study is to evaluate whether combining RT, anti-PDL-1 and STING agonist treatments can improve the immunogenicity of UPPL tumors. Methods: Seven-week-old male C57BL/6 mice were subcutaneously injected with 5.106 UPPL tumor cells. Once tumors reached 0.1-0.15 cm3, mice were randomized into the following treatment groups : 1) Untreated; 2) PDL-1; 3) STING; 4) PDL-1 + STING; 5) RT; 6) RT + PDL-1; 7) RT + STING; 8) RT + STING + PDL-1. Midpoint (~0.6-0.8 cm3, n=5) and endpoint (2 cm3, n=8) tumors, spleens and draining lymph nodes (dLN) were harvested and dissociated for flow cytometry analysis of TME composition and cytokine production. Results: We report that RT and RT-combined treatments delayed tumor growth and prolonged survival in vivo compared to untreated. Additionally, our combination approaches shifted immune infiltration : compared to untreated, each strategy changed myeloid and T cells infiltrating cells in tumors. Combination of RT with anti-PDL-1 and STING agonist favored macrophages infiltration as well as increased MHC-II expression in dendritic cells (DC). Furthermore, we observed increased infiltration of cytotoxic CD8+ T cells (Granzyme B+ and IFN-γ+ CD8+) in these groups. Conclusions: Our results suggest RT and RT combination treatments in cold tumor affect antigen presentation potency in DCs, fine-tune the functionality of infiltrating CD8+ T cells. This preliminary study shows immunologically cold tumors can be modulated through treatment combination, which has relevance in human treatment-resistant tumors. It adds to the very small body of literature taking a deep dive into the immune modulation of a conventionally used ‘cold’ tumor cell line. Citation Format: Eva Michaud, Gautier Marcq, Sabina Fehric, JiaMin Huang, Jose Joao Mansure, Ciriaco Piccirillo, Wassim Kassouf. Treatment combination strategies to improve radiation efficacy in immunologically cold tumors in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2060.

Abstract 2524: Investigating the role of neutrophils in muscle-invasive bladder cancer and response to radiation therapy

Abstract Background: Radiation therapy (RT) is an increasingly used bladder-sparing therapy for patients with muscle-invasive bladder cancer (MIBC). However, 20-30% of patients do not respond to RT. Neutrophils have previously been linked to radioresistance, but the specific mechanism is still unknown. Previous work by our team has shown that neutrophil extracellular trap (NET) formation plays a role in RT resistance in an immunologically hot tumor, with high T cell infiltration, using the MB49 murine bladder cancer cell line, in a vivo model. In MIBC, a recently developed cell line can be used to study cold, luminal-like tumors. However, work on this cell line and its response to RT is still in its initial stages. Immune checkpoint inhibitors such as anti-PD-L1 have been tested by our team in these cold tumors of MIBC and have not shown to be effective in treating or radiosensitizing the tumor. Consequently, we aim to determine how neutrophil migration and neutrophil extracellular traps (NET) impact tumor growth in a cold, luminal-like tumor model. Furthermore, we aim to understand how neutrophil migration and NETs impact immunological changes in the tumor microenvironment (TME). Methods: To determine the response to RT in these cold tumors, mice were injected with 5M cells on the right flank of our luminal-like cancer model (UPPL cell line). Once tumors reached 0.1-0.15 cm3, mice were randomized into the different treatment groups: 1) Control; 2) RT; 3) Anti-PDL1 4) DNAse I; 5) RT + Anti-PDL1; 6) RT + DNAse I; 7) Anti-PDL1 + DNAse I; 8) RT + Anti-PDL1 + DNAse I. Size of the tumor was monitored. 8 mice tumors per group were collected at the primary endpoint set at 1.5 cm3 and 5 mice tumors per group were collected after 21 days (midpoint). Tumors were collected analyzed by flow cytometry and immunohistochemistry (IHC). Preliminary Results: Prolonged survival was observed in the mice treated with the triple combination when compared to the other groups. Tumor growth in this group also showed delay early on. So far, the frequency of pro-tumorigenic neutrophils infiltration in tumors seems to happen early on (midpoints) in mice treated with RT, whereas the infiltration happens later (endpoint) in the mice treated with the triple combination. Currently, we are also evaluating the NET production in these tumors through IHC. Conclusion: This ongoing experiment using RT on the UPPL cell line will allow us to understand what happens in patients who are resistant to RT. Understanding the role of neutrophils and NETs, along with the changes in the TME caused by the manipulation of these is key to understanding radiation resistance. This knowledge will bring us one step closer to developing new bladder-sparring treatments and improving patient care in the clinic. Citation Format: Sabina Fehric, Eva Michaud, Surashri Shinde-Jadhav, JiaMin Huang, Jose J. Mansure, Roni Rayes, Jonathan Spicer, Wassim Kassouf. Investigating the role of neutrophils in muscle-invasive bladder cancer and response to radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2524.

