RESUMO
Reticulocytes and erythrocytes are the ultimate differentiated stages of erythropoiesis. In addition to being anucleate cells, they are characterized by the clearance of their mitochondrial pool or lack thereof. Given that for most research-oriented flow cytometry experiments erythrocytes and reticulocytes are often undesirable cell types, their identification and exclusion from analyses can be essential. Here, we describe a flow cytometric method based on cellular NAD(P)H-related autofluorescence, whose localization is mainly associated with mitochondria. By increasing the sensitivity of the specific NAD(P)H-fluorescence detector, we discovered a population with weak levels of NAD(P)H fluorescence signals whose immunophenotypical and physiological characterization in mouse bone marrow led to its identification as both erythrocytes and reticulocytes. Our method showed comparable sensitivity and specificity to the detection of red blood cells based on the absorption of light by oxyhemoglobin. This NAD(P)H-based approach consistently identified over 95% of the total pool of erythrocytes and reticulocytes in bone marrow samples and revealed robust as over 93% of these two erythropoietic subsets were identified in melanoma tumor samples with the same method. The measurement of cellular endogenous NAD(P)H fluorescence, therefore, offers a reliable and straightforward alternative to identify erythrocytes and reticulocytes without additional immunostaining or the need to modify the cytometer's optical configuration. © 2018 International Society for Advancement of Cytometry.
Assuntos
Eritrócitos/citologia , Eritrócitos/metabolismo , NAD/metabolismo , Reticulócitos/efeitos dos fármacos , Reticulócitos/metabolismo , Animais , Medula Óssea/metabolismo , Medula Óssea/fisiologia , Eritropoese/fisiologia , Citometria de Fluxo/métodos , Fluorescência , CamundongosRESUMO
Cutaneous melanoma is one of the most aggressive cancers characterized by a high plasticity, a propensity for metastasis, and drug resistance. Melanomas are composed of phenotypically diverse subpopulations of tumor cells with heterogeneous molecular profiles that reflect intrinsic invasive abilities. In an attempt to identify novel factors of the melanoma invasive cell state, we previously investigated the nature of the invasive secretome by using a comparative proteomic approach. Here, we have extended this analysis to show that PTX3, an acute phase inflammatory glycoprotein, is one such factor secreted by invasive melanoma to promote tumor cell invasiveness. Elevated PTX3 production was observed in the population of MITFlow invasive cells but not in the population of MITFhigh differentiated melanoma cells. Consistently, MITF knockdown increased PTX3 expression in MITFhigh proliferative and poorly invasive cells. High levels of PTX3 were found in tissues and blood of metastatic melanoma patients, and in BRAF inhibitor-resistant melanoma cells displaying a mesenchymal invasive MITFlow phenotype. Genetic silencing of PTX3 in invasive melanoma cells dramatically impaired migration and invasion in vitro and in experimental lung extravasation assay in xenografted mice. In contrast, addition of melanoma-derived or recombinant PTX3, or expression of PTX3 enhanced motility of low migratory cells. Mechanistically, autocrine production of PTX3 by melanoma cells triggered an IKK/NFκB signaling pathway that promotes migration, invasion, and expression of the EMT factor TWIST1. Finally, we found that TLR4 and MYD88 knockdown inhibited PTX3-induced melanoma cell migration, suggesting that PTX3 functions through a TLR4-dependent pathway. Our work reveals that tumor-derived PTX3 contributes to melanoma cell invasion via targetable inflammation-related pathways. In addition to providing new insights into the biology of melanoma invasive behavior, this study underscores the notion that secreted PTX3 represents a potential biomarker and therapeutic target in a subpopulation of MITFlow invasive and/or refractory melanoma.
Assuntos
Proteína C-Reativa/fisiologia , Melanoma/metabolismo , NF-kappa B/metabolismo , Metástase Neoplásica/fisiopatologia , Componente Amiloide P Sérico/fisiologia , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Melanoma/patologia , Camundongos , Invasividade Neoplásica , Neoplasias Cutâneas/patologia , Regulação para Cima , Melanoma Maligno CutâneoRESUMO
Aberrant expression of Secreted Protein Acidic and Rich in Cysteine (SPARC)/osteonectin has been associated with an invasive tumor cell phenotype and poor outcome in human melanomas. Although it is known that SPARC controls melanoma tumorigenesis, the precise role of SPARC in melanoma cell survival is still unclear. Here, we show that SPARC has a cell-autonomous survival activity, which requires Akt-dependent regulation of p53. Suppression of SPARC by RNA interference in several human melanoma cells and xenografted A375 tumors triggers apoptotic cell death through the mitochondrial intrinsic pathway and activation of caspase-3. Cell death induced by depletion of SPARC is dependent on p53 and induction of Bax, and results in the generation of ROS. Stabilization of p53 in SPARC-depleted cells is associated with a decrease in Akt-mediated activating phosphorylation of MDM2. Inhibition of Akt signaling pathway is important for the observed changes as overexpression of constitutively active Akt protects cells against apoptosis induced by SPARC depletion. Conversely, increased expression of SPARC stimulates Akt and MDM2 phosphorylation, thus facilitating p53 degradation. Finally, we show that overexpression of SPARC renders cells more resistant to the p53-mediated cytotoxic effects of the DNA-damaging drug actinomycin-D. Our study indicates that SPARC functions through activation of Akt and MDM2 to limit p53 levels and that acquired expression of SPARC during melanoma development would confer survival advantages through suppression of p53-dependent apoptotic pathways.