Cell Lung Cancer Xenograft Model Research Articles

Abstract 4465: In vivo antitumor characterization of the hypoxia-activated prodrug TH-302 in a preclinical SCLC xenograft model

Abstract The hypoxia activated prodrug TH-302, has showed anti-tumor activity in a variety of preclinical in vivo models. In hypoxic conditions, TH-302 acts as a DNA crosslinking agent by the reductase- and oxygen concentration-dependent release of bromo-IPM. In the present study, we investigated the anti-cancer mechanisms of TH-302 in a small cell lung cancer (SCLC) xenograft model with a focus on selective effects on the hypoxic compartment and tumor vasculature. The ectopic xenograft model was set up by s.c. implantation of 5×106 H82 human SCLC cells into the flank of nude mice. When tumor size reached 500 to 700mm3, animals were randomized (5-8 mice/group) and were injected i.p. with a single dose of 150mg/kg TH-302 or vehicle. Tumors were harvested for frozen sections 24, 48 and 72 hours after TH-302 treatment. Pimonidazole, for detection of hypoxia, was administered at 60mg/kg i.p. 1 hour before sacrifice, and to assess the effect of TH-302 on tumor vascular perfusion, Hoechst 33342 was injected at 10mg/kg i.v. into the tail vein 1 min before sacrifice. Anti-CD31 antibody was utilized as a marker for the microvasculature. Morphometric analysis was conducted by point counting and with Image-Pro Plus 6.0 software. Previous studies showed that TH-302 as a single agent, exhibited efficacy in the H82 xenograft model with tumor growth inhibition at about 55%. In the current study, anti-tumor activity was evaluated by assessment of tumor necrosis after H&E staining. The necrotic fraction in the vehicle treated tumor was 7.3% and reached 17.2%, 37.9% and 34.8% after 24, 48 and 72hrs of TH-302 treatment, respectively. A significant increase of necrosis occurred from 48hrs after the treatment (p<0.05). The pimonidazole-positive hypoxic fraction decreased after TH-302 treatment and reached a significant difference at 48hrs (6.3% ± 1.2% in vehicle vs. 1.8% ± 1.1% in the 48hr treatment group, p<0.05). Tumor vessel density and vascular perfusion in the non-necrotic area, as characterized by CD31-positive microvasculature cells and Hoechst 33342-positive cells, showed no significant difference between vehicle and any of the treated groups. There was significant correlation between tumor vessel density (CD31) and tumor vessel flow (Hoechst 33342) (R2=0.28, p<0.05). The results demonstrate an antitumor effect of TH-302 through cytotoxic activity on the hypoxic compartment in the tumor. In addition, a bystander effect is likely, given the observation that the necrosis fraction was 38% 48hrs after TH-302 treatment while the hypoxic fraction was less than 8%. Unlike tirapazamine, another hypoxia-activated prodrug which can cause vascular dysfunction, TH-302 appears to exhibit minor effects on the vasculature. The results support an in vivo tumor hypoxia targeting mechanism of TH-302. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4465.

Abstract B24: Establishment of human primary non-small cell lung cancer xenograft models to test anticancer drugs

Abstract Background: Human xenograft tumor models established by transplantation of human tumor cell lines into immunodifficient mice have been routinely used for preclinical test of anticancer agents. But such tumor models have a relatively low transplantability and limited correlation with clinical findings. Recently, we have developed human non-small lung cancer (NSCLC) xenograft models by transplanting human fresh lung cancer tissues into nude mice, which have been employed for test of clinically used chemotherapeutical drugs and are under way for new drug chemosensitivity in vivo screening. Methods: The fresh lung cancer samples were collected from local hospitals. The tumor fragments of 1–2 mm3 were subcutaneously implanted in the flanks of the Balb/c nude mice by trocar needle. Sixteen tumor fragments were grafted into four mice from one patient tumor tissue (passage 1). In the first passage, tumors derived from male patients were implanted into male mice, and tumors from women were inoculated into female mice. All therapeutic efficacy experiments used female mice. The drugs tested were clinically used drugs for NSCLC including cisplatin (4 mg/kg, q3d x 4), paclitaxel (15 mg/kg, q3d x 4), vinorelbine (2.5 mg/kg, q4d x 4), gemcitabine (120 mg/kg, q4d x 4), and the epidermal growth factor receptor (EGFR) inhibitor erlotinib (50 mg/kg/daily x 14, po). Results: A total of 42 NSCLC samples were implanted, and 13 tumor models were established (tumor taking rate 31%) for the first passage. The tumor taking rates were higher in the second and third passages (80–100%). Cisplatin, paclitaxel, vinorelbine, and gemcitatbine produced tumor growth inhibition rates of 48–55% regardless of the EGFR mutation status. While erlotinib demonstrated a significant antitumor activity only in the tumors bearing EGFR, which were consistent with their clinical findings. The tumor xenografts' architecture, the cell and histopathological morphology from the three generations mirrored the original patient cancers. Conclusions: These results suggest that human primary tumor xenograft models provide a unique renewable source of tumor material for test of novel anticancer agents. They may predict more relevant clinical response rates and higher correlation with clinical findings than use of xenograft models established from in vitro long-term cultured cancer cell lines, especially for test of target-oriented therapeutics in new drugs development programs. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B24

