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The bloodbrain barrier represents a significant challenge for the treatment of high-grade gliomas, and our understanding of drug transport across this critical biointerface remains limited. To advance preclinical therapeutic development for gliomas, there is an urgent need for predictive in vitro models with realistic bloodbrain-barrier vasculature. Here, we report a vascularized human glioblastoma multiforme (GBM) model in a microfluidic device that accurately recapitulates brain tumor vasculature with self-assembled endothelial cells, astrocytes, and pericytes to investigate the transport of targeted nanotherapeutics across the bloodbrain barrier and into GBM cells. Using modular layer-by-layer assembly, we functionalized the surface of nanoparticles with GBM-targeting motifs to improve trafficking to tumors. We directly compared nanoparticle transport in our in vitro platform with transport across mouse brain capillaries using intravital imaging, validating the ability of the platform to model in vivo bloodbrain-barrier transport. We investigated the therapeutic potential of functionalized nanoparticles by encapsulating cisplatin and showed improved efficacy of these GBM-targeted nanoparticles both in vitro and in an in vivo orthotopic xenograft model. Our vascularized GBM model represents a significant biomaterials advance, enabling in-depth investigation of brain tumor vasculature and accelerating the development of targeted nanotherapeutics.
Assuntos
Barreira Hematoencefálica , Neoplasias Encefálicas , Permeabilidade Capilar , Glioblastoma , Nanopartículas , Animais , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/metabolismo , Células Endoteliais/metabolismo , Glioblastoma/irrigação sanguínea , Glioblastoma/metabolismo , Humanos , Camundongos , Microfluídica , Nanopartículas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Secretion of C-C chemokine ligand 2 (CCL2) by mammary tumours recruits CCR2-expressing inflammatory monocytes to primary tumours and metastatic sites, and CCL2 neutralization in mice inhibits metastasis by retaining monocytes in the bone marrow. Here we report a paradoxical effect of CCL2 in four syngeneic mouse models of metastatic breast cancer. Surprisingly, interruption of CCL2 inhibition leads to an overshoot of metastases and accelerates death. This is the result of monocyte release from the bone marrow and enhancement of cancer cell mobilization from the primary tumour, as well as blood vessel formation and increased proliferation of metastatic cells in the lungs in an interleukin (IL)-6- and vascular endothelial growth factor (VEGF)-A-dependent manner. Notably, inhibition of CCL2 and IL-6 markedly reduced metastases and increased survival of the animals. CCL2 has been implicated in various neoplasias and adopted as a therapeutic target. However, our results call for caution when considering anti-CCL2 agents as monotherapy in metastatic disease and highlight the tumour microenvironment as a critical determinant of successful anti-metastatic therapy.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL2/metabolismo , Metástase Neoplásica , Neovascularização Patológica , Animais , Vasos Sanguíneos/citologia , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/crescimento & desenvolvimento , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Interleucina-6/antagonistas & inibidores , Interleucina-6/metabolismo , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos , Monócitos/citologia , Monócitos/metabolismo , Metástase Neoplásica/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Análise de Sobrevida , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The efficacy of implanted biomedical devices is often compromised by host recognition and subsequent foreign body responses. Here, we demonstrate the role of the geometry of implanted materials on their biocompatibility in vivo. In rodent and non-human primate animal models, implanted spheres 1.5 mm and above in diameter across a broad spectrum of materials, including hydrogels, ceramics, metals and plastics, significantly abrogated foreign body reactions and fibrosis when compared with smaller spheres. We also show that for encapsulated rat pancreatic islet cells transplanted into streptozotocin-treated diabetic C57BL/6 mice, islets prepared in 1.5-mm alginate capsules were able to restore blood-glucose control for up to 180 days, a period more than five times longer than for transplanted grafts encapsulated within conventionally sized 0.5-mm alginate capsules. Our findings suggest that the in vivo biocompatibility of biomedical devices can be significantly improved simply by tuning their spherical dimensions.
