RESUMO
Cell lines are applied on a large scale in the field of biomedicine, but they are susceptible to issues such as misidentification and cross-contamination. This situation is becoming worse over time due to the rapid growth of the biomedical field, and thus there is an urgent need for a more effective strategy to address the problem. As described herein, a cell coding method is established based on two types of uniform and stable glycan nanoparticles that are synthesized using the graft-copolymerization-induced self-assembly (GISA) method, which further exhibit distinct fluorescent properties due to elaborate modification with fluorescent labeling molecules. The different affinity between each nanoparticle and various cell lines results in clearly distinguishable differences in their endocytosis degrees, thus resulting in distinct characteristic fluorescence intensities. Through flow cytometry measurements, the specific signals of each cell sample can be recorded and turned into a map divided into different regions by statistical processing. Using this sensing array strategy, we have successfully identified six human cell lines, including one normal type and five tumor types. Moreover, cell contamination evaluation of different cell lines with HeLa cells as the contaminant in a semiquantitative analysis has also been successfully achieved. Notably, the whole process of nanoparticle fabrication and fluorescent testing is facile and the results are highly reliable.
Assuntos
Autenticação de Linhagem Celular/métodos , Quitosana/análogos & derivados , Dextranos/química , Corantes Fluorescentes/química , Nanopartículas/química , Carbocianinas/química , Carbocianinas/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quitosana/toxicidade , Dextranos/toxicidade , Endocitose/efeitos dos fármacos , Citometria de Fluxo , Fluoresceínas/química , Fluoresceínas/toxicidade , Corantes Fluorescentes/toxicidade , Células Endoteliais da Veia Umbilical Humana , Humanos , Nanopartículas/toxicidadeRESUMO
Renal cell carcinoma (RCC) is not a single disease but is made up of several different histologically defined subtypes that are associated with distinct genetic alterations which require subtype specific management and treatment. Papillary renal cell carcinoma (pRCC) is the second most common subtype after conventional/clear cell RCC (ccRCC), representing ~20% of cases, and is subcategorized into type 1 and type 2 pRCC. It is important for preclinical studies to have cell lines that accurately represent each specific RCC subtype. This study characterizes seven cell lines derived from both primary and metastatic sites of type 1 pRCC, including the first cell line derived from a hereditary papillary renal carcinoma (HPRC)-associated tumor. Complete or partial gain of chromosome 7 was observed in all cell lines and other common gains of chromosomes 16, 17, or 20 were seen in several cell lines. Activating mutations of MET were present in three cell lines that all demonstrated increased MET phosphorylation in response to HGF and abrogation of MET phosphorylation in response to MET inhibitors. CDKN2A loss due to mutation or gene deletion, associated with poor outcomes in type 1 pRCC patients, was observed in all cell line models. Six cell lines formed tumor xenografts in athymic nude mice and thus provide in vivo models of type 1 pRCC. These type 1 pRCC cell lines provide a comprehensive representation of the genetic alterations associated with pRCC that will give insight into the biology of this disease and be ideal preclinical models for therapeutic studies.
Assuntos
Carcinoma de Células Renais/genética , Autenticação de Linhagem Celular/métodos , Neoplasias Renais/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Instabilidade Cromossômica , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Neoplasias Renais/patologia , Camundongos , Mutação , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismoRESUMO
Study of vocal fold (VF) mucosal biology requires essential human vocal fold epithelial cell (hVFE) lines for use in appropriate model systems. We steadily transfected a retroviral construct containing human telomerase reverse transcriptase (hTERT) into primary normal hVFE to establish a continuously replicating hVFE cell line. Immortalized hVFE across passages have cobblestone morphology, express epithelial markers cytokeratin 4, 13 and 14, induced hTERT gene and protein expression, have similar RNAseq profiling, and can continuously grow for more than 8 months. DNA fingerprinting and karyotype analysis demonstrated that immortalized hVFE were consistent with the presence of a single cell line. Validation of the hVFE, in a three-dimensional in vitro VF mucosal construct revealed a multilayered epithelial structure with VF epithelial cell markers. Wound scratch assay revealed higher migration capability of the immortalized hVFE on the surface of collagen-fibronectin and collagen gel containing human vocal fold fibroblasts (hVFF). Collectively, our report demonstrates the first immortalized hVFE from true VFs providing a novel and invaluable tool for the study of epithelial cell-fibroblast interactions that dictate disease and health of this specialized tissue.