Pentastatin-1, a collagen IV derived 20-mer peptide, suppresses tumor growth in a small cell lung cancer xenograft model

BackgroundAngiogenesis is the formation of neovasculature from a pre-existing vascular network. Progression of solid tumors including lung cancer is angiogenesis-dependent. We previously introduced a bioinformatics-based methodology to identify endogenous anti-angiogenic peptide sequences, and validated these predictions in vitro in human umbilical vein endothelial cell (HUVEC) proliferation and migration assays.MethodsOne family of peptides with high activity is derived from the α-fibrils of type IV collagen. Based on the results from the in vitro screening, we have evaluated the ability of a 20 amino acid peptide derived from the α5 fibril of type IV collagen, pentastatin-1, to suppress vessel growth in an angioreactor-based directed in vivo angiogenesis assay (DIVAA). In addition, pentastatin-1 suppressed tumor growth with intraperitoneal peptide administration in a small cell lung cancer (SCLC) xenograft model in nude mice using the NCI-H82 human cancer cell line.ResultsPentastatin-1 decreased the invasion of vessels into angioreactors in vivo in a dose dependent manner. The peptide also decreased the rate of tumor growth and microvascular density in vivo in a small cell lung cancer xenograft model.ConclusionsThe peptide treatment significantly decreased the invasion of microvessels in angioreactors and the rate of tumor growth in the xenograft model, indicating potential treatment for angiogenesis-dependent disease, and for translational development as a therapeutic agent for lung cancer.

Abstract B255: Discovery and validation of a novel class of small molecule inhibitors of the CDC7 kinase: Modulation of tumor cell growth in vitro and in vivo

Abstract High throughput screening of compounds comprising the Memorial Sloan Kettering chemical library resulted in several confirmed hits against the recombinant Cdc7:Dbf4 heterodimeric kinase, a key regulator in the initiation of DNA replication and the G1 to S phase transition. Chemoinformatic analysis of the hits revealed an enrichment in one chemical cluster made up of several naturally occurring compounds, of which the most potent compound, CKI-7, was selected for further investigation. First, CKI-7 was found to be a non competitive inhibitor for ATP and prompted us to prolife it against a panel of 200 known kinases in order to assess its selectivity profile. The results were as predicted and very few kinases were specifically affected. Second, CKI-7 cytotoxic activity was assessed against a panel of well established cancer cell lines representing both hematopoietic and solid tumor malignancies as well as against a panel of primary hematopoietic cells derived from leukemia patients (both chemotherapy naive and relapsed/refractory samples) and was found to be a very effective agent with potencies in the low nanomolar range. Subsequent studies using an isogenic pair of cell lines with one over expressing the Bcl_xL anti-apoptotic protein further confirmed the induction of the intrinsic apoptotic pathway via caspase-3 activation in the absence and attenuation of the activity in the presence of Bcl_xL. This was further demonstrated through standard cell cycle synchronization studies revealing that exposure to the Cdc7 inhibitor results in an S phase arrest, cell cycle dependent caspase-3 activation, and apoptotic cell death. This cell death is the direct result of Cdc7 kinase inhibition by CKI-7 as demonstrated using a Cdc7 substrate biomarker assay. Third, the physicochemical properties of this class of naturally occurring compounds also prompted us to investigate their effect on several multidrug resistence (MDR) over-expressing cell lines. We found that CKI-7 was not a substrate for the efflux pumps demonstrating that this novel compound can overcome a major mechanism of chemotherapy resistence in human tumor cells. Based of the above observations, in vivo dose-dependent anti-tumor activity of CKI-7 was subsequently demonstrated in a SCID-Beige mouse systemic tumor model utilizing a recently isolated Philadelphia chromosome positive acute lymphoblastic leukemia cell line (PhALL3.1) in addition to melanoma and non small cell lung cancer xenograft models. Taken together, our data confirm that Cdc7 is a new promising target for cancer therapy, and that the newly discovered inhibitor CKI-7, a naturally occurring selective small molecule inhibitor of this enzyme, is an equally promising novel cancer therapeutic agent. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B255.