MP06-13 TARGETING THE ANDROGEN RECEPTOR AXIS TO IMPROVE RESPONSE TO BLADDER CANCER IMMUNOTHERAPY

You have accessJournal of UrologyCME1 May 2022MP06-13 TARGETING THE ANDROGEN RECEPTOR AXIS TO IMPROVE RESPONSE TO BLADDER CANCER IMMUNOTHERAPY Typhaine Gris, Marjorie Besançon, France-Hélène Joncas, Valérie Picard, Alain Bergeron, Yves Fradet, and Paul Toren Typhaine GrisTyphaine Gris More articles by this author , Marjorie BesançonMarjorie Besançon More articles by this author , France-Hélène JoncasFrance-Hélène Joncas More articles by this author , Valérie PicardValérie Picard More articles by this author , Alain BergeronAlain Bergeron More articles by this author , Yves FradetYves Fradet More articles by this author , and Paul TorenPaul Toren More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.13AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Men are 3-4 times more likely to be diagnosed with bladder cancer (BCa) than women, who often have more aggressive tumors. Intravesical instillation of BCG is one of the first immunotherapies in widespread use and is given to patients with non-muscle invasive bladder cancer (NMIBC). Newer PD-1/PD-L1 checkpoint inhibitors now also have proven efficacy against BCa. Nonetheless, approximately 35% of patients fail to respond to BCG, with response rates to checkpoint immunotherapy much lower. Based on prior work suggesting the importance of the androgen receptor (AR) in BCa, we investigated AR-targeting as a novel strategy to improve the response to BCa immunotherapy in the MBT-2 murine BCa model. METHODS: Subcutaneous injection of 106 MBT-2 cells into male and female C3H mice was performed, with tumors harvested at tumor size limits. The AR antagonist enzalutamide (ENZ) was given in alone or combination with anti-PD-1 or intra-tumoral BCG treatments as previously described. Selected human NMIBC were collected following transurethral resection. After digestion of fresh human and murine tumors, flow cytometry analyses characterized the immune cells present. The anti-proliferative effect of AR antagonists against human and murine BCa cell lines was tested in vitro. RESULTS: The CD45+/CD45- ratio, extent of tumor infiltrating lymphocytes, and expression of immune checkpoint markers from the MBT-2 model is comparable to the immune composition of NMIBC tumors resected from patients. Moreover, the model also recapitulates previously observed human tumor immune cell gender differences. AR antagonist ENZ demonstrated anti-proliferative effects in lower grade human and murine BCa cell lines, while limited differences were seen in human high grade BCa cell lines. While ENZ alone had no effect in vivo, its use in combination with either anti-PD-1 or BCG treatment in male mice synergized to improve response rates. Complete response was confirmed by re-challenge of mice, demonstrating immune memory. Notably, the proportion of complete responses in male mice treated with the combination treatment is similar to that observed in female mice with either immunotherapy alone. Flow cytometry analyses showed that these therapeutic benefits in male mice are related to an increase in pro-inflammatory tumor infiltrating dendritic cells as well as a decrease in anti-inflammatory tumor infiltrating dendritic cells and myeloid-derived suppressor cells. CONCLUSIONS: Our results suggest that combining AR antagonism with immunotherapy in male BCa patients may potentiate the antitumor immune response and increase response rates. Accordingly, a Phase II trial to evaluate the efficacy of AR antagonism in combination with BCG to decrease NMIBC tumor recurrences in men is planned. Further, the MBT-2 murine model appears relevant to investigate immunologic BCa sex differences. Source of Funding: Cancer Research Society (Upcycle Grant) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e82 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Typhaine Gris More articles by this author Marjorie Besançon More articles by this author France-Hélène Joncas More articles by this author Valérie Picard More articles by this author Alain Bergeron More articles by this author Yves Fradet More articles by this author Paul Toren More articles by this author Expand All Advertisement PDF DownloadLoading ...