Assuntos
Células Epiteliais/citologia , Mucosa Laríngea/citologia , Cultura Primária de Células/métodos , Prega Vocal/citologia , Idoso , Linhagem Celular , Autenticação de Linhagem Celular/métodos , Proliferação de Células , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Feminino , Humanos , Queratinas/genética , Queratinas/metabolismo , Masculino , Telomerase/genética , Telomerase/metabolismoRESUMO
Colorectal and glioblastoma cancer stem-like cells (CSCs) are essential for translational research. Cell line authentication by short tandem repeat (STR) profiling ensures reproducibility of results in oncology research. This technique enables to identify mislabeling or cross-contamination of cell lines. In our study, we provide a reference dataset for a panel of colorectal and glioblastoma CSCs that allows authentication. Each cell line was entered into the cell Line Integrated Molecular Authentication database 2.1 to be compared to the STR profiles of 4485 tumor cell lines. This article also provides clinical data of patients from whom CSCs arose and data on the parent tumor stage and mutations. STR profiles and information of our CSCs are also available in the Cellosaurus database (ExPASy) as identified by unique research resource identifier codes.
Assuntos
Autenticação de Linhagem Celular/métodos , Autenticação de Linhagem Celular/normas , Linhagem Celular Tumoral , Repetições de Microssatélites , Células-Tronco Neoplásicas , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/genética , Conjuntos de Dados como Assunto , Feminino , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , Glioblastoma/genética , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Sclerosing spindle cell rhabdomyosarcoma (SSRMS) is a rare rhabdomyosarcomas (RMS) subtype. Especially cases bearing a myogenic differentiation 1 (MYOD1) mutation are characterized by a high recurrence and metastasis rate, often leading to a fatal outcome. SSRMS cell lines are valuable in vitro models for studying disease mechanisms and for the preclinical evaluation of new therapeutic approaches. In this study, a cell line established from a primary SSRMS tumor of a 24-year-old female after multimodal chemotherapeutic pretreatment has been characterized in detail, including immunohistochemistry, growth characteristics, cytogenetic analysis, mutation analysis, evaluation of stem cell marker expression, differentiation potential, and tumorigenicity in mice. The cell line which was designated SRH exhibited a complex genomic profile, including several translocations and deletions. Array-comparative genomic hybridization (CGH) revealed an overall predominating loss of gene loci. The mesenchymal tumor origin was underlined by the expression of mesenchymal markers and potential to undergo adipogenic and osteogenic differentiation. Despite myogenic marker expression, terminal myogenic differentiation was inhibited, which might be elicited by the MYOD1 hotspot mutation. In vivo tumorigenicity could be confirmed after subcutaneous injection into NOD/SCID/γcnull mice. Summarized, the SRH cell line is the first adult SSRMS cell line available for preclinical research on this rare RMS subtype.
Assuntos
Genômica , Rabdomiossarcoma/patologia , Adipogenia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Autenticação de Linhagem Celular/métodos , Hibridização Genômica Comparativa , Feminino , Humanos , Cariotipagem , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína MyoD/genética , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/genética , Transdução de Sinais , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Adulto JovemRESUMO
The prevalence of nuclear cataracts was observed to be significantly higher among residents of tropical and subtropical regions compared to those of temperate and subarctic regions. We hypothesized that elevated environmental temperatures may pose a risk of nuclear cataract development. The results of our in silico simulation revealed that in temperate and tropical regions, the human lens temperature ranges from 35.0 °C to 37.5 °C depending on the environmental temperature. The medium temperature changes during the replacement regularly in the cell culture experiment were carefully monitored using a sensor connected to a thermometer and showed a decrease of 1.9 °C, 3.0 °C, 1.7 °C, and 0.1 °C, after 5 min when setting the temperature of the heat plate device at 35.0 °C, 37.5 °C, 40.0 °C, and 42.5 °C, respectively. In the newly created immortalized human lens epithelial cell line clone NY2 (iHLEC-NY2), the amounts of RNA synthesis of αA crystallin, protein expression, and amyloid ß (Aß)1-40 secreted into the medium were increased at the culture temperature of 37.5 °C compared to 35.0 °C. In short-term culture experiments, the secretion of Aß1-40 observed in cataracts was increased at 37.5 °C compared to 35.0 °C, suggesting that the long-term exposure to a high-temperature environment may increase the risk of cataracts.
