RESUMEN
BACKGROUND: The isolation of circulating tumor cells (CTCs) requires rapid processing of the collected blood due to their inherent fragility. The ability to recover CTCs from peripheral blood mononuclear cells (PBMCs) preserved from cancer patients could allow for retrospective analyses or multicenter CTC studies. METHODS: We compared the efficacy of CTC recovery and characterization using cryopreserved PMBCs vs fresh whole blood from patients with non-small cell lung cancer (NSCLC; n = 8) and sarcoma (n = 6). Two epithelial cellular adhesion molecule (EpCAM)-independent strategies for CTC enrichment, based on Parsortix® technology or immunomagnetic depletion of blood cells (AutoMACS®) were tested, followed by DEPArray™ single-cell isolation. Phenotype and genotype, assessed by copy number alterations analysis, were evaluated at a single-cell level. Detection of target mutations in CTC-enriched samples from frozen NSCLC PBMCs was also evaluated by digital PCR (dPCR). RESULTS: The use of cryopreserved PBMCs from cancer patients allowed for the retrospective enumeration of CTCs and their molecular characterization, using both EpCAM-independent strategies that performed equally in capturing CTC. Cells isolated from frozen PBMCs were representative of whole blood-derived CTCs in terms of number, phenotype, and copy number aberration profile/target mutations. Long-term storage (≥3 years) did not affect the efficacy of CTC recovery. Detection of target mutations was also feasible by dPCR in CTC-enriched samples derived from stored PBMCs. CONCLUSIONS: Isolating CTCs from longitudinally collected PBMCs using an unbiased selection strategy can offer a wider range of retrospective genomic/phenotypic analyses to guide patients' personalized therapy, paving the way for sample sharing in multicenter studies.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Células Neoplásicas Circulantes , Sarcoma , Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Molécula de Adhesión Celular Epitelial/genética , Humanos , Leucocitos Mononucleares/metabolismo , Neoplasias Pulmonares/patología , Células Neoplásicas Circulantes/patología , Estudios RetrospectivosRESUMEN
BACKGROUND: Cell therapy with autologous peripheral blood mononuclear cells (PB-MNCs) may help restore limb perfusion in patients with diabetes mellitus and critical limb-threatening ischemia (CLTI) deemed not eligible for revascularization procedures and consequently at risk for major amputation (no-option). Fundamental is to establish its clinical value and to identify candidates with a greater benefit over time. Assessing the frequency of PB circulating angiogenic cells and extracellular vesicles (EVs) may help in guiding candidate selection. METHODS: We conducted a prospective, non-controlled, observational study on no-option CLTI diabetic patients that underwent intramuscular PB-MNCs therapy, which consisted of more cell treatments repeated a maximum of three times. The primary endpoint was amputation rate at 1 year following the first treatment with PB-MNCs. We evaluated ulcer healing, walking capability, and mortality during the follow-up period. We assessed angiogenic cells and EVs at baseline and after each cell treatment, according to primary outcome and tissue perfusion at the last treatment [measured as transcutaneous oxygen pressure (TcPO2)]. RESULTS: 50 patients were consecutively enrolled and the primary endpoint was 16%. TcPO2 increased after PB-MNCs therapy (17.2 ± 11.6 vs 39.1 ± 21.8 mmHg, p < .0001), and ulcers healed with back-to-walk were observed in 60% of the study population (88% of survivors) during follow-up (median 1.5 years). Patients with a high level of TcPO2 (≥ 40 mmHg) after the last treatment showed a high frequency of small EVs at enrollment. CONCLUSIONS: In no-option CLTI diabetic patients, PB-MNCs therapy led to an improvement in tissue perfusion, a high rate of healing, and back-to-walk. Coupling circulating cellular markers of angiogenesis could help in the identification of patients with a better clinical benefit over time.
