RESUMO
Epithelial-mesenchymal transition (EMT) empowers epithelial cells with mesenchymal and stem-like attributes, facilitating metastasis, a leading cause of cancer-related mortality. Hybrid epithelial-mesenchymal (E/M) cells, retaining both epithelial and mesenchymal traits, exhibit heightened metastatic potential and stemness. The mesenchymal intermediate filament, vimentin, is upregulated during EMT, enhancing the resilience and invasiveness of carcinoma cells. The phosphorylation of vimentin is critical to its structure and function. Here, we identify that stabilizing vimentin phosphorylation at serine 56 induces multinucleation, specifically in hybrid E/M cells with stemness properties but not epithelial or mesenchymal cells. Cancer stem-like cells are especially susceptible to vimentin-induced multinucleation relative to differentiated cells, leading to a reduction in self-renewal and stemness. As a result, vimentin-induced multinucleation leads to sustained inhibition of stemness properties, tumor initiation, and metastasis. These observations indicate that a single, targetable phosphorylation event in vimentin is critical for stemness and metastasis in carcinomas with hybrid E/M properties.
Assuntos
Carcinoma , Filamentos Intermediários , Humanos , Vimentina/metabolismo , Fosforilação , Filamentos Intermediários/metabolismo , Filamentos Intermediários/patologia , Carcinoma/patologia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Células-Tronco Neoplásicas/metabolismo , Linhagem Celular Tumoral , Metástase Neoplásica/patologiaRESUMO
MASTL is a mitotic accelerator with an emerging role in breast cancer progression. However, the mechanisms behind its oncogenicity remain largely unknown. Here, we identify a previously unknown role and eminent expression of MASTL in stem cells. MASTL staining from a large breast cancer patient cohort indicated a significant association with ß3 integrin, an established mediator of breast cancer stemness. MASTL silencing reduced OCT4 levels in human pluripotent stem cells and OCT1 in breast cancer cells. Analysis of the cell-surface proteome indicated a strong link between MASTL and the regulation of TGF-ß receptor II (TGFBR2), a key modulator of TGF-ß signaling. Overexpression of wild-type and kinase-dead MASTL in normal mammary epithelial cells elevated TGFBR2 levels. Conversely, MASTL depletion in breast cancer cells attenuated TGFBR2 levels and downstream signaling through SMAD3 and AKT pathways. Taken together, these results indicate that MASTL supports stemness regulators in pluripotent and cancerous stem cells.
RESUMO
In breast cancer, the currently approved anti-receptor tyrosine-protein kinase erbB-2 (HER2) therapies do not fully meet the expected clinical goals due to therapy resistance. Identifying alternative HER2-related therapeutic targets could offer a means to overcome these resistance mechanisms. We have previously demonstrated that an endosomal sorting protein, sortilin-related receptor (SorLA), regulates the traffic and signaling of HER2 and HER3, thus promoting resistance to HER2-targeted therapy in breast cancer. This study aims to assess the feasibility of targeting SorLA using a monoclonal antibody. Our results demonstrate that anti-SorLA antibody (SorLA ab) alters the resistance of breast cancer cells to HER2 monoclonal antibody trastuzumab in vitro and in ovo. We found that SorLA ab and trastuzumab combination therapy also inhibits tumor cell proliferation and tumor cell density in a mouse xenograft model of HER2-positive breast cancer. In addition, SorLA ab inhibits the proliferation of breast cancer patient-derived explant three-dimensional cultures. These results provide, for the first time, proof of principle that SorLA is a druggable target in breast cancer.
