Injection Of Luciferin Research Articles

Abstract 4063: Adoptive transfer of NF-kB p50 knockout immature myeloid cells shows a trend towards slower glioblastoma tumor growth in an orthotopic mouse model

Abstract Background: Glioblastoma (GBM) is a universally fatal brain tumor. Tumor-associated macrophages (TAMs) comprise up to 40% of GBM tumors and predominantly demonstrate an M2 phenotype, favoring tumor growth while suppressing host immune response. Previously, we showed that in p50-/- mice lacking the repressive NF-κB subunit, GBM TAMs are biased toward the pro-inflammatory M1 phenotype, limiting GBM tumor growth by increasing activated tumor T cells. The present study aims to determine whether adoptively transferred immature myeloid cells lacking p50 (p50-IMC) localize to mouse GBM tumors and slow tumor growth. Methods: Lineage-negative bone marrow (BM) cells were isolated from C57BL/6 (B6) p50-/- mice, expanded, and treated with Macrophage-Colony Stimulating Factor (M-CSF) for 24h to produce p50-IMC. BM-derived myeloid cells from both wild-type (WT) and p50-/- mice were subjected to analysis of M1 (TNFα, IL-12b) and M2 (Fizz1, Mannose Receptor) markers via quantitative PCR after exposure to M-CSF for 24h or after M2-polarization via five days in M-CSF and then two days in IL-4. B6-derived GL261-Luc GBM cells were inoculated intracranially into wild-type B6 mice, and four days post-implantation, mice were given 5-fluorouracil to deplete endogenous leukocytes. Mice then received either no additional treatment or three doses of ten million p50-IMCs 9-, 11-, and 14-days post-implantation, and tumor growth was monitored via bioluminescence, measured following luciferin injection using the Spectrum in vivo imaging system. The number of p50-IMCs localizing to the GBM-containing brain was determined by CFSE-dye-labeling of IMC followed by flow cytometry analysis 24h after injection. Results: Both TNFα and IL-12b were increased in p50-/- cells after 5 days M-CSF, and increased IL-12b expression was maintained even after M2-polarization with IL-4. Of the 10 million injected p50-IMCs, 0.07% of those cells were found in the brain. Despite such a small number of p50-IMCs localizing to the tissue of interest, inhibition of GBM growth was observed. Mice receiving the p50-IMC treatments (n = 4) demonstrated a 2.3- and 2.4-fold reduction in tumor growth at 15 (p = 0.13) and 18 (p = 0.14) days post-implantation, respectively, compared to control mice (n = 5). At 21 days post-implantation, tumor size was effectively equivalent between the two groups (p=0.76). p50-IMC treatment did not correlate with improved survival. Conclusion: Loss of p50 skews myeloid cells toward a proinflammatory phenotype, even under M2-polarizing conditions. Adoptive transfer of p50-IMC transiently impairs GBM growth in an orthotopic mouse model. Although statistical significance was not attained, this may be due to the small number of animals used in this experiment. Further investigation is needed to evaluate the full potential of p50-IMC as an immunotherapy option for GBM patients. Citation Format: Patrick J. Beck, Theresa Barberi, Alan D. Friedman. Adoptive transfer of NF-kB p50 knockout immature myeloid cells shows a trend towards slower glioblastoma tumor growth in an orthotopic mouse model. [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 4063.

Abstract 1848: Targeting FZD7-positive cancers using a novel antibody-drug conjugate

Abstract WNT signaling is frequently dysregulated in cancers. However, pan-WNT inhibitors regularly induce adverse effects in patients, mostly notably in bone, creating a need for more specific WNT-pathway targeting strategies. Frizzled-7 (FZD7), a cell-surface receptor for WNT proteins, is a strong target candidate due to its high expression pattern in many tumor types (including but not limited to breast, ovarian, liver, gastric, and skin cancers) and low to modest expression in few normal adult tissues. We validated FZD7 protein expression in primary patient melanomas and breast and ovarian tumor samples, and developed an antibody-drug conjugate (ADC) that targets human FZD7, hereafter referred to as “FZD7 ADC.” Our ADC consists of a chimeric human-mouse IgG1 antibody conjugated to four molecules of antimitotic drug, monomethyl auristatin E (MMAE), by cleavable valine-citrulline linkers. By flow cytometry, we confirmed that the antibody component binds FZD7 and does not cross-react with the other nine human FZD receptors, FZD(1-6,8-10). We have identified MA-148 and PA-1 as human ovary-derived cancer cell lines responsive to our ADC. We also generated a negative control line, MA-148 FZD7-KO, by CRISPR/Cas9 knockout. In a cell viability assay, we demonstrated FZD7 ADC efficacy in inducing direct, FZD7-dependent cytotoxicity. A single dose of ADC killed MA-148 and PA-1 cells in vitro, with an IC50 of ~0.76 ug/mL (~5 nM) in both lines. MA-148 FZD7-KO cells exhibited an IC50 of ~9 ug/mL (~60 nM). Here, we established a therapeutic window in which our ADC specifically kills FZD7-positive cells. We are currently evaluating FZD7 ADC tumor-killing efficacy in vivo. Because our ADC only binds human FZD7, and not mouse Fzd7, we are utilizing a xenograft model in female nude mice. In a preliminary three-armed study, we established subcutaneous human MA-148-Luciferase (Luc) tumors in mice and treated with a PBS control, 1 mg/kg FZD7 ADC (~0.15 nmole), or 3 mg/kg FZD7 ADC (~0.5 nmole), n = 3-5 per group. We performed an identical study with mice bearing MA-148 FZD7-KO-Luc tumors in parallel. Treatments were delivered twice per week by tail vein injection. Tumor size was measured weekly by an IVIS Spectrum after intraperitoneal luciferin injection. After eight doses over 27 days, MA-148-Luc tumors treated with 3 mg/kg FZD7 ADC completely or partially regressed compared to the control tumors (p = 0.0101 by one-way ANOVA and Tukey's multiple comparisons test). The 1 mg/kg dose did not produce a therapeutic effect in the MA-148-Luc. Importantly, the negative-control MA-148 FZD7-KO-Luc tumors treated with 3- or 1 mg/kg of ADC were not statistically different from the control tumors. Here, we have established a therapeutic FZD7 ADC dose for tumor regression and demonstrated its specificity to FZD7-positive tumors in vivo. Our data show that the FZD7 ADC is an effective strategy to combat cancers expressing FZD7. Citation Format: Myan Do, Christina C. Wu, Stephen Adams, Dennis Carson, Sunil Advani, Karl Willert. Targeting FZD7-positive cancers using a novel antibody-drug conjugate [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 1848.