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Diabetes Mellitus , Pie Diabético , Amputación Quirúrgica , Pie Diabético/cirugía , Pie Diabético/terapia , Humanos , Isquemia/diagnóstico , Isquemia/cirugía , Leucocitos Mononucleares , Recuperación del Miembro/métodos , Oxígeno , Estudios Prospectivos , Resultado del TratamientoRESUMEN
Platinum-based chemotherapy remains widely used in advanced non-small cell lung cancer (NSCLC) despite experimental evidence of its potential to induce long-term detrimental effects, including the promotion of pro-metastatic microenvironments. In this study, we investigated the interconnected pathways underlying the promotion of cisplatin-induced metastases. In tumor-free mice, cisplatin treatment resulted in an expansion in the bone marrow of CCR2+CXCR4+Ly6Chigh inflammatory monocytes (IMs) and an increase in lung levels of stromal SDF-1, the CXCR4 ligand. In experimental lung metastasis assays, cisplatin-induced IMs promoted the extravasation of tumor cells and the expansion of CD133+CXCR4+ metastasis-initiating cells (MICs). Peptide R, a novel CXCR4 inhibitor designed as an SDF-1 mimetic peptide, prevented cisplatin-induced IM expansion, the recruitment of IMs into the lungs, and the promotion of metastasis. At the primary tumor site, cisplatin treatment reduced tumor size while simultaneously inducing tumor release of SDF-1, MIC expansion, and recruitment of pro-invasive CXCR4+ macrophages. Co-recruitment of MICs and CCR2+CXCR4+ IMs to distant SDF-1-enriched sites also promoted spontaneous metastases that were prevented by CXCR4 blockade. In clinical specimens from NSCLC patients SDF-1 levels were found to be higher in platinum-treated samples and related to a worse clinical outcome. Our findings reveal that activation of the CXCR4/SDF-1 axis specifically mediates the pro-metastatic effects of cisplatin and suggest CXCR4 blockade as a possible novel combination strategy to control metastatic disease.
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Antígeno AC133/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Quimiocina CXCL12/metabolismo , Cisplatino/administración & dosificación , Neoplasias Pulmonares/tratamiento farmacológico , Monocitos/metabolismo , Péptidos/administración & dosificación , Receptores CXCR4/metabolismo , Células A549 , Animales , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Cisplatino/farmacología , Interacciones Farmacológicas , Humanos , Neoplasias Pulmonares/inmunología , Masculino , Ratones , Metástasis de la Neoplasia , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/inmunología , Péptidos/farmacología , Células RAW 264.7 , Receptores CXCR4/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Adolescents and young adults (AYA) with rhabdomyosarcoma (RMS) form a subgroup of patients whose optimal clinical management and best possible access to care remain a challenge and whose survival rates lag behind that of children diagnosed with histologically similar tumors. A better understanding of tumor biology that differentiates children (PEDS-) from AYA-RMS could provide critical information and drive new initiatives to improve their final outcome. We investigated the functional role of miRNAs implicated in AYA-RMS development, as they have the potential to lead to discovery of new targets pathways for a more tailored treatment in these age groups of young RMS patients. MiR-223 and miR-486 were observed de-regulated in nine RMS tissues compared to their normal counterparts, yet only miR-223 replacement impaired proliferation and aggressiveness of AYA-RMS cell lines, while inducing apoptosis and determining cell cycle arrest. Interestingly, IGF1R resulted in the direct target of miR-223 in AYA-RMS cells, as demonstrated by IGF1R silencing. Our results highlight an exclusive functional role of miR-223 in AYA-RMS development and aggressiveness.
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MicroARNs , Rabdomiosarcoma , Niño , Humanos , Adulto Joven , Adolescente , Línea Celular Tumoral , Rabdomiosarcoma/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Apoptosis/genética , Tasa de Supervivencia , Receptor IGF Tipo 1/genéticaRESUMEN
BACKGROUND: Rhabdomyosarcoma (RMS), the most frequent soft-tissue sarcoma in childhood, shows extensive heterogeneity in histology, site and age of onset, clinical course, and prognosis. Adolescents and young adults (AYA) with RMS form a subgroup of patients whose survival lacks behind that of children while diagnosed with histologically similar tumors. PROCEDURES: A 67-gene prognostic signature related to chromosome integrity, mitotic control, and genome complexity in sarcomas (CINSARC) is considered a powerful tool for identifying tumors with a highly metastatic potential. With this study, we investigated the prognostic value of CINSARC signature on a cohort of 48 pediatric (PEDs) and AYAs-RMS. RESULTS: CINSARC resulted not significantly correlated with age, suggesting other determinants to be responsible for that difference in survival. It remained a significant prognostic variable in both the groups of PEDs and AYAs. Also, genomic grade index signature was tested on the same cohort and showed very similar results with CINSARC. CONCLUSIONS: Our study showed that CINSARC correlated with outcome in RMS patients and may be potentially considered a tool to predict outcome, and so stratify RMS patients.