Assuntos
Antineoplásicos , Neoplasias da Mama , Proteínas Adaptadoras de Transporte Vesicular , Animais , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Camundongos , Receptor ErbB-2/metabolismo , Receptor ErbB-3 , Trastuzumab/farmacologiaRESUMO
The human epidermal growth factor receptor 2 (HER2) is an oncogene targeted by several kinase inhibitors and therapeutic antibodies. While the endosomal trafficking of many other receptor tyrosine kinases is known to regulate their oncogenic signalling, the prevailing view on HER2 is that this receptor is predominantly retained on the cell surface. Here, we find that sortilin-related receptor 1 (SORLA; SORL1) co-precipitates with HER2 in cancer cells and regulates HER2 subcellular distribution by promoting recycling of the endosomal receptor back to the plasma membrane. SORLA protein levels in cancer cell lines and bladder cancers correlates with HER2 levels. Depletion of SORLA triggers HER2 targeting to late endosomal/lysosomal compartments and impairs HER2-driven signalling and in vivo tumour growth. SORLA silencing also disrupts normal lysosome function and sensitizes anti-HER2 therapy sensitive and resistant cancer cells to lysosome-targeting cationic amphiphilic drugs. These findings reveal potentially important SORLA-dependent endosomal trafficking-linked vulnerabilities in HER2-driven cancers.
Assuntos
Neoplasias da Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Carcinoma de Células de Transição/genética , Membrana Celular/metabolismo , Endossomos/metabolismo , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas de Membrana Transportadoras/genética , Receptor ErbB-2/metabolismo , Neoplasias da Bexiga Urinária/genética , Animais , Neoplasias da Mama/metabolismo , Carcinoma Intraductal não Infiltrante/metabolismo , Carcinoma de Células de Transição/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Proteínas Relacionadas a Receptor de LDL/metabolismo , Lisossomos/metabolismo , Células MCF-7 , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Transporte Proteico , Neoplasias da Bexiga Urinária/metabolismoRESUMO
ß1-integrins mediate cell-matrix interactions and their trafficking is important in the dynamic regulation of cell adhesion, migration and malignant processes, including cancer cell invasion. Here, we employ an RNAi screen to characterize regulators of integrin traffic and identify the association of Golgi-localized gamma ear-containing Arf-binding protein 2 (GGA2) with ß1-integrin, and its role in recycling of active but not inactive ß1-integrin receptors. Silencing of GGA2 limits active ß1-integrin levels in focal adhesions and decreases cancer cell migration and invasion, which is in agreement with its ability to regulate the dynamics of active integrins. By using the proximity-dependent biotin identification (BioID) method, we identified two RAB family small GTPases, i.e. RAB13 and RAB10, as novel interactors of GGA2. Functionally, RAB13 silencing triggers the intracellular accumulation of active ß1-integrin, and reduces integrin activity in focal adhesions and cell migration similarly to GGA2 depletion, indicating that both facilitate active ß1-integrin recycling to the plasma membrane. Thus, GGA2 and RAB13 are important specificity determinants for integrin activity-dependent traffic.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Neoplasias da Mama/patologia , Movimento Celular/fisiologia , Integrina beta1/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Animais Geneticamente Modificados , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Invasividade Neoplásica/patologia , Transplante de Neoplasias , Interferência de RNA , RNA Interferente Pequeno/genética , Transplante Heterólogo , Peixe-Zebra , Proteínas rab de Ligação ao GTP/genéticaRESUMO
BACKGROUND: Triple-negative breast cancers (TNBCs), which lack receptors for estrogen, progesterone, and amplification of epidermal growth factor receptor 2, are highly aggressive. Consequently, patients diagnosed with TNBCs have reduced overall and disease-free survival rates compared to patients with other subtypes of breast cancer. TNBCs are characterized by the presence of cancer cells with mesenchymal properties, indicating that the epithelial to mesenchymal transition (EMT) plays a major role in the progression of this disease. The EMT program has also been implicated in chemoresistance, tumor recurrence, and induction of cancer stem cell (CSC) properties. Currently, there are no targeted therapies for TNBC, and hence, it is critical to identify the novel targets to treat TNBC. METHODS: A library of compounds was screened for their ability to inhibit EMT in cells with mesenchymal phenotype as assessed using the previously described Z-cad reporters. Of the several drugs tested, GSK3ß inhibitors were identified as EMT inhibitors. The effects of GSK3ß inhibitors on the properties of TNBC cells with a mesenchymal phenotype