Intravesical High Dose BCG Tokyo and Low Dose BCG Tokyo with GMCSF+IFN α Induce Systemic Immunity in a Murine Orthotopic Bladder Cancer Model.

This study evaluates a short therapy schedule for bladder cancer using BCG Tokyo. BCG Tokyo was evaluated in vitro using bone marrow derived dendritic cells, neutrophils, RAW macrophages and the murine bladder cancer cell line, MB49PSA, and compared to other BCG strains. BCG Tokyo > BCG TICE at inducing cytokine production. In vivo, high dose (1 × 107 colony forming units (cfu)) and low dose (1 × 106 cfu) BCG Tokyo with and without cytokine genes (GMCSF + IFNα) were evaluated in C57BL/6J mice (n = 12–16 per group) with orthotopically implanted MB49PSA cells. Mice were treated with four instillations of cytokine gene therapy and BCG therapy. Both high dose BCG alone and low dose BCG combined with cytokine gene therapy were similarly effective. In the second part the responsive groups, mice (n = 27) were monitored by urinary PSA analysis for a further 7 weeks after therapy cessation. More mice were cured at day 84 than at day 42 confirming activation of the immune system. Cured mice resisted the re-challenge with subcutaneous tumors unlike naïve, age matched mice. Antigen specific T cells recognizing BCG, HY and PSA were identified. Thus, fewer intravesical instillations, with high dose BCG Tokyo or low dose BCG Tokyo with GMCSF + IFNα gene therapy, can induce effective systemic immunity.

Abstract 1516: 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 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 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, 4-1BB, GITR, ICOS, or OX40 costimulatory domains in the 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), 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. 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 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1516.

Bladder tumor metabolic alterations in response to IFNα gene therapy.

468 Background: Intravesical interferon-alpha (IFNα) gene therapy with Nadofaragene firadenovec has shown clinical efficacy in patients with non-muscle invasive bladder cancer (NMIBC) in a phase III clinical trial, highlighting the therapeutic potential of this approach in a disease with significant unmet clinical need. Optimizing the clinical efficacy of IFNα gene therapy requires an understanding of the underlying therapeutic mechanisms. Here, we investigate the impact of IFNα gene therapy on tumor metabolism using in vitro and orthotopic murine preclinical models and clinical trial data to elucidate mechanisms of tumor resistance and identify predictive biomarkers. Methods: In vitro murine bladder cancer cell lines treated with recombinant IFNα (rIFNα) and lentiviral IFNα (LV-IFNα) were analyzed by whole-transcriptome sequencing, glucose uptake, and lactate production. Preclinical murine bladder cancer models were treated with LV-IFNα (orthotopic tumor model) or Poly(I:C) (flank tumor model), a potent IFN inducer. Disease response was monitored by in vivo real-time luciferase imaging. Tumors were harvested and whole-transcriptome sequencing performed to assess effects of IFNα therapy on tumor metabolism and lipidomics. Lipidomic profiling was performed on patient urine samples from a phase II clinical trial of intravesical Nadofaragene firadenovec (7 clinical responders and 6 non-responders) to assess for clinically-relevant differences in lipid metabolism. Results: Following IFNα therapy in vitro and in murine orthotopic bladder cancer models, we identified downregulation of genes involved in fatty acid synthesis and upregulation of genes involved in glycolysis by whole-transcriptome sequencing. This was confirmed by higher glucose uptake and lactate production by IFNα-treated cells in vitro. These findings were recapitulated in whole-transcriptome sequencing data of human bladder tumors treated with intravesical Nadofaragene firadenovec. Lipidomics performed on murine MB49 tumors treated with poly(I:C) identified 79 upregulated lipids, including phosphotidyl choline, spingomyelin and phosphatidyl ethanolamine, and 12 downregulated lipids, notably the cardiolipin class. Lipidomics performed on patient urine samples collected pre- and post-treatment with intravesical Nadofaragene firadenovec detected >592 lipids with distinct expression profiles differentiating clinical responders and non-responders at both timepoints. Conclusions: We describe novel modulation of glucose and lipid metabolism by bladder tumor cells in response to IFNα gene therapy. These metabolic changes were reproducible across in vitro, in vivo and clinical trial studies and improve our mechanistic understanding of IFNα gene therapy, identify tumor escape pathways targetable with combination therapy regimens, and identify a new class of biomarkers for predicting clinical response of NMIBC to IFNα gene therapy.

