RESUMEN

Human mesenchymal stem/stromal cells (hMSCs) provide support for cancer progression, partly through their secretome that includes extracellular vesicles (EVs). Based on deep-sequencing of small RNA from EVs of MSCs, we now report the characterization of novel small RNA, named n-miR-G665, which exhibits typical properties of miRNAs. n-miR-G665 sequence is conserved and expressed in most cell types. Knockdown studies using anti-agomirs and shRNA studies demonstrated that n-miR-G665 plays an important role in cell proliferation. Functional assays to reveal the targets of n-miR-G665 showed that polycomb protein Suz12 is regulated by n-miR-G665, which in turn regulates the expression of n-miR-G665 through feedback loop mechanism. These data shed light on a previously unknown novel feedback regulatory mechanism for controlling Suz12 expression regulated by previously not described miRNA, which may highlight a new therapeutic approach to control the polycomb repressor complex 2 activity in cancers.

Asunto(s)

Regulación de la Expresión Génica , Células Madre Mesenquimatosas/química , Células Madre Mesenquimatosas/fisiología , MicroARNs/metabolismo , Complejo Represivo Polycomb 2/biosíntesis , Línea Celular , Proliferación Celular , Vesículas Extracelulares/química , Técnicas de Silenciamiento del Gen , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , MicroARNs/genética , MicroARNs/aislamiento & purificación , Proteínas de Neoplasias , Factores de Transcripción

RESUMEN

Mesenchymal stromal cells (hMSCs) have been used to understand the stromal cell properties in solid tumors because of their ablity to differentiate into most cell types. We investigated the role of EVs from hMSCs (hMSC-EVs) in breast cancer metastasis using MDA-MB-231 parental cell line and organotropic sub-lines. We demonstrated that hMSC-EVs significantly suppressed the metastatic potential of the parental cell line when compared to their organotropic sublines. hMSC-EVs induce dormancy in the parental cell line but not in their organotropic sub-lines and miR-205 and miR-31 from EV cargo played a role. Further, Ubiquitin Conjugating Enzyme E2 N (UBE2N/Ubc13) - metastasis-regulating gene, is a target of these miRNAs and silencing of UBE2N/Ubc13 expression significantly suppressed migration, invasion, and proliferation of breast cancer cells. To summarize, hMSC-EVs support primary breast tumor progression but suppress the metastasis of breast cancer cells that are not organ-committed through the UBE2N/Ubc13 pathway and play a role in premetastic niche formation.

RESUMEN

It is well recognized that one of the major drawbacks of using traditional two dimensional cultures to model the living systems is inaccurately reflecting the physiological manner in which modulators, nutrients, oxygen, and metabolites are applied and removed. Moreover, the two dimensional culture system poorly reflects how different cell types interact with each other in the same microenvironment. Since the first global development of three dimensional (3D) cell culture techniques in the late 1960s, this last decade has seen an explosion of studies to promote 3D models in the fields of regenerative medicine and cancer. The recent surge of interest in 3D cell culture in cancer research is attributable to the interest in developing closer to real life models. The ability to include various cell types and extracellular components reflect more the physiological conditions of tumor microenvironment. In this short review, we will discuss different approaches of 3D culture system models and techniques with a focus on the 3D interactions of cancer cells with stromal cells in the goal to reevaluate old and develop new therapeutics.

Asunto(s)

Técnicas de Cultivo de Célula , Neoplasias/patología , Evaluación Preclínica de Medicamentos/métodos , Humanos , Dispositivos Laboratorio en un Chip , Neoplasias de Tejido Conjuntivo/patología , Neoplasias Glandulares y Epiteliales/patología , Proyectos de Investigación , Esferoides Celulares , Andamios del Tejido , Células Tumorales Cultivadas , Microambiente Tumoral