were assessed using qRT-PCR, flow cytometry, western blot, mammosphere, and migration and cell viability assays. Publicly available datasets also were analyzed to examine if the expression of GSK3ß correlates with the overall survival of breast cancer patients. RESULTS: We identified a GSK3ß inhibitor, BIO, in a drug screen as one of the most potent inhibitors of EMT. BIO and two other GSK3ß inhibitors, TWS119 and LiCl, also decreased the expression of mesenchymal markers in several different cell lines with a mesenchymal phenotype. Further, inhibition of GSK3ß reduced EMT-related migratory properties of cells with mesenchymal properties. To determine if GSK3ß inhibitors target mesenchymal-like cells by affecting the CSC population, we employed mammosphere assays and profiled the stem cell-related cell surface marker CD44+/24- in cells after exposure to GSK3ß inhibitors. We found that GSK3ß inhibitors indeed decreased the CSC properties of cell types with mesenchymal properties. We treated cells with epithelial and mesenchymal properties with GSK3ß inhibitors and found that GSK3ß inhibitors selectively kill cells with mesenchymal attributes while sparing cells with epithelial properties. We analyzed patient data to identify genes predictive of poor clinical outcome that could serve as novel therapeutic targets for TNBC. The Wnt signaling pathway is critical to EMT, but among the various factors known to be involved in Wnt signaling, only the higher expression of GSK3ß correlated with poorer overall patient survival. CONCLUSIONS: Taken together, our data demonstrate that GSK3ß is a potential target for TNBCs and suggest that GSK3ß inhibitors could serve as selective inhibitors of EMT and CSC properties for the treatment of a subset of aggressive TNBC. GSK3ß inhibitors should be tested for use in combination with standard-of-care drugs in preclinical TNBC models.
Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Neoplasias de Mama Triplo Negativas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Conjuntos de Dados como Assunto , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Humanos , Concentração Inibidora 50 , Cloreto de Lítio/farmacologia , Cloreto de Lítio/uso terapêutico , Células-Tronco Neoplásicas/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Pirróis/farmacologia , Pirróis/uso terapêutico , Análise de Sobrevida , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/mortalidade , Via de Sinalização WntRESUMO
Expression of the transcription factor FOXC2 is induced and necessary for successful epithelial-mesenchymal transition, a developmental program that when activated in cancer endows cells with metastatic potential and the properties of stem cells. As such, identifying agents that inhibit the growth of FOXC2-transformed cells represents an attractive approach to inhibit chemotherapy resistance and metastatic dissemination. From a high throughput synthetic lethal screen, we identified a small molecule, FiVe1, which selectively and irreversibly inhibits the growth of mesenchymally transformed breast cancer cells and soft tissue sarcomas of diverse histological subtypes. FiVe1 targets the intermediate filament and mesenchymal marker vimentin (VIM) in a mode which promotes VIM disorganization and phosphorylation during metaphase, ultimately leading to mitotic catastrophe, multinucleation, and the loss of stemness. These findings illustrate a previously undescribed mechanism for interrupting faithful mitotic progression and may ultimately inform the design of therapies for a broad range of mesenchymal cancers.
Assuntos
Mitose/efeitos dos fármacos , Sarcoma/metabolismo , Vimentina/metabolismo , Vimentina/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Descoberta de Drogas , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Filamentos Intermediários/metabolismo , Células-Tronco Neoplásicas/patologia , Fosforilação , Sarcoma/patologia , Fatores de Transcrição/efeitos dos fármacos , Vimentina/químicaRESUMO
Cancer is a complex disease and progresses within a dynamically evolving extracellular matrix that controls virtually every aspect of the tumour and tumour-associated cells. Interactions with the extracellular microenvironment are predominately mediated by a family of cell-surface transmembrane receptors called integrins. Integrin-matrix engagement leads to the formation of adhesion plaques, consisting of signalling and adaptor proteins, at the plasma membrane that link the extracellular matrix to the regulation of the cell cytoskeleton. In this review, we will highlight exciting data that identify new roles for integrins and integrin-dependent signalling in cancer away from the plasma membrane, discuss the implications of integrin-dependent regulation of Met and ErbB2 growth factor receptors and highlight the role of specific integrins in different stages of cancer development including maintenance of cancer stem cells.
