Median Animal Survival Research Articles

Abstract 1258: Therapeutic potential of the cyclin- dependent kinase inhibitor on c-Myc overexpressing esophageal adenocarcinoma

Abstract Introduction: Esophageal adenocarcinoma (EAC) is now the fastest growing cancer in the western world and most EAC patients present with widespread metastatic disease, where current treatment is largely ineffective. Therefore, new therapeutic approaches are urgently needed. The transcription factor proto-oncogene c-Myc is a potent activator of tumorigenesis. Tumors with elevated c-Myc expression often exhibit highly aggressive phenotype. c-Myc amplification has been shown to be frequent in esophageal adenocarcinoma and has been implicated in Barrett's carcinogenesis. Emerging data suggests that synthetic lethal interactions between c-Myc pathway activation and small molecules inhibition involved in cell cycle signaling can be therapeutically exploited to preferentially kill tumor cells. In this study, we therefore investigated whether exploiting a synthetic-lethal approach dependent on elevated c-Myc signaling is effective in treating esophageal cancer with a cyclin-dependent kinase (CDK) inhibitor flavopiridol. Methods: Western blot analysis was done to see the expression of c-Myc and apoptotic signaling pathways in a panel of nine esophageal cancer cell lines. c-Myc overexpression and knockdown were performed using both genetic and novel chemical approaches. Cell viability assays were performed in 96-well plates using the colorimetric WST-1 reagent. Esophageal cancer tumors growth was measured in xenograft and a novel peritoneal disseminated metastatic survival model of immunodeficient mice. Results: Western blot analysis revealed frequent overexpression of c-Myc in EAC cell lines. In this panel of esophageal cancer cell lines tested more than 70% of EAC cell lines showed overexpression of c-Myc. When we tested these cell lines for their ability to form xenograft tumor and peritoneal dissemination, c-Myc overexpression correlated with accelerated EAC tumor growth in xenograft and peritoneal disseminated metastatic survival model of NOD/SCID mice. The xenograft tumor growth rate and formation rate of peritoneal cancer after injection of 5 million cells were highest in OE19 EAC cell line which showed the highest c-Myc expression. In addition, median animal survival with peritoneal dissemination was lowest for OE19 (55 days) whereas OACM5.1 C EAC cell line which had the lowest c-Myc expression didn't form any peritoneal tumor. EAC cell lines with elevated c-Myc expression are preferentially more sensitive to induction of apoptosis by CDK inhibitor flavopiridol compared to EAC cell lines with lower c-Myc expression. When we tested the role of c-Myc expression by upregulation/downregulation in this apoptotic effect we found that this effect is very much dependent on c-Myc expression. Conclusion: These results indicate that CDK inhibitor alone or in combination with other cytotoxic or targeted agents can be a potential therapy for c-Myc overexpressing EAC. Citation Format: Sazzad Hassan, Niranjan Awasthi, Margaret A. Schwarz, Roderich E. Schwarz, Urs V. Holzen. Therapeutic potential of the cyclin- dependent kinase inhibitor on c-Myc overexpressing esophageal adenocarcinoma. [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 1258.

Augmentation of response to nab-paclitaxel by inhibition of insulin-like growth factor (IGF) signaling in preclinical pancreatic cancer models.

Nab-paclitaxel has recently shown greater efficacy in pancreatic ductal adenocarcinoma (PDAC). Insulin like growth factor (IGF) signaling proteins are frequently overexpressed in PDAC and correlate with aggressive tumor phenotype and poor prognosis. We evaluated the improvement in nab-paclitaxel response by addition of BMS-754807, a small molecule inhibitor of IGF-1R/IR signaling, in preclinical PDAC models. In subcutaneous xenografts using AsPC-1 cells, average net tumor growth in different therapy groups was 248.3 mm3 in controls, 42.4 mm3 after nab-paclitaxel (p = 0.002), 93.3 mm3 after BMS-754807 (p = 0.01) and 1.9 mm3 after nab-paclitaxel plus BMS-754807 (p = 0.0002). In subcutaneous xenografts using Panc-1 cells, average net tumor growth in different therapy groups was: 294.3 mm3 in controls, 23.1 mm3 after nab-paclitaxel (p = 0.002), 118.2 mm3 after BMS-754807 (p = 0.02) and −87.4 mm3 (tumor regression) after nab-paclitaxel plus BMS-754807 (p = 0.0001). In peritoneal dissemination model using AsPC-1 cells, median animal survival was increased compared to controls (21 days) after therapy with nab-paclitaxel (40 days, a 90% increase, p = 0.002), BMS-754807 (27 days, a 29% increase, p = 0.01) and nab-paclitaxel plus BMS-754807 (47 days, a 124% increase, p = 0.005), respectively. Decrease in proliferation and increase in apoptosis by nab-paclitaxel and BMS-754807 therapy correlated with their in vivo antitumor activity. In vitro analysis revealed that the addition of IC25 dose of BMS-754807 decreased the nab-paclitaxel IC50 of PDAC cell lines. BMS-754807 therapy decreased phospho-IGF-1R/IR and phospho-AKT expression, and increased cleavage of caspase-3 and PARP-1. These results support the potential of BMS-754807 in combination with nab-paclitaxel as an effective targeting option for pancreatic cancer therapy.

Adding targeted inhibition of PI3K and MAPK signaling pathways to standard chemotherapy in experimental pancreatic cancer.