RESUMEN

Studies have shown that mesenchymal stem/stromal cells (MSCs) from bone marrow are involved in the growth and metastasis of solid tumors but the mechanism remains unclear in osteosarcoma (OS). Previous studies have raised the possibility that OS cells may receive support from associated MSCs in the nutrient deprived core of the tumors through the release of supportive macromolecules and growth factors either in vesicular or non-vesicular forms. In the present study, we used stressed mesenchymal stem cells (SD-MSCs), control MSCs and OS cells to examine the hypothesis that tumor-associated MSCs in nutrient deprived core provide pro-proliferative, anti-apoptotic, and metastatic support to nearby tumor cells. Assays to study of the effects of SD-MSC conditioned media revealed that OS cells maintained proliferation when compared to OS cells grown under serum-starved conditions alone. Furthermore, OS cells in MSCs and SD-MSC conditioned media were significantly resistant to apoptosis and an increased wound healing rate was observed in cells exposed to either conditioned media or EVs from MSCs and SD-MSCs. RT-PCR assays of OS cells incubated with extracellular vesicles (EVs) from SD-MSCs revealed microRNAs that could potentially target metabolism and metastasis associated genes as predicted by in silico algorithms, including monocarboxylate transporters, bone morphogenic receptor type 2, fibroblast growth factor 7, matrix metalloproteinase-1, and focal adhesion kinase-1. Changes in the expression levels of focal adhesion kinase, STK11 were confirmed by quantitative PCR assays. Together, these data indicate a tumor supportive role of MSCs in osteosarcoma growth that is strongly associated with the miRNA content of the EVs released from MSCs under conditions that mimic the nutrient deprived core of solid tumors.

Asunto(s)

Apoptosis , Comunicación Celular , Movimiento Celular , Vesículas Extracelulares/patología , Células Madre Mesenquimatosas/citología , Osteosarcoma/patología , Estrés Oxidativo , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Medios de Cultivo Condicionados , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , MicroARNs/genética , Microambiente Tumoral

RESUMEN

Chemical analysis of small extracellular vesicles (sEVs) circulating in body fluids holds potentials in noninvasive diagnosis of diseases and evaluation of therapeutic treatments. However, quantification of sEVs remains a challenge due to lacking of cost-effective analytical protocols. Herein we report a facile method based on size exclusion chromatography with fluorescence detection (SEC-FD) for sEVs quantification. After removal of cells and cell debris, a 0.50 mL sample (e.g., cell culture medium) is incubated with CM-Dil dye to fluorescently label sEVs. The incubation solution is then separated on a SEC column packed with Sepharose CL-4B. The eluent is monitored fluorescently at Ex553 nm/Em570 nm by using a fluorometer equipped with a 50-µL flow through cuvette. Separation efficiency of the proposed SEC-FD method was evaluated by analyzing 100 nm liposomes and albumin-FITC conjugate. Liposomes were eluted out in less than 6 min, about 10 min before albumin-FITC. A separation repeatability (RSD in retention time) of 1.4% (n = 5) was obtained for liposomes. In analysis of cell culture media, linear calibration curves based on SEC-FD peak height versus sEVs concentration were obtained with r2 value of 0.996. Intraday quantification repeatability (RSD in peak height) was 3.2% (n = 5). The detection limit was estimated to be 2.9 × 107 exosome particles/mL. The proposed assay was applied to the first study of sEVs secretion from TK6 cells cultured in serum-free medium for a culturing period from 1 to 48 h.

Asunto(s)

Carbocianinas/análisis , Cromatografía en Gel/métodos , Vesículas Extracelulares/química , Colorantes Fluorescentes/análisis , Línea Celular , Fluorescencia , Humanos , Liposomas/química , Tamaño de la Partícula