MP17-19 RECRUITED T CELLS PROMOTE THE BLADDER CANCER METASTASIS VIA UP-REGULATION OF THE ESTROGEN RECEPTOR β/IL-1/C-MET SIGNALS

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II (MP17)1 Apr 2020MP17-19 RECRUITED T CELLS PROMOTE THE BLADDER CANCER METASTASIS VIA UP-REGULATION OF THE ESTROGEN RECEPTOR β/IL-1/C-MET SIGNALS Le Tao, Jialin Meng*, Edward M. Messing, Jean V. Joseph, Chawnshang Chang, Jianxin Qiu, and ShuYuan Yeh Le TaoLe Tao More articles by this author , Jialin Meng*Jialin Meng* More articles by this author , Edward M. MessingEdward M. Messing More articles by this author , Jean V. JosephJean V. Joseph More articles by this author , Chawnshang ChangChawnshang Chang More articles by this author , Jianxin QiuJianxin Qiu More articles by this author , and ShuYuan YehShuYuan Yeh More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000842.020AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Bladder cancer (BCa) is the fourth most commonly occurring cancer in men and is the seventh leading cause of death from cancer among men and the eleventh among women in the United States. Previous studies have shown that higher ERβ expression rates in BCa are positively correlated to higher grades and stages, as well as muscle invasiveness, and that both ERα and ERβ signals affect BCa development. Immune cells may infiltrate into different types of cancers and influence cancer progression. An early study demonstrated that CD4+ T cells could promote prostate cancer invasion. In BCa, one report indicated that inflammation elicited from the tumor microenvironment (TME) may be able to influence tumor progression. METHODS: Tissue samples were obtained from 20 patients who were diagnosed with BCa. ERβ (PLKO.1-puro-shERβ) was constructed with the target sequence 5′ GCGAGTAACAAGGGCATGGAA-3′ into lentiviral pLKO.1 vector as ERβ sh-RNA. Chromatin immuno-precipitation (ChIP) assays were used to calculate the binding of ERβ-DNA complex. Female nude mice (6–8 week old) were purchased from NCI and implanted with 5637 cells that were engineered to express a luciferase reporter gene (lentiviral-luciferase). Images were detected 20 min after intraperitoneal injection of luciferin using a fluorescent imager (IVIS Spectrum, Caliper Life Sciences) at 5 different time points (3, 4, 5, 6 and 7 weeks after tumor cells implantation). RESULTS: Results from an in vitroco-culture system found that BCa recruited more CD4+ T cells than did normal bladder cells. The recruiting of T cells to BCa tissues may increase the proliferation and invasion of BCa cells. Mechanistic studies revealed that infiltrating T cells stimulate BCa estrogen receptor beta (ERβ) signaling and consequently increase the expression of the MET proto-oncogene, receptor tyrosine kinase (c-MET), through either direct binding to its promoter or viamodulation of IL-1 expression. Interruption of ERβ/c-MET or ERβ/IL-1/c-MET signaling viaERβ-shRNA, IL-1 antagonist, or the c-MET inhibitor, SU11274, could partially reverse the T cell-enhanced BCa cell invasion and proliferation. Finally, the mouse BCa model with xenografted BCa 5637 cells with T (HH) cells confirmed the results of in vitroco-culture studies. CONCLUSIONS: Infiltrating T cells could promote BCa metastasis viamodulation of the ERβ/c-MET or ERβ/IL-1/c-MET signaling pathways. Source of Funding: This work was partially supported by the University of Rochester CTSI (UL1 TR000042), and the National Natural Science Foundation of China. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e233-e233 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Le Tao More articles by this author Jialin Meng* More articles by this author Edward M. Messing More articles by this author Jean V. Joseph More articles by this author Chawnshang Chang More articles by this author Jianxin Qiu More articles by this author ShuYuan Yeh More articles by this author Expand All Advertisement Loading ...

P5437The BNP reporter mouse by knock-in technology is useful for the analysis of mechanism in reactivation of BNP in adult heart

Abstract Background It has been thought BNP is induced by undetermined stretch-activated receptors, however, which receptor is associated remains unknown. The stretch-activated receptors include mechanically gated channels, which can be activated by a mechanical stimulus alone, and mechanically modulated channels, which require nonmechanical stimuli such as agonists. It has been recently shown that 1.1kb segment of mouse NPPB promoter dose not reproduce the pattern of reactivation of BNP in adult heart, although it could monitor the expression of BNP in neonatal cardiomyocytes. Purpose Our aim is to develop a true BNP reporter mouse and examine whether this mouse is useful or not for the investigation of BNP reactivation mechanism in adult heart and for the measurement of serum-induced BNP expression in patients with heart failure. Methods We generated the BNP reporter mice by knocking luciferase cDNA in the initiation site of NPPB. In vivo imaging of luciferase was performed in the BNP reporter mice after the intraperitoneal injection of luciferin. The luciferase activity was examined in neonatal cardiomyocyte, isolated adult cardiomyocytes, adult cardiac dissected tissue with or without 120–150% stretch or angiotensin II stimulation. Left anterior descending (LAD) coronary artery was ligated to study myocardial infarction. Cardiac dissected tissue segments from the BNP reporter mouse were incubated for 8 hours with 20% serum from patients with or without heart failure and the luciferase activity was measured after homogenization. Results The in vivo imaging system showed the activity of BNP was high in 1 day-old neonates and the reactivation of BNP in the adult heart after LAD ligation could be monitored by the luciferase activity (figure). The treatment of Ang II could increase the activity of pBNP more than ten folds in heart tissue from adult mice. On the other hand, the 120–150% stretch did not show any effect on the activity of pBNP in this system. We could not observe any activation of pBNP in cultured neonatal or adult cardiomyocytes demonstrated by immunostain with antibodies against luciferase after 120–150% stretch. Interestingly, the luciferase activity was extensively higher in cultured heart tissue segments from the BNP reporter mice after the treatment of serum from patients with heart failure than without heart failure. In vivo imaging of BNP reporter mice Conclusion These results indicate the BNP reporter mouse by knock-in technology is useful for the analysis of mechanism in reactivation of BNP in adult heart and the elevation of BNP in patients of heart failure partly due to the serum-derived induction of BNP from heart.

