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Environmental chemicals, such as PFAS, exist as mixtures and are frequently encountered at varying concentrations, which can lead to serious health effects, such as cancer. Therefore, understanding the dose-dependent toxicity of chemical mixtures is essential for health risk assessment. However, comprehensive methods to assess toxicity and identify the mechanisms of these harmful mixtures are currently absent. In this study, the dose-dependent toxicity assessments of chemical mixtures are performed in three methodologically distinct phases. In the first phase, we evaluated our machine-learning method (AI-HNN) and pathophysiology method (CPTM) for predicting toxicity. In the second phase, we integrated AI-HNN and CPTM to establish a comprehensive new approach method (NAM) framework called AI-CPTM that is targeted at refining prediction accuracy and providing a comprehensive understanding of toxicity mechanisms. The third phase involved experimental validations of the AI-CPTM predictions. Initially, we developed binary, multiclass classification, and regression models to predict binary, categorical toxicity, and toxic potencies using nearly a thousand experimental mixtures. This empirical dataset was expanded with assumption-based virtual mixtures, compensating for the lack of experimental data and broadening the scope of the dataset. For comparison, we also developed machine-learning models based on RF, Bagging, AdaBoost, SVR, GB, KR, DT, KN, and Consensus methods. The AI-HNN achieved overall accuracies of over 80%, with the AUC exceeding 90%. In the final phase, we demonstrated the superior performance and predictive capability of AI-CPTM, including for PFAS mixtures and their interaction effects, through rigorous literature and statistical validations, along with experimental dose-response zebrafish-embryo toxicity assays. Overall, the AI-CPTM approach significantly improves upon the limitations of standalone AI models, showing extensive enhancements in identifying toxic chemicals and mixtures and their mechanisms. This study is the first to develop a hybrid NAM that integrates AI with a pathophysiology method to comprehensively predict chemical-mixture toxicity, carcinogenicity, and mechanisms.
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DiI is a lipophilic fluorescent dye frequently used to label and trace cells in cell cultures and xenograft models. However, DiI can also transfer from labeled to unlabeled cells, including host organism cells, and label dead cells obscuring interpretation of the results. These limitations of DiI labeling in xenograft models have not been thoroughly investigated. Here we labeled green fluorescent protein (GFP)-expressing MDA-MB-231 cells with DiI to directly compare tumor growth assessment in zebrafish xenografts using the DiI labeling and GFP fluorescence. Our results indicate that the DiI based assessment significantly overestimated tumor growth in zebrafish xenograft models compared to the GFP fluorescence based assessment. The imaging of DiI labeled GFP-expressing MDA-MB-231 cell cultures indicated that the DiI labeling of the membrane is uneven. Analysis of the DiI labeled GFP-expressing MDA-MB-231 cell cultures with flow cytometry indicated that the DiI labeling varied over time while the GFP fluorescence remained unchanged, suggesting that the GFP fluorescence is a more reliable signal for monitoring tumor progression than the DiI labeling. Taken together, our results demonstrate limitations of using DiI labeling for xenograft models and emphasize the need for validating the results based on DiI labeling with other orthogonal methods, such as the ones utilizing genetically encoded fluorophores.
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Neoplasias , Peixe-Zebra , Animais , Humanos , Proteínas de Fluorescência Verde/genética , Fluorescência , Peixe-Zebra/metabolismo , Xenoenxertos , Corantes Fluorescentes/metabolismoRESUMO
Natural killer (NK) cells play a critical role in physiologic and pathologic conditions such as pregnancy, infection, autoimmune disease and cancer. In cancer, numerous strategies have been designed to exploit the cytolytic properties of NK cells, with variable success. A major hurdle to NK-cell focused therapies is NK cell recruitment and infiltration into tumors. While the chemotaxis pathways regulating NK recruitment to different tissues are well delineated, the mechanisms human NK cells employ to physically migrate are ill-defined. We show for the first time that human NK cells express fibroblast activation protein (FAP), a cell surface protease previously thought to be primarily expressed by activated fibroblasts. FAP degrades the extracellular matrix to facilitate cell migration and tissue remodeling. We used novel in vivo zebrafish and in vitro 3D culture models to demonstrate that FAP knock out and pharmacologic inhibition restrict NK cell migration, extravasation, and invasion through tissue matrix. Notably, forced overexpression of FAP promotes NK cell invasion through matrix in both transwell and tumor spheroid assays, ultimately increasing tumor cell lysis. Additionally, FAP overexpression enhances NK cells invasion into a human tumor in immunodeficient mice. These findings demonstrate the necessity of FAP in NK cell migration and present a new approach to modulate NK cell trafficking and enhance cell-based therapy in solid tumors.