Assuntos
Reação a Corpo Estranho/imunologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , PrimatasRESUMO
Invadopodia are proteolytic membrane protrusions formed by highly invasive cancer cells, commonly observed on substrate(s) mimicking extracellular matrix. Although invadopodia are proposed to have roles in cancer invasion and metastasis, direct evidence has not been available. We previously reported that neural Wiskott-Aldrich syndrome protein (N-WASP), a member of WASP family proteins that regulate reorganization of the actin cytoskeleton, is an essential component of invadopodia. Here, we report that N-WASP-mediated invadopodium formation is essential in breast cancer invasion, intravasation and lung metastasis. We established stable cell lines based on MTLn3 rat mammary adenocarcinoma cells that either overexpressed a dominant-negative (DN) N-WASP construct or in which N-WASP expression was silenced by a pSuper N-WASP shRNA. Both the N-WASP shRNA and DN N-WASP cells showed a markedly decreased ability to form invadopodia and degrade extracellular matrix. In addition, formation of invadopodia in primary tumors and collagen I degradation were reduced in the areas of invasion (collagen-rich areas in the invasive edge of the tumor) and in the areas of intravasation (blood-vessel-rich areas). Our results suggest that tumor cells in vivo that have a decreased activity of N-WASP also have a reduced ability to form invadopodia, migrate, invade, intravasate and disseminate to lung compared with tumor cells with parental N-WASP levels.
Assuntos
Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Invasividade Neoplásica/fisiopatologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/secundário , Animais , Sequência de Bases , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Mamárias Experimentais/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos SCID , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , RNA Interferente Pequeno/genética , Ratos , Ratos Endogâmicos F344 , Proteína Neuronal da Síndrome de Wiskott-Aldrich/antagonistas & inibidores , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genéticaRESUMO
Drug-carrying nanoparticles are a promising strategy to deliver therapeutics into the brain, but their translation requires better characterization of interactions between nanomaterials and endothelial cells of the blood-brain barrier (BBB). Here, we use a library of 18 layer-by-layer electrostatically assembled nanoparticles (NPs) to independently assess the impact of NP core and surface materials on in vitro uptake, transport, and intracellular trafficking in brain endothelial cells. We demonstrate that NP core stiffness determines the magnitude of transport, while surface chemistry directs intracellular trafficking. Finally, we demonstrate that these factors similarly dictate in vivo BBB transport using intravital imaging through cranial windows in mice. We identify that hyaluronic acid surface chemistry increases transport across the BBB in vivo, and flow conditions are necessary to replicate this finding in vitro. Taken together, these findings highlight the importance of assay geometry, cell biology, and fluid flow in developing nanocarriers for delivery to the brain.
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Colony stimulating factor-1 (CSF-1) regulates macrophage morphology and motility, as well as mononuclear phagocytic cell proliferation and differentiation. The CSF-1 receptor (CSF-1R) transduces these pleiotropic signals through autophosphorylation of eight intracellular tyrosine residues. We have used a novel bone-marrow-derived macrophage cell line system to examine specific signaling pathways activated by tyrosine-phosphorylated CSF-1R in macrophages. Screening of macrophages expressing a single species of CSF-1R with individual tyrosine-to-phenylalanine residue mutations revealed striking morphological alterations upon mutation of Y721. Mâ»/â».Y721F cells were apolar and ruffled poorly in response to CSF-1. Y721-P-mediated CSF-1R signaling regulated adhesion and actin polymerization to control macrophage spreading and motility. Moreover, the reduced motility of Mâ»/â».Y721F macrophages was associated with their reduced capacity to enhance carcinoma cell invasion. Y721 phosphorylation mediated the direct association of the p85 subunit of phosphoinositide 3-kinase (PI3K) with the CSF-1R, but not that of phospholipase C (PLC) γ2, and induced polarized PtdIns(3,4,5)P3 production at the putative leading edge, implicating PI3K as a major regulator of CSF-1-induced macrophage motility. The Y721-P-motif-based motility signaling was at least partially independent of both Akt and increased Rac and Cdc42 activation but mediated the rapid and transient association of an unidentified ~170 kDa phosphorylated protein with either Rac-GTP or Cdc42-GTP. These studies identify CSF-1R-Y721-P-PI3K signaling as a major pathway in CSF-1-regulated macrophage motility and provide a starting point for the discovery of the immediate downstream signaling events.