An Inhibitory Ligand of Neuropilin 2 Blocks Pancreatic Cancer Progression and Impedes Tumor Angiogenesis

The proliferation and branching pattern of blood vessels and nerves is mediated during development, in part, by the interaction between the Neuropilin‐2 (NRP2) receptor and its endogenous secreted ligand, Semaphorin‐3F (SEMA3F). This interaction induces a repulsive signal, which participates in the guidance of both neuronal axons and blood vessels (veins and capillaries). Furthermore, upon binding vascular endothelial growth factor‐A (VEGFA), NRP2 forms a complex with the VEGF Receptor‐2 (VEGFR2), which mediates a potent pro‐angiogenic signal. Although NRP2 expression is dormant in adult endothelial tissue, its expression is upregulated in a variety of physiological and pathological conditions. Interestingly, pancreatic ductal adenocarcinoma (PDAC) induces the over‐expression of NRP2 in endothelial cells and in the pancreatic cancer cells themselves. Consequently, this project is aimed at studying the anti‐angiogenic and repulsive effects of SEMA3F in order to inhibit the growth of the primary tumor and the incidence of liver metastases, respectively, in PDAC. Our preclinical trials utilized an orthotopic, syngeneic luciferase‐labelled mouse pancreatic cancer xenograft model in C57BL/6J mice. The delivery of SEMA3F was performed through a systemic intravenous injection of SEMA3F‐encoding adenovirus. Next, we performed a series of three independent preclinical trials, in which we compared the disease progression of PDAC in SEMA3F‐virus treated mice (n=5) compared to control‐virus treated mice (n=5). In the Prevention Trial, the mice received the virus injection three days prior to receiving the PDAC implantation. The Experimental Prevention Trial followed the same timeline, but the PDAC cells were injected into the spleen in order to accelerate the incidence of liver metastases. Lastly, in the Intervention Trial the SEMA3F‐encoding virus was injected after PDAC tumors were established in order to simulate a more realistic clinical setting. Tumorigenicity was monitored during the trials via bioluminescence imaging following systemic luciferin injection. Mice were euthanized and necropsied 20 days after tumor injection, and livers and pancreata were weighed to asses primary tumor growth as well as metastatic burden. Furthermore, the tumors were stained with antibodies directed against CD31 and KI67, in order to quantify tissue microvessel density and cell proliferation respectively. Qualitative analysis of the livers consisted in staining them with antibodies directed against PDX1 and NRP2, in order to confirm that the metastases are of pancreatic origin and that they retain their NRP2 expression after migrating to the liver. Our results indicate a potent anti‐angiogenic effect of SEMA3F compared to control across all experimental groups. Furthermore, a reduced incidence of metastases to the liver supports the chemorepulsive effects of SEMA3F. Overall, SEMA3F shows great potential as a novel therapy in PDAC.Support or Funding InformationNIH CA155728This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Therapeutic Effect of Immune-Modifying Microparticles in an Acute Graft-Versus-Host Disease Model

Introduction Inflammatory monocytes are recruited to target organs during acute Graft versus Host Disease (aGVHD). As seen in other autoimmune disorders, inflammatory monocytes play an important role in antigen presentation and cytokine production. These actions allow for a sustained activation and proliferation signal to T-cells. Objective Previous studies have shown that IMP treatment in mouse models of colitis, encephalitis, myocardial infarction and peritonitis markedly reduced monocyte accumulation in the affected end-organs – promoting tissue repair; reducing disease symptoms and increasing survival. Therefore, our objective was to test clinical outcomes after IMP treatment in a mouse model of aGVHD. Methods Murine aGVHD model: BALB/c mice were given 800 cGy total body irradiation, irradiated BALB/c mice were transplanted with 5 × 106 C57BL/6 bone marrow cells and 1 × 106 C57BL/6 spleen cells via tail vein. IMP treatment: IMPs were made with PLGA (Phosphorex Inc, Hopkinton MA) was administered to the recipient mice (1.4 mg/kg body weight) by IV daily starting from day 5 to day 10 after bone marrow transplantation (BMT). PBS at the same volume was used as vehicle control. In vivo bioluminescence imaging: Mice were given an intraperitoneal injection of luciferin (150 mg/kg body weight) and then anesthetized and imaged using the IVIS Imaging system (Xenogen). Imaging data were analyzed and quantified with Living Image Software (Xenogen). Results IMP treated mice had significantly less severe acute GVHD symptoms (average score of 2.48) than the untreated BM+Sp group (average score 3.96) starting at the time of IMP treatment (days 5-10) and remained with significantly reduced symptoms for the 30 day course (Figure 1). IMP treatment also rescued BM+Sp mice from aGVHD associated mortality with a 30-day overall survival of 62% compared to 4% in the untreated BM+Sp group (Figure 2). Intestinal tissue from the IMP treated mice compared to the BM+Sp mice demonstrated less evidence of aGVHD (an average score of 1.25 and 2.75, respectively). Hepatic tissue from the IMP treated mice compared to the BM+Sp mice demonstrated less evidence of aGVHD (an average score of 1.5 and 2.42, respectively) (Figure 3). IMP treatment also significantly reduced INF-γ levels in the intestinal tissues of treated mice compared to untreated BM+Sp mice. In the mice infused with lymphoma cells (A20-luc), IMP treatment reduced aGVHD symptoms and death while preserving the GVL effect. (Figure 4) Conclusions Our results demonstrate that IMPs significantly reduce symptoms and mortality in a murine model of aGVHD while preserving GVL. The reduction in inflammatory monocytes with IMPs leads to a reduction in inflammatory cytokines, hepatic lymphocyte infiltration and intestinal mucosal denudation. These findings highlight the potential of IMP therapy as a specific and potentially safe treatment in acute GVHD.