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Rabdomiosarcoma , Adolescente , Biomarcadores de Tumor/genética , Niño , Genómica , Humanos , Pronóstico , Rabdomiosarcoma/genética , Rabdomiosarcoma Embrionario , Neoplasias de los Tejidos Blandos/genética , Adulto JovenRESUMEN
miRNAs play a central role in the complex signaling network of cancer cells with the tumor microenvironment. Little is known on the origin of circulating miRNAs and their relationship with the tumor microenvironment in lung cancer. Here, we focused on the cellular source and relative contribution of different cell types to circulating miRNAs composing our risk classifier of lung cancer using in vitro/in vivo models and clinical samples. A cell-type specific expression pattern and topography of several miRNAs such as mir-145 in fibroblasts, mir-126 in endothelial cells, mir-133a in skeletal muscle cells was observed in normal and lung cancer tissues. Granulocytes and platelets are the major contributors of miRNAs release in blood. miRNAs modulation observed in plasma of lung cancer subjects was consistent with de-regulation of the same miRNAs observed during immunosuppressive conversion of immune cells. In particular, activated neutrophils showed a miRNA profile mirroring that observed in plasma of lung cancer subjects. Interestingly mir-320a secreted by neutrophils of high-risk heavy-smokers promoted an M2-like protumorigenic phenotype through downregulation of STAT4 when shuttled into macrophages. These findings suggest a multifactorial and nonepithelial cell-autonomous origin of circulating miRNAs associated with risk of lung cancer and that circulating miRNAs may act in paracrine signaling with causative role in lung carcinogenesis and immunosuppression.
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MicroARN Circulante/metabolismo , Neoplasias Pulmonares/inmunología , Macrófagos/inmunología , MicroARNs/metabolismo , Escape del Tumor/genética , Animales , Carcinogénesis/inmunología , Línea Celular Tumoral , MicroARN Circulante/sangre , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Pulmón/patología , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Macrófagos/metabolismo , Masculino , Ratones , Ratones SCID , MicroARNs/sangre , Neutrófilos/inmunología , Neutrófilos/metabolismo , Factor de Transcripción STAT4/genética , Factor de Transcripción STAT4/metabolismo , Fumar Tabaco/sangre , Fumar Tabaco/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma of childhood and adolescence, is a rare but aggressive malignancy that originates from immature mesenchymal cells committed to skeletal muscle differentiation. Although RMS is, generally, responsive to the modern multimodal therapeutic approaches, the prognosis of RMS depends on multiple variables and for some patients the outcome remains dismal. Further comprehension of the molecular and cellular biology of RMS would lead to identification of novel therapeutic targets. MicroRNAs (miRNAs) are small non-coding RNAs proved to function as key regulators of skeletal muscle cell fate determination and to play important roles in RMS pathogenesis. The purpose of this review is to better delineate the role of miRNAs as a biomarkers or functional leaders in RMS development, so to possibly elucidate some of RMS molecular mechanisms and potentially therapeutically target them to improve clinical management of pediatric RMS.
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MicroARNs/genética , Rabdomiosarcoma/genética , Rabdomiosarcoma/patología , Animales , Biomarcadores de Tumor/genética , Diferenciación Celular/genética , Humanos , Músculo Esquelético/patología , ARN Pequeño no Traducido/genéticaRESUMEN
Lung cancer is the most frequent cause of cancer-related death worldwide, with limited therapeutic options and rapid development of drug resistance. MicroRNAs, a class of small non-coding RNAs that control different physiological processes, have been associated with cancer development, as either oncomiRNAs or tumor-suppressor miRNAs. In the present study we investigated the interaction between mir-486-5p and mir-660-5p, two independent tumor-suppressor miRNAs, to assess their possible role and synergistic effect in lung cancer treatment. Our data show that mir-660-5p over-expression in A549 lung cancer cells induced a remarkable increase in mir-486-5p expression level and activity, detected as a reduction of its target gene, p85. mir-486-5p expression was confirmed by microRNA in situ hybridization. mir-660-5p modulated mir-486-5p through the silencing of Mouse Double Minute 2 (MDM2), one of its direct target, and then through p53 stimulation. This regulatory pathway was effective in A549, but not in H1299; therefore, only in the context of a functional p53 protein. Our findings support the conclusion that mir-486-5p is positively regulated by mir-660-5p in lung cancer cell lines, through the mir-660-MDM2-p53 pathway, making mir-660-5p even more interesting for its potential successful use in lung cancer therapy.
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Carcinogénesis/genética , Redes Reguladoras de Genes , Neoplasias Pulmonares/genética , MicroARNs/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Antineoplásicos/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , MicroARNs/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismoRESUMEN
Reparative response by bone marrow (BM)-derived progenitor cells (PCs) to ischemia is a multistep process that comprises the detachment from the BM endosteal niche through activation of osteoclasts and proteolytic enzymes (such as matrix metalloproteinases (MMPs)), mobilization to the circulation, and homing to the injured tissue. We previously showed that intramyocardial nerve growth factor gene transfer (NGF-GT) promotes cardiac repair following myocardial infarction (MI) in mice. Here, we investigate the impact of cardiac NGF-GT on postinfarction BM-derived PCs mobilization and homing at different time points after adenovirus-mediated NGF-GT in mice. Immunohistochemistry and flow cytometry newly illustrate the temporal profile of osteoclast and activation of MMP9, PCs expansion in the BM, and liberation/homing to the injured myocardium. NGF-GT amplified these responses and increased the BM levels of active osteoclasts and MMP9, which were not observed in MMP9-deficient mice. Taken together, our results suggest a novel role for NGF in BM-derived PCs mobilization/homing following MI.
