RESUMO
Cellular therapy development and manufacturing has focused on providing novel therapeutic cell-based products for various diseases. The International Organization for Standardization (ISO) has provided guidance on critical quality attributes (CQAs) that shall be considered when testing and releasing cellular therapeutic products. Cell count and viability measurements are two of the CQAs that are determined during development, manufacturing, testing, and product release. The ISO Cell Counting Standard Part 1 and 2 addressed the needs for improving the quality of cell counting results. However, there is currently no guidance on the qualification and selection of a fit-for-purpose cell viability detection method. In this work, we present strategies for the characterization and comparison of AO/PI and AO/DAPI staining methods using the heat-killed (HK) and low temperature/nutrient-deprived (LT/ND) cell death models to evaluate the comparability of cell viability measurements and identify potential causes of differences. We compared the AO/PI and AO/DAPI staining methods using HK and LT/ND-generated dead cells, investigated the staining time effects on cell viability measurements, and determined their viability linearity with different mixtures of live and dead cells. Furthermore, we validated AO/PI and AO/DAPI cell viability measurement with a long-term cell proliferation assay. Finally, we demonstrate a practical example of cell viability measurement comparison using AO/PI and AO/DAPI on antibiotic-selected transduced Jurkat and THP-1 cells to select a fit-for-purpose method for functional genomics screening. The proposed strategies may potentially enable scientists to properly characterize, compare, and select cell viability detection methods that are critical for cellular therapeutic product development and manufacturing.
RESUMO
The surface proteoglycan/glycoprotein layer (glycocalyx) on tumor cells has been associated with cellular functions that can potentially enable invasion and metastasis. In addition, aggressive tumor cells with high metastatic potential have enhanced invasion rates in response to interstitial flow stimuli in vitro. Our previous studies suggest that heparan sulfate (HS) in the glycocalyx plays an important role in this flow mediated mechanostransduction and upregulation of invasive and metastatic potential. In this study, highly metastatic renal cell carcinoma cells were genetically modified to suppress HS production by knocking down its synthetic enzyme NDST1. Using modified Boyden chamber and microfluidic assays, we show that flow-enhanced invasion is suppressed in HS deficient cells. To assess the ability of these cells to metastasize in vivo, parental or knockdown cells expressing fluorescence reporters were injected into kidney capsules in SCID mice. Histological analysis confirmed that there was a large reduction (95%) in metastasis to distant organs by tumors formed from the NDST1 knockdown cells compared to control cells with intact HS. The ability of these cells to invade surrounding tissue was also impaired. The substantial inhibition of metastasis and invasion upon reduction of HS suggests an active role for the tumor cell glycocalyx in tumor progression.
Assuntos
Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Proteoglicanas de Heparan Sulfato/metabolismo , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Animais , Carcinoma de Células Renais/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Modelos Animais de Doenças , Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Neoplasias Renais/genética , Masculino , Camundongos , Camundongos SCID , Metástase Neoplásica , Fenótipo , Interferência de RNA , RNA Interferente Pequeno/genética , Esferoides Celulares , Sulfotransferases/genética , Sulfotransferases/metabolismo , Carga Tumoral , Células Tumorais CultivadasRESUMO
In cellular therapies chimeric antigen receptor (CAR) T or NK cells undergo phenotypic analysis at multiple stages during discovery and development of novel therapies. Patient samples are routinely analyzed via flow cytometry for population identification and distribution of CD3, CD4, and CD8 positive T cells. As an alternative or orthogonal method, image cytometry systems have been used to perform simple cell-based assays in lieu of flow cytometry. Recently, a new image cytometry system, the Cellaca® PLX (Revvity Health Sciences, Inc., Lawrence, MA), was developed for high-throughput cell counting and viability, immunophenotyping, transfection/transduction efficiency, and cell health assays. This novel instrument allows investigators to quickly assess several critical quality attributes (CQAs) such as cell identity, viability, and other relevant biological functions recommended by the International Organization for Standardization using the ISO Cell Characterization documents focused on cellular therapeutic products. In this work, we demonstrate a rapid and high-throughput image cytometry detection method for cellular immunophenotyping and viability using the Cellaca PLX system for samples throughout the cellular therapy workflow. Freshly isolated peripheral blood mononuclear cells (PBMCs) underwent red blood cell (RBC) lysis and CD3 enrichment. Samples were then subsequently stained with Hoechst/CD3/CD4/CD8 or Hoechst/CD3/CD8/RubyDead Dye surface marker kits and measured on the Cellaca PLX and three different flow cytometers for side-by-side comparison and assay validation. Acquisition and analysis of cell viability and cell populations was shown to be faster and more efficient process compared to flow while achieving highly comparable results between the two technology platforms. This data shows that the Cellaca PLX Image Cytometer may provide a rapid alternative or orthogonal method for PBMC immunophenotyping experiments, as well as potentially streamline the workflow to quickly move precious patient samples downstream within the development processes.