RESUMEN

Prostate cancer is known to frequently recur in bone; however, how dormant cells switch its phenotype leading to recurrent tumor remains poorly understood. We have isolated two syngeneic cell lines (indolent and aggressive) through in vivo selection by implanting PC3mm stem-like cells into tibial bones. We found that indolent cells retained the dormant phenotype, whereas aggressive cells grew rapidly in bone in vivo, and the growth rates of both cells in culture were similar, suggesting a role of the tumor microenvironment in the regulation of dormancy and recurrence. Indolent cells were found to secrete a high level of secreted protein acidic and rich in cysteine (SPARC), which significantly stimulated the expression of BMP7 in bone marrow stromal cells. The secreted BMP7 then kept cancer cells in a dormant state by inducing senescence, reducing "stemness," and activating dormancy-associated p38 MAPK signaling and p21 expression in cancer cells. Importantly, we found that SPARC was epigenetically silenced in aggressive cells by promoter methylation, but 5-azacytidine treatment reactivated the expression. Furthermore, high SPARC promoter methylation negatively correlated with disease-free survival of prostate cancer patients. We also found that the COX2 inhibitor NS398 down-regulated DNMTs and increased expression of SPARC, which led to tumor growth suppression in bone in vivo These findings suggest that SPARC plays a key role in maintaining the dormancy of prostate cancer cells in the bone microenvironment.

Asunto(s)

Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Proteínas de Neoplasias/metabolismo , Osteonectina/metabolismo , Neoplasias de la Próstata/metabolismo , Microambiente Tumoral , Animales , Azacitidina/farmacología , Proteína Morfogenética Ósea 7/genética , Proteína Morfogenética Ósea 7/metabolismo , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Línea Celular Tumoral , Metilación de ADN/efectos de los fármacos , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Humanos , Masculino , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Osteonectina/genética , Regiones Promotoras Genéticas , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

Mesenchymal stem/stromal cells (MSCs) have been extensively investigated for their potential to regenerate tissue, to modulate the immune system, and their wound healing properties in over 350 clinical trials worldwide. MSCs from various tissues such as adipose, bone, and others are currently being studied in clinical trials in indications for ischemic, inflammatory, autoimmune, and degenerative disorders. As a result, numerous isolation protocols have been published. This chapter provides a simple protocol whereby a total of 80-100 million human MSCs, with an average viability greater than 90 %, can be produced from a relatively small (1-3 mL) bone marrow aspirate in 14-20 days using double stack culture chambers. MSCs were originally referred to as fibroblastoid colony forming cells because one of their characteristic features is adherence to tissue culture plastic and generation of colonies when plated at low densities. The efficiency with which they form colonies still remains an important assay for the quality of cell preparations. To assess the quality of cell preparations, two different colony forming unit (CFU) assays are also provided.

Asunto(s)

Ensayo de Unidades Formadoras de Colonias/métodos , Células Madre Mesenquimatosas/citología , Recuento de Células , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Humanos

RESUMEN

Mesenchymal Stem/stromal cell (MSCs) transplantation procedures have been used since the 1960's to treat leukemia and other diseases, but due to the risks involved only patients with life threatening illnesses were typically subjected to the transplantation procedure until the last decade. Recent advancements in transplantation techniques have made it more feasible to use it for non-life-threatening diseases. However, the potential uses for stem cells are still limited by their rarity, and, in the case of allogeneic transplants, graft-vs.-host complications. An evolving alternative to conventional stem cell therapies is induced pluripotent stem-cell derived mesenchymal stem/stromal cells (iPSC- MSCs), which have a multi-lineage potential comparable to conventionally acquired MSCs with the added benefit of being less immunoreactive. However there are still many hurdles left to be overcome before they can be used regularly for personalized therapies. This review will focus on recent advancements that have been made regarding the role MSCs play in tumor development and the potential uses iPSC-MSCs may have in future cancer treatment.

Asunto(s)

Células Madre Pluripotentes Inducidas/trasplante , Trasplante de Células Madre Mesenquimatosas/tendencias , Células Madre Mesenquimatosas , Medicina de Precisión , Humanos

RESUMEN

In recent years, the knowledge about the control of tumor microenvironment has increased and emerged as an important player in tumorigenesis. The role of normal stromal cells in the tumor initiation and progression has brought our vision in to the forefront of cell-to-cell communication. In this review, we focus on the mechanism of communication between stromal and tumor cells, which is based on the exchange of extracellular vesicles (EVs). We describe several, evergrowing, pieces of evidence that EVs transfer messages through their miRNA, lipid, protein and nucleic acid contents. A better understanding of this sophisticated method of communication between normal cancer cells may lead to developing novel approaches for personalized diagnostics and therapeutics.