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Movilización de Célula Madre Hematopoyética/métodos , Células Madre Hematopoyéticas/citología , Infarto del Miocardio/genética , Miocardio/patología , Factor de Crecimiento Nervioso/metabolismo , Adenoviridae/genética , Animales , Trasplante de Médula Ósea , Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/patología , Factor de Crecimiento Nervioso/genética , Osteoclastos/citologíaRESUMEN
BACKGROUND: Research efforts for the management of cancer, in particular for lung cancer, are directed to identify new strategies for its early detection. MicroRNAs (miRNAs) are a new promising class of circulating biomarkers for cancer detection, but lack of consensus on data normalization methods has affected the diagnostic potential of circulating miRNAs. There is a growing interest in techniques that allow an absolute quantification of miRNAs which could be useful for early diagnosis. Recently, digital PCR, mainly based on droplets generation, emerged as an affordable technology for precise and absolute quantification of nucleic acids. RESULTS: In this work, we described a new interesting approach for profiling circulating miRNAs in plasma samples using a chip-based platform, the QuantStudio 3D digital PCR. The proposed method was validated using synthethic oligonucleotide at serial dilutions in plasma samples of lung cancer patients and in lung tissues and cell lines. CONCLUSION: Given its reproducibility and reliability, our approach could be potentially applied for the identification and quantification of miRNAs in other biological samples such as circulating exosomes or protein complexes. As chip-digital PCR becomes more established, it would be a robust tool for quantitative assessment of miRNA copy number for diagnosis of lung cancer and other diseases.
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Biomarcadores de Tumor/sangre , Neoplasias Pulmonares/sangre , MicroARNs/sangre , Células Neoplásicas Circulantes/metabolismo , Anciano , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Detección Precoz del Cáncer , Exosomas/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , MicroARNs/genética , Persona de Mediana Edad , Células Neoplásicas Circulantes/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Fumar/efectos adversosRESUMEN
RATIONALE: Circulating proangiogenic cells (PACs) support postischemic neovascularization. Cardiovascular disease and diabetes mellitus impair PAC regenerative capacities via molecular mechanisms that are not fully known. We hypothesize a role for microRNAs (miRs). Circulating miRs are currently investigated as potential diagnostic and prognostic biomarkers. OBJECTIVE: The objectives were the following: (1) to profile miR expression in PACs from critical limb ischemia (CLI) patients; (2) to demonstrate that miR-15a and miR-16 regulate PAC functions; and (3) to characterize circulating miR-15a and miR-16 and to investigate their potential biomarker value. METHODS AND RESULTS: Twenty-eight miRs potentially able to modulate angiogenesis were measured in PACs from CLI patients with and without diabetes mellitus and controls. miR-15a and miR-16 were further analyzed. CLI-PACs expressed higher level of mature miR-15a and miR-16 and of the primary transcript pri-miR-15a/16-1. miR-15a/16 overexpression impaired healthy PAC survival and migration. Conversely, miR-15a/16 inhibition improved CLI-PAC-defective migration. Vascular endothelial growth factor-A and AKT-3 were validated as direct targets of the 2 miRs, and their protein levels were reduced in miR-15a/16-overexpressing healthy PACs and in CLI-PACs. Transplantation of healthy PACs ex vivo-engineered with anti-miR-15a/16 improved postischemic blood flow recovery and muscular arteriole density in immunodeficient mice. miR-15a and miR-16 were present in human blood, including conjugated to argonaute-2 and in exosomes. Both miRs were increased in the serum of CLI patients and positively correlated with amputation after restenosis at 12 months postrevascularization of CLI type 2 diabetes mellitus patients. Serum miR-15a additionally correlated with restenosis at follow-up. CONCLUSIONS: Ex vivo miR-15a/16 inhibition enhances PAC therapeutic potential, and circulating miR-15a and miR-16 deserves further investigation as a prognostic biomarker in CLI patients undergoing revascularization.