A Novel Monoclonal Antibody Combination Plus DC/AML Fusion Vaccine Eradicates AML in an Immunocompetent Murine Model

Introduction: Acute myeloid leukemia (AML) is a lethal hematologic malignancy in which standard therapy is often inadequate in sustaining durable remissions. In part due to an immunosuppressive milieu characterized by the upregulation of checkpoint inhibitory pathways leading to T cell anergy, ineffective antigen (Ag) presentation, and increased numbers of myeloid derived suppressor cells (MDSCs). We have pioneered a novel vaccine that targets an array of leukemic antigens by fusing whole patient-derived AML cells to autologous dendritic cells (DCs) in the presence of polyethylene glycol (PEG). In a phase I/II clinical trial in AML, vaccination induced an expansion of tumor-specific T cells and resulted in prolonged remissions in a subset of patients. Checkpoint blockade is being investigated in combination with cancer vaccines to overcome the suppressive effects of the tumor microenvironment on cytotoxic tumor-specific T cells. Here we describe the addition of a novel combination of checkpoint inhibitors with DC/AML fusion vaccine in an immunocompetent murine model.Methods/Results: C57BL/6J mice underwent retro-orbital inoculation with 50,000 syngeneic luciferase and mCherry labeled TIB-49 AML cells. 24 hours later a cohort of mice was treated with 100,000 DC/AML irradiated fusion cells. An additional cohort was treated with 6 doses of a combination of anti-PD1/anti-TIM3/anti-RGMb mAbs administered IP every 3 days with and without vaccine. Control mice were treated with appropriate isotype control. AML burden was evaluated in peripheral blood (PB) 14 days post inoculation using flow cytometric analysis for mCherry expression. AML cells were present in PB of control mice and mice treated with check point inhibitors alone (mean 1.2% and 1.5% respectively). Lower disease burden was detected in mice treated with the DC/AML fusion vaccine (0.5%) or combination of fusion vaccine with check point inhibitors (0.5%).Furthermore, to measure tumor-specific T cell response, PB cells were cultured in the presence of autologous TIB-49 tumor lysate for 3 days and analyzed for intracellular IFN-γ expression using multicolor flow cytometric analysis. Mice treated with combination of DC/AML fusion vaccine and checkpoint inhibition showed a significant increase in IFN-γ expression by CD8+ T cells with mean values of 6.4%. Mice treated with vaccine, checkpoint inhibitors alone or IgG control demonstrated mean levels of 3.4%, 1.9% and 1.9% respectively (n=5; p=0.01).Additional immunologic assessment was performed from PB of surviving animals 37 days after tumor challenge. Combination treatment with DC/AML fusion vaccine and PD1/anti-TIM3/anti-RGMb mAbs led to a statistically significant increase in CD4/CD44+/CD62L- memory T cells with concurrent decrease in naïve CD8/ CD44-/CD62L+ T cells as compared to single agent treatments. Furthermore, a statistically significant decrease in CD4+CD25+FOXP3+ Tregs was detected after combination treatment compared to single treatment groups.The mice were followed for survival and disease progression using BLI imaging. All control mice developed visible AML by luminescence following luciferin injection as well as symptomatic disease requiring euthanasia by day 31 after initial challenge with tumor cells (Figure 1). Mice treated with the anti-PD1/anti-TIM3/anti-RGMb mAbs alone demonstrated a survival benefit but all required euthanasia by day 44. 3 of 5 mice treated with vaccine alone remain disease free at more than 65 days. And strikingly, the entire cohort of mice treated with the combination of DC/AML fusion vaccine and PD1/anti-TIM3/anti-RGMb mAbs remain alive and disease free in this aggressive AML model.Conclusion: In the current study we have demonstrated the capacity of a combination of PD-1, TIM-3 and RGMb checkpoint inhibition to create an enhanced environment for immune response to the DC/AML fusion vaccine in an immunocompetent murine AML model. Treatment with this combination led to an increase in tumor specific T cell immunity, decrease in circulating Tregs and shift toward a memory phenotype. Most significantly, mice who received the combination treatment remain disease-free several months post inoculation. This synergistic approach has promising translational potential and a phase 1 clinical trial is planned. [Display omitted] DisclosuresRosenblatt:Bristol-Myers Squibb: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding. Stone:Astellas: Consultancy; Otsuka: Consultancy; Arog: Consultancy, Research Funding; Argenx: Other: Data and Safety Monitoring Board; Pfizer: Consultancy; Fujifilm: Consultancy; Celgene: Consultancy, Other: Data and Safety Monitoring Board, Steering Committee; Orsenix: Consultancy; Amgen: Consultancy; Merck: Consultancy; Agios: Consultancy, Research Funding; AbbVie: Consultancy; Novartis: Consultancy, Research Funding; Jazz: Consultancy; Cornerstone: Consultancy; Ono: Consultancy; Sumitomo: Consultancy. Freeman:Roche: Patents & Royalties; Merck: Patents & Royalties; Origimed: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers-Squibb: Patents & Royalties; Xios: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers-Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Patents & Royalties; Dako: Patents & Royalties; Boehringer-Ingelheim: Patents & Royalties; Roche: Membership on an entity's Board of Directors or advisory committees; EMD-Serono: Patents & Royalties; AstraZeneca: Patents & Royalties.