278 Background: Treatment of pancreatic ductal adenocarcinoma (PDAC) remains a formidable challenge as gemcitabine (Gem) plus nab-paclitaxel (NPT) represent the current standard for systemic therapy of advanced PDAC. More than 90% of PDAC tumors harbor an activating mutation in the KRAS oncogene. Currently no therapeutics exist that can effectively target this oncogene product, but alternative strategies focus on inhibition of downstream effectors of KRAS signaling pathways. The RAF-MEK-ERK (MAPK) and the AKT-PI3K signaling pathways are well-described mediators of KRAS induced transformation and tumorigenesis and can be explored as targets for combination therapy. Methods: We evaluated combination treatment benefits of NPT+Gem with the MEK inhibitor trametinib (Tra) and the AKT inhibitor MK-2206 (MK) in experimental xenografts to test their therapeutic potential against PDAC. Results: Median animal survival in human intraperitoneal PDAC xenografts in mice revealed that the median survival was 21 days in controls; this was significantly improved by the NPT+Gem combination (35 days, a 67% increase over controls, p = 0.0007). Median survival was further increased by addition of MK-2206 or trametinib to NPT+Gem chemotherapy group: NPT+Gem+MK (39 days, a 86% increase over controls, p = 0.0003), NPT+Gem+Tra (43 days, a 105% increase over controls, p = 0.0003) and NPT+Gem+MK+Tra (48 days, a 129% increase over controls, p = 0.002). In human subcutaneous PDAC xenografts, MK-2206 or trametinib were able to enhance NPT+Gem effects. Compared to controls (100±34.8), the percent net local tumor growth in different therapy groups was: NPT+Gem 21.8±5.9 (p = 0.003), NPT+Gem+MK 17.2±4.8 (p = 0.002), NPT+Gem+Tra 7.1±11.2 (p = 0.002) and NPT+Gem+MK+Tra 5.7±9.1 (p = 0.001). In vivo effects of Tra and MK correlated with reduced expression of phospho-ERK and phospho-AKT in Western blots of tumor samples. Conclusions: These findings suggest that the effects of NPT+Gem can be enhanced through combined inhibition of MAPK and PI3K signaling pathways, which clinically could yield in improved antitumor results.

Inhibition of the Aurora A kinase augments the anti-tumor efficacy of oncolytic measles virotherapy.

Oncolytic measles virus (MV) strains have demonstrated broad spectrum preclinical anti-tumor, including breast cancer. Aurora A kinase controls mitotic spindle formation and plays a critical role in malignant transformation. We hypothesized that, by causing mitotic arrest, the Aurora A kinase inhibitor MLN8237 (alisertib) can increase MV oncolytic effect and efficacy. Alisertib enhanced MV oncolysis in vitro and significantly improved outcome in vivo against breast cancer xenografts. In a disseminated MDA-231-lu-P4 lung metastatic model, the MV/alisertib combination treatment markedly increased median survival to 82.5 days with 20% of the animals being long term survivors vs. 48 days median survival for the control animals. Similarly, in a pleural effusion model of advanced breast cancer, the MV/alisertib combination significantly improved outcome with a 74.5 day median survival versus the single agent groups (57 and 40 days respectively). Increased viral gene expression and IL-24 upregulation were demonstrated, representing possible mechanisms for the observed increase in antitumor effect. Inhibiting Aurora A kinase with alisertib represents a novel approach to enhance measles virus-mediated oncolysis and antitumor effect. Both oncolytic MV strains and alisertib are currently tested in clinical trials, this study therefore provides the basis for translational applications of this combinatorial strategy in the treatment of patients with advanced breast cancer.

Вплив супернатанта прогеніторних нейроклітин на цитотоксичну функцію лімфоцитів у щурів з гліомою

The impact of rat neurogenic progenitor cells supernatant (RPNS) on the cytotoxic function of lymphocytes in rats under conditions of physiological norm and experimentally modeled tumor (brain glioma strain 101.8) was studied. The research was carried out in animals with inoculated tumor without RPNS injection and with different regimes of RPNS injection (thrice repeated from 5th to 10th day after glioma inoculation as well as 1 week and 1 month before tumor inoculation). Comparison groups included rats without glioma who triple injected with RPNS; and intact animals (control). RPNS was received from suspension of neurogenic progenitor cells (NPC) of rat brain on 14th day of gestation and injected intraperitoneally (0,12 mg per animal). Cytotoxic function of lymphocytes of experimental rats was evaluated in MTT-colorimetric test with allogeneic glioma cells. RPNS administration increased the cytotoxic activity of lymphocytes in vitro tests with allogeneic tumor cells in intact animals (to 37-38%) as well as in rats with glioma (to 11-22%). Under the RPNS influence the life expectancy and median survival of tumor-bearing animals increased (an average of 3-4 days). RPNS input modes such as triple injection from 5th to 10th day after glioma inoculation and 1 week before inoculation were the most effective. Thus, indirect tumor-inhibiting effect under intraperitoneal. RPNS administration in rats with glioma is demonstrated, which is obviously due to increased efficiency of cytotoxic function of immune cells of animals with inoculated tumor under the influence of the factors produced by NPC.