Asunto(s)

Vesículas Extracelulares/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Microambiente Tumoral , Animales , Transporte Biológico , Comunicación Celular , Vesículas Extracelulares/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Lípidos , Células Madre Mesenquimatosas/metabolismo , MicroARNs , Metástasis de la Neoplasia , Neoplasias/genética , Proteínas , Transducción de Señal

RESUMEN

The unique properties of mesenchymal stromal/stem cells (MSCs) to self-renew and their multipotentiality have rendered them attractive to researchers and clinicians. In addition to the differentiation potential, the broad repertoire of secreted trophic factors (cytokines) exhibiting diverse functions such as immunomodulation, anti-inflammatory activity, angiogenesis and anti-apoptotic, commonly referred to as the MSC secretome, has gained immense attention in the past few years. There is enough evidence to show that the one important pathway by which MSCs participate in tissue repair and regeneration is through its secretome. Concurrently, a large body of MSC research has focused on characterization of the MSC secretome; this includes both soluble factors and factors released in extracellular vesicles, for example, exosomes and microvesicles. This review provides an overview of our current understanding of the MSC secretome with respect to their potential clinical applications.

Asunto(s)

Células Madre Mesenquimatosas/metabolismo , Regeneración , Movimiento Celular , Exosomas/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Nicho de Células Madre , Investigación Biomédica Traslacional

RESUMEN

Stem cells are proposed to continuously secrete trophic factors that potentially serve as mediators of autocrine and paracrine activities, associated with reprogramming of the tumor microenvironment, tissue regeneration, and repair. Hitherto, significant efforts have been made to understand the level of underlying paracrine activities influenced by stem cell secreted trophic factors, as little is known about these interactions. Recent findings, however, elucidate this role by reporting the effects of stem cell derived extracellular vesicles (EVs) that mimic the phenotypes of the cells from which they originate. Exchange of genetic information utilizing persistent bidirectional communication mediated by stem cell-EVs could regulate stemness, self-renewal, and differentiation in stem cells and their subpopulations. This review therefore discusses stem cell-EVs as evolving communication factors in stem cell biology, focusing on how they regulate cell fates by inducing persistent and prolonged genetic reprogramming of resident cells in a paracrine fashion. In addition, we address the role of stem cell-secreted vesicles in shaping the tumor microenvironment and immunomodulation and in their ability to stimulate endogenous repair processes during tissue damage. Collectively, these functions ensure an enormous potential for future therapies.

RESUMEN

Mesenchymal stem/stromal cells (MSCs) have been extensively investigated for their regenerative, immune-modulatory, and wound healing properties. While the laboratory studies have suggested that MSC's have a unique potential for modulating the etiopathology of multiple diseases, the results from clinical trials have not been encouraging or reproducible. One of the explanations for such variability is explained by the "art" of isolating and propagating MSCs. Therefore, establishing more than minimal criteria to define MSC would help understand best protocols to isolate, propagate and deliver MSCs. Developing a calibration standard, a database and a set of functional tests would be a better quality metric for MSCs. In this review, we discuss the importance of selecting a standard, issues associated with coming up with such a standard and how these issues can be mitigated.

Asunto(s)

Separación Celular/normas , Células Madre Mesenquimatosas/citología , Técnicas de Cultivo de Célula , Células Cultivadas , Humanos , Trasplante de Células Madre Mesenquimatosas/métodos , Estándares de Referencia

RESUMEN

Mutant p53 (mtp53) is an oncogene that drives cancer cell proliferation. Here we report that mtp53 associates with the promoters of numerous nucleotide metabolism genes (NMG). Mtp53 knockdown reduces NMG expression and substantially depletes nucleotide pools, which attenuates GTP-dependent protein activity and cell invasion. Addition of exogenous guanosine or GTP restores the invasiveness of mtp53 knockdown cells, suggesting that mtp53 promotes invasion by increasing GTP. In addition, mtp53 creates a dependency on the nucleoside salvage pathway enzyme deoxycytidine kinase for the maintenance of a proper balance in dNTP pools required for proliferation. These data indicate that mtp53-harbouring cells have acquired a synthetic sick or lethal phenotype relationship with the nucleoside salvage pathway. Finally, elevated expression of NMG correlates with mutant p53 status and poor prognosis in breast cancer patients. Thus, mtp53's control of nucleotide biosynthesis has both a driving and sustaining role in cancer development.