Assuntos
Integrinas/fisiologia , Terapia de Alvo Molecular/tendências , Neoplasias/patologia , Neoplasias/terapia , Animais , Humanos , Terapia de Alvo Molecular/métodos , Transdução de Sinais , Terapias em Estudo/métodos , Terapias em Estudo/tendências , Microambiente Tumoral/fisiologiaRESUMO
Metastasis can involve repeated cycles of epithelial-to-mesenchymal transition (EMT) and its reverse mesenchymal-to-epithelial transition. Cells can also undergo partial transitions to attain a hybrid epithelial/mesenchymal (E/M) phenotype that allows the migration of adhering cells to form a cluster of circulating tumour cells. These clusters can be apoptosis-resistant and possess an increased metastatic propensity as compared to the cells that undergo a complete EMT (mesenchymal cells). Hence, identifying the key players that can regulate the formation and maintenance of such clusters may inform anti-metastasis strategies. Here, we devise a mechanism-based theoretical model that links cell-cell communication via Notch-Delta-Jagged signalling with the regulation of EMT. We demonstrate that while both Notch-Delta and Notch-Jagged signalling can induce EMT in a population of cells, only Jagged-dominated Notch signalling, but not Delta-dominated signalling, can lead to the formation of clusters containing hybrid E/M cells. Our results offer possible mechanistic insights into the role of Jagged in tumour progression, and offer a framework to investigate the effects of other microenvironmental signals during metastasis.
Assuntos
Transição Epitelial-Mesenquimal , Proteína Jagged-1/metabolismo , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Humanos , Metástase Neoplásica , Neoplasias/patologiaRESUMO
Cancer cells with stem cell properties (CSCs) underpin the chemotherapy resistance and high therapeutic failure of triple-negative breast cancers (TNBCs). Even though CSCs are known to proliferate more slowly, they are sensitive to inhibitors of G2/M kinases such as polo-like kinase 1 (PLK1). Understanding the cell cycle regulatory mechanisms of CSCs will help target these cells more efficiently. Herein, we identify a novel role for the transcription factor FOXC2, which is mostly expressed in CSCs, in the regulation of cell cycle of CSC-enriched breast cancer cells. We demonstrate that FOXC2 expression is regulated in a cell cycle-dependent manner, with FOXC2 protein levels accumulating in G2, and rapidly decreasing during mitosis. Knockdown of FOXC2 in CSC-enriched TNBC cells delays mitotic entry without significantly affecting the overall proliferation rate of these cells. Moreover, PLK1 activity is important for FOXC2 protein stability, since PLK1 inhibition reduces FOXC2 protein levels. Indeed, FOXC2 expressing CSC-enriched TNBC cells are sensitive to PLK1 inhibition. Collectively, our findings demonstrate a novel role for FOXC2 as a regulator of the G2/M transition and elucidate the reason for the observed sensitivity of CSC-enriched breast cancer cells to PLK1 inhibitor.
Assuntos
Neoplasias da Mama/fisiopatologia , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Fatores de Transcrição Forkhead/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Fatores de Transcrição Forkhead/genética , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Células-Tronco/fisiologia , Quinase 1 Polo-LikeRESUMO
Sodium valproate (VPA) is a potentially hepatotoxic antiepileptic drug. Risk of VPA-induced hepatotoxicity is increased in patients with mitochondrial diseases and especially in patients with POLG1 gene mutations. We used a HepG2 cell in vitro model to investigate the effect of VPA on mitochondrial activity. Cells were incubated in glucose medium and mitochondrial respiration-inducing medium supplemented with galactose and pyruvate. VPA treatments were carried out at concentrations of 0-2.0mM for 24-72 h. In both media, VPA caused decrease in oxygen consumption rates and mitochondrial membrane potential. VPA exposure led to depleted ATP levels in HepG2 cells incubated in galactose medium suggesting dysfunction in mitochondrial ATP production. In addition, VPA exposure for 72 h increased levels of mitochondrial reactive oxygen species (ROS), but adversely decreased protein levels of mitochondrial superoxide dismutase SOD2, suggesting oxidative stress caused by impaired elimination of mitochondrial ROS and a novel pathomechanism related to VPA toxicity. Increased cell death and decrease in cell number was detected under both metabolic conditions. However, immunoblotting did not show any changes in the protein levels of the catalytic subunit A of mitochondrial DNA polymerase γ, the mitochondrial respiratory chain complexes I, II and IV, ATP synthase, E3 subunit dihydrolipoyl dehydrogenase of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and glutathione peroxidase. Our results show that VPA inhibits mitochondrial respiration and leads to mitochondrial dysfunction, oxidative stress and increased cell death, thus suggesting an essential role of mitochondria in VPA-induced hepatotoxicity.