Abstract C138: Anti-αv integrin monoclonal antibody intetumumab (CNTO 95) enhances the therapeutic efficacy of radiation therapy and reduces metastasis of human cancer xenografts in nude rats

Abstract Radiation therapy is used to treat cancer in over 50% of all cancer patients. Interaction of tumor cells with the extracellular matrix via adhesion molecules is important for survival of cancer stem cells, and may play a role in resistance to radiation therapy. Preclinical and clinical studies from our lab and others have demonstrated that a fully human anti- v integrin monoclonal antibody intetumumab (CNTO 95) may enhance the therapeutic index of radiotherapy by selective targeting of upregulated integrins on tumor cells and vascular endothelial cells of tumors. We previously reported that intetumumab is a radiosensitizer in mice with xenograft tumors. However, since intetumumab does not cross-react with mouse integrins, but has limited cross-reactivity with rat integrins, we next studied the potential of combined use of fractionated local tumor radiation therapy and intetumumab in human cancer xenograft models in nude rats in order to assess effects on both tumor cells and vasculatures. We found that intetumumab alone had a moderate effect on tumor growth in human head and neck cancer and non-small cell lung cancer xenograft models. When combined with fractionated radiation therapy, intetumumab significantly inhibited tumor growth and produced a tumor response rate that was significantly better than with radiation therapy alone (P < 0.01). The total tumor response rate (complete response and partial response) was 67% in A549 NSCLC model for intetumumab plus radiotherapy compared with 17% for radiotherapy alone. Treatment with intetumumab reduces the lung metastasis by inhibiting the homing of A549 NSCLC cells, as well as spontaneous metastasis to lung from subcutaneous tumors in A549 NSCLC xenograft model. The blood perfusion and blood volume in xenograft tumors measured by micro-bubble enhanced ultrasound image was substantially improved by treatment of intetumumab. Immunohistochemistry staining of xenograft tumor sections demonstrated that the combined use of intetumumab and radiation therapy significantly reduced the microvessel density and increased apoptosis in tumor cells and the tumor microenvironment. The oxygenation and hypoxic status of tumors treated with intetumumab with or without radiotherapy is under investigation and will be presented. Furthermore, intetumumab alone and in combination with radiation was well-tolerated and did not produce any obvious signs of systemic toxicity. An in vivo toxicity study of crypt stem cell survival in the gastrointestinal tract demonstrated that treatment with intetumumab did not alter the number of surviving crypt stem cells of duodenum, jejunum, or ileum following irradiation, suggesting that intetumumab did not sensitize the sensitive GI epithelium to the effect of radiation therapy. These results demonstrate that intetumumab can potentiate the efficacy of fractionated radiation therapy in human cancer xenograft tumors in nude rats. These findings are promising and may have high translational relevance for the treatment of patients with solid tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C138.

Establishment of human primary non-small cell lung cancer xenograft models for test of cytotoxic and targeted anticancer drugs

11008 Background: New oncology drug development has moved from general cytotoxic agents to molecular target-directed therapeutics. Consequently, there is a need to identify tumor types and individual patient tumors that express the target and could benefit from more selective therapies in clinical trials. Therefore, the in vivo models used in preclinical development should be“disease-oriented” and target-directed. Recently, we developed non-small cell lung cancer (NSCLC) xenograft models by transplanting human fresh tumor fragments into nude mice, which have been used for test of cytotoxic and targeted anticancer drugs. Methods: The fresh NSCLC tumors were collected from local hospitals. The tumor fragments were subcutaneously implanted into nude mice. The EGFR and K-ras mutation status of the tumors were investigated and compared with the efficacy results. The test drugs included paclitaxel, gemcitabine, and the epidermal growth factor receptor (EGFR) inhibitor erlotinib. Results: A total of 72 NSCLC samples were implanted, and 13 tumor models were established (tumor taking rate 18%) for the first passage. The tumor taking rates were higher in the second and third passages (80–100%). Paclitaxel and gemcitatbine produced tumor growth inhibition rates of 50–53% regardless of the EGFR and K-ras mutation status. While erlotinib demonstrated a significant antitumor activity only in the tumors bearing EGFR mutation with wild-type K-ras, which were consistent with their clinical findings. The tumor xenografts’ architecture, the cell and histopathological morphology from the three generations mirrored the original patient cancers. Conclusions: These results suggest that human primary tumor xenograft models provide a unique renewable source of tumor material for test of novel anticancer agents. They may predict more relevant clinical response rates and higher correlation with clinical findings than use of xenograft models established from long-term cultured cancer cell lines, especially for test of target-oriented therapeutics in new drugs development programs. No significant financial relationships to disclose.