Assuntos
Linfócitos T CD8-Positivos , Leucócitos Mononucleares , Humanos , Imunofenotipagem , Células Matadoras Naturais , BioensaioRESUMO
The migration of vascular smooth muscle cells (SMCs) and fibroblasts into the intima after vascular injury is a central process in vascular lesion formation. The elevation of transmural interstitial flow is also observed after damage to the vascular endothelium. We have previously shown that interstitial flow upregulates matrix metalloproteinase-1 (MMP-1) expression, which in turn promotes SMC and fibroblast migration in collagen I gels. In this study, we investigated further the mechanism of flow-induced MMP-1 expression. An ERK1/2 inhibitor PD-98059 completely abolished interstitial flow-induced SMC migration and MMP-1 expression. Interstitial flow promoted ERK1/2 phosphorylation, whereas PD-98059 abolished flow-induced activation. Silencing ERK1/2 completely abolished MMP-1 expression and SMC migration. In addition, interstitial flow increased the expression of activator protein-1 transcription factors (c-Jun and c-Fos), whereas PD-98059 attenuated flow-induced expression. Knocking down c-jun completely abolished flow-induced MMP-1 expression, whereas silencing c-fos did not affect MMP-1 expression. Taken together, our data indicate that interstitial flow induces MMP-1 expression and SMC migration in collagen I gels via an ERK1/2-dependent and c-Jun-mediated mechanism and suggest that interstitial flow, ERK1/2 MAPK, c-Jun, and MMP-1 may play important roles in SMC migration and neointima formation after vascular injury.
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Líquido Extracelular/fisiologia , Metaloproteinase 1 da Matriz/biossíntese , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Miócitos de Músculo Liso/fisiologia , Proteínas Proto-Oncogênicas c-jun/fisiologia , Animais , Western Blotting , Separação Celular , Colágeno Tipo I/química , Primers do DNA , Ativação Enzimática/fisiologia , Géis , Inativação Gênica , Genes fos/genética , Genes fos/fisiologia , Gliceraldeído-3-Fosfato Desidrogenases/fisiologia , Masculino , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , RNA/biossíntese , RNA/isolamento & purificação , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/fisiologiaRESUMO
Neointima formation often occurs in regions where the endothelium has been damaged and the transmural interstitial flow is elevated. Vascular smooth muscle cells (SMCs) and fibroblasts/myofibroblasts (FBs/MFBs) contribute to intimal thickening by migrating from the media and adventitia into the site of injury. In this study, for the first time, the direct effects of interstitial flow on SMC and FB/MFB migration were investigated in an in vitro three-dimensional system. Collagen I gels were used to mimic three-dimensional extracellular matrix (ECM) for rat aortic SMCs and FBs/MFBs. Exposure to interstitial flow induced by 1 cmH(2)O pressure differential (shear stress, approximately 0.05 dyn/cm(2); flow velocity, approximately 0.5 microm/s; and Darcy permeability, approximately 10(-11) cm(2)) substantially enhanced cell motility. Matrix metalloproteinase (MMP) inhibitor (GM-6001) abolished flow-induced migration augmentation, which suggested that the enhanced motility was MMP dependent. The upregulation of MMP-1 played a critical role for the flow-enhanced motility, which was further confirmed by silencing MMP-1 gene expression. Longer exposures to higher flows suppressed the number of migrated cells, although MMP-1 gene expression remained high. This suppression was a result of both flow-induced tissue inhibitor of metalloproteinase-1 upregulation and increased apoptotic and necrotic cell death. Interstitial flow did not affect MMP-2 gene expression or activity in the collagen I gel for any cell type. Our findings shed light on the mechanism by which vascular SMCs and FBs/MFBs contribute to intimal thickening in regions of vascular injury where interstitial flow is elevated.
Assuntos
Movimento Celular , Colágeno Tipo I/metabolismo , Fibroblastos/enzimologia , Metaloproteinase 1 da Matriz/metabolismo , Mecanotransdução Celular , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Fluxo Pulsátil , Animais , Apoptose , Pressão Sanguínea , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Dipeptídeos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Géis , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz , Mecanotransdução Celular/efeitos dos fármacos , Modelos Biológicos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Necrose , Permeabilidade , Inibidores de Proteases/farmacologia , Interferência de RNA , Ratos , Reologia , Estresse Mecânico , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Previous studies have demonstrated that the glycosaminoglycans (GAGs) heparan sulfate (HS) and hyaluronic acid (HA) are mechanosensors for interstitial flow on cancer cells. The proteins that link the GAGs to the cancer cell for mechanotransduction, however, are not known. OBJECTIVE: To assess whether the HS proteoglycan core proteins, Glypican-1 and Syndecan-1, or the HA receptor, CD44, provides the mechanical linkage to the cell. METHODS: The highly metastatic renal carcinoma cell line (SN12L1) and its companion low metastatic cell line (SN12C) were analyzed by Western blot, siRNA, and a 3-dimensional interstitial flow migration assay. RESULTS: There was significant elevation of Glypican-1 protein expression in the SN12L1 cells relative to the SN12C cells while there were no significant differences in Syndecan-1 or CD44. Knock down of Glypican-1 by siRNA completely blocked flow induced migration in SN12L1 cells. MAPK inhibitors also blocked flow induced migration in SN12L1 cells. CONCLUSIONS: Glypican-1 provides the mechanical linkage from HS (the flow sensor) to the SN12L1 cell where mechanotransduction leading to the enhancement of migration (metastasis) occurs. MAPKs downstream of Glypican-1 propagate the signal. The HS, Glypican-1, MAPK signaling axis suggests opportunities for pharmaceutical intervention.