Assuntos
Anticonvulsivantes/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Hepatócitos/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Doenças Mitocondriais/induzido quimicamente , Fosforilação Oxidativa/efeitos dos fármacos , Ácido Valproico/toxicidade , Trifosfato de Adenosina/metabolismo , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Relação Dose-Resposta a Droga , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Galactose/metabolismo , Glucose/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Estresse Oxidativo/efeitos dos fármacos , Ácido Pirúvico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Fatores de TempoRESUMO
The interplay between tumor stroma and breast cancer cells (BCCs) is thought to play a significant role in breast cancer. The current knowledge of human mesenchymal stromal cell (MSC) and BCC interaction is contradictory, and the donor sex issue is not addressed at all. We hypothesized that donor sex could have an effect on proliferation of MSCs or BCCs in co-culture in vitro. Three estrogen receptor-negative BCC lines, 19 primary human MSCs and breast tissue-derived fibroblasts from 4 donors were used. MSCs from female donors enhanced BCC proliferation (p = 0.005). The change in BCC proliferation was only partly due to soluble factors excreted by MSCs. The highly aggressive BCC line MDA-MB- 231 induced the proliferation of MSCs (p < 0.001) and fibroblasts (p = 0.037) in co-culture experiments. The magnitude in proliferation change was cell line dependent and partly sex dependent.
Assuntos
Neoplasias da Mama/patologia , Proliferação de Células , Células-Tronco Mesenquimais/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Mama/patologia , Diferenciação Celular , Movimento Celular , Técnicas de Cocultura , Feminino , Fibroblastos/patologia , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Fatores Sexuais , Células Tumorais Cultivadas , Adulto JovemRESUMO
The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell-cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells.
Assuntos
Anticorpos Monoclonais/metabolismo , Neoplasias da Mama/metabolismo , Comunicação Celular , Proteínas de Choque Térmico HSP70/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Pontos Quânticos , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Técnicas de Cocultura , Citosol/metabolismo , Feminino , Humanos , Células-Tronco Mesenquimais/ultraestrutura , Microscopia Eletrônica de Transmissão , Coloração e RotulagemRESUMO
Human mesenchymal stem cells (hMSCs) are multipotent cells that have aroused great expectations in regenerative medicine. They are assumed to originate from hypoxic stem cell niches, especially in the bone marrow. This suggests that O2 is of importance in their regulation. In order to characterize regulation of the oxygen sensing pathway in these cells, we studied hMSCs isolated from three origins, adult and pediatric bone marrow and umbilical cord blood (UCB). Surprisingly, pediatric bone marrow and UCB MSCs showed normoxic stabilization of hypoxia-inducible factor-1α (HIF-1α) that is normally degraded completely by HIF prolyl 4-hydroxylases in the presence of oxygen. This was due to a high expression level of HIF-1α mRNA rather than inappropriate post-translational degradation of HIF-1α protein. HIF-1α mRNA was also induced in normoxic adult bone marrow MSCs, but 40% less than in the pediatric cells, and this was apparently not enough to stabilize the protein. The high normoxic HIF expression in all the hMSCs studied was accompanied by increased expression of a large number of glycolytic HIF target genes and increased glycolysis. Osteogenic differentiation of bone marrow-derived hMSCs reduced HIF-1α mRNA and protein expression and the expression of glycolytic mRNAs, resulting in decreased glycolysis and induction of oxidative metabolism. Induced mitochondrial biogenesis, changes in mitochondrial morphology and size indicative of increased oxidative phosphorylation, and induction of extracellular matrix synthesis were observed following osteogenic differentiation. Altogether, these data suggest that HIF-1α is a general regulator controlling the metabolic fate and multipotency of the hMSCs.