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Cancer metastasis is the primary cause of the high mortality rate among human cancers. Efforts to identify therapeutic agents targeting cancer metastasis frequently fail to demonstrate efficacy in clinical trials despite strong preclinical evidence. Until recently, most preclinical studies used mouse models to evaluate anti-metastatic agents. Mouse models are time-consuming and expensive. In addition, an important drawback is that mouse models inadequately model the early stages of metastasis which plausibly leads to the poor correlation with clinical outcomes.Here, we report an in vivo model based on xenografted zebrafish embryos where we select for progressively invasive subpopulations of MDA-MB-231 breast cancer cells. A subpopulation analogous to circulating tumor cells found in human cancers was selected by injection of MDA-MB-231 cells into the yolk sacs of 2 days post-fertilized zebrafish embryos and selecting cells that migrated to the tail. The selected subpopulation derived from MDA-MB-231 cells were increasingly invasive in zebrafish. Isolation of these subpopulations and propagation in vitro revealed morphological changes consistent with activation of an epithelial-mesenchymal transition program. Differential gene analysis and knockdown of genes identified gene-candidates (DDIT4, MT1X, CTSD, and SERPINE1) as potential targets for anti-metastasis therapeutics. Furthermore, RNA-splicing analysis reinforced the importance of BIRC5 splice variants in breast cancer metastasis. This is the first report using zebrafish to isolate and expand progressively invasive populations of human cancer cells. The model has potential applications in understanding the metastatic process, identification and/or development of therapeutics that specifically target metastatic cells and formulating personalized treatment strategies for individual cancer patients.
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Environmental chemical (EC) exposures and our interactions with them has significantly increased in the recent decades. Toxicity associated biological characterization of these chemicals is challenging and inefficient, even with available high-throughput technologies. In this report, we describe a novel computational method for characterizing toxicity, associated biological perturbations and disease outcome, called the Chemo-Phenotypic Based Toxicity Measurement (CPTM). CPTM is used to quantify the EC "toxicity score" (Zts), which serves as a holistic metric of potential toxicity and disease outcome. CPTM quantitative toxicity is the measure of chemical features, biological phenotypic effects, and toxicokinetic properties of the ECs. For proof-of-concept, we subject ECs obtained from the Environmental Protection Agency's (EPA) database to the CPTM. We validated the CPTM toxicity predictions by correlating 'Zts' scores with known toxicity effects. We also confirmed the CPTM predictions with in-vitro, and in-vivo experiments. In in-vitro and zebrafish models, we showed that, mixtures of the motor oil and food additive 'Salpn' with endogenous nuclear receptor ligands such as Vitamin D3, dysregulated the nuclear receptors and key transcription pathways involved in Colorectal Cancer. Further, in a human patient derived cell organoid model, we found that a mixture of the widely used pesticides 'Tetramethrin' and 'Fenpropathrin' significantly impacts the population of patient derived pancreatic cancer cells and 3D organoid models to support rapid PDAC disease progression. The CPTM method is, to our knowledge, the first comprehensive toxico-physicochemical, and phenotypic bionetwork-based platform for efficient high-throughput screening of environmental chemical toxicity, mechanisms of action, and connection to disease outcomes.