Abstract 3533: Elevated expression of heme related proteins in viable tumor cells treated with vascular disrupting agent, combretastatin A-4 phosphate in xenograft models of NSCLC

Abstract Tumors cannot grow without a supply of oxygen and nutrients. Hence, targeting tumor vasculature with Vascular Disrupting Agents (VDAs) represents a promising therapeutic strategy, especially for the treatment of solid tumors. However, VDAs cause central necrosis, leaving a rim of viable tumor cells that often eventually lead to tumor recurrence and development of resistance. Here, we aim to better understand the underlying molecular mechanisms leading to the acquisition of VDA-resistance and its correlation with the extent of hypoxia and respiratory functions. A well-known VDA, Combretastatin A-4 phosphate (CA4P), was used for the study. First, a NSCLC cell line expressing luciferase was implanted into the right flanks of female SCID mice. Next, CA4P was administered intraperitoneally. The effect was monitored 3 and 24 hours post injection of CA4P through BLI (Perkin Elmer's IVIS Lumina III) after subcutaneous injection of luciferin. After sacrificing the mice, the tumors were harvested, formalin-fixed, and paraffin-embedded for Immunohistochemistry (IHC). Our BLI data indicated significant decrease in radiance 3 hour post treatment (total flux in photons per second) which remained so at 24 hours, when luciferin was re-administered. Our IHC data indicated enhanced levels of enzymes involved in heme biosynthesis, uptake and degradation, and hemoproteins involved in oxidative phosphorylation. Furthermore, we examined the effect of a mitochondrial targeting agent (MTA) that undermined the expression of these proteins, on the progression of subcutaneous tumor xenografts. Citation Format: Sanchareeka Dey, Sharda Kumari, Sarada Preeta Kalainayakan, Poorva Ghosh, Li Zhang, Li Liu. Elevated expression of heme related proteins in viable tumor cells treated with vascular disrupting agent, combretastatin A-4 phosphate in xenograft models of NSCLC [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 3533.

Application of fluorescent probe targeting transferrin receptor in glioma orthotopic animal models

Objective To validate the specificity of CRT-Cy5 peptide in glioma animal model and provide experimental basis for its clinical application. Methods The human glioma cell line U87 was transfected with lentivirus H113 to obtain U87-MG H113 that stably expressed luciferase. The U87-MG H113 cells were injected into the caudate nuclei of mice.After 3 weeks, the signal of luciferase was detected by small animal imaging instrument after intraperitoneal injection of luciferin to testify U87-MG H113 glioma model.Twelve animals of orthotopic glioma were divided into PBS, Cy5-CRT, Cy5-CG7C groups according to random number table. PBS, Cy5-CRT and Cy5-CG7C (all in a dose of 0.1 mol/L, 200 μl) were intravenously injected into the mice of each group.The signal of luciferase was detected by small animal imaging instrument after intraperitoneal injection of luciferin. The Cy5 signal was detected at 24 h, 36 h, 48 h post injection. Results (1) We successfully established cell lines that stably expressed luciferase whose intensity was positively correlated with the number of cells. (2) Three weeks later, 12 animals of orthotopic glioma were divided into PBS, Cy5-CRT, Cy5-CG7C groups. At 36 h post injection, the signal of luciferase of PBS, Cy5-CRT, Cy5-CG7C groups was 5.78±0.16、44.16±0.33、5.34±0.17, respectively. Signal analysis showed an 8 times increase of Cy5 signal intensity in Cy5-CRT group than the other 2 groups (P<0.005). Conclusion Cy5-CRT optical probe is enriched in glioma tissues rather than normal brain tissues and has potential values of clinical application. Key words: Glioma; Molecular probes; Models, animal; Transferrin; Molecular imaging

Murine breast cancer mastectomy model that predicts patient outcomes for drug development

BackgroundDespite massive expenditures in preclinical studies, many breast cancer agents show efficacy in murine models but fail in human trials. In humans, metastatic disease determines survival, but preclinical murine models only evaluate drug efficacy against the primary tumor. We hypothesized that evaluating efficacy against metastatic breast cancer would more efficiently predict efficacy in a murine model than evaluating the primary tumor alone. This study (1) critically evaluated a murine tumor removal model with metastatic tumor burden quantification for breast cancer preclinical trials and (2) validated the model with an agent that previously passed preclinical trials but failed human trials. Materials and methodsTumorectomy and Halsted (radical) mastectomy procedures after inoculation of 4T1-luc2 cells were compared. The effect of AZD0530, an oral Src inhibitor that passed preclinical trials but failed human trials, was evaluated using an inoculation model with/without Halsted mastectomy. ResultsSignificant amounts of residual disease were confirmed by bioluminescence (P = 0.003) and 100% developed local recurrence after tumorectomy versus 14% (P = 0.005) after Halsted mastectomy. Bioluminescence value at 15 min after luciferin injection highly correlated with peak except for 24 h after injection. AZD0530 significantly suppressed primary tumor burden compared with no treatment (P = 0.002); but not in lung metastases. In a Halsted mastectomy model, AZD0530 had no efficacy against lung metastases or difference in survival. ConclusionsWe critically evaluated and established a murine mastectomy model to evaluate metastatic tumors. It provides a new model for preclinical drug development that mimics the human adjuvant setting.

Abstract 1832: Bioluminescent pharmacokinetics of luciferin in preclinical brain metastases of breast cancer models