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células-Tronco Mesenquimais/metabolismo , Regulação para Cima/genética , Adulto , Antígenos de Superfície/metabolismo , Western Blotting , Diferenciação Celular/genética , Forma Celular , Criança , Glicólise/genética , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lactatos/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Modelos Biológicos , Osteogênese/genética , Reação em Cadeia da Polimerase , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Nicho de Células-Tronco/genéticaRESUMO
Systemic infusion of therapeutic cells would be the most practical and least invasive method of administration in many cellular therapies. One of the main obstacles especially in intravenous delivery of cells is a massive cell retention in the lungs, which impairs homing to the target tissue and may decrease the therapeutic outcome. In this study we showed that an alternative cell detachment of mesenchymal stromal/stem cells (MSCs) with pronase instead of trypsin significantly accelerated the lung clearance of the cells and, importantly, increased their targeting to an area of injury. Cell detachment with pronase transiently altered the MSC surface protein profile without compromising cell viability, multipotent cell characteristics, or immunomodulative and angiogenic potential. The transient modification of the cell surface protein profile was sufficient to produce effective changes in cell rolling behavior in vitro and, importantly, in the in vivo biodistribution of the cells in mouse, rat, and porcine models. In conclusion, pronase detachment could be used as a method to improve the MSC lung clearance and targeting in vivo. This may have a major impact on the bioavailability of MSCs in future therapeutic regimes.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Sobrevivência de Enxerto/fisiologia , Inflamação/terapia , Pulmão/citologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Animais , Antígenos de Superfície/metabolismo , Carragenina/toxicidade , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Humanos , Inflamação/induzido quimicamente , Inflamação/imunologia , Migração e Rolagem de Leucócitos/fisiologia , Pulmão/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Neovascularização Fisiológica/fisiologia , Pronase/metabolismo , Ratos , Suínos , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
The promising clinical effects of mesenchymal stromal/stem cells (MSCs) rely especially on paracrine and nonimmunogenic mechanisms. Delivery routes are essential for the efficacy of cell therapy and systemic delivery by infusion is the obvious goal for many forms of MSC therapy. Lung adhesion of MSCs might, however, be a major obstacle yet to overcome. Current knowledge does not allow us to make sound conclusions whether MSC lung entrapment is harmful or beneficial, and thus we wanted to explore MSC lung adhesion in greater detail. We found a striking difference in the lung clearance rate of systemically infused MSCs derived from two different clinical sources, namely bone marrow (BM-MSCs) and umbilical cord blood (UCB-MSCs). The BM-MSCs and UCB-MSCs used in this study differed in cell size, but our results also indicated other mechanisms behind the lung adherence. A detailed analysis of the cell surface profiles revealed differences in the expression of relevant adhesion molecules. The UCB-MSCs had higher expression levels of α4 integrin (CD49d, VLA-4), α6 integrin (CD49f, VLA-6), and the hepatocyte growth factor receptor (c-Met) and a higher general fucosylation level. Strikingly, the level of CD49d and CD49f expression could be functionally linked with the lung clearance rate. Additionally, we saw a possible link between MSC lung adherence and higher fibronectin expression and we show that the expression of fibronectin increases with MSC culture confluence. Future studies should aim at developing methods of transiently modifying the cell surface structures in order to improve the delivery of therapeutic cells.