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Neoplasias Colorretais , Neoplasias Pancreáticas , Praguicidas , Animais , Colecalciferol , Humanos , Praguicidas/toxicidade , Peixe-ZebraRESUMO
[This corrects the article DOI: 10.18632/oncotarget.26600.].
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Cancer cell vascular invasion and extravasation at metastatic sites are hallmarks of malignant progression of cancer and associated with poor disease outcome. Here we describe an in vivo approach to study the invasive ability of cancer cells into the vasculature and their hematogenous metastatic seeding in zebrafish (Danio rerio). In one approach, extravasation of fluorescently labeled cancer cells is monitored in zebrafish embryos whose vasculature is marked by a contrasting fluorescent reporter. After injection into the precardiac sinus of 2-day-old embryos, cancer cells can extravasate from the vasculature into tissues over the next few days. Extravasated cancer cells are identified and counted in live embryos via fluorescence microscopy. In a second approach, intravasation of cancer cells can be evaluated by changing their injection site to the yolk sac of zebrafish embryos. In addition to monitoring the impact of drivers of malignant progression, candidate inhibitors can be studied in this in vivo model system for their efficacy as well as their toxicity for the host.
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Modelos Animais de Doenças , Invasividade Neoplásica/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Migração Transendotelial e Transepitelial , Células Tumorais Cultivadas , Peixe-ZebraRESUMO
Cancer is the second leading cause of death in the world. Given that cancer is a highly individualized disease, predicting the best chemotherapeutic treatment for individual patients can be difficult. Ex vivo models such as mouse patient-derived xenografts (PDX) and organoids are being developed to predict patient-specific chemosensitivity profiles before treatment in the clinic. Although promising, these models have significant disadvantages including long growth times that introduce genetic and epigenetic changes to the tumor. The zebrafish xenograft assay is ideal for personalized medicine. Imaging of the small, transparent fry is unparalleled among vertebrate organisms. In addition, the speed (5-7 days) and small patient tissue requirements (100-200 cells per animal) are unique features of the zebrafish xenograft model that enable patient-specific chemosensitivity analyses.
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Antineoplásicos/farmacologia , Descoberta de Drogas/métodos , Neoplasias/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Antineoplásicos/uso terapêutico , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Embrião não Mamífero , Ensaios de Triagem em Larga Escala/métodos , Humanos , Camundongos , Neoplasias/genética , Neoplasias/patologia , Medicina de Precisão/métodos , Fatores de Tempo , Quimeras de TransplanteRESUMO
BACKGROUND: Cell migration and invasion are essential processes for metastatic dissemination of cancer cells. Significant progress has been made in developing new therapies against oncogenic signaling to eliminate cancer cells and shrink tumors. However, inherent heterogeneity and treatment-induced adaptation to drugs commonly enable subsets of cancer cells to survive therapy. In addition to local recurrence, these cells escape a primary tumor and migrate through the stroma to access the circulation and metastasize to different organs, leading to an incurable disease. As such, therapeutics that block migration and invasion of cancer cells may inhibit or reduce metastasis and significantly improve cancer therapy. This is particularly more important for cancers, such as triple negative breast cancer, that currently lack targeted drugs. METHODS: We used cell migration, 3D invasion, zebrafish metastasis model, and phosphorylation analysis of 43 protein kinases in nine triple negative breast cancer (TNBC) cell lines to study effects of fisetin and quercetin on inhibition of TNBC cell migration, invasion, and metastasis. RESULTS: Fisetin and quercetin were highly effective against migration of all nine TNBC cell lines with up to 76 and 74% inhibitory effects, respectively. In addition, treatments significantly reduced 3D invasion of highly motile TNBC cells from spheroids into a collagen matrix and their metastasis in vivo. Fisetin and quercetin commonly targeted different components and substrates of the oncogenic PI3K/AKT pathway and significantly reduced their activities. Additionally, both compounds disrupted activities of several protein kinases in MAPK and STAT pathways. We used molecular inhibitors specific to these signaling proteins to establish the migration-inhibitory role of the two phytochemicals against TNBC cells. CONCLUSIONS: We established that fisetin and quercetin potently inhibit migration of metastatic TNBC cells by interfering with activities of oncogenic protein kinases in multiple pathways.
