Pancreatic Carcinoma Xenografts Research Articles

P0157 Preclinical evaluation of NBN-paclitaxel in pancreatic cancer xenograft models

<h3>Background</h3> Despite being the standard of care, gemcitabine has limited clinical benefits in pancreatic cancer. The goal of this study was to determine whether non-biological, nanoparticle paclitaxel (NBN-Pac/IG-001) exhibits sufficiently robust preclinical activity to qualify for clinical development. <h3>Methods</h3> We tested NBN-paclitaxel in various pancreatic cancer cell lines and xenograft models. <h3>Findings</h3> The maximum-tolerated dose of NBN-paclitaxel was calculated to be 60mg/kg versus 20mg/kg for standard paclitaxel. NBN-paclitaxel was generally more efficacious than standard paclitaxel or gemcitabine. In vitro, enhanced activity was shown by NBN-paclitaxel as compared to standard paclitaxel or gemcitabine against four pancreatic cell lines (MIA Paca-2, Capan-1, PANC-1, and AsPC-1). This increased activity was also demonstrated in vivo in female athymic nude mice using two pancreatic cancer cell lines (MIA PaCa-2 and PANC-1) as well as in male nude BALB/c mice following the orthotopic (pancreatic) implantation of AsPC-1 pancreatic cancer cells. NBN-paclitaxel produced superior anti-tumour activity against the human pancreatic carcinoma xenograft MIA PaCa-2. The rank order (% complete regression) was as follows: NBN-paclitaxel (60, 40, 25mg/kg), standard paclitaxel (25mg/kg), and gemcitabine (140mg/kg). NBN-paclitaxel produced a marked, dose-related anti-tumour effect in the PANC-1 xenograft model, with complete remission in all animals at 50mg/kg per dose and significant partial remission at 20mg/kg per dose. Overall, NBN-paclitaxel was shown to be more efficacious than either standard paclitaxel or gemcitabine, at equitoxic dose. NBN-paclitaxel produced dose-dependent primary tumour suppression (41%, 37% and 26%) and reduced the number of metastasised tumour nodules (22%, 19% and 13%) in the AsPC-1 metastasis model at doses of 20, 35, and 50mg/kg, respectively. Anti-tumour activity of NBN-paclitaxel at 50mg/kg was superior to standard paclitaxel and gemcitabine at an equitoxic dose. <h3>Interpretation</h3> The preclinical data suggest that NBN-paclitaxel could be superior to standard paclitaxel at an equitoxic dose. The data support further development of NBN-paclitaxel.

Effect of treatment with elemene on expression of apoptosis-related proteins in pancreatic carcinoma xenografts in nude mice

Effect of treatment with elemene on expression of apoptosis-related proteins in pancreatic carcinoma xenografts in nude mice

MicroPET-CT evaluation of interstitial brachytherapy in pancreatic carcinoma xenografts

Interstitial brachytherapy (IBT) has been introduced as treatment for unresectable pancreatic cancers to maximize local dose and minimize irradiation of surrounding normal tissue. MicroPET-CT systems provide excellent anatomic and molecular information. To use 18F-FDG micro-positron emission tomography (PET)/computed tomography (CT) to evaluate the therapeutic effect of (125)I interstitial brachytherapy on transplantation tumor of human pancreatic carcinoma in Balb/c-nu mice. Xenograft models were created by subcutaneous injection of Swl990 human pancreatic cancer cell suspensions into the immunodeficient Balb/c-nu mice. The study was randomly divided into three groups: control group (n = 6), empty seed implant group (n = 6), and (125)I seed implant group (n = 6), respectively. Before and 1 week after treatment, 18F-FDG microPET-CT scan was performed. In-vivo cell proliferation and apoptosis were monitored by thymidine kinase 1 (TK1) immunostaining and Dutp-biotin nick end labeling (TUNEL) assay. Our results showed that before treatment the SUVmax and SUVmean values among three groups had no significant statistical difference. One week after treatment the SUVmax and SUVmean in (125)I seed implant group were significantly lower than before, while for the empty seed group and control group there were no significant difference compared with before treatment. Immunohistochemical analysis of tumor tissue revealed significantly less TK1 positive cells in (125)I seed implant group than in empty seed group and control group. The index of apoptosis was slightly higher in (125)I seed implant group than in empty seed group and control group as evaluated by TUNEL assay. These results suggest that 18F-FDG microPET-CT may be useful as a non-invasive imaging modality to assess early response to (125)I seed brachytherapy in a pancreatic carcinoma Xenograft.