Abstract 3491: Enhancing nab-paclitaxel antitumor activity through addition of BMS-754807, a small-molecule inhibitor of IGF-1R/IR, in experimental pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, and is expected to become the second-leading cause of cancer death by 2030. Gemcitabine, the standard of care agent over the past 16 years, has limited clinical benefits. Recently, nab-paclitaxel has shown greater efficacy against advanced PDAC in combination with gemcitabine. Our lab has demonstrated superior antitumor activity of nab-paclitaxel compared with gemcitabine or docetaxel in preclinical PDAC studies. Insulin like growth factor (IGF) signaling proteins are frequently overexpressed in PDAC and correlate with aggressive tumor phenotype and poor prognosis indicating that the IGF system is an important therapeutic target in PDAC patients. BMS-754807, a small molecule inhibitor of IGF-type 1 receptor (IGF-1R) and insulin receptor (IR), has shown efficacy in PDAC. We evaluated combination treatment benefits of nab-paclitaxel with BMS-754807 in experimental PDAC. In vitro cell proliferation and protein expression were measured by WST-1 assay and immunoblotting. Heterotopic tumor growth and animal survival experiments were performed in murine xenografts. In vitro analysis of four PDAC cell lines (AsPC-1, BxPC-3, MIA PaCa-2, Panc-1) revealed that single agent nab-paclitaxel and BMS-754807 both inhibited cell proliferation in a dose-dependent manner. To evaluate the combination treatment benefits of nab-paclitaxel and BMS-754807, the IC25 dose of BMS-754807 was combined with increasing doses of nab-paclitaxel. Addition of BMS-754807 decreased the nab-paclitaxel IC50 from 7.2 μM to 490 nM for AsPC-1, 430 nM to 50 nM for BxPC-3, 740 nM to 540 nM for MIA PaCa-2, and 690 nM to 280 nM for Panc-1 cell lines. These data suggest that BMS-754807 combines well with nab-paclitaxel and efficiently decreases the nab-paclitaxel IC50 in PDAC cell lines. In a heterotopic AsPC-1 PDAC model, relative to controls (100±21.3), percent net local tumor growth was 17.1±21.1 with nab-paclitaxel (p = 0.002), 37.6±16.2 with BMS-754807 (p = 0.01) and 0.76±9.1 with nab-paclitaxel plus BMS-754807 (p = 0.0002). In Panc-1 subcutaneous xenografts, compared with controls (100±31.5), percent net tumor growth was 7.8±27.2 with nab-paclitaxel (p = 0.002), 40.2±24.1 with BMS-754807 (p = 0.02) and -29.7±16.8 with nab-paclitaxel plus BMS-754807 (p = 0.0001). In an animal survival study, median animal survival was increased compared to controls (21 days) after nab-paclitaxel therapy (40 days, a 90% increase, p = 0.002), BMS-754807 therapy (27 days, a 29% increase, p = 0.01), and nab-paclitaxel plus BMS-754807 therapy (47 days, a 124% increase, p = 0.005). These results highlight measurable antitumor activity of BMS-754807 in experimental PDAC and support the potential of BMS-754807 in combination with nab-paclitaxel as promising targeted therapy for clinical pancreatic cancer therapy. Citation Format: Niranjan Awasthi, Margaret A. Schwarz, Roderich E. Schwarz. Enhancing nab-paclitaxel antitumor activity through addition of BMS-754807, a small-molecule inhibitor of IGF-1R/IR, in experimental pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3491. doi:10.1158/1538-7445.AM2015-3491

Abstract 1385: TGF beta regulates tumor resistance to antiangiogenic therapy through POSTN in glioma stem cell models

Abstract Periostin (osteoblast-specific factor 2, POSTN) is a 90-kDa ECM (extracellular matrix) protein containing an amino-terminal EMI, tandem repeat of four fascilin-domains and carboxyl-terminal domain including a heparin-binding site. Stromal POSTN plays a key role in regulating cancer stem cell (CSC) maintenance and expansion during metastatic colonization. Recently, it has been reported that POSTN functions as a progression associated and prognostic biomarker in glioma via inducing an invasive and proliferative phenotype. POSTN mRNA expression was significantly higher in grade IV gliomas than in grade II and grade III tumors. POSTN interacts with several integrin receptors such as αvβ1 and αvβ3 to regulate cellular response including cell proliferation, EMT (epithelial-mesenchymal transition) and cell migration. We demonstrated in previous studies that bevacizumab (Bevacizumab, Roche/Genentech) increased glioblastoma invasion in vivo. In the present study, we investigated the role of POSTN and its receptor in tumor invasion and resistance to antiangiogenic therapy. POSTN expression after treatment with avastin in vivo was increased as measured by Western blot and immunofluorescence. Stable knockdown of POSTN expression using specific shRNA abrogated expression of EMT (CAMK2N1, COL1A2, KRT14, COL3A1 and MMP-9) and angiogenesis-related (ANGPTL4, VEGFA, CXCL5, HPSE and EFNA3) genes compared to controls. VEGF expression was decreased in POSTN shRNA, and POSTN shRNA infected cells decreased invasion in both GSC11 and GSC272 glioma stem cell lines. TGF beta1 increased secretion of POSTN and phosphorylation of smad3 through a decrease in POSTN binding to smad3. Moreover, recombinant POSTN increased glioma cell invasion in an intergrin β1 receptor-dependent fashion. In animal experiments, median survival of animals implanted with GSC272-control cells was 63 days, while GSC272-bevacizumab was 84.5 days and knock down of POSTN was 84 days (p<0.05). Treatment with bevacizumab increased survival of POSTN shPOSTN tumors to 114.5 days compare to control (p<0.01). Tumor volume was decreased in the bevacizumab treatment (1.35 mm3) and POSTN shRNA (0.05 mm3) groups compared to controls (7.04 mm3). However, no tumor was observed in mice treated with both POSTN shRNA and bevacizumab. Bevacizumab increased TGF beta expression in GSC272 tumor tissue whereas tumors from POSTN shRNA mice treated with bevacizumab did not show evidence of TGF beta expression. Collectively, our data suggests that TGF beta increases POSTN secretion which appears to play an important role in glioma invasion and resistance to antiangiogenic therapy. Citation Format: Soon Young Park, Yuji Piao, Ningyi Tiao, Verlene Henry, Jianwen Dong, John Frederick de Groot. TGF beta regulates tumor resistance to antiangiogenic therapy through POSTN in glioma stem cell models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1385. doi:10.1158/1538-7445.AM2015-1385