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Antineoplásicos Fitogênicos/farmacologia , Compostos Fitoquímicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Antineoplásicos Fitogênicos/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Compostos Fitoquímicos/química , Inibidores de Proteínas Quinases/química , Proteoma , Proteômica/métodos , Transdução de Sinais/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/metabolismo , Peixe-ZebraRESUMO
Glioblastoma (GBM; grade 4 glioma) is a highly aggressive and incurable tumor. GBM has recently been characterized as highly dependent on alternative splicing, a critical driver of tumor heterogeneity and plasticity. Estrogen-related receptor ß (ERR-ß) is an orphan nuclear receptor expressed in the brain, where alternative splicing of the 3' end of the pre-mRNA leads to the production of 3 validated ERR-ß protein products: ERR-ß short form (ERR-ßsf), ERR-ß2, and ERR-ß exon 10 deleted. Our prior studies have shown the ERR-ß2 isoform to play a role in G2/M cell cycle arrest and induction of apoptosis, in contrast to the function of the shorter ERR-ßsf isoform in senescence and G1 cell cycle arrest. In this study, we sought to better define the role of the proapoptotic ERR-ß2 isoform in GBM. We show that the ERR-ß2 isoform is located not only in the nucleus but also in the cytoplasm. ERR-ß2 suppresses GBM cell migration and interacts with the actin nucleation-promoting factor cortactin, and an ERR-ß agonist is able to remodel the actin cytoskeleton and similarly suppress GBM cell migration. We further show that inhibition of the splicing regulatory cdc2-like kinases in combination with an ERR-ß agonist shifts isoform expression in favor of ERR-ß2 and potentiates inhibition of growth and migration in GBM cells and intracranial tumors.-Tiek, D. M., Khatib, S. A., Trepicchio, C. J., Heckler, M. M., Divekar, S. D., Sarkaria, J. N., Glasgow, E., Riggins, R. B. Estrogen-related receptor ß activation and isoform shifting by cdc2-like kinase inhibition restricts migration and intracranial tumor growth in glioblastoma.
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Neoplasias Encefálicas/prevenção & controle , Movimento Celular , Glioblastoma/prevenção & controle , Hidrazinas/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores de Estrogênio/metabolismo , Tiazóis/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Apoptose , Biomarcadores Tumorais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Ciclo Celular , Proliferação de Células , Quimioterapia Combinada , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Isoformas de Proteínas , Receptores de Estrogênio/química , Receptores de Estrogênio/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
CD133, known as prominin1, is a penta-span transmembrane glycoprotein presumably a cancer stem cell marker for carcinomas, glioblastomas, and melanomas. We showed that CD133(+) 'melanoma-initiating cells' are associated with chemoresistance, contributing to poor patient outcome. The current study investigates the role(s) of CD133 in invasion and metastasis. Magnetic-activated cell sorting of a melanoma cell line (BAKP) followed by transwell invasion assays revealed that CD133(+) cells are significantly more invasive than CD133(-) cells. Conditional reprogramming of BAKP CD133(+) cells maintained stable CD133 overexpression (BAK-R), and induced cancer stem cell markers, melanosphere formation, and chemoresistance to kinase inhibitors. BAK-R cells showed upregulated CD133 expression, and consequently were more invasive and metastatic than BAK-P cells in transwell and zebrafish assays. CD133 knockdown by siRNA or CRISPR-Cas9 (BAK-R-T3) in BAK-R cells reduced invasion and levels of matrix metalloproteinases MMP2/MMP9. BAK-R-SC cells, but not BAK-R-T3, were metastatic in zebrafish. While CD133 knockdown by siRNA or CRISPR-Cas9 in BAK-P cells attenuated invasion and diminished MMP2/MMP9 levels, doxycycline-induced CD133 expression in BAK-P cells enhanced invasion and MMP2/MMP9 concentrations. CD133 may therefore play an essential role in invasion and metastasis via upregulation of MMP2/MMP9, leading to tumor progression, and represents an attractive target for intervention in melanoma.
