RESUMO
The fetal oncogene 5T4 is a cell surface protein, with overexpression observed in a variety of cancers as compared to normal adult tissue. The ability to select patients with tumors that express high levels of 5T4 may enrich a clinical trial cohort with patients most likely to respond to 5T4 targeted therapy. To that end, we developed assays to measure 5T4 in both tumors and in circulating tumor cells (CTCs). We identified the presence of 5T4 in both adenocarcinoma and squamous cell carcinoma of lung, in all clinical stages and grades of disease. CTCs were identified in peripheral blood from the majority of patients with NSCLC, and 5T4 was detectable in most samples. Although 5T4 was present in both CTCs and tumors in most patients, there was no concordance between relative amount in either sample type. Clinical response rates of patients treated with the therapies directed against 5T4 in early stage clinical trials, as determined by these assays, may provide important insights into the biology of 5T4 in tumors and the mechanisms of action of 5T4-targeting therapy.
Assuntos
Adenocarcinoma/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Pulmonares/metabolismo , Glicoproteínas de Membrana/metabolismo , Células Neoplásicas Circulantes/metabolismo , Adenocarcinoma/patologia , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Transplante de Neoplasias , Células Neoplásicas Circulantes/patologiaRESUMO
Noninvasive imaging has been widely applied for monitoring antiangiogenesis therapy in cancer drug discovery. In this report, we used different imaging modalities including high-frequency ultrasound (HFUS), dynamic contrast enhanced-MR (DCE-MR), and fluorescence molecular tomography (FMT) imaging systems to monitor the changes in the tumor vascular properties after treatment with γ-secretase inhibitor PF-03084014. Sunitinib was tested in parallel for comparison. In the MDA-MB-231Luc model, we demonstrated that antiangiogenesis was one of the contributing mechanisms for the therapeutic effect of PF-03084014. By immunohistochemistry and FITC-lectin perfusion assays, we showed that the vascular defects upon treatment with PF-03084014 were associated with Notch pathway modulation, evidenced by a decrease in the HES1 protein and by the changes in VEGFR2 and HIF1α levels, which indicates down-stream effects. Using a 3D power Doppler scanning method, ultrasound imaging showed that the% vascularity in the MDA-MB-231Luc tumor decreased significantly at 4 and 7 days after the treatment with PF-03084014. A decrease in the tumor vessel function was also observed through contrast-enhanced ultrasound imaging with microbubble injection. These findings were consistent with the PF-03084014-induced functional vessel changes measured by suppressing the K(trans) values using DCE-MRI. In contrast, the FMT imaging with the AngioSence 680EX failed to detect any treatment-associated tumor vascular changes. Sunitinib demonstrated an outcome similar to PF-03084014 in the tested imaging modalities. In summary, ultrasound and DCE-MR imaging successfully provided longitudinal measurement of the phenotypic and functional changes in tumor vasculature after treatment with PF-03084014 and sunitinib.