Triptolide abrogates growth of colon cancer and induces cell cycle arrest by inhibiting transcriptional activation of E2F

Despite significant progress in diagnostics and therapeutics, over 50 thousand patients die from colorectal cancer annually. Hence, there is urgent need for new lines of treatment. Triptolide, a natural compound isolated from the Chinese herb Tripterygium wilfordii, is effective against multiple cancers. We have synthesized a water soluble analog of triptolide, named Minnelide, which is currently in phase I trial against pancreatic cancer. The aims of the current study were to evaluate whether triptolide/Minnelide is effective against colorectal cancer and to elucidate the mechanism by which triptolide induces cell death in colorectal cancer. Efficacy of Minnelide was evaluated in subcutaneous xenograft and liver metastasis model of colorectal cancer. For mechanistic studies, colon cancer cell lines HCT116 and HT29 were treated with triptolide and the effect on viability, caspase activation, annexin positivity, lactate dehydrogenase release, and cell cycle progression was evaluated. Effect of triptolide on E2F transcriptional activity, mRNA levels of E2F-dependent genes, E2F1- retinoblastoma protein (Rb) binding, and proteins levels of regulator of G1–S transition was also measured. DNA binding of E2F1 was evaluated by chromatin immunoprecipitation assay. Triptolide decreased colon cancer cell viability in a dose- and time-dependent fashion. Minnelide markedly inhibited the growth of colon cancer in the xenograft and liver metastasis model of colon cancer and more than doubles the median survival of animals with liver metastases from colon cancer. Mechanistically, we demonstrate that at low concentrations triptolide induces apoptotic cell death but at higher concentrations it induces cell cycle arrest. Our data suggest that triptolide is able to induce G1 cell cycle arrest by inhibiting transcriptional activation of E2F1. Our data also show that triptolide downregulates E2F activity by potentially modulating events downstream of DNA binding. Therefore, we conclude that Triptolide and Minnelide are effective against colon cancer in multiple pre-clinical models.

286. Systemic Delivery of AAV9-IFNβ Vectors for Glioblastoma Gene Therapy

Introduction: Glioblastoma multiforme (GBM) is the most prevalent primary brain tumor, with a median survival (MS) of only 14.6 months from diagnosis despite providing multimodal treatment including maximal surgical resection, radiation and chemotherapy. Due to the highly invasive properties of GBM cells, complete surgical resection is practically impossible, leading to tumor recurrence and death. Thus, new approaches are needed to prevent tumor recurrence.IFNb is a pleiotropic cytokine with potent anti-tumor effects. Previous studies have shown that local delivery of AAV-IFNb in a non-invasive GBM mouse model had a remarkable therapeutic effect. However local treatment may be insufficient to treat GBM where long distance migration is a common feature. Therefore the therapeutic approach should be matched to the tumor properties, which may be accomplished with a disperse therapeutic network generated by global brain gene delivery. Here we tested the therapeutic efficacy of an AAV9-IFNb vector infused via the tail vein in an orthotopic xenograft GBM model in athymic nude mice implanted in the striatum with 50,000 highly invasive GBM8 human glioblastoma cells stably expressing firefly luciferase (GBM8-fluc) for in vivo bioluminescence monitoring of tumor growth. AAV9-IFNb vector was infused via the tail vein at different times after tumor implantation.We performed a dose response study by infusing AAV9-IFNb vectors at different doses (1E11vg to 1E12vg/mouse) on d14 posttumor implantation. The improvement in survival with AAV9-IFNb was dose dependent and ranged from an increase in median survival of 111 days to complete tumor regression compared to 46 days in PBS-treated controls. Treatment with 3e11vg AAV9-IFNb resulted in the survival of 87.5% of animals for at least 8 months with no remaining evidence of GBM cells in the brain upon histological examination. This dose was considered the minimal effective dose (MED) and used in subsequent experiments. Next, we examined the impact of time between tumor implantation and treatment by infusion AAV9-IFNb at MED on d14, d21, d25 or d28. We found that d21 is the longest interval showing 100% long-term survival. Interestingly, the survival benefit was drastically reduced when treated on d25 or later, corroborated by Fluc imaging results and histological analysis showing untreated GBM growth accelerates considerably after d21.Finally, we tested the importance of global gene delivery in a model of multifocal GBM model with tumors implanted in both hemispheres. On d14, AAV9-IFNb was infused systemically at MED or injected intratumorally on one side of the brain. We found that systemic delivery resulted in reduction of tumor size in both hemispheres while intra-tumoral injection only impacted tumor growth on the injected side. All animals treated systemically with AAV9-IFNb survived long-term, while the median survival for animals receiving a unilateral injection of AAV9-IFNb was 57 days compared to 46 days for PBS-treated controls.Conclusion: Global delivery of AAV9-IFNb can be beneficial particularly to treat invasive or multifocal GBM and can provide long-term survival in the xenograft GBM model when treated before the onset of highly accelerated tumor growth.

Highly specific in vivo gene delivery for p53-mediated apoptosis and genetic photodynamic therapies of tumour.