Abstract Background: Approximately 20% of breast cancer patients with disseminated disease will develop brain metastases. Preclinical models of brain metastases of breast cancer rely on ex-vivo histology to evaluate drug efficacy. However, bioluminescence imaging allows more precise quantification of tumor burden and progression through counting photons per second without requiring animal sacrifice. This methodology has not readily been applied to hematogenously derived brain metastases models given the heterogeneity of tumor burden and growth. Herein we demonstrate repeatable methodology to quantify brain metastases of breast cancer progression which is verified with histology. Methods: Approximately 1.75 x 105 JIMT-1 (n=3) and 231-BrLuc (n=4) brain seeking subclones were injected intracardially to produce metastatic brain lesions. Mice were given 150 mg/kg of luciferin IP and luminescence was captured every 2-minutes for 60 minutes starting 24 hours after cell implantation. Radiance (photons per second per square centimeter per steridian) was plotted to observe peak luminescence. Imaging was repeated twice weekly to evaluate progression until euthanasia. Results: Following intracardiac injection, both brain seeking metastatic models produced a maximum luminescence signal between 14-20 minutes after luciferin injection. For all subsequent imaging, 5 minute imaging between 15-20 minutes after luciferin injection was used. Longitudinally, both tumor cell lines produce bioluminescence 24 hours after cell injection, which is used to ensure tumor implantation and randomization. The BLI signal decreases to undetectable limits in both models between 3-7 days after cell implantation. Re-emergence of signal occurs on day 14 for the JIMT-1 line and day 21 for 231-Br line. JIMT-1 bioluminescence on day 14 begins at ~105 photons/sec/cm2/sr and then over time signal increases with an ~slope of 5.6x105 until reaching a maximum of 107 units on day 28. 231-Br bioluminescence on day 21 begins at ~104 photons/sec/cm2/sr and then over time signal increases with an ~ slope of 1.1x106 until reaching a maximum of ~107 on day 53. Conclusion: To accurately measure bioluminescence as a surrogate for tumor burden in hematogenously implanted metastases, the optimal circulation time is approximately 15 minutes with a 5 minute imaging period. Bioluminescence of the 231Br line initially starts at a lower magnitude and later in time than the JIMT-1 line, but slopes of both intracardiac models are similar and result in similar bioluminescent curves. Citation Format: Neal Shah, Chris E. Adkins, Afroz S. Mohammad, Paul R. Lockman. Bioluminescent pharmacokinetics of luciferin in preclinical brain metastases of breast cancer models [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 1832. doi:10.1158/1538-7445.AM2017-1832

Abstract 5898: The consomic xenograft model identifies genetic changes in the tumor microenvironment that alter the growth and metastasis of head and neck cancers

Abstract Background: The tumor microenvironment (TME) is known to impact tumor growth, metastatic potential, and treatment response. Nearly all studies of head and neck cancer (HNC) have focused on somatic mutations in the malignant cells. We hypothesized that genetic determinants limited to the microenvironment would influence HNC growth and metastatic potential. Approach: To demonstrate the impact of genetic differences in the TME on HNC cell line in vivo growth we utilized a novel tool, the consomic xenograft model (CXM). A consomic rat has an entire chromosome substituted into the isogenic background of another inbred strain by selective breeding. Use of immunodeficient (IL2Rγ-/-) consomic rats allows one to study the influence of stromal genetics on tumor biology without the confounding effect of differences in the immune system through the orthotopic implantation of cancer cells into different consomic rat strains. In this system, any differences in tumor growth or metastases are due to differences in the TME rather than cancer cells or immune response. We utilized SS and SS.BN3 consomic rat strains, previously shown to affect the growth of breast tumors, to study the effects of the TME on HNC tumor growth using two well-characterized HPV negative HNC cell lines, SCC-6 (base of tongue derived) and SCC-22b (derived from a hypopharyngeal cancer that had metastasized to lymph nodes). Both cell lines were modified to stably express luciferase. HNC cells were inoculated into the tongue of SS and SS.BN3 animals and tumor growth was monitored by biophotonic imaging after luciferin injection. Results: A significant difference in the tumor growth was seen between rat strains for both cell lines, with the SS.BN3 rats exhibiting less tumor growth and metastasis. Median luciferase activity from baseline increased by 4.1-fold vs. 1.1 fold in SCC-6 tumors in SS vs SS.BN3 rats, respectively (p&amp;lt;0.03). SCC-22B tumors demonstrated a significant difference in tumor size as well, with median luciferase activity from baseline 12.7 vs 4.4 fold on day 26, for SS vs SS.BN3, respectively (p&amp;lt;0.05). A significant differences in lung metastases was also seen between strains. Lung metastases were seen in 88% of SS and 0% of SS.BN3 rats (n=6 per group) inoculated with SCC-6 (p&amp;lt;0.02) and in 75% of SS and 30% of SS.BN3 rats (n=10 per group) inoculated with SCC-22b (p&amp;lt;0.08). Despite a non-statistically significant difference in the number of SCC-22b inoculated animals with lung metastases, there was a significantly higher metastatic burden as measured by luciferase signal, with the median signal 6.8 fold higher in SS as compared to SS.BN3 (p&amp;lt;0.03). Conclusions: The use of the CXM model demonstrates an important role for the TME in the growth and metastatic spread of HNC cell lines. This model allows for future congenic mapping to identify the causative genetic variants in the TME mediating the HNC changes in tumor growth and metastasis. Citation Format: Michael W. Straza, Amy Rymaszewski, Kwangok P. Nickel, Anne Frei, Anirban Chatterjee, Rachel Schlaak, Amit Joshi, Michael Flister, Randy J. Kimple, Carmen Bergom. The consomic xenograft model identifies genetic changes in the tumor microenvironment that alter the growth and metastasis of head and neck cancers [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 5898. doi:10.1158/1538-7445.AM2017-5898

Abstract 3499: Triptolide pro-drug decreases tumor burden and halts tumor progression in a murine model of acute myeloid leukemia