Assuntos
Imageamento por Ressonância Magnética , Neovascularização Patológica/diagnóstico por imagem , Neovascularização Patológica/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Inibidores da Angiogênese/administração & dosagem , Linhagem Celular Tumoral , Meios de Contraste/química , Humanos , Indóis/administração & dosagem , Imagem Molecular , Neovascularização Patológica/patologia , Pirróis/administração & dosagem , Radiografia , Sunitinibe , Tetra-Hidronaftalenos/administração & dosagem , Ultrassonografia , Valina/administração & dosagem , Valina/análogos & derivados , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Notch signaling mediates breast cancer cell survival and chemoresistance. In this report, we aimed to evaluate the antitumor efficacy of PF-03084014 in combination with docetaxel in triple-negative breast cancer models. The mechanism of action was investigated. PF-03084014 significantly enhanced the antitumor activity of docetaxel in multiple xenograft models including HCC1599, MDA-MB-231Luc, and AA1077. Docetaxel activated the Notch pathway by increasing the cleaved Notch1 intracellular domain and suppressing the endogenous Notch inhibitor NUMB. PF-03084014 used in combination with docetaxel reversed these effects and demonstrated early-stage synergistic apoptosis. Docetaxel elicited chemoresistance by elevating cytokine release and expression of survivin and induced an endothelial mesenchymal transition (EMT) phenotype by increasing the expressions of Snail, Slug, and N-cadherin. When reimplanted, the docetaxel-residual cells not only became much more tumorigenic, as evidenced by a higher fraction of tumor-initiating cells (TICs), but also showed higher metastatic potential compared with nontreated cells, leading to significantly shortened survival. In contrast, PF-03084014 was able to suppress expression of survivin and MCL1, reduce ABCB1 and ABCC2, upregulate BIM, reverse the EMT phenotype, and diminish the TICs. Additionally, the changes to the ALDH(+) and CD133(+)/CD44(+) subpopulations following therapy corresponded with the TIC self-renewal assay outcome. In summary, PF-03084014 demonstrated synergistic effects with docetaxel through multiple mechanisms. This work provides a strong preclinical rationale for the clinical utility of PF-03084014 to improve taxane therapy.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Antígeno AC133 , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Docetaxel , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Glicoproteínas/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos SCID , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Taxoides/administração & dosagem , Tetra-Hidronaftalenos/administração & dosagem , Fatores de Tempo , Valina/administração & dosagem , Valina/análogos & derivados , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: We aimed to assess the biologic activity of PF-03084014 in breast xenograft models. The biomarkers for mechanism and patient stratification were also explored. EXPERIMENTAL DESIGN: The in vitro and in vivo properties of PF-03084014 were investigated. The mRNA expressions of 40 key Notch pathway genes at baseline or after treatment were analyzed to link with the antitumor efficacy of PF-03084014 in a panel of breast cancer xenograft models. RESULTS: In vitro, PF-03084014 exhibited activity against tumor cell migration, endothelial cell tube formation, and mammosphere formation. In vivo, we observed apoptosis, antiproliferation, reduced tumor cell self-renewal ability, impaired tumor vasculature, and decreased metastasis activity after the treatment of PF-03084014. PF-03084014 treatment displayed significant antitumor activity in 10 of the 18 breast xenograft models. However, the antitumor efficacy in most models did not correlate with the in vitro antiproliferation results in the corresponding cell lines, suggesting the critical involvement of tumor microenvironment during Notch activation. In the tested breast xenograft models, the baseline expressions of the Notch receptors, ligands, and the cleaved Notch1 failed to predict the antitumor response to PF-03084014, whereas several Notch pathway target genes, including HEY2, HES4, and HES3, strongly corresponded with the response with a P value less than 0.01. Many of the best molecular predictors of response were also significantly modulated following PF-03084014 treatment. CONCLUSIONS: PF-03084014 showed antitumor and antimetastatic properties via pleiotropic mechanisms. The Notch pathway downstream genes may be used to predict the antitumor activity of PF-03084014 and enrich for responders among breast cancer patients.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Tetra-Hidronaftalenos/farmacologia , Valina/análogos & derivados , Secretases da Proteína Precursora do Amiloide/metabolismo , Antineoplásicos/administração & dosagem , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Metástase Neoplásica/tratamento farmacológico , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tetra-Hidronaftalenos/administração & dosagem , Valina/administração & dosagem , Valina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: [(18)F]FLT (3'-Fluoro-3' deoxythymidine)-PET imaging was proposed as a tool for measuring in vivo tumor cell proliferation. The aim of this article was to validate the use of [(18)F]FLT-PET imaging for measuring xenograft proliferation and subsequent monitoring of targeted therapy. EXPERIMENTAL DESIGN: In exponentially growing xenografts, factors that could impact the outcome of [(18)F]FLT-PET imaging, such as nucleoside transporters, thymidine kinase 1, the relative contribution of DNA salvage pathway, and the ratio of FLT to thymidine, were evaluated. The [(18)F]FLT tracer avidity was compared with other proliferation markers. RESULTS: In a panel of proliferating xenografts, [(18)F]FLT or [(3)H]thymidine tracer avidity failed to reflect the tumor growth rate across different tumor types, despite the high expressions of Ki67 and TK1. When FLT was injected at the same dose level as used in the preclinical [(18)F]FLT-PET imaging, the plasma exposure ratio of FLT to thymidine was approximately 1:200. Thymidine levels in different tumor types seemed to be variable and exhibited an inverse relationship with the FLT tracer avidity. In contrast, high-dose administration of bromdeoxyuridine (BrdUrd; 50 mg/kg) yielded a plasma exposure of more than 4-fold higher than thymidine and leads to a strong correlation between the BrdUrd uptake and the tumor proliferation rate. In FLT tracer-avid models, [(18)F]FLT-PET imaging as a surrogate biomarker predicted the therapeutic response of CDK4/6 inhibitor PD-0332991. CONCLUSIONS: Tumor thymidine level is one of the factors that impact the correlation between [(18)F]FLT uptake and tumor cell proliferation. With careful validation, [(18)F]FLT-PET imaging can be used to monitor antiproliferative therapies in tracer-avid malignancies.