Expression of RECK in pancreatic carcinoma and its relationship with prognosis

Objective To study the expression of reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) in human pancreatic carcinoma tissues and pancreatic carcinoma cell lines; the effects of recombinant lentiviruses carrying RECK gene(LV-RECK) therapy on human pancreatic carcinoma xenograft in nude mice; and to find out the relationship between the expression of RECK and the prognosis of pancreatic carcinoma.Methods Immunohistochemical method was used to detect the expression of RECK in the resected specimens of pancreatic carcinoma and their corresponding normal pancreatic tissues in 42 patients.Western blotting was used to examine the expression of RECK in human pancreatic carcinoma cell lines (PANC-1,MIAPaCa-2,AsPC-1).Statistical analyses were performed to determine the relationship between RECK expression and the clinicopathological characteristics and prognosis in pancreatic carcinoma.Subcutaneous xenograft tumor models of human pancreatic carcinoma were established in nude mice.These nude mice were then divided into the experimental group,the negative control group and the blank control group randomly.The three groups of nude mice were intratumorally injected with LV-RECK,LV-EGFP and normal saline (NS) respectively.The antitumor effect was studied.Immunohistochemical method was used to detect the expression of RECK and microvessel density (MVD).Terminal deoxynucleotidyl transferase mediated dUTP-DIG nick end labeling (TUNEL) was used to detect the apoptosis of tumor cells.Survival analysis was performed.Results All three pancreatic carcinoma cell lines did not express RECK.The overall positive rate of RECK expression was 45.2 ％ (19/42) in pancreatic carcinoma,and 88.1 ％ (37/42) in normal pancreatic tissue.The expression level of RECK was significantly lower in the tumor tissues than in the normal tissues (P＜0.01).The expression of RECK was significantly associated with TNM stage,lymph node metastasis and local infiltration of pancreatic carcinoma (P＜0.05).Kaplan-Meier survival analysis revealed that the survival time was significantly longer in the RECK positive patient group than in the RECK negative patient group.Univariate Cox regression analysis revealed that RECK expression,TNM stage,lymph node metastasis and local infiltration were significantly related with prognosis for pancreatic carcinoma (P＜0.05).Multivariate Cox regression analysis revealed that only RECK expression remained as an independent significant factor in predicting the prognosis of pancreatic carcinoma (P ＜ 0.001).When compared with the negative control and the blank control groups,the volume of subcutaneous xenograft tumor in the experimental group was significantly decreased (P＜0.05).RECK protein in the experimental group was re-expressed.MVD of the experimental group was significantly less than those of the control groups (P＜0.05).Apoptotic index (AI)of the experimental group was significantly higher than those of the control groups (P＜0.05).The survival time of nude mice in the experimental group was significantly longer than those in the control groups (P＜0.05).Conclusions RECK expression was closely related to invasion,metastasis and prognosis of pancreatic carcinoma and it was an independent prognostic marker.RECK gene over-expression inhibited neovascularization of pancreatic carcinoma,induced apoptosis of tumor cells,inhibited the growth of tumor xenograft and improved the prognosis of tumor-bearing mice.These results suggest a possible new treatment for pancreatic carcinoma. Key words: Pancreatic neoplasms; Matrix metalloproteinases ; Prognosis ; Reversion-inducing-cysteine-rich protein with Kazal motifs

Tumor Selective Hyperthermia Induced by Short-Wave Capacitively-Coupled RF Electric-Fields

There is a renewed interest in developing high-intensity short wave capacitively-coupled radiofrequency (RF) electric-fields for nanoparticle-mediated tumor-targeted hyperthermia. However, the direct thermal effects of such high-intensity electric-fields (13.56 MHZ, 600 W) on normal and tumor tissues are not completely understood. In this study, we investigate the heating behavior and dielectric properties of normal mouse tissues and orthotopically-implanted human hepatocellular and pancreatic carcinoma xenografts. We note tumor-selective hyperthermia (relative to normal mouse tissues) in implanted xenografts that can be explained on the basis of differential dielectric properties. Furthermore, we demonstrate that repeated RF exposure of tumor-bearing mice can result in significant anti-tumor effects compared to control groups without detectable harm to normal mouse tissues.