Anticancer therapies are often compromised by nonspecific effects and challenged by tumour environments’ inherent physicochemical and biological characteristics. Often, therapeutic effect can be increased by addressing multiple parameters simultaneously. Here we report on exploiting extravasation due to inherent vascular leakiness for the delivery of a pH-sensitive polymer carrier. Tumours’ acidic microenvironment instigates a charge reversal that promotes cellular internalization where endosomes destabilize and gene delivery is achieved. We assess our carrier with an aggressive non-small cell lung carcinoma (NSCLC) in vivo model and achieve >30% transfection efficiency via systemic delivery. Rejuvenation of the p53 apoptotic pathway as well as expression of KillerRed protein for sensitization in photodynamic therapy (PDT) is accomplished. A single administration greatly suppresses tumour growth and extends median animal survival from 28 days in control subjects to 68 days. The carrier has capacity for multiple payloads for greater therapeutic response where inter-individual variability can compromise efficacy.

Increasing Bioavailability of Cytotoxic Agents through Prolonged Therapy and Addition of Polymechanistic Antiangiogenic Agents Enhances Antitumor Response in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis. Meaningful therapeutic options for unresectable PDAC remain limited and therefore more efficient therapeutic strategies are urgently required. We explored the approach of manipulating dose intensity of cytotoxic agents through prolonged therapy and by combination with antiangiogenic agents in experimental PDAC. Median animal survival over controls (19 days) was increased after prolonged therapy (up to 6 weeks) with gemcitabine to 29 days (a 53% increase, p = 0.008). Addition of two antiangiogenic agents, bevacizumab and EMAP, further extended median survival to 46 days (a 142% increase, p = 0.0001). Corresponding survival extension after therapy with similar agents limited to 2 weeks was 37% for gemcitabine and 127% after gemcitabine plus antiangiogenic agents. Pharmacokinetic analysis revealed that the addition of antiangiogenic agents caused a 4.4-fold increase in gemcitabine plasma concentration and 8-fold increase in tumor concentration compared with gemcitabine monotherapy group. Prolonged therapy of docetaxel also increased animal survival (38 days, a 100% increase, p = 0.0004) that was further extended by the addition of the antiangiogenic combination (49 days, a 158% increase, p = 0.0001). In vitro evaluation of exposure time effects on cell proliferation revealed that in PDAC cells (AsPC-1) longer exposure (120 h) of gemcitabine or docetaxel caused greater relative inhibition in cell proliferation compared with 72 h exposure. At 1i­M concentration, inhibition in cell proliferation after 72 h and 120 h was 27% and 55% (gemcitabine); 64% and 84% (docetaxel). Only in endothelial cells and fibroblasts but not in PDAC cells did the combination with antiangiogenic agents create additive effects over each cytotoxic agent alone. These findings demonstrate that increasing bioavailability of cytotoxic agents, both via increased exposure through prolonged therapy or greater intratumoral dose intensity by combination with antiangiogenic agents could enhance in vivo antitumor efficacy and thus warrants further investigation.

Nintedanib, a triple angiokinase inhibitor, enhances cytotoxic therapy response in pancreatic cancer

Angiogenesis remains a sensible target for pancreatic ductal adenocarcinoma (PDAC) therapy. VEGF, PDGF, FGF and their receptors are expressed at high levels and correlate with poor prognosis in human PDAC. Nintedanib is a triple angiokinase inhibitor that targets VEGFR1/2/3, FGFR1/2/3 and PDGFRα/β signaling. We investigated the antitumor activity of nintedanib alone or in combination with the cytotoxic agent gemcitabine in experimental PDAC. Nintedanib inhibited proliferation of cells from multiple lineages found in PDAC, with gemcitabine enhancing inhibitory effects. Nintedanib blocked PI3K/MAPK activity and induced apoptosis in vitro and in vivo. In a heterotopic model, net local tumor growth compared to controls (100%) was 60.8 ± 10.5% in the gemcitabine group, −2.1 ± 9.9% after nintedanib therapy and −12.4 ± 16% after gemcitabine plus nintedanib therapy. Effects of therapy on intratumoral proliferation, microvessel density and apoptosis corresponded with tumor growth inhibition data. In a PDAC survival model, median animal survival after gemcitabine, nintedanib and gemcitabine plus nintedanib was 25, 31 and 38 days, respectively, compared to 16 days in controls. The strong antitumor activity of nintedanib in experimental PDAC supports the potential of nintedanib-controlled mechanisms as targets for improved clinical PDAC therapy.

NT-36 * FOCUSED ULTRASOUND ENHANCED CHEMOTHERAPEUTIC AGENT (TEMOZOLOMIDE) DELIVERY FOR BRAIN TUMOR TREATMENT: A PRECLINICAL INVESTIGATION

Malignant brain tumor is highly aggressive disease with high recurrence and motility rate. Current therapeutic approaches including surgery, radiation and chemotherapy only provide limited disease progression control. One of the major issues results its poor outcome is the blockade of chemotherapeutic agent delivery by the blood-brain barrier (BBB). Recently, focused ultrasound (FUS) with the presence of microbubbles has been confirmed its feasibility in transient and local BBB opening, demonstrated the potential in performing noninvasive brain drug delivery. Therefore, the use of FUS for enhancement of drug delivery to brain parenchyma may serve as a possible treatment of brain tumors. The objective of this study is to investigate the potential in combining the FUS-induced BBB opening with the Temozolomide (TMZ), the first-line brain tumor chemotherapeutic agent, to improve the brain tumor treatment. Brain tumor bearing mice were served as the animal model. Evans-blue was administered to demonstrate the blood-brain BBB-opened site, quantification of TMZ concentration in brain and tumor tissue was performed by high performance liquid chromatography (HPLC) and electrospray ion trap mass spectrum. Our results revealed the accumulation of TMZ in tissue or cerebrospinal fluid of FUS exposed groups is significantly increased as compared with controlled group, which demonstrates the efficacy of FUS-BBB opening for enhancement of local TMZ delivery in brain. Furthermore, FUS-BBB opening combines TMZ treatment significantly inhibits the tumor growth rate and prolongs the median survival of brain tumor animals. Our study provide evidences supporting the effect of FUS-BBB opening in enhancement of local delivery of chemotherapeutic agent to brain and the potential for future clinical application.