Asunto(s)

Neoplasias Encefálicas/genética , Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica/genética , Nucleótidos/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Western Blotting , Neoplasias Encefálicas/secundario , Neoplasias de la Mama/metabolismo , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/genética , Desoxicitidina Quinasa , Femenino , Técnicas de Silenciamiento del Gen , Guanosina Trifosfato , Humanos , Inmunoprecipitación , Estimación de Kaplan-Meier , Ratones , Mutación , Invasividad Neoplásica/genética , Trasplante de Neoplasias , Nucleósidos/metabolismo , Pronóstico , Regiones Promotoras Genéticas , Modelos de Riesgos Proporcionales , Ensayo de Tumor de Célula Madre , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

Brain is one of the major sites of metastasis in breast cancer; however, the pathological mechanism of brain metastasis is poorly understood. One of the critical rate-limiting steps of brain metastasis is the breaching of blood-brain barrier, which acts as a selective interface between the circulation and the central nervous system, and this process is considered to involve tumor-secreted proteinases. We analyzed clinical significance of 21 matrix metalloproteinases on brain metastasis-free survival of breast cancer followed by verification in brain metastatic cell lines and found that only matrix metalloproteinase 1 (MMP1) is significantly correlated with brain metastasis. We have shown that MMP1 is highly expressed in brain metastatic cells and is capable of degrading Claudin and Occludin but not Zo-1, which are key components of blood-brain barrier. Knockdown of MMP1 in brain metastatic cells significantly suppressed their ability of brain metastasis in vivo, whereas ectopic expression of MMP1 significantly increased the brain metastatic ability of the cells that are not brain metastatic. We also found that COX2 was highly up-regulated in brain metastatic cells and that COX2-induced prostaglandins were directly able to promote the expression of MMP1 followed by augmenting brain metastasis. Furthermore, we found that COX2 and prostaglandin were able to activate astrocytes to release chemokine (C-C motif) ligand 7 (CCL7), which in turn promoted self-renewal of tumor-initiating cells in the brain and that knockdown of COX2 significantly reduced the brain metastatic ability of tumor cells. Our results suggest the COX2-MMP1/CCL7 axis as a novel therapeutic target for brain metastasis.

Asunto(s)

Neoplasias Encefálicas/genética , Neoplasias de la Mama/genética , Ciclooxigenasa 2/genética , Metaloproteinasa 1 de la Matriz/genética , Transducción de Señal/genética , Animales , Barrera Hematoencefálica/metabolismo , Western Blotting , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundario , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Ciclooxigenasa 2/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Metaloproteinasa 1 de la Matriz/metabolismo , Ratones Desnudos , Trasplante de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores CCR7/genética , Receptores CCR7/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Supervivencia , Trasplante Heterólogo

RESUMEN

Human mesenchymal stem/stromal cells (hMSCs) have been shown to support breast cancer cell proliferation and metastasis, partly through their secretome. hMSCs have a remarkable ability to survive for long periods under stress, and their secretome is tumor supportive. In this study, we have characterized the cargo of extracellular vesicular (EV) fraction (that is in the size range of 40-150nm) of serum deprived hMSCs (SD-MSCs). Next Generation Sequencing assays were used to identify small RNA secreted in the EVs, which indicated presence of tumor supportive miRNA. Further assays demonstrated the role of miRNA-21 and 34a as tumor supportive miRNAs. Next, proteomic assays revealed the presence of ≈150 different proteins, most of which are known tumor supportive factors such as PDGFR-ß, TIMP-1, and TIMP-2. Lipidomic assays verified presence of bioactive lipids such as sphingomyelin. Furthermore, metabolite assays identified the presence of lactic acid and glutamic acid in EVs. The co-injection xenograft assays using MCF-7 breast cancer cells demonstrated the tumor supportive function of these EVs. To our knowledge this is the first comprehensive -omics based study that characterized the complex cargo of extracellular vesicles secreted by hMSCs and their role in supporting breast cancers.