Assuntos
Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/metabolismo , Neoplasias Mamárias Experimentais/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Adesão Celular , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Feminino , Regulação da Expressão Gênica , Humanos , Fator Estimulador de Colônias de Macrófagos/genética , Macrófagos/citologia , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Invasividade Neoplásica , Paxilina/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Ratos , Transdução de SinaisRESUMO
We have shown previously that distinct Mena isoforms are expressed in invasive and migratory tumor cells in vivo and that the invasion isoform (Mena(INV)) potentiates carcinoma cell metastasis in murine models of breast cancer. However, the specific step of metastatic progression affected by this isoform and the effects on metastasis of the Mena11a isoform, expressed in primary tumor cells, are largely unknown. Here, we provide evidence that elevated Mena(INV) increases coordinated streaming motility, and enhances transendothelial migration and intravasation of tumor cells. We demonstrate that promotion of these early stages of metastasis by Mena(INV) is dependent on a macrophage-tumor cell paracrine loop. Our studies also show that increased Mena11a expression correlates with decreased expression of colony-stimulating factor 1 and a dramatically decreased ability to participate in paracrine-mediated invasion and intravasation. Our results illustrate the importance of paracrine-mediated cell streaming and intravasation on tumor cell dissemination, and demonstrate that the relative abundance of Mena(INV) and Mena11a helps to regulate these key stages of metastatic progression in breast cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular , Proteínas do Citoesqueleto/metabolismo , Migração Transendotelial e Transepitelial , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Fator Estimulador de Colônias de Macrófagos/biossíntese , Macrófagos/metabolismo , Camundongos , Camundongos SCID , Proteínas dos Microfilamentos , Invasividade Neoplásica , Metástase Neoplásica , Isoformas de Proteínas/metabolismo , RatosRESUMO
A fundamental hallmark of cancer is progression to metastasis and the growth of breast cancer metastases in lung, bone, liver and/or brain causes fatal complications. Unfortunately, the cellular and biochemical mechanisms of the metastatic process remain ill-defined. Recent application of intravital multiphoton microscopy (MP-IVM) to image fluorescently labeled cells in mouse models of cancer has allowed dynamic observation of this multi-step process at the cellular and subcellular levels. In this article, we discuss the use of MP-IVM in studies of breast cancer metastasis, as well as surgical techniques for exposing tumors prior to imaging. We also describe a versatile multiphoton microscope for imaging tumor-stroma interactions.
Assuntos
Neoplasias da Mama/patologia , Proteínas de Fluorescência Verde/metabolismo , Metástase Linfática/patologia , Glândulas Mamárias Animais/patologia , Proteínas de Neoplasias/metabolismo , Animais , Neoplasias da Medula Óssea/patologia , Neoplasias da Medula Óssea/secundário , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/genética , Hospedeiro Imunocomprometido , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Proteínas de Neoplasias/genética , Transplante de Neoplasias/patologia , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
INTRODUCTION: Neu (HER2/ErbB2) is overexpressed in 25% to 30% of human breast cancer, correlating with a poor prognosis. Researchers in previous studies who used the mouse mammary tumor virus Neu-transgenic mouse model (MMTV-Neu) demonstrated that the Neu-YB line had increased production of CXCL12 and increased metastasis, whereas the Neu-YD line had decreased metastasis. In this study, we examined the role of increased production of CXCL12 in tumor cell invasion and malignancy. METHODS: We studied invasion in the tumor microenvironment using multiphoton intravital imaging, in vivo invasion and intravasation assays. CXCL12 signaling was altered by using the CXCR4 inhibitor AMD3100 or by increasing CXCL12 expression. The role of macrophage signaling in vivo was determined using a colony-stimulating factor 1 receptor (CSF-1R) blocking antibody. RESULTS: The Neu-YD strain was reduced in invasion, intravasation and metastasis compared to the Neu-YB and Neu deletion mutant (activated receptor) strains. Remarkably, in the Neu-YB strain, in vivo invasion to epidermal growth factor was dependent on both CXCL12-CXCR4 and CSF1-CSF-1R signaling. Neu-YB tumors had increased macrophage and microvessel density. Overexpression of CXCL12 in rat mammary adenocarcinoma cells increased in vivo invasion as well as microvessel and macrophage density. CONCLUSIONS: Expression of CXCL12 by tumor cells results in increased macrophage and microvessel density and in vivo invasiveness.