Abstract 1025: Antitumor activity of nintedanib (BIBF 1120), a triple angiokinase inhibitor, in combination with gemcitabine in experimental pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States with extremely poor prognosis. Gemcitabine (Gem), the standard of care agent over the past 15 years, has limited clinical benefits. There is an urgent requirement for novel therapeutic strategies to improve overall survival of patients with pancreatic cancer. Angiogenesis, an essential process for tumor growth and metastasis, remains a sensible target for PDAC therapy. VEGF, PDGF, FGF and their receptors are all overexpressed and have been correlated with poor prognosis in human PDAC. Nintedanib (BIBF 1120, Nin) is a triple angiokinase inhibitor that targets VEGFR1/2/3, FGFR1/2/3 and PDGFRα/β signaling. Nintedanib is currently under clinical investigation for several solid tumors. The present study investigated the combination treatment benefits of gemcitabine and nintedanib in experimental PDAC. Cell proliferation, migration and protein expression were analyzed by WST-1 assay, wound healing assay and Western blotting. Tumor growth and animal survival experiments were performed in murine xenografts. Percent inhibition of cell proliferation in the Gem, Nin and Gem+Nin groups was 22%, 25% and 53% (AsPC-1 PDAC cells, Gem 50 nM, Nin 100 nM); 55%, 11% and 80% (endothelial cells HUVECs, Gem 100 nM, Nin 100 nM); 91%, 68% and 94% (fibroblast WI-38 cells, Gem 100 nM, Nin 100 nM). Nintedanib increased apoptosis, indicated by the cleavage of caspase-3 protein in all three cell-types tested. In PDAC cells, antitumor activity was accompanied by higher level of cell cycle inhibitor protein p27. Nintedanib (10 μM) inhibited in vitro wound closure by 84% in endothelial cells. In a heterotopic PDAC model, compared to the percent net local tumor growth in controls (100±29), a reduced growth of 60.8±10.5 (p=0.02) was observed in the Gem group, while a net reduction of -2.1±9.9 (p=0.0001) and -12.4±16.2 (p=0.0001) was observed in Nin and Gem+Nin groups, respectively. Compared with controls (16 days) in an AsPC-1 intraperitoneal model, median animal survival in Gem, Nin and Gem+Nin treatment groups was 25 days, 31 days and 38 days. The strong antitumor activity of nintedanib in experimental PDAC supports the potential of nintedanib-controlled mechanisms as targets for improved clinical PDAC therapy. Citation Format: Niranjan Awasthi, Stefan Hinz, Rolf A. Brekken, Margaret A. Schwarz, Roderich E. Schwarz. Antitumor activity of nintedanib (BIBF 1120), a triple angiokinase inhibitor, in combination with gemcitabine in experimental pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1025. doi:10.1158/1538-7445.AM2014-1025

An autologous in situ tumor vaccination approach for hepatocellular carcinoma. 2. Tumor-specific immunity and cure after radio-inducible suicide gene therapy and systemic CD40-ligand and Flt3-ligand gene therapy in an orthotopic tumor model.

Diffuse hepatocellular carcinoma (HCC) is a lethal disease that radiation therapy (RT) currently has a limited role in treating because of the potential for developing fatal radiation-induced liver disease. However, recently diffuse HCC, "radio-inducible suicide gene therapy" has been shown to enhance local tumor control and residual microscopic disease within the liver for diffuse HCC, by using a combination of chemoactivation and molecular radiosensitization. We have demonstrated that the addition of recombinant adenovirus-expressing human Flt3 ligand (Adeno-Flt3L) after radio-inducible suicide gene therapy induced a Th1-biased, immune response and enhanced tumor control in an ectopic model of HCC. We hypothesized that sequential administration of recombinant adenovirus-expressing CD40L (Adeno-CD40L) could further potentiate the efficacy of our trimodal therapy with RT + HSV-TK + Adeno-Flt3L. We examined our hypothesis in an orthotopic model of diffuse HCC using BNL1ME A.7R.1 (BNL) cells in Balb/c mice. BNL murine hepatoma cells (5 × 10(4)) transfected with an expression vector of HSV-TK under the control of a radiation-inducible promoter were injected intraportally into BALB/cJ mice. Fourteen days after the HCC injection, mice were treated with a 25 Gy dose of radiation to the whole liver, followed by ganciclovir (GCV) treatment and systemic adenoviral cytokine gene therapy (Flt3L or CD40L or both). Untreated mice died in 27 ± 4 days. Radiation therapy alone had a marginal effect on survival (median = 35 ± 7 days) and the addition of HSV-TK/GCV gene therapy improved the median survival to 47 ± 6 days. However, the addition of Adeno-Flt3L to radiation therapy and HSV-TK/GCV therapy significantly (P = 0.0005) increased survival to a median of 63 ± 20 days with 44% (7/16) of the animals still alive 116 days after tumor implantation. The curative effect of Flt3L was completely abolished when using immunodeficient nude mice or mice depleted for CD4, CD8 and natural killer cells. The addition of Adeno-CD40L further improved the median survival of animals to 80 ± 15 days and this effect was abolished only when using anti-CD8 antibodies. Chromium-51 (51Cr) release assay showed cytotoxic T lymphocyte (CTL) activation, suggesting efficient dendritic cell (DC) activation with CTL activation after the treatment. Furthermore, when surviving mice were rechallenged with BNL-ETK cells on the foot pad, RT + HSV-TK/GCV + Flt3L + CD40L-treated mice developed a small tumor on day 56 but the tumor eventually disappeared after 105 days. Mice treated with RT + HSV-TK/GCV + Flt3L showed a slowed tumor growth curve compared with untreated mice. Therefore, combination therapy using Flt3L to induce DC proliferation and CD40L to enhance DC maturation holds great promise for immunomodulation of radiation therapy to enhance HCC tumor control and prevent progression of disease in patients with diffuse HCC.