Polyisoprenoyl gemcitabine conjugates self assemble as nanoparticles, useful for cancer therapy

A series of new polyisoprenoyl prodrugs of gemcitabine, which can be formulated as nanoassemblies are described. These prodrugs were designed to improve gemcitabine efficacy and to overcome the limitations due to the systemic toxicity of this anticancer compound. In vitro biological assessment showed that these polyisoprenoyl gemcitabine nanoassemblies displayed notable cytotoxicity on several cancer cell lines, including murine melanoma cell line B16F10, human pancreatic carcinoma cell line MiaPaCa-2, human lung carcinoma cell line A549 and human breast adenocarcinoma cell line MCF7. Interestingly, it was observed that the anticancer efficacy of these nanoassemblies was dependant on the size of polyisoprenoyl moiety. The polyisoprenoyl prodrug of gemcitabine containing three isoprene units (2d) was the more active on all the cancer cell lines tested. The antitumor efficacy of the nanoassemblies (NAs) constructed with the most active prodrug 2d was further evaluated on a human pancreatic (MiaPaCa-2) carcinoma xenograft model in mice. The prodrug 2d NAs showed an increased antitumor efficacy as compared to free gemcitabine or to squalene-gemcitabine (SQ-gem, 2a) nanoassemblies. Interestingly, MiaPaCa-2 tumors that did not respond to gemcitabine were inhibited by 76% after treatment with prodrug 2d NAs, whereas SQ-gem-treated MiaPaCa-2 tumor xenografts decreased only by 41% compared to saline or to gemcitabine-treated mice. Together, these findings demonstrated that the modulation of the length of nanoassemblies polyisoprenoyl moiety made tumor cells more sensitive to gemcitabine treatment without flagrant toxicity, thus providing a significant improvement in the drug therapeutic index.

Abstract 2839: Antitumor activity on colorectal and pancreatic tumors of a new strategy based on ROS generation by non-thermal plasma

Abstract Background. Local treatments of tumors are mainly based on surgical resection and/or treatment such as photodynamic therapy (PDT) or ionizing radiation (IR). Action mechanisms of IR and PDT are based on the generation of ROS in the vicinity of the cells. In this context, cold or Non Thermal Plasma (NTP), a new technology which can generate in situ ROS, is currently under investigations. NTP is a cold (&amp;lt;40°C) ionized gas (air or noble gas) thank to an electric discharge. It contains free charges (electrons, ions), free radicals and excited molecules. NTP can be generated at the end of a catheter (plasma jet) allowing a local treatment that is compatible with usual endoscopes for dysplasia or non resecable tumors. Previous studies have shown an in vitro antitumor activity of NTP on various cells lines and in vivo on subcutaneous xenografts. The aim of this work was to assess the antitumor potential of fibred NTP when applied directly on in situ orthotopic human pancreatic or colorectal carcinoma xenografts. Methods. For in vitro experiments, HCT116-Luc and Mia Paca-Luc cells were seeded in 24-well plate and then treated with a plasma jet device called plasma gun. The effects on cells were assessed by bioluminescence imaging (BLI). For in vivo experiments, HCT116-Luc cells were xenografted in the caecum wall of nude mice and tumors were treated by a single NTP exposure 7 days post-induction. Concerning pancreatic tumors, MIA PaCa2-Luc cells were injected in the pancreas head and NTP treatments were started 4 days after implantation. Two others exposures were performed on D14 and D24. For both models, tumor growth was monitored by BLI once a week. Results and Discussion. NTP treatment showed a significant antitumor effect on HCT116 and Mia Paca cells in vitro and in vivo. In vitro plasma induced a decrease of BLI resulting from a massive cell death. Localized treatments of tumor using plasma gun induced significant tumor stabilization. On colorectal tumors, a BLI difference of 90% between treated and CTRL tumors were observed at D22. This effect was confirmed by tumor weight measurement. Moreover, a significant metastasis decrease was observed. Similar results were observed on pancreatic tumors with also a stabilization of tumor growth and a BLI decrease following each treatment leading finally at D36 to a significant difference of tumor volume (84%). Comparison of NTP with the references drug of these pathologies is ongoing. The results obtained on two different tumor types show the potential antitumor activity of NTP on implanted small tumors. These effects were linked to a high rate generation of ROS in the vicinity of tumors, inducing lethal DNA damages, a multiphase cell cycle arrest and finally apoptosis. Such results demonstrate the interest of NTP for treatment of dysplasia and non-resecable tumors that could be associated to real time per operative near infrared fluorescence imaging via fibro/coelioscopy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2839. doi:1538-7445.AM2012-2839

Abstract 2763: Combination therapy of an acid-sensitive albumin-binding prodrug of doxorubicin (INNO-206) and an albumin-binding prodrug of methotrexate (AW054) that is cleaved by cathepsin B and plasmin induces complete remissions against pancreatic cancer in vivo (MiaPaca-2)