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Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with limited and, very often, ineffective medical and surgical therapeutic options. The treatment of patients with advanced unresectable PDAC is restricted to systemic chemotherapy, a therapeutic intervention to which most eventually develop resistance. Recently, nab-paclitaxel (n-PTX) has been added to the arsenal of first-line therapies, and the combination of gemcitabine and n-PTX has modestly prolonged median overall survival. However, patients almost invariably succumb to the disease, and little is known about the mechanisms underlying n-PTX resistance. Using the conditionally reprogrammed (CR) cell approach, we established and verified continuously growing cell cultures from treatment-naïve patients with PDAC. To study the mechanisms of primary drug resistance, nab-paclitaxel-resistant (n-PTX-R) cells were generated from primary cultures and drug resistance was verified in vivo, both in zebrafish and in athymic nude mouse xenograft models. Molecular analyses identified the sustained induction of c-MYC in the n-PTX-R cells. Depletion of c-MYC restored n-PTX sensitivity, as did treatment with either the MEK inhibitor, trametinib, or a small-molecule activator of protein phosphatase 2a. IMPLICATIONS: The strategies we have devised, including the patient-derived primary cells and the unique, drug-resistant isogenic cells, are rapid and easily applied in vitro and in vivo platforms to better understand the mechanisms of drug resistance and for defining effective therapeutic options on a patient by patient basis.
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Albuminas/farmacologia , Carcinoma Ductal Pancreático/genética , Resistencia a Medicamentos Antineoplásicos , Paclitaxel/farmacologia , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Regulação para Cima , Idoso , Idoso de 80 Anos ou mais , Albuminas/uso terapêutico , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Paclitaxel/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Cultura Primária de Células , Células Tumorais Cultivadas , Peixe-Zebra , Neoplasias PancreáticasRESUMO
RARRES1, a retinoic acid regulated carboxypeptidase inhibitor associated with fatty acid metabolism, stem cell differentiation and tumorigenesis is among the most commonly methylated loci in multiple cancers but has no known mechanism of action. Here we show that RARRES1 interaction with cytoplasmic carboxypeptidase 2 (CCP2) inhibits tubulin deglutamylation, which in turn regulates the mitochondrial voltage dependent anion channel (VDAC1), mitochondrial membrane potential, AMPK activation, energy balance and metabolically reprograms cells and zebrafish to a more energetic and anabolic phenotype. Depletion of RARRES1 also increases expression of stem cell markers, promotes anoikis, anchorage independent growth and insensitivity to multiple apoptotic stimuli. As depletion of CCP2 or inhibition of VDAC1 reverses the effects of RARRES1 depletion on energy balance and cell survival we conclude that RARRES1 modulation of CCP2-modulated tubulin-mitochondrial VDAC1 interactions is a fundamental regulator of cancer and stem cell metabolism and survival.
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[This corrects the article DOI: 10.18632/genesandcancer.151.].
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PRAJA, a RING-H2 E3 ligase, is abundantly expressed in brain tissues such as the cerebellum and frontal cortex, amongst others, and more specifically in neural progenitor cells as well as in multiple cancers that include glioblastomas. However, the specific role that Praja plays in neural development and gliomas remains unclear. In this investigation, we performed bioinformatic analyses to examine Praja1 and Praja2 expression across 29 cancer types, and observed raised levels of Praja1 and Praja2 in gliomas with an inverse relationship between Praja1 and apoptotic genes and Praja substrates such as Smad3. We analyzed the role of Praja in the developing brain through loss of function studies, using morpholinos targeting Praja1 in embryonic zebrafish, and observed that Praja1 is expressed prominently in regions enriched with neural precursor cell subtypes. Antisense Praja morpholinos resulted in multiple embryonic defects including delayed neural development likely through increased apoptosis. Further studies revealed high levels of Cdk1 with loss of Praja1 in TGF-ß or insulin treated cells, supporting the link between Praja1 and cell cycle regulation. In summary, these studies underscore Praja's role in mammalian brain development and Praja1 deregulation may lead to gliomas possibly through the regulation of cell cycle and/or apoptosis.