Assuntos
Adenocarcinoma/secundário , Quimiocina CXCL12/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/patologia , Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/metabolismo , Animais , Movimento Celular , Quimiocina CXCL12/fisiologia , Feminino , Humanos , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Macrófagos/fisiologia , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Microscopia de Vídeo , Invasividade Neoplásica , Transplante de Neoplasias , Células Neoplásicas Circulantes/patologia , Comunicação Parácrina , Ratos , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais , Carga Tumoral , Células Tumorais Cultivadas , Microambiente TumoralRESUMO
Invasion of cancer cells into surrounding tissue and the vasculature is an initial step in tumor metastasis. This requires chemotactic migration of cancer cells, steered by protrusive activity of the cell membrane and its attachment to the extracellular matrix. Recent advances in intravital imaging and the development of an in vivo invasion assay have provided new insights into how cancer cell migration is regulated by elements of the local microenvironment, including the extracellular matrix architecture and other cell types found in primary tumors. These results, combined with new findings from in vitro studies, have led to new insights into the molecular mechanisms of cell protrusive activity and chemotactic migration during invasion and metastasis.
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Movimento Celular/fisiologia , Neoplasias/patologia , Matriz Extracelular/fisiologia , Feminino , Humanos , Macrófagos/metabolismo , Macrófagos/fisiologia , Modelos Biológicos , Neoplasias/metabolismo , Pseudópodes/metabolismo , Pseudópodes/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Access to electron acceptors supports oxidized biomass synthesis and can be limiting for cancer cell proliferation, but how cancer cells overcome this limitation in tumors is incompletely understood. Nontransformed cells in tumors can help cancer cells overcome metabolic limitations, particularly in pancreatic cancer, where pancreatic stellate cells (PSCs) promote cancer cell proliferation and tumor growth. However, whether PSCs affect the redox state of cancer cells is not known. By taking advantage of the endogenous fluorescence properties of reduced nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide cofactors we use optical imaging to assess the redox state of pancreatic cancer cells and PSCs and find that direct interactions between PSCs and cancer cells promote a more oxidized state in cancer cells. This suggests that metabolic interaction between cancer cells and PSCs is a mechanism to overcome the redox limitations of cell proliferation in pancreatic cancer.
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Neoplasias Pancreáticas , Células Estreladas do Pâncreas , Linhagem Celular Tumoral , Humanos , Oxirredução , Neoplasias Pancreáticas/patologia , Células Estreladas do Pâncreas/metabolismo , Células Estromais , Neoplasias PancreáticasRESUMO
We report a technique to evaluate the same tumor microenvironment over multiple intravital imaging sessions in living mice. We optically marked individual tumor cells expressing photoswitchable proteins in an orthotopic mammary carcinoma and followed them for extended periods through a mammary imaging window. We found that two distinct microenvironments in the same orthotopic mammary tumor affected differently the invasion and intravasation of tumor cells.