Assuntos
Didesoxinucleosídeos , Neoplasias/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Bromodesoxiuridina/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Didesoxinucleosídeos/farmacocinética , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Nus , Camundongos SCID , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Piperazinas/administração & dosagem , Piperazinas/farmacologia , Piridinas/administração & dosagem , Piridinas/farmacologia , Timidina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: P-cadherin is a membrane glycoprotein that functionally mediates tumor cell adhesion, proliferation, and invasiveness. We characterized the biological properties of PF-03732010, a human monoclonal antibody against P-cadherin, in cell-based assays and tumor models. EXPERIMENTAL DESIGN: The affinity, selectivity, and cellular inhibitory activity of PF-03732010 were tested in vitro. Multiple orthotopic and metastatic tumor models were used for assessing the antitumor and antimetastatic activities of PF-03732010. Treatment-associated pharmacodynamic changes were also investigated. RESULTS: PF-03732010 selectively inhibits P-cadherin-mediated cell adhesion and aggregation in vitro. In the P-cadherin-overexpressing tumor models, including MDA-MB-231-CDH3, 4T1-CDH3, MDA-MB-435HAL-CDH3, HCT116, H1650, PC3M-CDH3, and DU145, PF-03732010 inhibited the growth of primary tumors and metastatic progression, as determined by bioluminescence imaging. Computed tomography imaging, H&E stain, and quantitative PCR analysis confirmed the antimetastatic activity of PF-03732010. In contrast, PF-03732010 did not show antitumor and antimetastatic efficacy in the counterpart tumor models exhibiting low P-cadherin expression. Mechanistic studies via immunofluorescence, immunohistochemical analyses, and 3'-[(18)F]fluoro-3'-deoxythymidine-positron emission tomography imaging revealed that PF-03732010 suppressed P-cadherin levels, caused degradation of membrane ß-catenin, and concurrently suppressed cytoplasmic vimentin, resulting in diminished metastatic capacity. Changes in the levels of Ki67, caspase-3, and 3'-[(18)F]fluoro-3'-deoxythymidine tracer uptake also indicated antiproliferative activity and increased apoptosis in the tested xenografts. CONCLUSIONS: These findings suggest that interrupting the P-cadherin signaling pathway may be a novel therapeutic approach for cancer therapy. PF-03732010 is presently undergoing evaluation in Phase 1 clinical trials.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Caderinas/imunologia , Metástase Neoplásica/tratamento farmacológico , Neoplasias/tratamento farmacológico , Animais , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Metástase Neoplásica/prevenção & controle , Transplante de Neoplasias , Neoplasias/patologia , Transplante Heterotópico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Phase II attrition of clinical candidates in the drug development cycle is currently a major issue facing the pharmaceutical industry. To decrease phase II attrition, there is an increased emphasis on validation of mechanism of action, development of efficacy models and measurement of drug levels at the site of action. PD 0332991, a highly specific inhibitor of cyclin-dependent kinase 4 (CDK-4) is currently in clinical development for the treatment of solid tumor. A clinical presurgical study will be required to better understand how PD 0332991 affects signaling pathways and how the intratumoral concentration of PD 0332991 correlates with plasma PK parameters and molecular alterations in breast cancer tissues after PD 0332991 treatment. Before conducting such a clinical study, it is important to evaluate PD 0332991 levels