Antibodies targeting hepatoma-derived growth factor as a novel strategy in treating lung cancer

Hepatoma-derived growth factor (HDGF) is overexpressed in lung cancer and the overexpression correlates with aggressive biological behaviors and poor clinical outcomes. We developed anti-HDGF monoclonal antibodies and tested their antitumor activity in lung cancer xenograft models. We also determined biological effects in tumors treated with the antibody alone or in combination with bevacizumab/avastin (an anti-vascular endothelial growth factor antibody) and/or gemcitabine (a chemotherapeutic agent). We found the anti-HDGF was effective to inhibit tumor growth in non-small cell lung cancer xenograft models. In the A549 model, compared with control IgG, tumor growth was substantially inhibited in animals treated with anti-HDGF antibodies, particularly HDGF-C1 (P = 0.002) and HDGF-H3 (P = 0.005). When HDGF-H3 was combined with either bevacizumab or gemcitabine, we observed enhanced tumor growth inhibition, particularly when the three agents were used together. HDGF-H3-treated tumors exhibited significant reduction of microvessel density with a pattern distinctive from the microvessel reduction pattern observed in bevacizumab-treated tumors. HDGF-H3-treated but not bevacizumab-treated tumors also showed a significant increase of apoptosis. Interestingly, many of the apoptotic cells in HDGF-H3-treated tumors are stroma cells, suggesting that the mechanism of the antitumor activity is, at least in part, through disrupting formation of tumor-stroma structures. Our results show that HDGF is a novel therapeutic target for lung cancer and can be effectively targeted by an antibody-based approach.

Molecular pharmacodynamics of PM02734 (elisidepsin) as single agent and in combination with erlotinib; synergistic activity in human non-small cell lung cancer cell lines and xenograft models

PM02734 (elisidepsin) is a novel marine-derived cyclic peptide belonging to the Kahalalide family of compounds currently under phase I development with early evidence of a positive therapeutic index. The cytotoxicity of PM02734 has been determined in a panel of human NSCLC (non-small cell lung cancer) cell lines. Western blot analysis showed a direct correlation between ErbB3 expression and cell sensitivity to PM02734. Furthermore, PM02734 was more effective in the induction of ErbB3 degradation and dephosphorylation than in that of ErbB2 and ErbB1 in human NSCLC cell lines. The combination of PM02734 and erlotinib was synergistic in all NSCLC cell lines tested, including erlotinib resistant cell lines, with combination indexes ranging between 0.59 and 0.81. The combination of PM02734 and erlotinib was more effective than either drug alone in mice inoculated intravenously (i.v.) with A549 cells. The combination of PM02734 and erlotinib was more effective in inhibiting AKT than either single agent alone in H322 cells. These results have provided a rational basis for an ongoing clinical trial to explore this combination in patients with advanced malignant solid tumours.

Rational Optimization of a Bispecific Ligand Trap Targeting EGF Receptor Family Ligands

The human epidermal growth factor (EGF) receptor (HER) family members cooperate in malignancy. Of this family, HER2 does not bind growth factors and HER3 does not encode an active tyrosine kinase. This diversity creates difficulty in creating pan-specific therapeutic HER family inhibitors. We have identified single amino acid changes in epidermal growth factor receptor (EGFR) and HER3 which create high affinity sequestration of the cognate ligands, and may be used as receptor decoys to downregulate aberrant HER family activity. In silico modeling and high throughput mutagenesis were utilized to identify receptor mutants with very high ligand binding activity. A single mutation (T15S; EGFR subdomain I) enhanced affinity for EGF (two-fold), TGF-alpha (twenty-six-fold), and heparin-binding (HB)-EGF (six-fold). This indicates that T15 is an important, previously undescribed, negative regulatory amino acid for EGFR ligand binding. Another mutation (Y246A; HER 3 subdomain II) enhanced neuregulin (NRG)1-beta binding eight-fold, probably by interfering with subdomain II-IV interactions. Further work revealed that the HER3 subunit of an EGFR:HER3 heterodimer suppresses EGFR ligand binding. Optimization required reversing this suppression by mutation of the EGFR tether domain (G564A; subdomain IV). This mutation resulted in enhanced ligand binding (EGF, ten-fold; TGF-alpha, thirty-four-fold; HB-EGF, seventeen-fold; NRG1-beta, thirty-one-fold). This increased ligand binding was reflected in improved inhibition of in vitro tumor cell proliferation and tumor suppression in a human non-small cell lung cancer xenograft model. In conclusion, amino acid substitutions were identified in the EGFR and HER3 ECDs that enhance ligand affinity, potentially enabling a pan-specific therapeutic approach for downregulating the HER family in cancer.