Abstract Pancreatic cancer is the fourth most common cause of cancer death across the globe. Pancreatic cancer has a poor prognosis. For locally advanced and for metastatic disease, which collectively represent over 80 % of individuals, median survival is approximately 10 and 6 months, respectively. Gemcitabine and 5-fluorouracil that have been approved for this indication have a poor response rate with a benefit in overall survival for a small population of treated patients of up to 2 months on average. Thus there is an unmet medical need for treating this tumor indication more effectively. INNO-206, the (6-maleimidocaproyl)hydrazone of doxorubicin (DOXO-EMCH) is an albumin-binding prodrug of doxorubicin with acid-sensitive properties that has shown objective responses in a phase I study, and phase II studies are scheduled in sarcoma for 2011/2012 (see www.cytrx.com). For treating essentially chemoresistant pancreatic cancer, we preclinically investigated a combination of two albumin-binding prodrugs, i.e. the albumin-binding prodrug INNO-206 and an albumin-binding prodrug of methotrexate, AW054, that is cleaved by two proteases that are over-expressed in solid tumors, cathepsin B and plasmin. In the pancreatic carcinoma xenograft model MiaPaca-2 both INNO-206 and AW054 were administered simultaneously (i.v.) in a three weekly schedule at half the maximum tolerated dose (MTD) of the respective prodrug, i.e. 3 x 12 mg/kg for INNO-206 (doxorubicin equivalents) and 3 x 10 mg/kg (methotrexate equivalents). Compared to doxorubicin (dosed at the MTD of 3 x 6 mg/kg) or methotrexate (dosed at the MTD of 3 x 125 mg/kg) or the combination of the two free drugs (3 x 3 mg/kg for doxorubicin and 3 x 62.5 mg/kg for doxorubicin) which only showed moderate antitumor effects over control animals treated with saline, the combination of the two albumin-binding prodrugs produced complete remissions and an acceptable body weight loss of −13 %. Immunohistochemical staining with CD31 that makes up a large portion of endothelial cell intercellular junctions was moderate to good in control tumors indicating a sufficient vascularity for the albumin-bound forms of the prodrugs to accumulate in the tumor via the EPR effect. This encouraging in vivo result is an impetus for investigating further combinations of albumin-binding prodrugs with other anticancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2763. doi:1538-7445.AM2012-2763

Therapeutic Potential of Amanitin-Conjugated Anti-Epithelial Cell Adhesion Molecule Monoclonal Antibody Against Pancreatic Carcinoma

Human epithelial cell adhesion molecule (EpCAM) is overexpressed in many cancers. Anti-EpCAM antibodies have shown promise in preclinical studies, but showed no tumor regression in a recent phase II clinical trial. Therefore, we generated a novel anti-EpCAM antibody-drug conjugate and assessed whether it showed enhanced antitumor effects. Chemical cross-linking was conducted to covalently conjugate α-amanitin, a toxin known to inhibit DNA transcription, with chiHEA125, a chimerized anti-EpCAM monoclonal antibody, to generate the antibody-drug conjugate α-amanitin-glutarate-chiHEA125 (chiHEA125-Ama). Antiproliferative activity of chiHEA125-Ama was tested in human pancreatic (BxPc-3 and Capan-1), colorectal (Colo205), breast (MCF-7), and bile duct (OZ) cancer cell lines in vitro using [(3)H]-thymidine incorporation assay. Antitumor activity of chiHEA125-Ama was assessed in vivo in immunocompromised mice bearing subcutaneous human BxPc-3 pancreatic carcinoma xenograft tumors (n = 66 mice). Cell proliferation and apoptosis were evaluated in xenograft tumors by immunohistochemistry. All statistical tests were two-sided. In all cell lines, chiHEA125-Ama reduced cell proliferation (mean half maximal inhibitory concentration [IC(50)] = 2.5 × 10(-10) to 5.4 × 10(-12) M). A single dose of chiHEA125-Ama inhibited BxPc-3 xenograft tumor growth (chiHEA125 [control, n = 4 mice] vs. chiHEA125-Ama [n = 6 mice], dose of 15 mg/kg with respect to IgG and 50 μg/kg with respect to α-amanitin, mean relative increase in tumor volume on day 16 = 884% vs. -79%, difference = 963%, 95% CI = 582% to 1344%, P = .019). Two higher doses of chiHEA125-Ama (100 μg/kg with respect to α-amanitin), administered 1 week apart (n = 10 mice per group), led to complete tumor regression in nine of 10 (90%) mice compared with chiHEA125, during the observation period of 16 days; increased apoptosis and reduced cell proliferation were observed in mice treated with chiHEA125-Ama. This preclinical study suggests that anti-EpCAM antibody conjugates with α-amanitin have the potential to be highly effective therapeutic agents for pancreatic carcinomas and various EpCAM-expressing malignancies.