Assuntos
Cristalinas/metabolismo , Células Epiteliais/metabolismo , Peptídeos beta-Amiloides/metabolismo , Autenticação de Linhagem Celular/métodos , Proliferação de Células , Células Cultivadas , Simulação por Computador , Cristalinas/genética , Meios de Cultura/química , Células Epiteliais/citologia , Células Epiteliais/patologia , Humanos , Cristalino/citologia , Cristalino/metabolismo , Temperatura , Cadeia A de alfa-Cristalina/genética , Cadeia A de alfa-Cristalina/metabolismoRESUMO
Although short tandem repeat (STR) analysis is available as a reliable method for the determination of the genetic origin of cell lines, the occurrence of misauthenticated cell lines remains an important issue. Reasons include the cost, effort and time associated with STR analysis. Moreover, there are currently no methods for the discrimination between isogenic cell lines (cell lines of the same genetic origin, e.g. different cell lines derived from the same organism, clonal sublines, sublines adapted to grow under certain conditions). Hence, additional complementary, ideally low-cost and low-effort methods are required that enable (1) the monitoring of cell line identity as part of the daily laboratory routine and 2) the authentication of isogenic cell lines. In this research, we automate the process of cell line identification by image-based analysis using deep convolutional neural networks. Two different convolutional neural networks models (MobileNet and InceptionResNet V2) were trained to automatically identify four parental cancer cell line (COLO 704, EFO-21, EFO-27 and UKF-NB-3) and their sublines adapted to the anti-cancer drugs cisplatin (COLO-704rCDDP1000, EFO-21rCDDP2000, EFO-27rCDDP2000) or oxaliplatin (UKF-NB-3rOXALI2000), hence resulting in an eight-class problem. Our best performing model, InceptionResNet V2, achieved an average of 0.91 F1-score on tenfold cross validation with an average area under the curve (AUC) of 0.95, for the 8-class problem. Our best model also achieved an average F1-score of 0.94 and 0.96 on the authentication through a classification process of the four parental cell lines and the respective drug-adapted cells, respectively, on a four-class problem separately. These findings provide the basis for further development of the application of deep learning for the automation of cell line authentication into a readily available easy-to-use methodology that enables routine monitoring of the identity of cell lines including isogenic cell lines. It should be noted that, this is just a proof of principal that, images can also be used as a method for authentication of cancer cell lines and not a replacement for the STR method.
Assuntos
Autenticação de Linhagem Celular/métodos , Cisplatino/farmacologia , Oxaliplatina/farmacologia , Área Sob a Curva , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Aprendizado Profundo , Humanos , Processamento de Imagem Assistida por Computador , Modelos Teóricos , Redes Neurais de ComputaçãoRESUMO
Cross-contamination of cell lines is a highly relevant and pervasive problem. The analysis of short tandem repeats (STR) is a simple and commercially available technique to authenticate cell lines for more than two decades. At present, STR multiple amplification kits have been developed up to 21 loci while the current STR databases only provide 9-loci STR profiles. Here, we compared the advantages of 21-loci STR methodology using the same algorithm as 9-loci method. The 21-loci method reduced the uncertainty ratio for authentications by 97.5% relative to the 9-loci method and exclude effectively false positive. We show that the additional 12 loci helped to greatly reduce sample-site marker specificity arising from genetic isolation and the occurrence of null alleles, suggesting that inclusion of additional loci in these databases will ultimately improve the efficiency and accuracy of authentication of cell lines. Taken together, we demonstrate the utility of a 21-loci method in human cells, providing a novel marker panel for use as a valuable alternative to 9-loci analyses to minimize cell line authentication errors and reduce costs due to erroneous experiments.