Enhancement of Nab-Paclitaxel Antitumor Activity through Addition of Multitargeting Antiangiogenic Agents in Experimental Pancreatic Cancer

Nanoparticle albumin-bound paclitaxel (nab-paclitaxel, NPT) has recently shown efficacy in pancreatic ductal adenocarcinoma (PDAC). Targeting tumor angiogenesis is a sensible combination therapeutic strategy for cancer, including PDAC. We tested the hypothesis that NPT response in PDAC can be enhanced by the mechanistically different antiangiogenic agents bevacizumab (Bev) or sunitinib (Su), despite its inherently increased tumor penetration and drug delivery. Compared with controls (19 days), median animal survival was increased after NPT therapy (32 days, a 68% increase, P = 0.0008); other regimens with enhanced survival were NPT+Bev (38 days, a 100% increase, P = 0.0004), NPT+Su (37 days, a 95% increase, P = 0.0004), and NPT+Bev+Su (49 days, a 158% increase, P = 0.0001) but not bevacizumab, sunitinib, or Bev+Su therapy. Relative to controls (100 ± 22.8), percentage net local tumor growth was 28.2 ± 23.4 with NPT, 55.6 ± 18 (Bev), 38.8 ± 30.2 (Su), 11 ± 7.2 (Bev+Su), 32.8 ± 29.2 (NPT+Bev), 6.6 ± 10.4 (NPT+Su), and 13.8 ± 12.5 (NPT+Bev+Su). Therapeutic effects on intratumoral proliferation, apoptosis, microvessel density, and stromal density corresponded with tumor growth inhibition data. In AsPC-1 PDAC cells, NPT IC(50) was reduced >6-fold by the addition of sunitinib (IC(25)) but not by bevacizumab. In human umbilical vein endothelial cells (HUVEC), NPT IC(50) (82 nmol/L) was decreased to 41 nmol/L by bevacizumab and to 63 nmol/L by sunitinib. In fibroblast WI-38 cells, NPT IC(50) (7.2 μmol/L) was decreased to 7.8 nmol/L by sunitinib, but not by bevacizumab. These findings suggest that the effects of one of the most active cytotoxic agents against PDAC, NPT, can be enhanced with antiangiogenic agents, which clinically could relate to greater responses and improved antitumor results.

The dual PI3K/mTOR inhibitor NVP-BEZ235 enhances nab-paclitaxel antitumor response in experimental gastric cancer

Gastric cancer is the second most common cause of cancer-related deaths worldwide. Taxanes have shown therapeutic effects against gastric cancer while also activating the PI3K/mTOR signaling pathway. We investigated the effects of NVP-BEZ235 (BEZ235), a novel dual PI3K/mTOR inhibitor, alone and in combination with nanoparticle albumin-bound (nab)-paclitaxel in experimental gastric cancer. Cell proliferation and protein expression were measured by WST-1 assay and immunoblotting. Tumor growth and survival studies were performed in murine xenografts. Phosphorylated mTOR and 4E-BP1 levels were elevated in gastric cancer cells and tumor tissues by nab-paclitaxel. BEZ235 effectively inhibited cell proliferation in vitro and provided additive effects in combination with nab-paclitaxel. Furthermore, BEZ235 blocked the activated PI3K/mTOR pathway either alone or in combination with nab-paclitaxel in gastric cancer cells. BEZ235 and nab-paclitaxel caused an increase in PARP-1 and caspase-3 cleavage. Net local tumor growth inhibition for the BEZ235, nab-paclitaxel and BEZ235+nab-paclitaxel groups was 45.1, 77.9 and 97% compared to controls. The effects of therapy on intratumoral proliferation and apoptosis corresponded with tumor growth inhibition data. BEZ235 also caused a decrease in phospho-mTOR and phospho-Akt in tumor tissue lysates. Median animal survival (controls, 23 days) was 26.5 days after BEZ235 (p=0.227), 90.5 days after nab-paclitaxel (p=0.001) and 97 days in the BEZ235+nab-paclitaxel combination treatment group (p=0.001). Our findings suggest that BEZ235 exerts some antitumor effects against gastric cancer and enhances effects of nab-paclitaxel through inhibition of cell proliferation and modulation of the PI3K/mTOR pathway. This approach may represent a promising combination targeted therapy for gastric cancer.