Assuntos
Células da Medula Óssea/citologia , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Sangue Fetal/citologia , Pulmão/citologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Animais , Biomarcadores/metabolismo , Células da Medula Óssea/metabolismo , Adesão Celular , Diferenciação Celular , Feminino , Sangue Fetal/metabolismo , Expressão Gênica , Meia-Vida , Humanos , Infusões Intravenosas , Integrina alfa4/genética , Integrina alfa4/metabolismo , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Integrina alfa6/genética , Integrina alfa6/metabolismo , Integrina alfa6beta1/genética , Integrina alfa6beta1/metabolismo , Marcação por Isótopo , Pulmão/imunologia , Pulmão/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Compostos de Tecnécio , Transplante HeterólogoRESUMO
INTRODUCTION: One of the most challenging safety issues in the manufacture of cell based medicinal products is the control of microbial risk as cell-based products cannot undergo terminal sterilization. Accordingly, sensitive and reliable methods for detection of microbial contamination are called for. As mitochondrial function has been shown to correlate with the viability and functionality of human mesenchymal stem cells (hMSCs) we have studied the use of a mitochondrial inner membrane potential sensitive dye for detecting changes in the function of mitochondria following infection by bacteria. METHODS: The effect of bacterial contamination on the viability of bone marrow-derived mesenchymal stem cells (BMMSCs) was studied. BMMSC lines were infected with three different bacterial species, namely two strains of Pseudomonas aeruginosa, three strains of Staphylococcus aureus, and three strains of Staphylococcus epidermidis. The changes in viability of the BMMSCs after bacterial infection were studied by staining with Trypan blue, by morphological analysis and by monitoring of the mitochondrial inner membrane potential. RESULTS: Microscopy and viability assessment by Trypan blue staining showed that even the lowest bacterial inocula caused total dissipation of BMMSCs within 24 hours of infection, similar to the effects seen with bacterial loads which were several magnitudes higher. The first significant signs of damage induced by the pathogens became evident after 6 hours of infection. Early changes in mitochondrial inner membrane potential of BMMSCs were evident after 4 hours of infection even though no visible changes in viability of the BMMSCs could be seen. CONCLUSIONS: Even low levels of bacterial contamination can cause a significant change in the viability of BMMSCs. Moreover, monitoring the depolarization of the mitochondrial inner membrane potential may provide a rapid tool for early detection of cellular damage induced by microbial infection. Accordingly, mitochondrial analyses offer sensitive tools for quality control and monitoring of safety and efficacy of cellular therapy products.
Assuntos
Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Mitocôndrias/metabolismo , Pseudomonas aeruginosa/patogenicidade , Staphylococcus aureus/patogenicidade , Staphylococcus epidermidis/patogenicidade , Idoso , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Feminino , Humanos , Masculino , Potencial da Membrana Mitocondrial , Células-Tronco Mesenquimais/microbiologia , Pessoa de Meia-IdadeRESUMO
Human mesenchymal stem cells (hMSCs) display immunosuppressive properties in vitro and the potential has also been transferred successfully to clinical trials for treatment of autoimmune diseases. OX-2 (CD200), a member of the immunoglobulin superfamily, is widely expressed in several tissues and has recently been found from hMSCs. The CD200 receptor (CD200R) occurs only in myeloid-lineage cells. The CD200-CD200R is involved in down-regulation of several immune cells, especially macrophages. The present study on 20 hMSC lines shows that the CD200 expression pattern varied from high (CD200Hi) to medium (CD200Me) and low (CD200Lo) in bone marrow-derived mesenchymal stem cell (BMMSC) lines, whereas umbilical cord blood derived mesenchymal stem cells (UCBMSCs) were constantly negative for CD200. The role of the CD200-CD200R axis in BMMSCs mediated immunosuppression was studied using THP-1 human macrophages. Interestingly, hMSCs showed greater inhibition of TNF-α secretion in co-cultures with IFN-γ primed THP-1 macrophages when compared to LPS activated cells. The ability of CD200Hi BMMSCs to suppress TNF-α secretion from IFN-γ stimulated THP-1 macrophages was significantly greater when compared to CD200Lo whereas UCBMSCs did not significantly reduce TNF-α secretion. The interference of CD200 binding to the CD200R by anti-CD200 antibody weakened the capability of BMMSCs to inhibit TNF-α secretion from IFN-γ activated THP-1 macrophages. This study clearly demonstrated that the efficiency of BMMSCs to suppress TNF-α secretion of THP-1 macrophages was dependent on the type of stimulus. Moreover, the CD200-CD200r axis could have a previously unidentified role in the BMMSC mediated immunosuppression.