Abstract A12: Bladder cancer-derived extracellular vesicles and immune activation

Abstract Background: Bladder tumors occur on the luminal surface of the bladder, and cancer cells and cellular components are shed in urine, which permits the isolation of an enriched population of bladder cancer-specific extracellular vesicles (EVs). EVs contain proteins, miRNA, mRNA, and cellular metabolites, which may serve as stimulators of immune cells. The objective of this study was to evaluate the immune potential of tumor-derived EVs (TEVs) produced on exposure to BCG using a murine bladder cancer cell line and immune cells. Methods: TEVs were isolated by differential high-speed centrifugation of cell culture supernatants. They were confirmed to be extracellular vesicles by electron microscopy and size measurement using nanoparticle tracking analysis. Unstimulated murine bladder cancer cells (MB49) were used to prepare TEV1. The cells were then exposed to BCG Tokyo (MOI 4) and TEVs were isolated after 2 hours (TEV2). BCG was then washed off and the cells were allowed to grow in complete media for 2 days before isolation of the third sample, TEV3. EVs from BCG (BEV) were collected to serve as a control. Bone marrow-derived cells were obtained from C57BL/6J mice to generate dendritic cells (DC) and neutrophils. These cells were untreated or treated with 1ug of TEVs or BEV or BCG (MOI 5) for 24 hours. The supernatants were analyzed for cytokine production and the DCs and neutrophils were analyzed for activation markers by flow cytometry. The institutional animal care and use committee approved the use of mice. Results: DCs treated with TEV2 had significantly higher expression of CD86 (at similar levels when DCs were treated with BCG) and MHC2, both of which enhanced the DC's capacity to present antigens to T cells, when compared to TEV1 or TEV3. While neutrophils induced by BCG expressed significantly higher levels of costimulatory molecules CD80, CD83, CD86, and MHC2, TEV2 induced significant expression of CD80 and CD86. BCG caused a massive release of proinflammatory cytokines (IL6, TNFa, and IL12p70) and the anti-inflammatory cytokine IL10. Cytokine production induced by TEV2 was not as high as that induced by direct BCG stimulation, but it was significantly higher than that produced by untreated cells or TEV1 and TEV3. Unlike BCG, TEV2 did not significantly stimulate IL10 production. Conclusion: Our results demonstrate that tumor cells treated with BCG for 2 hours produce EVs that are able to cause significant modulation of DCs and neutrophils. TEVs generated during BCG immunotherapy have immunostimulatory potential. Therefore, TEVs represent a promising source of material to increase immune activation and efficacy of BCG therapy. This could reduce the number of instillations and adverse effects associated with BCG therapy. Citation Format: Sin Mun Tham, Kesavan Esuvaranathan, Ratha Mahendran. Bladder cancer-derived extracellular vesicles and immune activation [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A12.

Analysis of chemotherapy-related modulation of the immune microenvironment in muscle invasive bladder cancer.