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Adenoid cystic carcinomas (ACC) are rare salivary gland cancers with a high incidence of metastases. In order to study this tumor type, a reliable model system exhibiting the molecular features of this tumor is critical, but none exists, thereby inhibiting in-vitro studies and the analysis of metastatic behavior. To address this deficiency, we have coupled an efficient method to establish tumor cell cultures, conditional reprogramming (CR), with a rapid, reproducible and robust in-vivo zebrafish model. We have established cell cultures from two individual ACC PDX tumors that maintain the characteristic MYB translocation. Additional mutations found in one ACC culture also seen in the PDX tumor. Finally, the CR/zebrafish model mirrors the PDX mouse model and identifies regorafenib as a potential therapeutic drug to treat this cancer type that mimic the drug sensitivity profile in PDX model, further confirming the unique advantages of multiplex system.
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Antineoplásicos/farmacologia , Carcinoma Adenoide Cístico/tratamento farmacológico , Compostos de Fenilureia/farmacologia , Piridinas/farmacologia , Neoplasias das Glândulas Salivares/tratamento farmacológico , Animais , Biomarcadores Tumorais , Carcinoma Adenoide Cístico/genética , Carcinoma Adenoide Cístico/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Camundongos , Repetições de Microssatélites , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Neoplasias das Glândulas Salivares/genética , Neoplasias das Glândulas Salivares/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression. Traditional in vitro models of cancer cell invasion of endothelia typically lack the fluid dynamics that invading cells are otherwise exposed to in vivo. However, in vivo systems such as mouse models, though more physiologically relevant, require longer experimental timescales and present unique challenges associated with monitoring and data analysis. Here we describe a zebrafish assay that seeks to bridge this technical gap by allowing for the rapid assessment of cancer cell vascular invasion and extravasation. The approach involves injecting fluorescent cancer cells into the precardiac sinus of transparent 2-day old zebrafish embryos whose vasculature is marked by a contrasting fluorescent reporter. Following injection, the cancer cells must survive in circulation and subsequently extravasate from vessels into tissues in the caudal region of the embryo. Extravasated cancer cells are efficiently identified and scored in live embryos via fluorescence imaging at a fixed timepoint. This technique can be modified to study intravasation and/or competition amongst a heterogeneous mixture of cancer cells by changing the injection site to the yolk sac. Together, these methods can evaluate a hallmark behavior of cancer cells and help uncover mechanisms indicative of malignant progression to the metastatic phenotype.
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Invasividade Neoplásica , Peixe-Zebra , Animais , Bioensaio , Modelos Animais de Doenças , Embrião não Mamífero , Fluorescência , Humanos , Neoplasias , Saco VitelinoRESUMO
Stem cell antigen Sca-1 is implicated in murine cancer stem cell biology and breast cancer models, but the role of its human homologs Ly6K and Ly6E in breast cancer are not established. Here we report increased expression of Ly6K/E in human breast cancer specimens correlates with poor overall survival, with an additional specific role for Ly6E in poor therapeutic outcomes. Increased expression of Ly6K/E also correlated with increased expression of the immune checkpoint molecules PDL1 and CTLA4, increased tumor-infiltrating T regulatory cells, and decreased natural killer (NK) cell activation. Mechanistically, Ly6K/E was required for TGFß signaling and proliferation in breast cancer cells, where they contributed to phosphorylation of Smad1/5 and Smad2/3. Furthermore, Ly6K/E promoted cytokine-induced PDL1 expression and activation and binding of NK cells to cancer cells. Finally, we found that Ly6K/E promoted drug resistance and facilitated immune escape in this setting. Overall, our results establish a pivotal role for a Ly6K/E signaling axis involving TGFß in breast cancer pathophysiology and drug response, and highlight this signaling axis as a compelling realm for therapeutic invention. Cancer Res; 76(11); 3376-86. ©2016 AACR.