Assuntos
Antígenos CD/metabolismo , Antígenos de Superfície/metabolismo , Macrófagos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Receptores de Superfície Celular/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Anticorpos/imunologia , Antígenos CD/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Linhagem Celular , Técnicas de Cocultura , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Terapia de Imunossupressão , Interferon gama/farmacologia , Interleucina-10/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Receptores de OrexinaRESUMO
Human mesenchymal stem cells (hMSCs) are an attractive choice for a variety of cellular therapies. hMSCs can be isolated from many different tissues and possess unique mitochondrial properties that can be used to determine their differentiation potential. Mitochondrial properties may possibly be used as a quality measure of hMSC-based products. Accordingly, the present work focuses on the mitochondrial function of hMSCs from umbilical cord blood (UCBMSC) cells and bone marrow cells from donors younger than 18 years of age (BMMSC <18) and those more than 50 years of age (BMMSC >50). Changes of ultrastructure and energy metabolism during osteogenic differentiation in all hMSC types were studied in detail. Results show that despite similar surface antigen characteristics, the UCBMSCs had smaller cell surface area and possessed more abundant rough endoplasmic reticulum than BMMSC >50. BMMSC <18 were morphologically more UCBMSC-like. UCBMSC showed dramatically higher mitochondrial-to-cytoplasm area ratio and elevated superoxide and manganese superoxide dismutase (MnSOD) levels as compared with BMMSC >50 and BMMSC <18. All hMSCs types showed changes indicative of mitochondrial activation after 2 weeks of osteogenic differentiation, and the increase in mitochondrial-to-cytoplasm area ratio appears to be one of the first steps in the differentiation process. However, BMMSC >50 showed a lower level of mitochondrial maturation and differentiation capacity. UCBMSCs and BMMSCs also showed a different pattern of exocytosed proteins and glycoproteoglycansins. These results indicate that hMSCs with similar cell surface antigen expression have different mitochondrial and functional properties, suggesting different maturation levels and other significant biological variations of the hMSCs. Therefore, it appears that mitochondrial analysis presents useful characterization criteria for hMSCs intended for clinical use.
Assuntos
Células da Medula Óssea/metabolismo , Metabolismo Energético/fisiologia , Sangue Fetal/metabolismo , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Sangue Fetal/citologia , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Osteogênese/fisiologiaRESUMO
Mesenchymal stem cells (MSCs) are widely used in experimental treatments for various conditions that involve normal tissue regeneration via inflammatory repair. It is known that MSCs can secrete multiple soluble factors and suppress inflammation. Even though the effect of MSCs on inflammation has been extensively studied, the effect of inflammation on MSCs is poorly understood. One of the major cytokines released at the site of inflammation is tumor necrosis factor alpha (TNF-α) which is known to induce MSC invasion and proliferation. Therefore, we wanted to test the effects of TNF-α exposure on MSCs derived from human bone marrow. We found, as expected, that cell proliferation was significantly enhanced during TNF-α exposure. However, according to the cell surface marker analysis, the intensity of several antigens in the minimum criteria panel for MSCs proposed by International Society of Cellular Therapy (ISCT) was decreased dramatically, and in certain cases, the criteria for MSCs were not fulfilled. In addition, TNF-α exposure resulted in a significant but transient increase in human leukocyte antigen and CD54 expression. Additional proteomic analysis by two-dimensional difference gel electrophoresis and mass spectrometry revealed three proteins whose expression levels decreased and 8 proteins whose expression levels increased significantly during TNF-α exposure. The majority of these proteins could be linked to immunosuppressive and signalling pathways. These results strongly support reactive and immunosuppressive activation of MSCs during TNF-α exposure, which might influence MSC differentiation stage and capacity.