5049 Background: Novel immune checkpoint inhibitors provide significant clinical benefits for patients with metastatic bladder cancer. It is known that chemotherapy administered to muscle invasive patients prior to radical cystectomy (neoadjuvant chemotherapy) improves survival. However, it is unknown whether immune checkpoint inhibitor therapy in combination with chemotherapy can provide further clinical benefits as neoadjuvant therapy. Here, we test the hypothesis that treatment of bladder cancer with certain chemotherapy agents can modulate bladder tumor immune microenvironment (TIME) for optimal combination with immune checkpoint therapy. Methods: Time course and dose response experiments were performed using eight human bladder cancer cell lines (UMUC3, RT4, 253J, RT112, J82, HT1376, T24, and HT1197) and two murine bladder cancer cell lines (MB49, MBT2). Conventional chemotherapy agents and combinations (MVAC, GemCis, PemVin) were used to treat bladder cancer cell lines. Flow cytometry analysis was used to measure immune cell subsets and PD-L1 expression. For in vivo studies, the subcutaneous MB49 murine bladder cancer model was used to evaluate responses to chemotherapy and anti-PD-L1 combinations. Pre- and post-treatment bladder tumors from patients who received neoadjuvant MVAC and GemCis are selected to evaluate changes in TIME. Results: Our data demonstrate that chemotherapy agents varies in their ability to up-regulate PD-L1 expression on bladder cancer cell lines. Vinblastine, gemcitabine, and pemetrexed treatment each resulted in significant upregulation of PD-L1 expression. Combination regimens with GemCis or PemVin demonstrated induction of PD-L1 across different cell lines. In in-vivo studies, GemCis + anti-PD-L1 had a synergistic activity in causing tumor regression. We also found that sequential versus concurrent treatment with chemotherapy and anti-PD-L1 had a similar outcome. Tissue analyses show that combination chemotherapies increased CD4 Th cell infiltration while decreasing Treg cells in TIME. Consistent with the in vitro data, PD-L1 expression was also up-regulated with combination treatment. The evaluation of TIME modulation in human bladder tumors treated with neoadjuvant MVAC or GemCis is ongoing. Conclusions: Our data suggest that chemotherapy could favorably modulate TIME and thus, may be combined with immune checkpoint inhibitor to improve anti-tumor responses in the neoadjuvant setting for patients with muscle invasive bladder cancer.

Lentiviral interferon with immune checkpoint blockade: A novel method for gene therapy in bladder cancer.

33 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. LV can infect non-dividing cells and integrate into the host’s genome, making it more efficient gene delivery vectors. As treatment with IFN upregulates checkpoint inhibitors, we also wanted to investigate the role of checkpoint inhibitors with and without IFN gene therapy. Methods: Murine BLCA cell lines were transduced in-vitro with LV-IFNα (MOI 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, the MB49 intravesical tumor model and p53+/- C57/B6 mouse model were employed. We also assessed the role of combination therapy with immune checkpoint blockade using PD1 antibody using our MB49 intravesical model. Results: Efficient LV-IFNα transduction of BLCA cells resulted in increased expression of IFNα and its target genes and reduced cell viability vs. controls (p<0.001). Mechanistically, TRAIL dependent cytotoxicity in the LV-IFNα cells was rescued by Caspase8 inhibition. Urinary IFNα levels were elevated in mice receiving LV-IFNα compared with control virus. Overall survival improved in the MB49 model and BBN model in treated mice. LV-IFN induced intratumoral CD8+ T cell infiltration, high expression of PD-L1, and inhibited angiogenesis in BBN model whereas in the MB49 tumor response was mediated by TRAIL. Combination therapy with PD1 resulted in further improved survival. Conclusions: LV-IFNα effectively upregulated IFNα target genes, was cytotoxic to murine BLCA cells, and improved the survival in mouse models. Combining it with PD1 therapy appears to further improve survival.

Biomolecular study and conjugation of two para-aminobenzoic acid derivatives with serum proteins: drug binding efficacy and protein structural analysis

Two aminobenzoic acid derivatives DAB-0 and DAB-1 showed distinct biological properties on murine bladder cancer (BCa) cell line MB49-I. In contrast to DAB-1, DAB-0 does not possess any anti-inflammatory activity and is less toxic. Furthermore, DAB-0 does not interfere with INFγ-induced STAT1 activation and TNFα-induced IκB phosphorylation, while DAB-1 does. In order to rationalize these results, the binding efficacy of DAB-0 and DAB-1 with serum proteins such a human serum albumin (HSA), bovine serum albumin (BSA) and beta-lactoglobulin (β-LG) was investigated in aqueous solution at physiological pH. Multiple spectroscopic methods and thermodynamic analysis were used to determine the binding efficacy of DAB-0 and DAB-1 with serum proteins. Drug-protein conjugation was observed via through ionic contacts. DAB-1 forms stronger adducts than DAB-0, while β-LG shows more affinity with the order of stability β-LG > BSA > HSA. The stronger complexation of DAB-1 with serum proteins might account for its biological potential and transport in the blood. The binding efficacy ranged from 40 to 60%. Major alterations of protein secondary structures were detected upon drug complexation. Serum proteins are capable of delivering DAB-1 in vitro.Communicated by Ramaswamy H. Sarma

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<0.0001) and this was reversed through addition of GLZ (p<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<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<0.001), and reduced cell viability vs. controls (p<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 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.