In Pancreatic Carcinoma, Dual EGFR/HER2 Targeting with Cetuximab/Trastuzumab Is More Effective than Treatment with Trastuzumab/Erlotinib or Lapatinib Alone: Implication of Receptors' Down-regulation and Dimers' Disruption

We previously demonstrated the synergistic therapeutic effect of the cetuximab (anti-epidermal growth factor receptor [EGFR] monoclonal antibody, mAb)-trastuzumab (anti-HER2 mAb) combination (2mAbs therapy) in HER2low human pancreatic carcinoma xenografts. Here, we compared the 2mAbs therapy, the erlotinib (EGFR tyrosine kinase inhibitor [TKI])-trastuzumab combination and lapatinib alone (dual HER2/EGFR TKI) and explored their possible mechanisms of action. The effects on tumor growth and animal survival of the three therapies were assessed in nude mice xenografted with the human pancreatic carcinoma cell lines Capan-1 and BxPC-3. After therapy, EGFR and HER2 expression and AKT phosphorylation in tumor cells were analyzed by Western blot analysis. EGFR/HER2 heterodimerization was quantified in BxPC-3 cells by time-resolved FRET. In K-ras-mutated Capan-1 xenografts, the 2mAbs therapy gave significantly higher inhibition of tumor growth than the erlotinib/trastuzumab combination, whereas in BxPC-3 (wild-type K-ras) xenografts, the erlotinib/trastuzumab combination showed similar growth inhibition but fewer tumor-free mice. Lapatinib showed no antitumor effect in both types of xenografts. The efficacy of the 2mAbs therapy was partly Fc-independent because F(ab′)2 fragments of the two mAbs significantly inhibited BxPC-3 growth, although with a time-limited therapeutic effect. The 2mAbs therapy was associated with a reduction of EGFR and HER2 expression and AKT phosphorylation. BxPC-3 cells preincubated with the two mAbs showed 50% less EGFR/HER2 heterodimers than controls. In pancreatic carcinoma xenografts, the 2mAbs therapy is more effective than treatments involving dual EGFR/HER2 TKIs. The mechanism of action may involve decreased AKT phosphorylation and/or disruption of EGFR/HER2 heterodimerization.

Recombinant Human Arginase Toxicity in Mice Is Reduced by Citrulline Supplementation

Human recombinant arginase I cobalt coupled to polyethylene glycol 5000 (HuArg I [Co]-PEG5000) achieved potent in vitro depletion of arginine from tissue culture medium and cytotoxicity to many cancer cell lines. The recombinant enzyme also produced tumor growth inhibition of hepatocellular carcinoma and pancreatic carcinoma xenografts. Although these results were promising, the therapeutic index was narrow. Toxicities were seen in normal cells in tissue culture. In vivo normal tissue injury occurred at doses twice the effective dose. The current study was conducted to define, in greater detail, the maximum tolerated dose (MTD), pharmacodynamics, and dose-limiting toxicities (DLTs) of twice-weekly intraperitoneal HuArg I [Co]-PEG5000 in Balb/c mice. Animal weight and survival were monitored, serum arginine levels measured, and complete blood cell counts, chemistries, necropsies, and histologies were performed. In addition, methods to ameliorate the HuArg I [Co]-PEG5000 adverse effects were tested. Supplemental l-citrulline was given concurrently with the arginase drug. The HuArg I [Co]-PEG5000 MTD in mice was 5 mg/kg twice weekly, and DLTs included weight loss and marrow necrosis. No other organ damage or changes in blood cell counts or chemistries were observed. Arginase reduced serum arginine levels from 60 µM to 4 to 6 µM. Supplemental l-citrulline given per os or daily subcutaneously reduced and delayed toxicities, and l-citrulline given twice daily subcutaneously completely prevented animal toxicities. On the basis of these results, we hypothesize that HuArg I [Co]-PEG5000, particularly with supplemental l-citrulline, may be an attractive therapeutic agent for argininosuccinate synthetase-deficient tumors.

Intervention of Mirtazapine on gemcitabine-induced mild cachexia in nude mice with pancreatic carcinoma xenografts

To investigate the effect of mirtazapine on tumor growth, food intake, body weight, and nutritional status in gemcitabine-induced mild cachexia. Fourteen mice with subcutaneous xenografts of a pancreatic cancer cell line (SW1990) were randomly divided into mirtazapine and control groups. Either mirtazapine (10 mg/kg) or saline solution was orally fed to the mice every day after tumor implantation. A model of mild cachexia was then established in both groups by intraperitoneal injection of gemcitabine (50 mg/kg) 10 d, 13 d, and 16 d after tumor implantation. Tumor size, food intake, body weight, and nutritional status were measured during the experiment. All mice were sacrificed at day 28. (1) After 7 d of gemcitabine administration, body-weight losses of 5%-7% which suggested mild cachexia were measured; (2) No significant difference in tumor size was detected between the mirtazapine and control groups (P > 0.05); and (3) During the entire experimental period, food intake and body weight were slightly greater for the mirtazapine group compared with controls (although these differences were not statistically significant). After 21 d, mice in the mirtazapine group consumed significantly more food than control mice (3.95 ± 0.14 g vs 3.54 ± 0.10 g, P = 0.004). After 25 d, mice in the mirtazapine group were also significantly heavier than control mice (17.24 ± 0.53 g vs 18.05 ± 0.68 g, P = 0.014). Mild cachexia model was successfully established by gemcitabine in pancreatic tumor-bearing mice. Mirtazapine can improve gemcitabine-induced mild cachexia in pancreatic tumor-bearing mice. It was believed to provide a potential therapeutic perspective for further studies on cachexia.