Establishment of Patient-Derived Non–Small Cell Lung Cancer Xenografts as Models for the Identification of Predictive Biomarkers

It was the aim of our study to establish an extensive panel of non-small cell lung cancer (NSCLC) xenograft models useful for the testing of novel compounds and for the identification of biomarkers. Starting from 102 surgical NSCLC specimens, which were obtained from primarily diagnosed patients with early-stage tumors (T(2)/T(3)), 25 transplantable xenografts were established and used for further investigations. Early passages of the NSCLC xenografts revealed a high degree of similarity with the original clinical tumor sample with regard to histology, immunohistochemistry, as well as mutation status. The chemotherapeutic responsiveness of the xenografts resembled the clinical situation in NSCLC with tumor shrinkage obtained with paclitaxel (4 of 25), gemcitabine (3 of 25), and carboplatin (3 of 25) and lower effectiveness of etoposide (1 of 25) and vinorelbine (0 of 11). Twelve of 25 NSCLC xenografts were >50% growth inhibited by the anti-epidermal growth factor receptor (EGFR) antibody cetuximab and 6 of 25 by the EGFR tyrosine kinase inhibitor erlotinib. The response to the anti-EGFR therapies did not correlate with mutations in the EGFR or p53, but there was a correlation of K-ras mutations and erlotinib resistance. Protein analysis revealed a heterogeneous pattern of expression. After treatment with cetuximab, we observed a down-regulation of EGFR in 2 of 6 sensitive xenograft models investigated but never in resistant models. An extensive panel of patient-derived NSCLC xenografts has been established. It provides appropriate models for testing marketed as well as novel drug candidates. Additional expression studies allow the identification of stratification biomarkers for targeted therapies.

Activity of the Bcl-2 Family Inhibitor ABT-263 in a Panel of Small Cell Lung Cancer Xenograft Models

The purpose of this study was to characterize the activity of the Bcl-2 protein family inhibitor ABT-263 in a panel of small cell lung cancer (SCLC) xenograft models. A panel of 11 SCLC xenograft models was established to evaluate the efficacy of ABT-263. Single agent activity was examined on a continuous dosing schedule in each of these models. The H146 model was used to further evaluate dose and schedule, comparison to standard cytotoxic agents, and induction of apoptosis. ABT-263 exhibited a range of antitumor activity, leading to complete tumor regression in several models. Significant regressions of tumors as large as 1 cc were also observed. The efficacy of ABT-263 was also quite durable; in several cases, minimal tumor regrowth was noted several weeks after the cessation of treatment. Antitumor effects were equal or superior to that of several clinically approved cytotoxic agents. Regression of large established tumors was observed through several cycles of therapy and efficacy was retained in a Pgp-1 overexpressing line. Significant efficacy was observed on several dose and therapeutic schedules and was associated with significant induction of apoptosis. ABT-263 is a potent, orally bioavailable inhibitor of Bcl-2 family proteins that has recently entered clinical trials. The efficacy data reported here suggest that SCLC is a promising area of clinical investigation with this agent.

Antitumor activity of noscapine in human non-small cell lung cancer xenograft model