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Complicaciones de la Diabetes/sangre , Miembro Posterior/irrigación sanguínea , Isquemia/sangre , MicroARNs/efectos adversos , Neovascularización Patológica/sangre , Animales , Movimiento Celular/genética , Supervivencia Celular/genética , Trasplante de Células/métodos , Células Cultivadas , Complicaciones de la Diabetes/genética , Complicaciones de la Diabetes/patología , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Células HEK293 , Miembro Posterior/patología , Humanos , Isquemia/genética , Ratones , Ratones Desnudos , MicroARNs/biosíntesis , Neovascularización Patológica/genéticaRESUMEN
RATIONALE: The impact of diabetes mellitus on bone marrow (BM) structure is incompletely understood. OBJECTIVE: Investigate the effect of type-2 diabetes mellitus (T2DM) on BM microvascular and hematopoietic cell composition in patients without vascular complications. METHODS AND RESULTS: Bone samples were obtained from T2DM patients and nondiabetic controls (C) during hip replacement surgery and from T2DM patients undergoing amputation for critical limb ischemia. BM composition was assessed by histomorphometry, immunostaining, and flow cytometry. Expressional studies were performed on CD34(pos) immunosorted BM progenitor cells (PCs). Diabetes mellitus causes a reduction of hematopoietic tissue, fat deposition, and microvascular rarefaction, especially when associated with critical limb ischemia. Immunohistochemistry documented increased apoptosis and reduced abundance of CD34(pos)-PCs in diabetic groups. Likewise, flow cytometry showed scarcity of BM PCs in T2DM and T2DM+critical limb ischemia compared with C, but similar levels of mature hematopoietic cells. Activation of apoptosis in CD34(pos)-PCs was associated with upregulation and nuclear localization of the proapoptotic factor FOXO3a and induction of FOXO3a targets, p21 and p27(kip1). Moreover, microRNA-155, which regulates cell survival through inhibition of FOXO3a, was downregulated in diabetic CD34(pos)-PCs and inversely correlated with FOXO3a levels. The effect of diabetes mellitus on anatomic and molecular end points was confirmed when considering background covariates. Furthermore, exposure of healthy CD34(pos)-PCs to high glucose reproduced the transcriptional changes induced by diabetes mellitus, with this effect being reversed by forced expression of microRNA-155. CONCLUSIONS: We provide new anatomic and molecular evidence for the damaging effect of diabetes mellitus on human BM, comprising microvascular rarefaction and shortage of PCs attributable to activation of proapoptotic pathway.
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Células de la Médula Ósea/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Angiopatías Diabéticas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Células Madre Hematopoyéticas/metabolismo , MicroARNs/metabolismo , Microvasos/metabolismo , Transducción de Señal , Nicho de Células Madre , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD34/metabolismo , Apoptosis , Biomarcadores/metabolismo , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/patología , Examen de la Médula Ósea , Estudios de Casos y Controles , Linaje de la Célula , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Angiopatías Diabéticas/genética , Angiopatías Diabéticas/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Citometría de Flujo , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/patología , Humanos , Inmunohistoquímica , Isquemia/genética , Isquemia/metabolismo , Isquemia/patología , Masculino , MicroARNs/genética , Microvasos/inmunología , Microvasos/patología , Persona de Mediana Edad , Enfermedad Arterial Periférica/genética , Enfermedad Arterial Periférica/metabolismo , Enfermedad Arterial Periférica/patología , TransfecciónRESUMEN
Lung cancer is the most common cause of cancer deaths worldwide and numerous ongoing research efforts are directed to identify new strategies for its early detection. The development of non-invasive blood-based biomarkers for cancer detection in its preclinical phases is crucial to improve the outcome of this deadly disease. MicroRNAs (miRNAs) are a new promising class of circulating biomarkers for cancer detection and prognosis definition, but lack of consensus on data normalization methods for circulating miRNAs and the critical issue of haemolysis, has affected the identification of circulating miRNAs with diagnostic potential. We describe here an interesting approach for profiling circulating miRNAs in plasma samples based on the evaluation of reciprocal miRNA levels measured by quantitative Real-Time PCR. By monitoring changes of plasma miRNA-ratios, it is possible to assess the deregulation of tumor-related miRNAs and identify signatures with diagnostic and prognostic value. In addition, to avoid bias due to the release of miRNAs from blood cells, a miRNA-ratios signature distinguishing haemolyzed samples was identified. The method described was validated in plasma samples of lung cancer patients, but given its reproducibility and reliability, could be potentially applied for the identification of diagnostic circulating miRNAs in other diseases.