Asunto(s)

Neoplasias de la Mama/metabolismo , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , MicroARNs/metabolismo , Animales , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/fisiología , Vesículas Extracelulares/patología , Femenino , Xenoinjertos , Humanos , Metabolismo de los Lípidos , Células MCF-7 , Ratones , Ratones Desnudos , Osteosarcoma/metabolismo , Osteosarcoma/patología , Proteoma/metabolismo , Microambiente Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

It is well known that acidic microenvironment promotes tumorigenesis, however, the underlying mechanism remains largely unknown. In the present study, we show that acidosis promotes invasiveness of breast cancer cells through a series of signaling events. First, our study indicates that NF-κB is a key factor for acidosis-induced cell invasion. Acidosis activates NF-κB without affecting STAT3 activity; knockdown of NF-κB p65 abrogates the acidosis-induced invasion activity. Next, we show that the activation of NF-κB is mediated through phosphorylation and degradation of IκBα; and phosphorylation and nuclear translocation of p65. Upstream to NF-κB signaling, AKT is activated under acidic conditions. Moreover, acidosis induces generation of reactive oxygen species (ROS) which can be suppressed by ROS scavengers, reversing the acidosis-induced activation of AKT and NF-κB, and invasiveness. As a negative regulator of AKT, PTEN is oxidized and inactivated by the acidosis-induced ROS. Finally, inhibition of NADPH oxidase (NOX) suppresses acidosis-induced ROS production, suggesting involvement of NOX in acidosis-induced signaling cascade. Of considerable interest, acidosis-induced ROS production and activation of AKT and NF-κB can be only detected in cancer cells, but not in non-malignant cells. Together, these results demonstrate a cancer specific acidosis-induced signaling cascade in breast cancer cells, leading to cell invasion.

Asunto(s)

Acidosis/complicaciones , Neoplasias de la Mama/patología , FN-kappa B/metabolismo , Invasividad Neoplásica/patología , Proteína Oncogénica v-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Western Blotting , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Humanos , Microscopía Confocal , Transducción de Señal/fisiología , Transfección

RESUMEN

INTRODUCTION: Exosomes are 30-100 nm membrane vesicles of endocytic origin, mediating diverse biological functions including tumor cell invasion, cell-cell communication and antigen presentation through transfer of proteins, mRNAs and microRNAs. Recent evidence suggests that microRNAs can be released through ceramide-dependent secretory machinery regulated by neutral sphingomyelinase 2 (nSMase2) enzyme encoded by the smpd3 gene that triggers exosome secretion. However, whether exosome-mediated microRNA transfer plays any role in cell invasion remains poorly understood. Thus, the aim of this study was to identify the exosomal microRNAs involved in breast cancer invasion. METHODS: The expression level of endogenous and exosomal miRNAs were examined by real time PCR and the expression level of target proteins were detected by western blot. Scanning electron and confocal microscopy were used to characterize exosomes and to study its uptake and transfer. Luciferase reporter plasmids and its mutant were used to confirm direct targeting. Furthermore, the functional significance of exosomal miR-10b was estimated by invasion assay. RESULTS: In this study, we demonstrate that microRNA carrying exosomes can be transferred among different cell lines through direct uptake. miR-10b is highly expressed in metastatic breast cancer MDA-MB-231 cells as compared to non-metastatic breast cancer cells or non-malignant breast cells; it is actively secreted into medium via exosomes. In particular, nSMase2 or ceramide promotes the exosome-mediated miR-10b secretion whereas ceramide inhibitor suppresses this secretion. Moreover, upon uptake, miR-10b can suppress the protein level of its target genes such as HOXD10 and KLF4, indicating its functional significance. Finally, treatment with exosomes derived from MDA-MB-231 cells could induce the invasion ability of non-malignant HMLE cells. CONCLUSION: Together, our results suggest that a set of specific microRNAs may play an important role in modulating tumor microenvironment through exosomes. Thus, a better understanding of this process may aid in the development of novel therapeutic agents.