Abstract Introduction Standard treatment for acute myelogenous leukemia (AML) has not changed over the past few decades and relies primarily on the traditional “7 + 3″ regimen of daunorubicin, administered over 3 days and Cytarabine, administered over 7 days. This chemo-intensive regimen is poorly tolerated by patients and is characterized by a high rate of relapse and treatment failure. Triptolide is a diterpenoid tri-epoxide compound isolated from the Chinese herb Tripterygium wilfordii. The water-soluble pro-drug of triptolide, Minnelide has been shown by our group to be highly effective in a number of pre-clinical models of solid tumors and is currently undergoing Phase I Clinical trial for gastrointestinal tumors. Interim analysis of the Phase I data has shown that the equivalent mouse dose of 0.2 mg/kg/day is well tolerated by patients with no reported dose limiting toxicity. Methods Primary AML apheresis samples from patients with acute myeloid leukemia and multiple AML cell Lines (THP1, KG1, Kasumi1, HL-60) were treated with Triptolide at doses from of 2.5 nM to 50nM and cell viability was measured using a formazan based colorimetric assay. Apoptosis was measured using Annexin V via flowcytometry and colony forming ability of AML cell lines was measured using a methylcellulose based assay. To generate an engraftment model of AML, NOD.Rag1-/-;γcnull (NRG) animals expressing human interleukin-3 (IL-3) and human GM-CSF (NRGS) were injected luciferase expressing THP1 cells after sublethal irradiation of 250 cGy. These animals were then serially evaluated using In Vivo Imaging System (IVIS) and leukemic burden was calculated the total bioluminescent signal after intra-periotneal injection of luciferin. Treatment with vehicle or Minnelide at a dose of 0.1mg/kg/day and 0.15 mg/kg/day was started on the 10th day after confirming engraftment using IVIS. Results Triptolide at a dose range of 2.5 nM to 50 nM produced a dose and time dependent killing of leukemic cells in both cell lines and primary AML patient samples. The IC-50 for THP1, KG1, Kasumi1 and HL-60 cell lines with triptolide treatment at 48 hours were 5 ± 0.8 nM, 8 ± 0.7 nM, 7.2 ± 0.6 nM, 10 nM ± 2 nM respectively. Treatment with triptolide at a dose of 2.5 nM induced cell death and apoptosis as measured by Annexin V posiitvity in primary AML apheresis samples. Colony formation by THP1 cells and KG1 cells was completely abrogated by treatment with Triptolide at 2.5 nM and 25 nM respectively. Vehicle treated mice showed a rapid progression of disease burden when compared to Minnelide treated mice. At a dose of 0.1 mg/kg/day and 0.15 mg/kg/day, these mice had no appreciable increase in leukemic burden over normal mice when assessed by IVIS. Conclusion We show that Minnelide induces cell death at therapeutically relevant concentrations in vitro and decreased leukemic burden in a murine model of AML. Minnelide may emerge as a novel therapeutic strategy in treating patients with AML. Citation Format: Bhuwan Giri, Sulagna Banerjee, John George, Shrey Modi, Vineet Kumar Gupta, Mahendra K. Singh, Vikas Dudeja, Ashok K. Saluja. Triptolide pro-drug decreases tumor burden and halts tumor progression in a murine model of acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3499.

Abstract 4409: Assessment of sonic hedgehog pathway inhibition in a novel orthotopic xenograft bladder cancer murine model

Abstract Purpose of the study: To develop a reliable and reproducible bladder cancer murine model for the evaluation of the SHH pathway. Introduction: The sonic hedgehog (SHH) signaling pathway has been shown to play an integral role in the maintenance and progression of bladder cancer. Smoothened inhibitors are currently used in the clinic for treatment of some skin cancers, however they have failed to be effective in bladder cancers in vitro. Blocking alternative downstream molecules of the SHH pathway may be an efficacious strategy for bladder cancer treatment. In vitro assays possess a number of limitations such as environmental differences, loss of heterogenicity and vascularization and artificial levels of growth factors and cytokines within the cell culture media. To overcome this animal models are needed to facilitate the study of carcinogenesis mechanisms. A new bladder cancer model is now required to evaluate experimental therapeutics as previous animal models have utilized the mistakenly identified cervical cancer cell line KU7. Methods: A novel orthotopic murine bladder cancer model was developed to assess the role of the SHH signalling pathway in tumor growth and disease progression. The model comprises the instillation of luciferase-expressing UM-UC3 human transitional cell carcinoma cells into the bladder of athymic nude mice following a poly-L-lysine irrigation of the bladder. Tumors are measured by bioluminescent imaging after intra-peritoneal injection of luciferin. Antisense oligonucleotides (ASO) targeted to the SHH pathway transcription factors, Gli family zinc fingers 1 and 2 (Gli1, Gli2), were assessed in this model. Immunohistochemistry (IHC) of proteins in the SHH pathway were evaluated and compared to our in-house tissue microarray (TMA) of clinical bladder cancer samples. Results: Tumors in this model grew in an average period of time of 40 days with high rates of engraftment and reproducibility. In vitro analysis of SHH pathway inhibition showed a decrease in bladder cancer cell viability and increase in apoptosis following Gli ASO treatment. This effect bypasses smoothened protein regulation which was found to be ineffective in some cell lines. Gli ASO treatment reduced tumor size, and prevented further growth, of UM-UC3 bladder cancers in this mouse model. IHC confirmed Gli2 knock-down and showed increased apoptosis and decreased proliferation in the in vivo tumors. Comparison of modelled tumors to clinical samples showed an increased and diffused Gli2 staining throughout the tissue in both cases. Conclusions: A murine intravesical model consisting of human tumors provides a robust and effective in vivo system for the assessment of cancer inhibiting treatments. Gli2 ASO proved to be a promising treatment for bladder cancer by inhibiting tumor growth and disease progression. Citation Format: Peter A. Raven, Sebastian Frees, Betty Zhou, Claudia Chavez-Munoz, Michael Cox, Alan So. Assessment of sonic hedgehog pathway inhibition in a novel orthotopic xenograft bladder cancer murine model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4409.

Quantification of adipose transfer viability using a novel, bioluminescent murine model

Fat grafting has highly variable long-term results. Research efforts to improve the reliability of fat grafting are limited by inefficient methods for evaluation of fat engraftment. In this work, we describe a novel animal model for the quantitative evaluation of fat grafting using invivo bioluminescence of adipocytes from luciferase-expressing mice. Subcutaneous adipose tissue from GFP and luciferase-expressing FVB mice were obtained. The samples were homogenized, decanted, and injected into the dorsal skin folds of wild-type FVB mice. Viability of the transferred tissue was examined over a 28-day time period with quantitative bioluminescence after luciferin injection. All animals demonstrated viable adipose transfer with bioluminescence detectable on days 0, 1, 7, 14, and 28. This animal model may be used for noninvasive, longitudinal studies for quantification of the fat engraftment process.