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Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/genética , MicroARNs/sangre , MicroARNs/genética , Biomarcadores de Tumor , Análisis por Conglomerados , Perfilación de la Expresión Génica , Hemólisis , Humanos , Control de Calidad , Reproducibilidad de los ResultadosRESUMEN
Over the past three decades, cell therapy development has fallen short of expectations, with many cellular sources demonstrating a 'Janus effect' and raising safety concerns. Extracellular vesicles (EVs), supported by advanced technologies, present a promising avenue in regenerative medicine, offering benefits such as immune tolerance and avoidance of negative aspects associated with cell transplants. Our previous research showcased enhanced and organized subcutaneous vascularization using three-dimensional bioprinted patches containing HUVEC-derived EVs in immunodeficient animal models. In this context, stress conditions on the cells of origin further boosted the EVs' neoangiogenic potential. Since neovascularization is the first regenerative target requiring restoration, the present study aims to complement our previous work by employing an injectable gelatin methacrylate (GelMA) hydrogel functionalized with HUVEC-derived EVs in a pathological condition of acute myocardial infarction. This bioactive hydrogel resulted in reduced fibrosis, improved contractility, and promoted angiogenesis, showing promise in countering tissue deterioration and addressing vascular deficits. Moreover, the molecular characterization of EVs through miRNome and proteomic analyses further supports their potential as bio-additives for hydrogel functionalization. This cell-free approach mitigates immune rejection and oncogenic risks, offering innovative therapeutic advantages.
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Vesículas Extracelulares , Células Endoteliales de la Vena Umbilical Humana , Hidrogeles , Infarto del Miocardio , Neovascularización Fisiológica , Humanos , Animales , Infarto del Miocardio/terapia , Infarto del Miocardio/patología , Hidrogeles/química , Neovascularización Fisiológica/efectos de los fármacos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/química , Vesículas Extracelulares/trasplante , Metacrilatos/química , Gelatina/química , Inyecciones , MasculinoRESUMEN
BACKGROUND: Pain triggers a homeostatic alarm reaction to injury. It remains unknown, however, whether nociceptive signaling activated by ischemia is relevant for progenitor cells (PC) release from bone marrow. To this end, we investigated the role of the neuropeptide substance P (SP) and cognate neurokinin 1 (NK1) nociceptor in PC activation and angiogenesis during ischemia in mice and in human subjects. METHODS AND RESULTS: The mouse bone marrow contains sensory fibers and PC that express SP. Moreover, SP-induced migration provides enrichment for PC that express NK1 and promote reparative angiogenesis after transplantation in a mouse model of limb ischemia. Acute myocardial infarction and limb ischemia increase SP levels in peripheral blood, decrease SP levels in bone marrow, and stimulate the mobilization of NK1-expressing PC, with these effects being abrogated by systemic administration of the opioid receptor agonist morphine. Moreover, bone marrow reconstitution with NK1-knockout cells results in depressed PC mobilization, delayed blood flow recovery, and reduced neovascularization after ischemia. We next asked whether SP is instrumental to PC mobilization and homing in patients with ischemia. Human PC express NK1, and SP-induced migration provides enrichment for proangiogenic PC. Patients with acute myocardial infarction show high circulating levels of SP and NK1-positive cells that coexpress PC antigens, such as CD34, KDR, and CXCR4. Moreover, NK1-expressing PC are abundant in infarcted hearts but not in hearts that developed an infarct after transplantation. CONCLUSIONS: Our data highlight the role of SP in reparative neovascularization. Nociceptive signaling may represent a novel target of regenerative medicine.
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Isquemia/fisiopatología , Neovascularización Fisiológica , Nocicepción/fisiología , Transducción de Señal/fisiología , Células Madre/fisiología , Sustancia P/fisiología , Animales , Movilización de Célula Madre Hematopoyética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Péptido Relacionado con el Gen de Calcitonina/fisiología , Receptores de Neuroquinina-1/fisiologíaRESUMEN
RATIONALE: Homing of proangiogenic cells (PACs) is guided by chemoattractants and requires proteases to disrupt the extracellular matrix. The possibility that PAC recruitment involves an interaction between proteases and chemotactic factor receptors remains largely unexplored. OBJECTIVE: To determine the role of human tissue kallikrein (hK1) in PAC invasion and its dependency on kinin receptor signaling. METHODS AND RESULTS: Human mononuclear cells (MNCs) and culture-selected PACs express and release mature hK1 protein. HK1 gene (KLK1) silencing reduced PACs migratory, invasive, and proangiogenic activities. KLK1-knockout mouse bone marrow-derived MNCs showed similar impairments and were unable to support reparative angiogenesis in a mouse model of peripheral ischemia. Conversely, adenovirus-mediated KLK1 (Ad.KLK1) gene transfer enhanced PAC-associated functions, whereas the catalytically inactive variant R53H-KLK1 was ineffective. HK1-induced effects are mediated by a kinin B(2) receptor (B(2)R)-dependent mechanism involving inducible nitric oxide synthase and metalloproteinase-2 (MMP2). Lower hK1 protein levels were observed in PACs from type 2 diabetic (T2D) patients, whereas KLK1 mRNA levels were similar to those of healthy subjects, suggesting a post-transcriptional defect. Furthermore, B(2)R is normally expressed on T2D-PACs but remains uncoupled from downstream signaling. Importantly, whereas Ad.KLK1 alone could not restore T2D-PAC invasion capacity, combined KLK1 and B(2)R expression rescued the diabetic phenotype. CONCLUSIONS: This study reveals new interactive components of the PACs invasive machinery, acting via protease- and kinin receptor-dependent mechanisms.