Superior Antitumor Activity of Nanoparticle Albumin-Bound Paclitaxel in Experimental Gastric Cancer

Gastric cancer is the second common cause of cancer related death worldwide and lacks highly effective treatment for advanced disease. Nab-paclitaxel is a novel microtubule-inhibitory cytotoxic agent that has not been tested in gastric cancer as of yet. In this study, human gastric cancer cell lines AGS, NCI-N87 and SNU16 were studied. Nab-paclitaxel inhibited cell proliferation with an IC50 of 5 nM in SNU16, 23 nM in AGS and 49 nM in NCI-N87 cells after 72-hour treatment, which was lower than that of oxaliplatin (1.05 μM to 1.51 μM) and epirubicin (0.12 μM to 0.25 μM). Nab-paclitaxel treatment increased expression of the mitotic-spindle associated phospho-stathmin irrespective of the baseline total or phosphorylated stathmin level, and induced mitotic cell death as confirmed through increased expression of cleaved-PARP and caspase-3. After a two-week nab-paclitaxel, oxaliplatin or epirubicin treatment, the average in vivo local tumor growth inhibition rate was 77, 17.2 and 21.4 percent, respectively (p = 0.002). Effects of therapy on tumoral proliferative and apoptotic indices corresponded with tumor growth inhibition data, while expression of phospho-stathmin also increased in tissues. There was an increase in median animal survival after nab-paclitaxel treatment (93 days) compared to controls (31 days, p = 0.0007), oxaliplatin (40 days, p = 0.0007) or to docetaxel therapy (81 days, p = 0.0416). The strong antitumor activity of nab-paclitaxel in experimental gastric cancer supports such microtubule-inhibitory strategy for clinical application. Nab-paclitaxel benefits were observed independent from phosphorylated stathmin expression at baseline, putting into question the consideration of nab-paclitaxel use in gastric cancer based on this putative biomarker.

Comparative benefits of nab-paclitaxel over gemcitabine or polysorbate-based docetaxel in experimental pancreatic cancer.

192 Background: Gemcitabine (Gem), a standard cytotoxic therapy for pancreatic cancer, has shown limited clinical benefits. Nanoparticle albumin-bound (nab) paclitaxel (NPT), an approved treatment for breast cancer, has shown efficacy as mono- and combination therapy in multiple tumor types including pancreatic, lung and ovarian cancer. We evaluated the NPT treatment benefits compared with Gem or solvent-based taxane docetaxel (DT) in experimental pancreatic cancer. Methods: In vitro cell proliferation and protein expression were measured by WST-1 assay and immunoblotting. Tumor growth and animal survival studies were performed in murine xenografts. Intratumoral proliferative activity was measured using Ki67 nuclear antigen staining. Results: For AsPC-1, BxPC-3, MIA PaCa-2 and Panc-1 cells in vitro, Gem IC50 levels were 23.9 mM, 506 nM, 332 nM and 14.5 nM; DT IC50 levels were 30 nM, 4.6 nM, 37.5 nM and 27 nM; and NPT IC50 levels were 7.6 mM, 208 nM, 519 nM and 526 nM. NPT addition decreased Gem IC50 to 1.7 mM, 189 nM, 123 nM and 913 nM; DT addition decreased Gem IC50 to 436 nM, 470 nM, 124 nM and 0.2 nM in AsPC-1, BxPC-3, MIA PaCa-2 and Panc-1 cells, respectively. NPT and DT treatment increased stathmin phosphorylation and decreased tubulin expression in vitro. In a heterotopic in vivo model, net tumor growth inhibition after Gem, DT and NPT was 67, 31 and 72 percent, while intratumoral proliferative index inhibition was 41, 53 and 68 percent, respectively. In an intraperitoneal model, median animal survival was significantly longer in the NPT treatment group (41 days, p<0.002 vs. control and Gem) compared to Gem (32 days, p=0.005 vs. control), DT (32 days, p=0.005 vs. control) and controls (20 days). Animal survival in NPT-Gem and DT-Gem sequential treatment groups was 43 and 40 days, and thus not superior to NPT alone. Conclusions: Nab-paclitaxel has significantly superior antitumor activity as a single agent in experimental pancreatic cancer compared with gemcitabine or docetaxel. These findings provide a strong rationale for considering nab-paclitaxel as first-line monotherapy in patients with pancreatic cancer.

Antitumor activity of nanoparticle albumin-bound paclitaxel in experimental gastric cancer.

33 Background: Gastric cancer is the second most common cause of cancer related death worldwide and lacks highly effective adjuvant or definitive systemic treatment for advanced disease. Nab-paclitaxel is a novel microtubule-targeting cytotoxic agent and not tested in gastric cancer as of yet. Methods: Human gastric cancer cell lines AGS, NCI-N87 and SNU16 were studied for treatment effects on cell proliferation, mitotic arrests and apoptosis in vitro and vivo. Tumor growth and survival studies were performed in murine xenografts. Results: Nab-paclitaxel inhibited cell proliferation with an IC50 of 2.01 nM in SNU16, 23.3 nM in AGS and 48.69 nM in NCI-N87 cells after 72-hour treatment, which was lower than that of oxaliplatin (1.05 μM to 1.51μM) and epirubicin (0.12 μM to 0.25 μM). Nab-paclitaxel treatment caused increased expression of the mitotic-spindle associated phospho-stathmin, nuclear fragmentation or karyopyknosis, and apoptotic events as confirmed through increased expression of cleaved-PARP and caspase-3. After a two-week nab-paclitaxel, oxaliplatin or epirubicin treatment, the local tumor growth inhibition rate was 77, 17.2 and 21.4 percent, respectively (p=0.002). Effects of therapy on tumoral proliferative and apoptotic indices corresponded with tumor growth inhibition data, while expression of phospho-stathmin also increased in tissues. There was an increase in median animal survival after nab-paclitaxel treatment (86 days) compared to controls (24 days, p=0.0004) or to oxaliplatin therapy (37.5 days, p=0.0005). Conclusions: The strong antitumor activity of nab-paclitaxel in experimental gastric cancer supports such microtubule-targeting therapy for clinical application. Nab-paclitaxel benefits were observed independent from phosphorylated stathmin expression at baseline, putting into question the consideration of nab-paclitaxel use in gastric cancer based on this putative biomarker.