Bioengineered Human Arginase I with Enhanced Activity and Stability Controls Hepatocellular and Pancreatic Carcinoma Xenografts

Hepatocellular carcinoma (HCC) and pancreatic carcinoma (PC) cells often have inherent urea cycle defects rendering them auxotrophic for the amino acid L-arginine (L-arg). Most HCC and PC require extracellular sources of L-arg and undergo cell cycle arrest and apoptosis when L-arg is restricted. Systemic, enzyme-mediated depletion of L-arg has been investigated in mouse models and human trials. Non-human enzymes elicit neutralizing antibodies, whereas human arginases display poor pharmacological properties in serum. Co2+ substitution of the Mn2+ metal cofactor in human arginase I (Co-hArgI) was shown to confer more than 10-fold higher catalytic activity (kcat/Km) and 5-fold greater stability. We hypothesized that the Co-hArgI enzyme would decrease tumor burden by systemic elimination of L-arg in a murine model. Co-hArgI was conjugated to 5-kDa PEG (Co-hArgI-PEG) to enhance circulation persistence. It was used as monotherapy for HCC and PC in vitro and in vivo murine xenografts. The mechanism of cell death was also investigated. Weekly treatment of 8 mg/kg Co-hArgI-PEG effectively controlled human HepG2 (HCC) and Panc-1 (PC) tumor xenografts (P = .001 and P = .03, respectively). Both cell lines underwent apoptosis in vitro with significant increased expression of activated caspase-3 (P < .001). Furthermore, there was evidence of autophagy in vitro and in vivo. We have demonstrated that Co-hArgI-PEG is effective at controlling two types of L-arg-dependent carcinomas. Being a nonessential amino acid, arginine deprivation therapy through Co-hArgI-PEG holds promise as a new therapy in the treatment of HCC and PC.

Combination radioimmunotherapy and chemoimmunotherapy involving different or the same targets improves therapy of human pancreatic carcinoma xenograft models.

Chemoimmunotherapy with antibody-drug conjugates (ADC) is emerging as a promising therapy for solid tumors, whereas radioimmunotherapy (RAIT) of solid tumors has been relatively ineffective because of their resistance to radiation. We developed antibody-SN-38 conjugates that have significant antitumor activity in xenograft models at nontoxic doses. The goal of this study was to determine if an ADC could be combined with RAIT to enhance efficacy without a commensurate increase in host toxicity. Nude mice bearing human pancreatic cancer xenografts (Capan-1 and BxPC-3) were treated with a single dose of 90Y-labeled antimucin antibody (hPAM4; clivatuzumab tetraxetan) alone or in combination with an anti-Trop-2-SN-38 conjugate, typically administered twice weekly over 4 weeks. The combination, even at RAIT's maximum tolerated dose, controlled tumor progression and cured established xenografts significantly better than the individual treatments without appreciable toxicity. The ADC could be started 1 week after or up to 2 weeks before RAIT with similar enhanced responses, but delaying RAIT for 2 weeks after the ADC was less effective. A nonspecific ADC provided additional benefit over using free drug (irinotecan), but the response was enhanced with the specific ADC. When targeting Capan-1 with ample mucin, hPAM4 could be used as the RAIT and the ADC agent without losing effectiveness, but in BxPC-3 with less mucin, targeting of different antigens was preferred. These studies show the feasibility of combining ADC and RAIT for improved efficacy without increased toxicity.

Cell apoptosis and proliferation inhibition of pancreatic cancer induced by sub-threshold focused ultrasound (FUS)