An antitussive plant alkaloid, Noscapine HCl (Nos) displays anticancer activity and has a safe pharmacological profile in humans. The current study was aimed to investigate the in vitro and in vivo anti tumor activity of Nos to determine possible mechanisms of anti tumor activity for treatment of non-small cell lung cancer (NSCLC). In vitro cytotoxicity of Nos was studied in H460 cells treated with different doses of Nos (10-160 microM) for 72 h and cell viability was determined using crystal violet assay. Apoptosis in H460 cells was evaluated by TUNEL assay after treatment of cells for 72 h with 30 and 40 microM doses of Nos. For in vivo studies, female athymic Nu/nu mice were xenografted with H460 tumors and on day 4 onwards Nos was administered orally at dose of 300, 450 and 550 mg/kg/day for 24 days. As a control, xenografted tumors were separately treated with Docetaxel (10 mg/kg i.v. bolus on day 5, 11, 17, 23). The tumor volumes were measured every five days. Expression of PARP, Bcl(2, )Bax, and caspase-3 families of proteins was measured by Western Blotting (WB), while TUNEL and Immunohistochemical methods were utilized to determine DNA fragmentation and cleaved caspase-3 levels respectively. Nos inhibited growth of H460 cells with the IC50 values of 34.7 +/- 2.5 microM. Nos at 30 and 40 microM doses caused apoptosis as evidenced by nuclear condensation in treated H460 cells. Nos caused 49, 65 and 86% reduction in the xenografted tumor volumes at a dose of 300 (P < 0.05), 450 (P < 0.01), 550 mg/kg/day (P < 0.01), respectively, when compared to controls. Nos-dependent suppression of xenografted tumor growth involved up regulation of PARP, Bax, caspase-3 and repression of Bcl(2) expression. An increase in Bax/Bcl(2) ratio suggests involvement of a mitochondrial mediated apoptotic processes. Our studies revealed a non significant (P > 0.05) increase in Bax/Bcl(2) ratio with Nos at a dose of 300 mg/kg/day, while a significant (P < 0.001) increase in Bax/Bcl(2) ratio was observed with Nos doses of 450 and 550 mg/kg/day. Further, Nos caused elevated apoptosis in tumor xenografts as evidenced by enhanced expression of caspase-3 and positive TUNEL staining in regressed tumor tissues, thus suggesting induction of apoptosis by mitochondrial pathway. Our studies suggest that potent antitumor activity of Nos against NSCLC cells. Oral administration of Nos showed significant reduction in tumor volume in human non-small cell lung tumor xenograft in nude mice in a dose dependant manner. Thus, Nos is a promising novel chemotherapeutic agent for the treatment of human lung cancer.

SNX2112, a Synthetic Heat Shock Protein 90 Inhibitor, Has Potent Antitumor Activity against HER Kinase–Dependent Cancers

The heat shock protein 90 (Hsp90) chaperone plays an important role in transformation by regulating the conformational maturation and stability of oncogenic kinases and transcription factors. Ansamycins, such as 17-(allylamino)-17-demethoxygeldanmycin (17-AAG), inhibit Hsp90 function; induce the degradation of Hsp90 client proteins such as HER2, and have shown activity in early clinical trials. However, the utility of these drugs has been limited by their hepatotoxicity, poor solubility, and poorly tolerated formulations. We determined the pharmacodynamic and antitumor properties of a novel, synthetic Hsp90 inhibitor, SNX-2112, in cell culture and xenograft models of HER kinase-dependent cancers. We show in a panel of tumor cell lines that SNX-2112 and its prodrug SNX-5542 are Hsp90 inhibitors with properties and potency similar to that of 17-AAG, including: degradation of HER2, mutant epidermal growth factor receptor, and other client proteins, inhibition of extracellular signal-regulated kinase and Akt activation, and induction of a Rb-dependent G(1) arrest with subsequent apoptosis. SNX-5542 can be administered to mice orally on a daily schedule. Following oral administration, SNX-5542 is rapidly converted to SNX-2112, which accumulates in tumors relative to normal tissues. A single dose of SNX-5542 causes HER2 degradation and inhibits its downstream signaling for up to 24 h, and daily dosing results in regression of HER2-dependent xenografts. SNX-5542 also shows greater activity than 17-AAG in a non-small cell lung cancer xenograft model expressing mutant EGFR. These results suggest that Hsp90 inhibition with SNX-2112 (delivered as a prodrug) may represent a promising therapeutic strategy for tumors whose growth and survival is dependent on Hsp90 clients.

Inhalation Delivery and Anti-tumor Activity of Celecoxib in Human Orthotopic Non-Small Cell Lung Cancer Xenograft Model