Assuntos
Aumento da Imagem/instrumentação , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/secundário , Microscopia/instrumentação , Animais , Linhagem Celular Tumoral , Desenho de Equipamento , Análise de Falha de Equipamento , CamundongosRESUMO
Understanding the mechanisms controlling cancer cell invasion and metastasis constitutes a fundamental step in setting new strategies for diagnosis, prognosis, and therapy of metastatic cancers. LIM kinase1 (LIMK1) is a member of a novel class of serine-threonine protein kinases. Cofilin, a LIMK1 substrate, is essential for the regulation of actin polymerization and depolymerization during cell migration. Previous studies have made opposite conclusions as to the role of LIMK1 in tumor cell motility and metastasis, claiming either an increase or decrease in cell motility and metastasis as a result of LIMK1 over expression (Zebda, N., O. Bernard, M. Bailly, S. Welti, D.S. Lawrence, and J.S. Condeelis. 2000. J. Cell Biol. 151:1119-1128; Davila, M., A.R. Frost, W.E. Grizzle, and R. Chakrabarti. 2003. J. Biol. Chem. 278:36868-36875; Yoshioka, K., V. Foletta, O. Bernard, and K. Itoh. 2003. Proc. Natl. Acad. Sci. USA. 100:7247-7252; Nishita, M., C. Tomizawa, M. Yamamoto, Y. Horita, K. Ohashi, and K. Mizuno. 2005. J. Cell Biol. 171:349-359). We resolve this paradox by showing that the effects of LIMK1 expression on migration, intravasation, and metastasis of cancer cells can be most simply explained by its regulation of the output of the cofilin pathway. LIMK1-mediated decreases or increases in the activity of the cofilin pathway are shown to cause proportional decreases or increases in motility, intravasation, and metastasis of tumor cells.
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Cofilina 1/metabolismo , Neoplasias Mamárias Experimentais/genética , Proteínas Quinases/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Extensões da Superfície Celular/genética , Extensões da Superfície Celular/fisiologia , Quimiotaxia/genética , Quimiotaxia/fisiologia , Fator de Crescimento Epidérmico/farmacologia , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Quinases Lim , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Mutação , Invasividade Neoplásica , Metástase Neoplásica , Fosforilação , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/genética , Ratos , Ratos Endogâmicos F344 , Análise de Sobrevida , TransfecçãoRESUMO
INTRODUCTION: The actin binding protein Mammalian enabled (Mena), has been implicated in the metastatic progression of solid tumors in humans. Mena expression level in primary tumors is correlated with metastasis in breast, cervical, colorectal and pancreatic cancers. Cells expressing high Mena levels are part of the tumor microenvironment for metastasis (TMEM), an anatomical structure that is predictive for risk of breast cancer metastasis. Previously we have shown that forced expression of Mena adenocarcinoma cells enhances invasion and metastasis in xenograft mice. Whether Mena is required for tumor progression is still unknown. Here we report the effects of Mena deficiency on tumor progression, metastasis and on normal mammary gland development. METHODS: To investigate the role of Mena in tumor progression and metastasis, Mena deficient mice were intercrossed with mice carrying a transgene expressing the polyoma middle T oncoprotein, driven by the mouse mammary tumor virus. The progeny were investigated for the effects of Mena deficiency on tumor progression via staging of primary mammary tumors and by evaluation of morbidity. Stages of metastatic progression were investigated using an in vivo invasion assay, intravital multiphoton microscopy, circulating tumor cell burden, and lung metastases. Mammary gland development was studied in whole mount mammary glands of wild type and Mena deficient mice. RESULTS: Mena deficiency decreased morbidity and metastatic dissemination. Loss of Mena increased mammary tumor latency but had no affect on mammary tumor burden or histologic progression to carcinoma. Elimination of Mena also significantly decreased epidermal growth factor (EGF) induced in vivo invasion, in vivo motility, intravasation and metastasis. Non-tumor bearing mice deficient for Mena also showed defects in mammary gland terminal end bud formation and branching. CONCLUSIONS: Deficiency of Mena decreases metastasis by slowing tumor progression and reducing tumor cell invasion and intravasation. Mena deficiency during development causes defects in invasive processes involved in mammary gland development. These findings suggest that functional intervention targeting Mena in breast cancer patients may provide a valuable treatment option to delay tumor progression and decrease invasion and metastatic spread leading to an improved prognostic outcome.