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Antígenos Ly/metabolismo , Antígenos de Superfície/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Resistencia a Medicamentos Antineoplásicos , Fator de Crescimento Transformador beta/metabolismo , Evasão Tumoral/imunologia , Animais , Antígenos Ly/genética , Antígenos de Superfície/genética , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/imunologia , Carcinoma Ductal de Mama/metabolismo , Proliferação de Células , Progressão da Doença , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Nus , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fator de Crescimento Transformador beta/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ezrin is a member of the ERM (ezrin, radixin, moesin) family of proteins and functions as a linker between the plasma membrane and the actin cytoskeleton. Ezrin is a key driver of tumor progression and metastatic spread of osteosarcoma. We discovered a quinoline-based small molecule, NSC305787, that directly binds to ezrin and inhibits its functions in promoting invasive phenotype. NSC305787 possesses a very close structural similarity to commonly used quinoline-containing antimalarial drugs. On the basis of this similarity and of recent findings that ezrin has a likely role in the pathogenesis of malaria infection, we screened antimalarial compounds in an attempt to identify novel ezrin inhibitors with better efficacy and drug properties. Screening of Medicines for Malaria Venture (MMV) Malaria Box compounds for their ability to bind to recombinant ezrin protein yielded 12 primary hits with high selective binding activity. The specificity of the hits on ezrin function was confirmed by inhibition of the ezrin-mediated cell motility of osteosarcoma cells. Compounds were further tested for phenocopying the morphologic defects associated with ezrin suppression in zebrafish embryos as well as for inhibiting the lung metastasis of high ezrin-expressing osteosarcoma cells. The compound MMV667492 exhibited potent anti-ezrin activity in all biologic assays and had better physicochemical properties for drug-likeness than NSC305787. The drug-like compounds MMV020549 and MMV666069 also showed promising activities in functional assays. Thus, our study suggests further evaluation of antimalarial compounds as a novel class of antimetastatic agents for the treatment of metastatic osteosarcoma.
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Adamantano/análogos & derivados , Antineoplásicos/farmacologia , Proteínas do Citoesqueleto/antagonistas & inibidores , Osteossarcoma/tratamento farmacológico , Quinolinas/farmacologia , Adamantano/farmacologia , Animais , Antimaláricos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Immunoblotting , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Camundongos Endogâmicos BALB C , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Bibliotecas de Moléculas Pequenas/farmacologia , Ressonância de Plasmônio de Superfície , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismoRESUMO
Dysregulation of the pathways that preserve mitochondrial integrity hallmarks many human diseases including diabetes, neurodegeration, aging and cancer. The mitochondrial citrate transporter gene, SLC25A1 or CIC, maps on chromosome 22q11.21, a region amplified in some tumors and deleted in developmental disorders known as velo-cardio-facial- and DiGeorge syndromes. We report here that in tumor cells CIC maintains mitochondrial integrity and bioenergetics, protects from mitochondrial damage and circumvents mitochondrial depletion via autophagy, hence promoting proliferation. CIC levels are increased in human cancers and its inhibition has anti-tumor activity, albeit with no toxicity on adult normal tissues. The knock-down of the CIC gene in zebrafish leads to mitochondria depletion and to proliferation defects that recapitulate features of human velo-cardio-facial syndrome, a phenotype rescued by blocking autophagy. Our findings reveal that CIC maintains mitochondrial homeostasis in metabolically active, high proliferating tissues and imply that this protein is a therapeutic target in cancer and likely, in other human diseases.