in tumor tissue samples from a xenograft mouse model for the determination of drug exposure at the site of action. Therefore, the objectives of this study were (1) to develop and validate a sensitive LC-MS/MS method to quantify PD 0332991 in mouse tumor tissues from MDA-MB-231-Luc human breast tumor xenografts in SCID-beige mice; (2) to quantify PD 0332991 levels in mouse tumor tissues after oral administration of PD 0332991 at 10 and 100mg/kg using the validated LC-MS/MS method. Both liquid-liquid extraction (LLE) and supported liquid extraction (SLE) in a 96-well format were developed and evaluated to achieve optimal extraction recovery with minimal matrix effects. The newly developed SLE method is more efficient (speed and ease) and demonstrates comparable recovery (93.1-100% at three different concentrations) compared to the traditional LLE method. The validated LC-MS/MS for PD 032291 in mouse tumor tissue homogenate method exhibited a linear dynamic range of 0.1-100 ng/mL with inter-day accuracy and precision within 15%. The validated method was successfully applied to measure PD 0332991 levels in tumor tissues in MDA-MB-231-Luc human breast tumor xenografts in SCID beige mice. The mean tumor concentrations at 6h post-oral PD 0332991 administration at 10 and 100mg/kg were 1793 (+/-1008) and 25,163 (+/-3959) ng/g, respectively.
Assuntos
Cromatografia Líquida/métodos , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Neoplasias Experimentais/química , Piperazinas/análise , Inibidores de Proteínas Quinases/análise , Piridinas/análise , Espectrometria de Massas em Tandem/métodos , Animais , Estabilidade de Medicamentos , Feminino , Humanos , Camundongos , Camundongos SCID , Transplante de Neoplasias , Sensibilidade e Especificidade , Transplante HeterólogoRESUMO
PURPOSE: Checkpoint kinase 1 (Chk1) plays a critical role in the activation of mitotic spindle checkpoint and DNA damage checkpoint. We examined the preclinical use of the Chk1 inhibitor PF-00477736 as a docetaxel-sensitizing agent. Specifically, we investigated the correlation between PF-00477736-mediated modulation of biomarkers and the sensitization of docetaxel efficacy. EXPERIMENTAL DESIGN: In vitro and in vivo studies using COLO205 and other cell lines were done to assess PF-00477736-induced enhancement of docetaxel efficacy and effects on associated biomarkers. RESULTS: PF-00477736 significantly enhanced the docetaxel-induced efficacy in tumor cells and xenografts. Docetaxel induced dose- and time-dependent increase in the levels of phosphorylated Chk1 (Ser(345)), phosphorylated histone H3 (Ser(10)), and gammaH2AX foci and promoted the cytoplasmic localization of phosphorylated Cdc25C (Ser(216)). PF-00477736 cotreatment suppressed docetaxel-induced changes in phosphorylated histone H3 and cytoplasmic phosphorylated Cdc25C (Ser(216)) levels and concurrently sensitized the docetaxel-induced apoptosis. Docetaxel alone or in combination with PF-00477736 induced significant antiproliferative activity in xenografts, shown via [18F]FLT-PET imaging. However, changes in [18F]FLT uptake did not reflect the potentiation of docetaxel efficacy. In contrast, bioluminescence imaging showed that PF-00477736 sensitized docetaxel-induced suppression of tumor survival. CONCLUSIONS: Docetaxel triggers mitotic spindle checkpoint activation at low concentrations and activates both the DNA damage checkpoint and the spindle checkpoint at high concentrations. In combination with docetaxel, PF-00477736 abrogates the mitotic checkpoint, as well as the DNA damage checkpoint, and results in sensitization to docetaxel. Chk1 inhibitor PF-00477736 offers a therapeutic potential for the enhancement of taxane therapy.