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Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/fisiologia , Neoplasias Mamárias Experimentais/genética , Animais , Antígenos Transformantes de Poliomavirus/genética , Proteínas do Citoesqueleto/genética , Progressão da Doença , Fator de Crescimento Epidérmico/metabolismo , Feminino , Expressão Gênica , Neoplasias Pulmonares/secundário , Macrófagos , Glândulas Mamárias Animais/anatomia & histologia , Glândulas Mamárias Animais/embriologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos SCID , Camundongos Transgênicos , Proteínas dos Microfilamentos , Invasividade Neoplásica , Metástase Neoplásica , Reação em Cadeia da Polimerase , Microambiente TumoralRESUMO
Invasion of neighboring extracellular matrix tissue, the lymphatic system and blood vessels is a key element of tumor cell metastasis in many epithelial tumors. Understanding the cell motility pathways that contribute to invasion can provide new approaches and targets for anticancer therapy. The recent convergence of technologies for expression profiling and intravital imaging has revealed the identities of some of the genes that contribute to motility and chemotaxis of cancer cells in tumors. In particular, the genes encoding a minimum motility machine are coordinately upregulated in tumor cells collected by an in vivo invasion assay. These results support a "tumor microenvironment invasion model" and provide new target opportunities for cancer therapy.
Assuntos
Movimento Celular , Neoplasias/patologia , Animais , Quimiotaxia , Regulação Neoplásica da Expressão Gênica , Humanos , Modelos Biológicos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/metabolismo , Análise de Sequência com Séries de OligonucleotídeosRESUMO
PURPOSE: The epidermal growth factor receptor (ERBB1) and related family member HER-2/neu (ERBB2) are often overexpressed in aggressive breast cancers and their overexpression is correlated with poor prognosis. Clinical studies using ERBB inhibitors have focused on tumor growth effects, but ERBBs can contribute to malignancy independent of their effects on tumor growth. Our studies were designed to evaluate the effect of ERBB inhibition on tumor cell motility and intravasation in vivo using clinically relevant small-molecule inhibitors. EXPERIMENTAL DESIGN: Using in vivo mouse models of breast cancer, we test the effects of ERBB1 and ERBB2 inhibitors AC480 and lapatinib, ERBB1 inhibitor gefitinib, and ERBB2 inhibitor AG825 on in vivo tumor cell invasive properties in mammary fat pad tumors. RESULTS: ERBB1 and ERBB2 inhibition rapidly (within 3 h) inhibits both tumor cell motility and intravasation. Using gefitinib, ERBB1 inhibition rapidly inhibits tumor cell motility and invasion but not intravasation, whereas ERBB2 inhibition by AG825 rapidly blocks intravasation. CONCLUSIONS: ERBB1 and ERBB2 inhibition can rapidly block tumor cell invasive properties. In addition, we differentiate for the first time the contributions of ERBB1 and ERBB2 to the key metastatic properties of in vivo tumor cell invasion and intravasation. These experiments temporally and molecularly separate two key stages in tumor cell entry into blood vessels: invasion and intravasation. These results indicate that ERBB inhibition should be considered for blocking other tumor cell malignant properties besides growth.
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Receptores ErbB/metabolismo , Neoplasias Mamárias Experimentais/patologia , Receptor ErbB-2/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Benzotiazóis/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/fisiologia , Feminino , Gefitinibe , Humanos , Lapatinib , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos SCID , Invasividade Neoplásica , Metástase Neoplásica , Fosforilação/efeitos dos fármacos , Quinazolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/fisiologia , Tirfostinas/farmacologiaRESUMO
Tumor cells invading three-dimensional matrices need to remodel the extracellular matrix (ECM) in their path. Many studies have focused on the role of extracellular proteases; however, cells with amoeboid or rounded morphologies are able to invade even when these enzymes are inhibited. Here, we describe the mechanism by which cells move through a dense ECM without proteolysis. Amoeboid tumor cells generate sufficient actomyosin force to deform collagen fibers and are able to push through the ECM. Force generation is elevated in metastatic MTLn3E cells, and this correlates with increased invasion and altered myosin light chain (MLC) organization. In metastatic cells, MLC is organized perpendicularly to the direction of movement behind the invading edge. Both the organization of MLC and force generation are dependent upon ROCK function. We demonstrate that ROCK regulates the phosphorylation of MLC just behind the invading margin of the cell. Imaging of live tumors shows that MLC is organized in a similar ROCK-dependent fashion in vivo and that inhibition of ROCK but not matrix-metalloproteases reduces cancer cell motility in vivo.
