RESUMEN
Human embryonic stem cells (hESCs) provide an important resource for novel regenerative medicine therapies and have been used to derive diverse cell populations, including hematopoietic and endothelial cells. However, it remains a challenge to achieve significant engraftment of hESC-derived blood cells when transplanted into animal models. To better understand mechanisms that enhance or limit the in vivo developmental potential of hESC-derived cells, we utilized hESCs that express firefly luciferase (luc) to allow noninvasive, real-time bioluminescent imaging of hESC-derived CD34(+) cells transplanted into the liver of neonatal immunodeficient mice. Serial imaging demonstrated stable engraftment and expansion of the luc(+) hESC-derived cells in vivo over several months. While we found that these hESC-derived CD34(+) cells have bipotential ability to generate both hematopoietic and endothelial lineages in vitro, these studies demonstrate preferential differentiation into endothelial cells in vivo, with only low levels of hematopoietic cell engraftment. Therefore, these studies reveal key differences in the developmental potential of hESC-derived cells using in vitro and in vivo analyses. Although transplanted hESC-derived CD34(+) cells are well-suited for revascularization therapies, additional measures are needed to provide higher levels of long-term hematopoietic engraftment.
Asunto(s)
Antígenos CD34/metabolismo , Diferenciación Celular/fisiología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Hematopoyesis/fisiología , Animales , Animales Recién Nacidos , Diferenciación Celular/genética , Células Cultivadas , Células Madre Embrionarias/trasplante , Sangre Fetal/citología , Citometría de Flujo , Humanos , Inmunohistoquímica , Hígado/metabolismo , Mediciones Luminiscentes , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
PURPOSE: The purpose of our study was to characterize the epidemiology of blood stream infection (BSI) in adult extracorporeal membrane oxygenation (ECMO) patients at a single tertiary care academic medical center with standardized post-cannulation antibiotic prophylaxis practices. METHODS: A single-center retrospective cohort study was performed over a five-year period. BSI incidence was characterized and patients who developed BSI during ECMO were compared with those who did not. RESULTS: Nineteen of 145 VV ECMO patients (13.1%) developed BSI while 7 of 123 VA ECMO patients (5.7%) developed BSI. When accounting for total ECMO days, the incidence rate was 8 BSIs per 1,000 ECMO days for both VV and VA ECMO patients. VV ECMO patients with BSI had longer ECMO runs and more red blood cell transfusion (both p<0.05). VA ECMO patients who developed BSI had longer ECMO runs and more platelet transfusion (both p<0.05). In VV ECMO patients there was an association between renal failure and BSI and in VA ECMO patients there was an association between hepatic failure and BSI. CONCLUSIONS: BSIs are common in ECMO patients even with post-cannulation antimicrobial prophylaxis and are associated with ECMO duration, blood transfusion, and organ failure. Further work is needed to clarify the optimal duration and type of antimicrobial prophylaxis, as well as surveillance strategies for BSIs during adult ECMO.
Asunto(s)
Bacteriemia/epidemiología , Oxigenación por Membrana Extracorpórea/efectos adversos , Adulto , Profilaxis Antibiótica/métodos , Bacteriemia/etiología , Estudios de Cohortes , Femenino , Humanos , Incidencia , Masculino , Maryland/epidemiología , Persona de Mediana Edad , Estudios Retrospectivos , Choque Cardiogénico/terapiaRESUMEN
OBJECTIVE: Cancer-associated fibroblasts (CAFs) play an important role in the progression of pancreatic ductal adenocarcinoma (PDAC) by promoting tumor cell migration and drug resistance. We determined the impact of CAFs on PDAC cancer stem cells (CSCs). METHODS: Fibroblast cell lines from patients' tumors were cocultured with PDAC cells and examined for clonogenic growth and self-renewal using colony-forming assays and migration in vitro. Changes in the frequency of CSCs was determined by flow cytometry. The effect of integrin-focal adhesion kinase (FAK) signaling on CAF-mediated clonogenic growth was evaluated using short hairpin RNAs against ß1 integrin and FAK as well as a small-molecule FAK inhibitor. RESULTS: Cancer-associated fibroblasts enhanced PDAC clonogenic growth, self-renewal, and migration that was associated with an increase in the frequency of CSCs. These fibroblast cells were activated by PDAC cells and increased collagen synthesis resulting in FAK activation in PDAC cells. Knockdown of ß1-integrin and FAK or the inhibition of FAK kinase activity in PDAC cells abrogated the impact of CAFs on clonogenic growth. CONCLUSION: Therefore, CAFs enhance PDAC clonogenic growth, self-renewal, and the frequency of CSCs through type I collagen production that enhances integrin-FAK signaling in PDAC cells.