Asunto(s)
Movimiento Celular/fisiología , Calicreínas/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Neovascularización Fisiológica/fisiología , Adulto , Anciano , Animales , Estudios de Casos y Controles , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Regulación hacia Abajo , Femenino , Miembro Posterior/irrigación sanguínea , Humanos , Isquemia/metabolismo , Calicreínas/genética , Cininas/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Modelos Animales , Óxido Nítrico Sintasa/metabolismo , ARN Mensajero/metabolismo , Receptor de Bradiquinina B2/metabolismo , Transducción de Señal/fisiologíaRESUMEN
RATIONALE: Pericytes are key regulators of vascular maturation, but their value for cardiac repair remains unknown. OBJECTIVE: We investigated the therapeutic activity and mechanistic targets of saphenous vein-derived pericyte progenitor cells (SVPs) in a mouse myocardial infarction (MI) model. METHODS AND RESULTS: SVPs have a low immunogenic profile and are resistant to hypoxia/starvation (H/S). Transplantation of SVPs into the peri-infarct zone of immunodeficient CD1/Foxn-1(nu/nu) or immunocompetent CD1 mice attenuated left ventricular dilatation and improved ejection fraction compared to vehicle. Moreover, SVPs reduced myocardial scar, cardiomyocyte apoptosis and interstitial fibrosis, improved myocardial blood flow and neovascularization, and attenuated vascular permeability. SVPs secrete vascular endothelial growth factor A, angiopoietin-1, and chemokines and induce an endogenous angiocrine response by the host, through recruitment of vascular endothelial growth factor B expressing monocytes. The association of donor- and recipient-derived stimuli activates the proangiogenic and prosurvival Akt/eNOS/Bcl-2 signaling pathway. Moreover, microRNA-132 (miR-132) was constitutively expressed and secreted by SVPs and remarkably upregulated, together with its transcriptional activator cyclic AMP response element-binding protein, on stimulation by H/S or vascular endothelial growth factor B. We next investigated if SVP-secreted miR-132 acts as a paracrine activator of cardiac healing. In vitro studies showed that SVP conditioned medium stimulates endothelial tube formation and reduces myofibroblast differentiation, through inhibition of Ras-GTPase activating protein and methyl-CpG-binding protein 2, which are validated miR-132 targets. Furthermore, miR-132 inhibition by antimiR-132 decreased SVP capacity to improve contractility, reparative angiogenesis, and interstitial fibrosis in infarcted hearts. CONCLUSION: SVP transplantation produces long-term improvement of cardiac function through a novel paracrine mechanism involving the secretion of miR-132 and inhibition of its target genes.
Asunto(s)
Trasplante de Células Madre Mesenquimatosas/métodos , MicroARNs/biosíntesis , Infarto del Miocardio/cirugía , Neovascularización Fisiológica/fisiología , Pericitos/trasplante , Células Madre , Animales , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Desnudos , MicroARNs/metabolismo , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Pericitos/metabolismo , Ratas , Células Madre/metabolismoRESUMEN
OBJECTIVE: The p75 neurotrophin receptor (p75(NTR)) contributes to diabetes mellitus-induced defective postischemic neovascularization. The interleukin-33 receptor ST2 is expressed as transmembrane (ST2L) and soluble (sST2) isoforms. Here, we studied the following: (1) the impact of p75(NTR) in the healing of ischemic and diabetic calf wounds; (2) the link between p75(NTR) and ST2; and (3) circulating sST2 levels in critical limb ischemia (CLI) patients. METHODS AND RESULTS: Diabetes mellitus was induced in p75(NTR) knockout (p75KO) mice and wild-type (WT) littermates by streptozotocin. Diabetic and nondiabetic p75KO and WT mice received left limb ischemia induction and a full-thickness wound on the ipsilateral calf. Diabetes mellitus impaired wound closure and angiogenesis and increased ST2 expression in WT, but not in p75KO wounds. In cultured endothelial cells, p75(NTR) promoted ST2 (both isoforms) expression through p38(MAPK)/activating transcription factor 2 pathway activation. Next, sST2 was measured in the serum of patients with CLI undergoing either revascularization or limb amputation and in the 2 nondiabetic groups (with CLI or nonischemic individuals). Serum sST2 increased in diabetic patients with CLI and was directly associated with higher mortality at 1 year from revascularization. CONCLUSIONS: p75(NTR) inhibits the healing of ischemic lower limb wounds in diabetes mellitus and promotes ST2 expression. Circulating sST2 predicts mortality in diabetic CLI patients.