To evaluate the effects of sub-threshold focused ultrasound (FUS) sonication on the pancreatic cancer cell. The human pancreatic carcinoma cell line PaTu 8988t suspension and pancreatic carcinoma xenograft in nude mice were sonicated by FUS using sub-threshold doses. The temperature at the focus was controlled at below 60°C. The cell apoptosis in vitro was tested by flow cytometer at 3, 6, 12, 24 and 48h after FUS sonication. Colony formation was used to evaluate the cell growth inhibition of FUS in vitro. The tumor volume of the xenograft was measured before and after FUS sonication. Then the slides of the tumor were under hematoxylin-eosin (H&E) staining and TdT-mediated dUTP nick end labeling (TUNEL) to evaluate the effect of FUS on pancreatic carcinoma xenograft in vivo. The maximum cell suspension temperature of the FUS sonication group was 55.8±2.17°C. The cell apoptosis rate peaked at 24h after FUS sonication, the differences between the FUS sonication group and control group were statistically significant (P<0.05). Colony formation rates were 50.40±3.81% for the control group and 26.82±2.88% for FUS sonication group (P<0.05). For the xenografts in nude mice, the mean tumor volumes of the control group and the FUS sonication group 15 days after sonication were 1746.58±312.77mm(3) and 1085.23±217.13mm(3) (P<0.05). H&E staining and TUNEL assay showed both necrotic and apoptotic cells. Sub-threshold FUS sonication could induce cell apoptosis and inhibit the proliferation of pancreatic cancer cells.

Noninvasive Radiofrequency Field Destruction of Pancreatic Adenocarcinoma Xenografts Treated with Targeted Gold Nanoparticles

Pancreatic carcinoma is one of the deadliest cancers with few effective treatments. Gold nanoparticles (AuNP) are potentially therapeutic because of the safety demonstrated thus far and their physiochemical characteristics. We used the astounding heating rates of AuNPs in nonionizing radiofrequency (RF) radiation to investigate human pancreatic xenograft destruction in a murine model. Weekly, Panc-1 and Capan-1 human pancreatic carcinoma xenografts in immunocompromised mice were exposed to an RF field 36 hours after treatment (intraperitoneal) with cetuximab- or PAM4 antibody-conjugated AuNPs, respectively. Tumor sizes were measured weekly, whereas necrosis and cleaved caspase-3 were investigated with hematoxylin-eosin staining and immunofluorescence, respectively. In addition, AuNP internalization and cytotoxicity were investigated in vitro with confocal microscopy and flow cytometry, respectively. Panc-1 cells demonstrated increased apoptosis with decreased viability after treatment with cetuximab-conjugated AuNPs and RF field exposure (P = 0.00005). Differences in xenograft volumes were observed within 2 weeks of initiating therapy. Cetuximab- and PAM4-conjugated AuNPs demonstrated RF field-induced destruction of Panc-1 and Capan-1 pancreatic carcinoma xenografts after 6 weeks of weekly treatment (P = 0.004 and P = 0.035, respectively). There was no evidence of injury to murine organs. Cleaved caspase-3 and necrosis were both increased in treated tumors. This study demonstrates a potentially novel cancer therapy by noninvasively inducing intracellular hyperthermia with targeted AuNPs in an RF field. While the therapy is dependent on the specificity of the targeting antibody, normal tissues were without toxicity despite systemic therapy and whole-body RF field exposure.

Rapid optical imaging of EGF receptor expression with a single-chain antibody SNAP-tag fusion protein

The epidermal growth factor receptor (EGFR) is overexpressed in several types of cancer and its inhibition can effectively inhibit tumour progression. The purpose of this study was to design an EGFR-specific imaging probe that combines efficient tumour targeting with rapid systemic clearance to facilitate non-invasive assessment of EGFR expression. Genetic fusion of a single-chain antibody fragment with the SNAP-tag produced a 48-kDa antibody derivative that can be covalently and site-specifically labelled with substrates containing 0 (6)-benzylguanine. The EGFR-specific single-chain variable fragment (scFv) fusion protein 425(scFv)SNAP was labelled with the near infrared (NIR) dye BG-747, and its accumulation, specificity and kinetics were monitored using NIR fluorescence imaging in a subcutaneous pancreatic carcinoma xenograft model. The 425(scFv)SNAP fusion protein accumulates rapidly and specifically at the tumour site. Its small size allows efficient renal clearance and a high tumour to background ratio (TBR) of 33.2 +/- 6.3 (n = 4) 10 h after injection. Binding of the labelled antibody was efficiently competed with a 20-fold excess of unlabelled probe, resulting in an average TBR of 6 +/- 1.35 (n = 4), which is similar to that obtained with a non-tumour-specific probe (5.44 +/- 1.92, n = 4). When compared with a full-length antibody against EGFR (cetuximab), 425(scFv)SNAP-747 showed significantly higher TBRs and complete clearance 72 h post-injection. The 425(scFv)SNAP fusion protein combines rapid and specific targeting of EGFR-positive tumours with a versatile and robust labelling technique that facilitates the attachment of fluorophores for use in optical imaging. The same approach could be used to couple a chelating agent for use in nuclear imaging.