To determine the in vivo anti-tumor effect of aerosolized Celecoxib (Cxb) in combination with i.v Docetaxel (Doc) and compare the anti-tumor effect with oral Cxb combined with i.v Doc in human orthotopic non-small cell lung cancer (NSCLC) xenograft model. Female Nu/Nu mice were implanted with orthotopic tumors by injecting A549 cells into the lung parenchyma. Seven day after tumor implantation the mice were treated with aerosolized Cxb (30 min exposure/day, 5 mg/ml solution) + i.v Doc (10 mg/kg) and the effect was compared with oral Cxb (150 mg/kg/day) + i.v Doc (10 mg/kg), for 28 days. Small-animal nose only inhalation chamber (CH Technologies, Westwood, NJ) was utilized for aerosol exposure. Therapeutic activity of Cxb (aerosol/oral) + Doc was estimated by differences in lung weight, tumor area and animal body weight. Lung tumor samples isolated from mice were analyzed for (a) PGE2 levels by enzyme immunoassay (EIA) (b) expression of Fas and Factor VIII by immunohistochemistry (c) IL-8 expression using EIA kits and (d) mRNA expression for caspase-3 by Real-Time PCR. Mice treated with Cxb (aerosol/oral) + Doc showed significant reduction (P < 0.001) in lung weight and tumor area as compared to Cxb or Doc treatments. Cxb (aerosol/oral) + Doc showed increased apoptosis mediated via increased Fas and caspase-3 (P < 0.001) expression as compared to untreated control. Further, the combination treatment showed antiangiogenic effect as demonstrated by reduced expression of Factor VIII, IL-8 (P < 0.001) and PGE2 (P < 0.001) in lung tumors as compared to untreated control. Aerosolized Cxb at a significantly lower therapeutic dose (4.56 mg/kg/day) demonstrated comparable anti-tumor efficacy to orally administered Cxb (150 mg/kg/day). Cxb was formulated and effectively delivered via aerosolization to treat orthotopic lung tumors in combination with i.v Doc. Cxb when administered by aerosol produced same therapeutic effect as oral Cxb, but at lower therapeutic dose and thus shows promise for the treatment of lung cancer.

146 E7080, a novel multi-targeted tyrosine kinase inhibitor, exhibits anti-angiogenic activity via inhibition of KIT signalling in a small cell lung cancer xenograft model

Vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis by stimulating the proangiogenic signaling of endothelial cells via activation of VEGF receptor (VEGFR) tyrosine kinases. Therefore, VEGFRs are an attractive therapeutic target for cancer treatment. In the present study, we show that a quinoline-urea derivative, KRN951, is a novel tyrosine kinase inhibitor for VEGFRs with antitumor angiogenesis and antigrowth activities. KRN951 potently inhibited VEGF-induced VEGFR-2 phosphorylation in endothelial cells at in vitro subnanomolar IC50 values (IC50 = 0.16 nmol/L). It also inhibited ligand-induced phosphorylation of platelet-derived growth factor receptor-beta (PDGFR-β) and c-Kit (IC50 = 1.72 and 1.63 nmol/L, respectively). KRN951 blocked VEGF-dependent, but not VEGF-independent, activation of mitogen-activated protein kinases and proliferation of endothelial cells. In addition, it inhibited VEGF-mediated migration of human umbilical vein endothelial cells. Following p.o. administration to athymic rats, KRN951 decreased the microvessel density within tumor xenografts and attenuated VEGFR-2 phosphorylation levels in tumor endothelium. It also displayed antitumor activity against a wide variety of human tumor xenografts, including lung, breast, colon, ovarian, pancreas, and prostate cancer. Furthermore, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) analysis revealed that a significant reduction in tumor vascular hyperpermeability was closely associated with the antitumor activity of KRN951. These findings suggest that KRN951 is a highly potent, p.o. active antiangiogenesis and antitumor agent, and that DCE-MRI would be useful in detecting early responses to KRN951 in a clinical setting. KRN951 is currently in phase I clinical development for the treatment of patients with advanced cancer.

Antitumor activity of ZD6126, a novel vascular-targeting agent, is enhanced when combined with ZD1839, an epidermal growth factor receptor tyrosine kinase inhibitor, and potentiates the effects of radiation in a human non-small cell lung cancer xenograft model