Assuntos
Movimento Celular/fisiologia , Matriz Extracelular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cadeias Leves de Miosina/metabolismo , Invasividade Neoplásica/fisiopatologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Colágeno/metabolismo , Microscopia Eletrônica de Varredura , Fosforilação , Ratos , Quinases Associadas a rhoRESUMO
Correlating tumor cell behavior in vivo with patterns of gene expression has led to new insights into the microenvironment of tumor cells in the primary tumor. Until now, these studies have been done with cell line-derived tumors. In the current study, we have analyzed, in polyoma middle T oncogene (PyMT)-derived mammary tumors, tumor cell behavior and gene expression patterns of the invasive subpopulation of tumor cells by multiphoton-based intravital imaging and microarray-based expression profiling, respectively. Our results indicate that the patterns of cell behavior that contribute to invasion and metastasis in the PyMT tumor are similar to those seen previously in rat MTLn3 cell line-derived mammary tumors. The invasive tumor cells collected from PyMT mouse mammary tumors, like their counterparts from rat xenograft mammary tumors, are a population that is relatively nondividing and nonapoptotic but chemotherapy resistant and chemotactic. Changes in the expression of genes that occur uniquely in the invasive subpopulation of tumor cells in the PyMT mammary tumors that fall on the Arp2/3 complex, capping protein and cofilin pathways show a pattern like that seen previously in invasive tumor cells from the MTLn3 cell line-derived tumors. These changes predict an enhanced activity of the cofilin pathway, and this was confirmed in isolated invasive PyMT tumor cells. We conclude that changes in gene expression and their related changes in cell behavior, which were identified in the invasive tumor cells of cell line-derived tumors, are conserved in the invasive tumor cells of PyMT-derived mouse mammary tumors, although these tumor types have different genetic origins.
Assuntos
Movimento Celular/genética , Quimiotaxia/genética , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Animais , Antígenos Transformantes de Poliomavirus/genética , Cofilina 1/biossíntese , Cofilina 1/genética , Cofilina 1/metabolismo , Regulação Neoplásica da Expressão Gênica , Quinases Lim , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica , Invasividade Neoplásica , Proteínas Quinases/biossíntese , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , RatosRESUMO
Although the presence of macrophages in tumors has been correlated with poor prognosis, until now there was no direct observation of how macrophages are involved in hematogenous metastasis. In this study, we use multiphoton microscopy to show, for the first time, that tumor cell intravasation occurs in association with perivascular macrophages in mammary tumors. Furthermore, we show that perivascular macrophages of the mammary tumor are associated with tumor cell intravasation in the absence of local angiogenesis. These results show that the interaction between macrophages and tumor cells lying in close proximity defines a microenvironment that is directly involved in the intravasation of cancer cells in mammary tumors.
Assuntos
Vasos Sanguíneos/patologia , Comunicação Celular/fisiologia , Macrófagos/patologia , Neoplasias Mamárias Experimentais/patologia , Animais , Movimento Celular/fisiologia , Endotélio Vascular/patologia , Feminino , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica , Células Neoplásicas Circulantes/patologiaRESUMO
A better understanding of how epidermal growth factor receptor family members (ErbBs) contribute to metastasis is important for evaluating ErbB-directed therapies. Activation of ErbB3/ErbB2 heterodimers can affect both proliferation and motility. We find that increasing ErbB3-dependent signaling in orthotopic injection models of breast cancer can enhance intravasation and lung metastasis with no effect on primary tumor growth or microvessel density. Enhanced metastatic ability due to increased expression of ErbB2 or ErbB3 correlated with stronger chemotaxis and invasion responses to heregulin beta1. Suppression of ErbB3 expression reduced both intravasation and metastasis. A human breast cancer tumor tissue microarray showed a significant association between ErbB3 and ErbB2 expression and metastasis independent of tumor size. These results indicate that ErbB3-dependent signaling through ErbB3/ErbB2 heterodimers can contribute to metastasis through enhancing tumor cell invasion and intravasation in vivo and that ErbB-directed therapies may be useful for the inhibition of invasion independent of effects on tumor growth.