Asunto(s)
Fibroblastos Asociados al Cáncer/patología , Comunicación Celular , Células Madre Neoplásicas/patología , Neoplasias Pancreáticas/patología , Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Células Cultivadas , Técnicas de Cocultivo , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Células Madre Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Interferencia de ARN , Transducción de Señal/genéticaRESUMEN
Self-renewal maintains the long-term clonogenic growth that is required for cancer relapse and progression, but the cellular processes regulating this property are not fully understood. In many diseases, self-renewal is enhanced in cancer stem cells (CSC), and in pancreatic ductal adenocarcinoma (PDAC), CSCs are characterized by the surface expression of CD44. In addition to cell adhesion, CD44 impacts cell shape and morphology by modulating the actin cytoskeleton via Ezrin, a member of the Ezrin/Radixin/Moesin (ERM) family of linker proteins. We examined the expression of Ezrin in PDAC cells and found higher levels of both total and activated Ezrin in CSCs compared with bulk tumor cells. We also found that the knockdown of Ezrin in PDAC cells decreased clonogenic growth, self-renewal, cell migration, and CSC frequency in vitro as well as tumor initiation in vivo. These effects were associated with cytoskeletal changes that are similar to those occurring during the differentiation of normal stem cells, and the inhibition of actin remodeling reversed the impact of Ezrin loss. Finally, targeting Ezrin using a small-molecule inhibitor limited the self-renewal of clinically derived low-passage PDAC xenografts. Our findings demonstrate that Ezrin modulates CSCs properties and may represent a novel target for the treatment of PDAC. IMPLICATIONS: Our findings demonstrate that Ezrin modulates CSCs' properties and may represent a novel target for the treatment of PDAC.
Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Proteínas del Citoesqueleto/biosíntesis , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Actinas/metabolismo , Adamantano/análogos & derivados , Adamantano/farmacología , Animales , Línea Celular Tumoral , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas del Citoesqueleto/metabolismo , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Quinolinas/farmacologíaRESUMEN
Cancer stem cells (CSCs) play an important role in the clonogenic growth and metastasis of pancreatic ductal adenocarcinoma (PDAC). A hallmark of PDAC is the desmoplastic reaction, but the impact of the tumor microenvironment (TME) on CSCs is unknown. In order to better understand the mechanisms, we examined the impact of extracellular matrix (ECM) proteins on PDAC CSCs. We quantified the effect of ECM proteins, ß1-integrin, and focal adhesion kinase (FAK) on clonogenic PDAC growth and migration in vitro and tumor initiation, growth, and metastasis in vivo in nude mice using shRNA and overexpression constructs as well as small molecule FAK inhibitors. Type I collagen increased PDAC tumor initiating potential, self-renewal, and the frequency of CSCs through the activation of FAK. FAK overexpression increased tumor initiation, whereas a dominant negative FAK mutant or FAK kinase inhibitors reduced clonogenic PDAC growth in vitro and in vivo. Moreover, the FAK inhibitor VS-4718 extended the anti-tumor response to gemcitabine and nab-paclitaxel in patient-derived PDAC xenografts, and the loss of FAK expression limited metastatic dissemination of orthotopic xenografts. Type I collagen enhances PDAC CSCs, and both kinase-dependent and independent activities of FAK impact PDAC tumor initiation, self-renewal, and metastasis. The anti-tumor impact of FAK inhibitors in combination with standard chemotherapy support the clinical testing of this combination.