Asunto(s)

Neoplasias de la Mama/genética , Exosomas/genética , MicroARNs/genética , Invasividad Neoplásica/genética , Línea Celular Tumoral , Movimiento Celular/genética , Femenino , Proteínas de Homeodominio/genética , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción/genética

RESUMEN

Overall prognosis for osteosarcoma (OS) is poor despite aggressive treatment options. Limited access to primary tumors, technical challenges in processing OS tissues, and the lack of well-characterized primary cell cultures has hindered our ability to fully understand the properties of OS tumor initiation and progression. In this study, we have isolated and characterized cell cultures derived from four central high-grade human OS samples. Furthermore, we used the cell cultures to study the role of CD49f in OS progression. Recent studies have implicated CD49f in stemness and multipotency of both cancer stem cells and mesenchymal stem cells. Therefore, we investigated the role of CD49f in osteosarcomagenesis. First, single cell suspensions of tumor biopsies were subcultured and characterized for cell surface marker expression. Next, we characterized the growth and differentiation properties, sensitivity to chemotherapy drugs, and anchorage-independent growth. Xenograft assays showed that cell populations expressing CD49f(hi) /CD90(lo) cell phenotype produced an aggressive tumor. Multiple lines of evidence demonstrated that inhibiting CD49f decreased the tumor-forming ability. Furthermore, the CD49f(hi) /CD90(lo) cell population is generating more aggressive OS tumor growth and indicating this cell surface marker could be a potential candidate for the isolation of an aggressive cell type in OSs.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Neoplasias Óseas/metabolismo , Integrina alfa6/metabolismo , Osteosarcoma/metabolismo , Adolescente , Adulto , Animales , Antineoplásicos/farmacología , Neoplasias Óseas/patología , Movimiento Celular , Proliferación Celular , Niño , Cisplatino/farmacología , Progresión de la Enfermedad , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Femenino , Humanos , Masculino , Ratones Desnudos , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , Osteosarcoma/patología , Cultivo Primario de Células , Células Tumorales Cultivadas

RESUMEN

This study examines the antitumor potential of curcumin and C6 ceramide (C6) against osteosarcoma (OS) cell lines when both are encapsulated in the bilayer of liposomal nanoparticles. Three liposomal formulations were prepared: curcumin liposomes, C6 liposomes and C6-curcumin liposomes. Curcumin in combination with C6 showed 1.5 times enhanced cytotoxic effect in the case of MG-63 and KHOS OS cell lines, in comparison with curcumin liposomes alone. Importantly, C6-curcumin liposomes were found to be less toxic on untransformed primary human cells (human mesenchymal stem cells) in comparison to OS cell lines. In addition, cell cycle assays on a KHOS cell line after treatment revealed that curcumin only liposomes induced G2/M arrest by upregulation of cyclin B1, while C6 only liposomes induced G1 arrest by downregulation of cyclin D1. C6-curcumin liposomes induced G2/M arrest and showed a combined effect in the expression levels of cyclin D1 and cyclin B1. The efficiency of the preparations was tested in vivo using a human osteosarcoma xenograft assay. Using pegylated liposomes to increase the plasma half-life and tagging with folate (FA) for targeted delivery in vivo, a significant reduction in tumor size was observed with C6-curcumin-FA liposomes. The encapsulation of two water insoluble drugs, curcumin and C6, in the lipid bilayer of liposomes enhances the cytotoxic effect and validates the potential of combined drug therapy.

Asunto(s)

Ceramidas/administración & dosificación , Curcumina/administración & dosificación , Liposomas/química , Nanopartículas/química , Osteosarcoma/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ceramidas/química , Ceramidas/farmacología , Curcumina/química , Curcumina/farmacología , Sistemas de Liberación de Medicamentos , Citometría de Flujo , Humanos , Concentración 50 Inhibidora , Ratones , Microscopía Electrónica de Transmisión , Estructura Molecular , Nanopartículas/uso terapéutico