Optimization of 6,6-dimethyl pyrrolo[3,4- c]pyrazoles: Identification of PHA-793887, a potent CDK inhibitor suitable for intravenous dosing

We have recently reported CDK inhibitors based on the 6-substituted pyrrolo[3,4- c]pyrazole core structure. Improvement of inhibitory potency against multiple CDKs, antiproliferative activity against cancer cell lines and optimization of the physico-chemical properties led to the identification of highly potent compounds. Compound 31 (PHA-793887) showed good efficacy in the human ovarian A2780, colon HCT-116 and pancreatic BX-PC3 carcinoma xenograft models and was well tolerated upon daily treatments by iv administration. It was identified as a drug candidate for clinical evaluation in patients with solid tumors.

Combined cetuximab and trastuzumab are superior to gemcitabine in the treatment of human pancreatic carcinoma xenografts

BackgroundPancreatic carcinoma remains a treatment-refractory cancer with a poor prognosis. Here, we compared anti-epidermal growth factor receptor (EGFR) and anti-HER2 monoclonal antibodies (2mAbs) injections with standard gemcitabine treatment on human pancreatic carcinoma xenografts. Materials and methodsNude mice, bearing human pancreatic carcinoma xenografts, were treated with either combined anti-EGFR (cetuximab) and anti-HER2 (trastuzumab) or gemcitabine, and tumor growth was observed. Results and conclusionIn first-line therapy, mice survival was significantly longer in the 2mAbs group compared with gemcitabine (P < 0.0001 for BxPC-3, P = 0.0679 for MiaPaCa-2 and P = 0.0019 for Capan-1) and with controls (P < 0.0001). In second-line therapy, tumor regressions were observed after replacing gemcitabine by 2mAbs treatment, resulting in significantly longer animal survival compared with mice receiving continuous gemcitabine injections (P = 0.008 for BxPC-3, P = 0.05 for MiaPaCa-2 and P < 0.001 for Capan-1). Therapeutic benefit of 2mAbs was observed despite K-Ras mutation. Interestingly, concerning the mechanism of action, coinjection of F(ab′)2 fragments from 2mAbs induced significant tumor growth inhibition, compared with controls (P = 0.001), indicating that the 2mAbs had an Fc fragment-independent direct action on tumor cells. This preclinical study demonstrated a significant improvement of survival and tumor regression in mice treated with anti-EGFR/anti-HER2 2mAbs in first- and second-line treatments, compared with gemcitabine, independently of the K-Ras status.

Abstract B192: Preclinical characterization of BMS-833923 (XL139), a hedgehog (HH) pathway inhibitor in early clinical development

Abstract Background: Aberrant HH pathway signaling has been implicated in human malignancies ranging from semi-malignant tumors of the skin to highly aggressive cancers of the brain, lung, pancreas, breast, prostate, and lymphohematopoietic lineages. Dysregulation of this pathway contributes to uncontrolled proliferation, invasion, metastasis, evasion of apoptosis, and resistance to chemotherapy. Results: BMS-833923 is a potent inhibitor of SMO, a GPCR-like 7-transmembrane receptor that is a critical regulator of the HH pathway. In vitro, BMS-833923 inhibits the expression of downstream effectors in the HH pathway (GLI1 and PTCH1) in cell lines that express wild-type SMO and those which express activated mutant forms of SMO (IC50 values of 6–35 nM). In FACS-based binding assays, BMS-833923 inhibits BODIPY cyclopamine binding to SMO in a dose-dependent manner with an IC50 of 21 nM. Prior work has demonstrated that HH pathway blockade inhibits the in vitro clonal expansion of multiple myeloma (MM) precursor cells in both established cell lines and in clinical samples from patients with MM (Peacock et al 2007, PNAS 104:4048). Consistent with these observations, BMS-833923 inhibited the in vitro growth of MM clones and proportion of ALDH+ cancer stem cells derived from bone marrow samples from subjects with MM. Furthermore, BMS-833923 inhibited the clonogenic growth of multiple tumor cell lines derived from patients with hematological malignancies including CML, ALL and AML, suggesting that targeting the HH pathway may have broad therapeutic utility for patients with hematologic malignancies. In vivo pharmacodynamic studies show that BMS-833923 robustly inhibits HH pathway activity with a long duration of action after a single oral dose in medulloblastoma and pancreatic carcinoma xenograft models. The pharmacodynamic effects of BMS-833923 observed in these models translate into tumor growth inhibition at well-tolerated doses. These results are consistent with pharmacokinetic parameters demonstrated in nonclinical studies. Conclusions: BMS-833923 is an orally bioavailable, potent and selective inhibitor of the HH pathway as measured by in vitro cell-based assays and in vivo pharmacodynamic and efficacy models, supporting the clinical development of BMS-833923 in both hematological and non-hematological malignancies. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B192.