Assuntos
Movimento Celular/fisiologia , Líquido Extracelular/fisiologia , Glicocálix/metabolismo , Glipicanas/metabolismo , Mecanotransdução Celular/fisiologia , Metástase Neoplásica/fisiopatologia , Carcinoma de Células Renais/fisiopatologia , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Neoplasias Renais/fisiopatologia , Sindecana-1/metabolismoRESUMO
Mammalian cells are covered by a surface proteoglycan (glycocalyx) layer, and it is known that blood vessel-lining endothelial cells use the glycocalyx to sense and transduce the shearing forces of blood flow into intracellular signals. Tumor cells in vivo are exposed to forces from interstitial fluid flow that may affect metastatic potential but are not reproduced by most in vitro cell motility assays. We hypothesized that glycocalyx-mediated mechanotransduction of interstitial flow shear stress is an un-recognized factor that can significantly enhance metastatic cell motility and play a role in augmentation of invasion. Involvement of MMP levels, cell adhesion molecules (CD44, α3 integrin), and glycocalyx components (heparan sulfate and hyaluronan) was investigated in a cell/collagen gel suspension model designed to mimic the interstitial flow microenvironment. Physiological levels of flow upregulated MMP levels and enhanced the motility of metastatic cells. Blocking the flow-enhanced expression of MMP activity or adhesion molecules (CD44 and integrins) resulted in blocking the flow-enhanced migratory activity. The presence of a glycocalyx-like layer was verified around tumor cells, and the degradation of this layer by hyaluronidase and heparinase blocked the flow-regulated invasion. This study shows for the first time that interstitial flow enhancement of metastatic cell motility can be mediated by the cell surface glycocalyx - a potential target for therapeutics.
Assuntos
Movimento Celular , Glicocálix/fisiologia , Mecanotransdução Celular/fisiologia , Adesão Celular , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Colágeno/metabolismo , Endotélio Vascular/patologia , Gelatina/metabolismo , Regulação Neoplásica da Expressão Gênica , Heparitina Sulfato/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Integrina alfa3/metabolismo , Metaloproteinases da Matriz/metabolismo , Metástase Neoplásica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Resistência ao Cisalhamento , Estresse MecânicoRESUMO
BACKGROUND: Glioma cells are exposed to elevated interstitial fluid flow during the onset of angiogenesis, at the tumor periphery while invading normal parenchyma, within white matter tracts, and during vascular normalization therapy. Glioma cell lines that have been exposed to fluid flow forces in vivo have much lower invasive potentials than in vitro cell motility assays without flow would indicate. METHODOLOGY/PRINCIPAL FINDINGS: A 3D Modified Boyden chamber (Darcy flow through collagen/cell suspension) model was designed to mimic the fluid dynamic microenvironment to study the effects of fluid shear stress on the migratory activity of glioma cells. Novel methods for gel compaction and isolation of chemotactic migration from flow stimulation were utilized for three glioma cell lines: U87, CNS-1, and U251. All physiologic levels of fluid shear stress suppressed the migratory activity of U87 and CNS-1 cell lines. U251 motility remained unaltered within the 3D interstitial flow model. Matrix Metalloproteinase (MMP) inhibition experiments and assays demonstrated that the glioma cells depended on MMP activity to invade, and suppression in motility correlated with downregulation of MMP-1 and MMP-2 levels. This was confirmed by RT-PCR and with the aid of MMP-1 and MMP-2 shRNA constructs. CONCLUSIONS/SIGNIFICANCE: Fluid shear stress in the tumor microenvironment may explain reduced glioma invasion through modulation of cell motility and MMP levels. The flow-induced migration trends were consistent with reported invasive potentials of implanted gliomas. The models developed for this study imply that flow-modulated motility involves mechanotransduction of fluid shear stress affecting MMP activation and expression. These models should be useful for the continued study of interstitial flow effects on processes that affect tumor progression.