Asunto(s)
Carcinoma Ductal Pancreático/enzimología , Carcinoma Ductal Pancreático/patología , Matriz Extracelular/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Células Madre Neoplásicas/metabolismo , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/patología , Transducción de Señal , Aldehído Deshidrogenasa/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Autorrenovación de las Células/efectos de los fármacos , Células Clonales , Colágeno Tipo I/metabolismo , Matriz Extracelular/efectos de los fármacos , Humanos , Integrina beta1/metabolismo , Ratones Desnudos , Metástasis de la Neoplasia , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Neoplasias PancreáticasRESUMEN
Despite improved outcomes in newly diagnosed multiple myeloma, virtually all patients relapse and ultimately develop drug-resistant disease. Aberrant RAS/MAPK signaling is activated in the majority of relapsed/refractory multiple myeloma patients, but its biological consequences are not fully understood. Self-renewal, as defined by the long-term maintenance of clonogenic growth, is essential for disease relapse, and we examined the role of RAS/MAPK activation on multiple myeloma self-renewal by targeting IQ motif-containing GTPase-activating protein 1 (IQGAP1), an intracellular scaffold protein required for mutant RAS signaling. We found that loss of IQGAP1 expression decreased MAPK signaling, cell-cycle progression, and tumor colony formation. Similarly, a peptide mimicking the WW domain of IQGAP1 that interacts with ERK inhibited the clonogenic growth and self-renewal of multiple myeloma cell lines and primary clinical specimens in vitro as well as tumor-initiating cell frequency in immunodeficient mice. During multiple myeloma progression, self-renewal may be enhanced by aberrant RAS/MAPK signaling and inhibited by targeting IQGAP1. Mol Cancer Ther; 15(11); 2733-9. ©2016 AACR.
Asunto(s)
Autorrenovación de las Células , Evolución Clonal , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Células Madre Neoplásicas/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Animales , Mimetismo Biológico , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Autorrenovación de las Células/efectos de los fármacos , Autorrenovación de las Células/genética , Evolución Clonal/efectos de los fármacos , Evolución Clonal/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/mortalidad , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Péptidos/química , Péptidos/farmacología , Dominios y Motivos de Interacción de Proteínas , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Activadoras de ras GTPasa/antagonistas & inhibidores , Proteínas Activadoras de ras GTPasa/químicaRESUMEN
Cancer stem cells (CSC) have been identified in an ever-increasing number of human malignancies on the basis of their ability to recapitulate tumors in the ectopic setting and maintain long-term tumorigenic potential. In addition, in pancreatic adenocarcinoma, CSCs may display additional properties, such as relative drug resistance and enhanced invasive and migratory potential that implicate a role in disease pathogenesis spanning initial tumor formation to metastatic disease progression. Importantly, these findings also indicate that the development of novel therapeutic strategies capable of inhibiting or eliminating CSCs will improve clinical outcomes. Preclinical studies have already described a wide array of potential approaches that target CSC-specific surface antigens and cellular pathways involved in cell survival, adhesion, self-renewal, and differentiation. Further, progress in this area should continue to move forward as the unique biology of CSCs is better understood. All preclinical studies to date have focused on targeting specific and phenotypically defined CSCs, but multiple cell populations with the ability to form tumors and self-renew have been identified in pancreatic carcinoma. As the clinical efficacy of CSC-directed therapies will depend on the inhibition of all sources of tumor self-renewal, better understanding of how specific CSC populations are related to one another and whether each possesses specific functional properties will be critical. In this CCR Focus article, we discuss the potential relationships between different pancreatic CSC populations and strategies to identify novel targeting approaches.
Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , Células Madre Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Humanos , Terapia Molecular Dirigida , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológicoRESUMEN
Mesenchymal stem/stromal cells (MSCs) have been isolated from various tissues and utilized for an expanding number of therapies. The developmental pathways involved in producing MSCs and the phenotypic precursor/progenitor cells that give rise to human MSCs remain poorly defined. Human embryonic stem cells (hESCs) have the capability to generate functional hemato-endothelial cells and other mesoderm lineage cells. hESC-derived CD73(+) cells have been isolated and found to have similar phenotypic and functional characteristics as adult MSCs. Here we demonstrate hESC-derived CD34(+)CD73(-) cells can serve as MSC progenitor cells with the ability to differentiate into adipocytes, osteoblasts and chondrocytes. Additionally, gene array analysis of hESC-derived MSCs show substantially different gene expression compared to bone marrow (BM)-derived MSCs, especially with increased expression of pluripotent and multipotent stem cell and endothelial cell-associated genes. The isolation of functional MSCs from hESC-derived CD34(+)CD73(-) cells provides improved understanding of MSC development and utilization of pluripotent stem cells to produce MSCs suited for novel regenerative therapies.