DDEL-01EVALUATION OF A MULTIFUNCTIONAL NANOVESICLE IN A MPNST MOUSE MODEL

Peripheral nerve sheath tumors are benign tumors that have the potential to transform to malignant peripheral nerve sheath tumor (MPNST). In our initial study we found that majority of the MPNST cells and tissues express IL13Rα2 receptor, which could be a potential target for therapy. We have also observed earlier that the IL13 conjugated liposomal doxorubicin was effectively endocytosed by established MPNST cells like ST88-14 and sNF96.2 and induces cytotocity. To determine the in vivo efficacy, sciatic nerve tumor mouse model was developed by injecting the luciferase transfected STS26T cells in the sciatic nerve. The tumor progression was monitored following the injection of luciferin intraperitoneally and imaging with intravital imaging spectroscopy (IVIS). All the mice developed tumor in 2 weeks after implantation of tumor cells in the sciatic nerve as evidenced by IVIS imaging. Once the tumor was visualized the mice were treated with IL-13 conjugated and non-conjugated liposomal doxorubicin for 7 weeks at a low dose of 4 mg/kg body weight weekly once. Similarly the tumor sections from the mice which were treated with liposomes were analyzed for apoptotic markers like Caspase 3. The tumor volume, weight of the mice and the serum chemistry were recorded. Although the tumor progression was controlled initially, the efficacy was reduced with increasing tumor volume. So currently we are adopting a more frequent dosage regimen instead of weekly once strategy. Furthermore, as Ras activation has also been reported to promote the Schwann cell to MPNST transformation, we combined the doxorubicin with farnesyl thiosalicylic acid (FTS, a Ras inhibitor) in the liposome formulation to study their tumor regression property in such models. In conclusion we developed a successful MPNST animal model to monitor the tumor progression by non-invasive method and currently utilizing this model for investigating the therapeutic efficacy of multifunctional nanovesicles.

Platform Technologies for Directly Reconstructing 3D Living Biomaterials.

Bio-electrospraying and cell electrospinning is explored for reconstructing living biomaterials for regenerative biology and medicine. The investigations carried out in this study demonstrate these approaches as platform biotechnologies for tissue reconstruction for repair, replacement, and rejuvenation of damaged and/or ageing tissues and/or organs.

Remote Ischemic Preconditioning Improves the Viability of Donor Lipoaspirate during Murine Fat Transfer

Variable results associated with fat grafting have been attributed to local trauma, inconsistencies in transfer, and ischemia before the development of recipient circulation. Remote ischemic preconditioning is an inexpensive noninvasive technique that has been used in animal models and multicenter clinical trials to protect organ systems. In this work, the authors describe a novel animal model for analyzing the efficacy of fat grafting, and investigate the effect of remote ischemic preconditioning on volume retention. Subcutaneous adipose tissue samples from green fluorescent protein/luciferase-expressing FVB mice were obtained with or without pretreatment with a temporary hind-limb tourniquet. The samples were injected into the dorsal skin folds of wild-type FVB mice. The viability of the transferred tissue was examined over a 28-day period with quantitative bioluminescence after luciferin injection. Transferred tissue was also explanted for histologic analysis. The remote ischemic preconditioning group had significantly increased bioluminescence at days 0, 1, and 28. Histologic analysis at day 28 confirmed the presence of vascularized adipose in both groups. However, significant amounts of interstitial fibrosis were found in the control group, whereas substantially less was found in the remote ischemic preconditioning group. The remote ischemic preconditioning group retained a substantially greater amount of green fluorescent protein, suggesting increased survival of donor adipocytes. In this work, the authors describe a novel animal model for quantitative evaluation of fat grafting using in vivo bioluminescence of adipocytes from luciferase-expressing mice. The authors also demonstrate that remote ischemic preconditioning increases the viability of fat transfer and decreases interstitial fibrosis.

IUGR in rats is associated with changes in placental expression of MTAs

IUGR in rats is associated with changes in placental expression of MTAs

Single hind limb burn injury to mice alters nuclear factor-κB expression and [¹⁸F] 2-fluoro-2-deoxy-D-glucose uptake.

Burn trauma to the extremities can produce marked systemic effects in mice. Burn injury to the dorsal surface of mice is also associated with changes in glucose metabolism ([18F] 2-fluoro-2-deoxy-D-glucose [18FDG] uptake) by brown adipose tissue (BAT) and nuclear factor (NF)-κB activity in several tissues including skeletal muscle. This study examined the effect of a single hind limb burn in mice on 18FDG uptake by NF-κB activity in vivo, and blood flow was determined by laser Doppler techniques. Male NF-κB luciferase reporter mice (28-30 g) were anesthetized, both legs were shaven, and the right leg was subjected to scald injury by immersion in 90°C water for 5 seconds. Sham-treated animals were used as controls. Each burned and sham mouse was resuscitated with saline (2 mL, i.p.). The individual animals were placed in wire bottom cages with no food and free access to water. After 24 hours, the animals were imaged with laser Doppler for measuring blood flow in the hind limb. The animals were then unanesthetized with 50 μCi of FDG or luciferin (1.0 mg, i.v.) via tail vein. Five minutes after luciferin injection, NF-κB mice were studied by bioluminescence imaging with a charge-coupled device camera. One hour after 18FDG injection, the animals were killed with carbon dioxide overdose, and 18FDG biodistribution was measured. Tissues were also analyzed for NF-κB luciferase activity. The scalding procedure used here produced a full-thickness burn injury to the leg with sharp margins. 18FDG uptake by the burned leg was lower than that in the contralateral limb. Similarly, luciferase activity and blood flow in the burned leg were lower than those in the contralateral leg. 18FDG uptake by BAT and heart increased, whereas that by brain decreased. In conclusion, the present study suggests that burn injury to a single leg decreased FDG uptake by skeletal muscle but increased 18FDG uptake by BAT. The injury to the leg reduced NF-κB expression compared with the contralateral leg and the uninjured skeletal muscle of the sham but activated NF-κB expression in a number of other organs. These findings are consistent with the hypothesis that burn trauma to the extremities can produce marked systemic effects, including activation of NF-κB expression and activation of 18FDG uptake by BAT.