Targeting the tumor vasculature may offer an alternative or complementary therapeutic approach to targeting growth factor signaling in lung cancer. The aim of these studies was to evaluate the antitumor effects in vivo of the combination of ZD6126, a tumor-selective vascular-targeting agent; ZD1839 (gefitinib, Iressa), an epidermal growth factor receptor tyrosine kinase inhibitor; and ionizing radiation in the treatment of non-small cell lung cancer xenograft model. Athymic nude mice with established flank A549 human non-small cell lung cancer xenograft model xenografts were treated with fractionated radiation therapy, ZD6126, ZD1839, or combinations of each treatment. ZD6126 (150 mg/kg) was given i.p. the day after each course of radiation. Animals treated with ZD1839 received 100 mg/kg per dose per animal, 5 or 7 days/wk for 2 weeks. Immunohistochemistry was done to evaluate the effects on tumor growth using an anti-Ki67 monoclonal antibody. Effects on tumor-induced vascularization were quantified using an anti-factor VIII-related antigen monoclonal antibody. ZD6126 attenuated the growth of human A549 flank xenografts compared with untreated animals. Marked antitumor effects were observed when animals were treated with a combination of ZD6126 and fractionated radiation therapy with protracted tumor regression. ZD6126 + ZD1839 resulted in a greater tumor growth delay than either agent alone. Similar additive effects were seen with ZD1839 + fractionated radiation. Finally, the addition of ZD6126 to ZD1839 and radiation therapy seemed to further improve tumor growth control, with a significant tumor growth delay compared with animals treated with single agent or with double combinations. Immunohistochemistry showed that ZD1839 induced a marked reduction in A549 tumor cell proliferation. Both ZD1839 and ZD6126 treatment substantially reduced tumor-induced angiogenesis. ZD6126 caused marked vessel destruction through loss of endothelial cells and thrombosis, substantially increasing the level of necrosis seen when combined with radiation therapy. The combination of radiation therapy, ZD6126, and ZD1839 induced the greatest effects on tumor growth and angiogenesis. This first report shows that a selective vascular-targeting agent (ZD6126) + an anti-epidermal growth factor receptor agent (ZD1839) and radiation have additive in vivo effects in a human cancer model. Targeting the tumor vasculature offers an excellent strategy to enhance radiation cytotoxicity. Polytargeted therapy with agents that interfere with both growth factor and angiogenic signaling warrants further investigation.

Gene expression imaging with radiolabeled peptides.

An approach to image radiolabeled peptide localization at tumor sites by inducing tumor cells to synthesize membrane expressed human somatostatin receptor subtype 2 (hSSTr2) with a high affinity for radiolabeled somatostatin analogues is described. The use of gene transfer technology to induce expression of high affinity membrane hSSTr2 can enhance the specificity and degree of radiolabeled peptide localization in tumors. Employing this strategy, induction of high levels of hSSTr2 expression with selective tumor uptake of radiolabeled peptides was achieved in both subcutaneous non-small cell lung cancer and intraperitoneal ovarian cancer mouse human tumor xenograft models. The features of this genetic transduction imaging approach are: (1) constitutive expression of a tumor-associated receptor is not required; (2) tumor cells are altered to express a new target receptor or increased quantities of a constitutive receptor at levels which may significantly increase tumor targeting of radiolabeled peptides compared to uptake in normal tissues; (3) gene transfer can be accomplished by local or regional injection of adenoviral vectors; (4) it is feasible to target adenovirus vectors to tumor cells by modifying adenoviral tropism (binding) or by the use of tumor-specific promoters such that the hSSTr2 will be specifically expressed in the desired tumor; and (5) this technique can be used to image expression of a second therapeutic gene.

2-deoxy-D-glucose increases the efficacy of adriamycin and paclitaxel in human osteosarcoma and non-small cell lung cancers in vivo.

Slow-growing cell populations located within solid tumors are difficult to target selectively because most cells in normal tissues also have low replication rates. However, a distinguishing feature between slow-growing normal and tumor cells is the hypoxic microenvironment of the latter, which makes them extraordinarily dependent on anaerobic glycolysis for survival. Previously, we have shown that hypoxic tumor cells exhibit increased sensitivity to inhibitors of glycolysis in three distinct in vitro models. Based on these results, we predicted that combination therapy of a chemotherapeutic agent to target rapidly dividing cells and a glycolytic inhibitor to target slow-growing tumor cells would have better efficacy than either agent alone. Here, we test this strategy in vivo using the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) in combination with Adriamycin (ADR) or paclitaxel in nude mouse xenograft models of human osteosarcoma and non-small cell lung cancer. Nude mice implanted with osteosarcoma cells were divided into four groups as follows: (a) untreated controls; (b) mice treated with ADR alone; (c) mice treated with 2-DG alone; or (d) mice treated with a combination of ADR + 2-DG. Treatment began when tumors were either 50 or 300 mm(3) in volume. Starting with small or large tumors, the ADR + 2-DG combination treatment resulted in significantly slower tumor growth (and therefore longer survival) than the control, 2-DG, or ADR treatments (P < 0.0001). Similar beneficial effects of combination treatment were found with 2-DG and paclitaxel in the MV522 non-small cell lung cancer xenograft model. In summary, the treatment of tumors with both the glycolytic inhibitor 2-DG and ADR or paclitaxel results in a significant reduction in tumor growth compared with either agent alone. Overall, these results, combined with our in vitro data, provide a rationale for initiating clinical trials using glycolytic inhibitors in combination with chemotherapeutic agents to increase their therapeutic effectiveness.