Assuntos
Benzodiazepinonas/farmacologia , Neoplasias/tratamento farmacológico , Proteínas Quinases/metabolismo , Pirazóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Benzodiazepinonas/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Didesoxinucleosídeos , Docetaxel , Sinergismo Farmacológico , Radioisótopos de Flúor , Histonas/metabolismo , Humanos , Camundongos , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Pirazóis/administração & dosagem , Taxoides/administração & dosagem , Taxoides/farmacologia , Tomografia Computadorizada de Emissão , Carga Tumoral/efeitos dos fármacos , Fosfatases cdc25/metabolismoRESUMO
PURPOSE: Tumors grafted s.c. or under the mammary fat pad (MFP) rarely develop efficient metastasis. By applying bioluminescence imaging (BLI) technology, the MDA-MB-435-HAL-Luc subrenal capsule (SRC) model was compared with the MFP model for disease progression, metastatic potential, and response to therapy. EXPERIMENTAL DESIGN: The luciferase-expressing MDA-MB-435-HAL-Luc cell line was used in both MFP and SRC models. BLI technology allowed longitudinal assessment of disease progression and the therapeutic response to PD-0332991, Avastin, and docetaxel. Immunohistochemical analysis of Ki67 and CD31 staining in the primary tumors was compared in these models. Caliper measurement was used in the MFP model to validate the BLI quantification of primary tumors. RESULTS: The primary tumors in MDA-MB-435-HAL-Luc MFP and SRC models displayed comparable growth rates and vascularity. However, tumor-bearing mice in the SRC model developed lung metastases much earlier (4 weeks) than in the MFP model (>7 weeks), and the metastatic progression contributed significantly to the survival time. In the MFP model, BLI and caliper measurements were comparable for quantifying palpable tumors, but BLI offered an advantage for detecting the primary tumors that fell below a palpable threshold and for visualizing metastases. In the SRC model, BLI allowed longitudinal assessment of the antitumor and antimetastatic effects of PD-0332991, Avastin, and docetaxel, and the results correlated with the survival benefits of these agents. CONCLUSIONS: The MDA-MB-435-HAL-Luc SRC model and the MFP model displayed differences in disease progression. BLI is an innovative approach for developing animal models and creates opportunities for improving preclinical evaluations of anticancer agents.
Assuntos
Proteínas Luminescentes , Transplante de Neoplasias/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Bevacizumab , Linhagem Celular Tumoral , Docetaxel , Feminino , Humanos , Rim , Neoplasias Pulmonares/secundário , Glândulas Mamárias Humanas , Camundongos , Camundongos Nus , Camundongos SCID , Neoplasias Experimentais/tratamento farmacológico , Piperazinas/uso terapêutico , Piridinas/uso terapêutico , Taxoides/uso terapêuticoRESUMO
2-D gel electrophoresis has been used for more than three decades to study the protein complement of organisms, tissues, and cells. Three issues are holding back large-scale proteomics studies: low-throughput, high technical variation, and study designs lacking statistical power. We identified image analysis as the central factor connecting these three issues. By developing an improved image analysis workflow we shortened project timelines, decreased technical variation, and thus enabled large-scale proteomics studies that are statistically powered. Rather than detecting protein spots on each gel image and matching spots across gel images, the improved workflow is based on aligning images first, then creating a consensus spot pattern and finally propagating the consensus spot pattern to all gel images for quantitation. This results in a data table without gaps. As an example we show here a study aimed at discovering circulating biomarkers for chronic obstructive pulmonary disease (COPD). Eight candidate biomarkers were identified by comparing plasma from 24 smokers with COPD and 24 smokers without COPD. Among the candidates are proteins such as plasma retinal-binding protein (RETB) and fibrinogen that had previously been linked to the disease and are frequently monitored in COPD patients, as well as other proteins such as apolipoprotein E (ApoE), inter-alpha-trypsininhibitor heavy chain H4 (ITIH4), and glutathione peroxidase.