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus/mortalidad , Isquemia/fisiopatología , Extremidad Inferior/irrigación sanguínea , Proteínas del Tejido Nervioso/fisiología , Receptores de Superficie Celular/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Factor de Crecimiento Nervioso/fisiología , Factor de Transcripción Activador 2/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores/metabolismo , Células Cultivadas , Complicaciones de la Diabetes/complicaciones , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatología , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/metabolismo , Modelos Animales de Enfermedad , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Femenino , Humanos , Proteína 1 Similar al Receptor de Interleucina-1 , Isquemia/etiología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Proteínas del Tejido Nervioso/farmacología , Valor Predictivo de las Pruebas , Receptores de Factor de Crecimiento Nervioso/deficiencia , Receptores de Factor de Crecimiento Nervioso/genética , Estreptozocina/efectos adversos , Cicatrización de Heridas/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Lung cancer is the deadliest cancer in the world, with the majority of patients presenting with advanced or metastatic disease at first diagnosis. The lungs are also one of the most common sites of metastasis from lung cancer and other tumors. Understanding the mechanisms that regulate metastasis formation from primary lung cancer and in the lungs is therefore fundamental unmet clinical need. One of the first steps during the establishment of lung cancer metastases includes the formation of the pre-metastatic niche (PMN) at distant organs, which may occur even during the early phases of cancer development. The PMN is established through intricate cross-talk between primary tumor-secreted factors and stromal components at distant sites. Mechanisms controlling primary tumor escape and seeding of distant organs rely on specific properties of tumor cells but are also tightly regulated by interactions with stromal cells at the metastatic niche that finally dictate the success of metastasis establishment. Here, we summarize the mechanisms underlying pre-metastatic niche formation starting from how lung primary tumor cells modulate distant sites through the release of several factors, focusing on Extracellular Vesicles (EVs). In this context, we highlight the role of lung cancer-derived EVs in the modulation of tumor immune escape. Then, we illustrate the complexity of Circulating Tumor Cells (CTCs) that represent the seeds of metastasis and how interactions with stromal and immune cells can help their metastatic dissemination. Finally, we evaluate the contribution of EVs in dictating metastasis development at the PMN through stimulation of proliferation and control of disseminated tumor cell dormancy. Overall, we present an overview of different steps in the lung cancer metastatic cascade, focusing on the EV-mediated interactions between tumor cells and stromal/immune cells.
RESUMEN
Chronic obstructive pulmonary disease (COPD) is a risk factor for lung cancer development. COPD induces activation of hypoxia-induced signaling, causing remodeling of surrounding microenvironmental cells also modulating the release and cargo of their extracellular vesicles (EVs). We aimed to evaluate the potential role of circulating EVs from COPD subjects in lung cancer onset. Plasma-EVs were isolated by ultracentrifugation from heavy smoker volunteers with (COPD-EVs) or without (heavy smoker-EVs, HS-EV) COPD and characterized following MISEV guidelines. Immortalized human bronchial epithelial cells (CDK4, hTERT-HBEC3-KT), genetically modified with different oncogenic alterations commonly found in lung cancer (sh-p53, KRASV12), were used to test plasma-EVs pro-tumorigenic activity in vitro. COPD-EVs mainly derived from immune and endothelial cells. COPD-EVs selectively increased the subset of CD133+CXCR4+ metastasis initiating cells (MICs) in HBEC-sh-p53-KRASV12high cells and stimulated 3D growth, migration/invasion, and acquisition of mesenchymal traits. These effects were not observed in HBEC cells bearing single oncogenic mutation (sh-p53 or KRASV12). Mechanistically, hypoxia-inducible factor 1-alpha (HIF-1α) transferred from COPD-EVs triggers CXCR4 pathway activation that in turn mediates MICs expansion and acquisition of pro-tumorigenic effects. Indeed, HIF-1α inhibition or CXCR4 silencing prevented the acquisition of malignant traits induced by COPD-EVs alone. Hypoxia recapitulates the effects observed with COPD-EVs in HBEC-sh-p53-KRASV12high cells. Notably, higher levels of HIF-1α were observed in EVs from COPD subjects who subsequently developed cancer compared to those who remained cancer-free. Our findings support a role of COPD-EVs to promote the expansion of MICs in premalignant epithelial cells through HIF-1α-CXCR4 axis activation thereby potentially sustaining lung cancer progression.