Assuntos
Autenticação de Linhagem Celular/métodos , Repetições de Microssatélites , Linhagem Celular , Autenticação de Linhagem Celular/normas , Linhagem Celular Tumoral , Loci Gênicos , Marcadores Genéticos , Humanos , Tipagem Molecular/métodos , Tipagem Molecular/normasRESUMO
MALT lymphomas with API2(BIRC3)-MALT1 translocation usually have an indolent clinical course and rarely transform into aggressive lymphoma, and there have been no lymphoma cell lines carrying API2-MALT1 translocation reported to date. We established a novel lymphoma cell line named BMA19, carrying the API2-MALT1 translocation from a patient with histologic transformation of intestinal MALT lymphoma. The cells were suggested to carry API2-MALT1 and MYC-IGH translocations by chromosomal analysis, and these translocations were confirmed by polymerase chain reaction analysis. The expression of MYC was shown to be enhanced as a result of the MYC-IGH translocation, and it is considered to have played a role in the histologic transformation of MALT lymphoma. Whole exome sequencing of BMA19 identified several nucleotide variations in genes reported to be mutated in previous studies of marginal zone lymphomas. The MALT1 inhibitor MI-2 specifically decreased cell growth, and the BMA19 cell line was suggested to be still dependent on the API2-MALT1 signal. Subtractive microarray analysis showed that one of the earliest events resulting from MALT1 inhibition is increased susceptibility to endoplasmic reticulum stress-induced apoptosis. The BMA19 cell line is considered to conserve the biological properties of MALT lymphoma and is expected to be a valuable tool for research into the pathogenesis of MALT lymphoma with an API2-MALT1 translocation.
Assuntos
Autenticação de Linhagem Celular/métodos , Linfoma/patologia , Proteínas de Fusão Oncogênica/genética , Cultura Primária de Células/métodos , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Estresse do Retículo Endoplasmático , Humanos , Linfoma/genética , Linfoma/metabolismo , Masculino , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Proteínas de Fusão Oncogênica/metabolismo , Células Tumorais CultivadasRESUMO
Renal medullary carcinoma (RMC) is a rare, aggressive disease that predominantly afflicts individuals of African or Mediterranean descent with sickle cell trait. RMC comprises 1% of all renal cell carcinoma diagnoses with a median overall survival of 13 months. Patients are typically young (median age-22) and male (male:female ratio of 2:1) and tumors are characterized by complete loss of expression of the SMARCB1 tumor suppressor protein. Due to the low incidence of RMC and the disease's aggressiveness, treatment decisions are often based on case reports. Thus, it is critical to develop preclinical models of RMC to better understand the pathogenesis of this disease and to identify effective forms of therapy. Two novel cell line models, UOK353 and UOK360, were derived from primary RMCs that both demonstrated the characteristic SMARCB1 loss. Both cell lines overexpressed EZH2 and other members of the polycomb repressive complex and EZH2 inhibition in RMC tumor spheroids resulted in decreased viability. High throughput drug screening of both cell lines revealed several additional candidate compounds, including bortezomib that had both in vitro and in vivo antitumor activity. The activity of bortezomib was shown to be partially dependent on increased oxidative stress as addition of the N-acetyl cysteine antioxidant reduced the effect on cell proliferation. Combining bortezomib and cisplatin further decreased cell viability both in vitro and in vivo that single agent bortezomib treatment. The UOK353 and UOK360 cell lines represent novel preclinical models for the development of effective forms of therapy for RMC patients.
Assuntos
Carcinoma Medular/patologia , Neoplasias Renais/patologia , Cultura Primária de Células/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Bortezomib/farmacologia , Bortezomib/uso terapêutico , Carcinoma Medular/tratamento farmacológico , Carcinoma Medular/genética , Autenticação de Linhagem Celular/métodos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Camundongos , Camundongos Nus , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Células Tumorais CultivadasRESUMO
Despite an increased awareness of the problematic of cell line cross-contamination and misidentification, it remains nowadays a major source of erroneous experimental results in biomedical research. To prevent it, researchers are expected to frequently test the authenticity of the cell lines they are working on. STR profiling was selected as the international reference method to perform cell line authentication. While the experimental protocols and manipulations for generating a STR profile are well described, the available tools and workflows to analyze such data are lacking. The Cellosaurus knowledge resource aimed to improve the situation by compiling all the publicly available STR profiles from the literature and other databases. As a result, it grew to become the largest database in terms of human STR profiles, with 6,474 distinct cell lines having an associated STR profile (release July 31, 2019). Here we present CLASTR, the Cellosaurus STR similarity search tool enabling users to compare one or more STR profiles with those available in the Cellosaurus cell line knowledge resource. It aims to help researchers in the process of cell line authentication by providing numerous functionalities. The tool is publicly accessible on the SIB ExPASy server (https://web.expasy.org/cellosaurus-str-search) and its source code is available on GitHub under the GPL-3.0 license.
Assuntos
Autenticação de Linhagem Celular/métodos , Mineração de Dados/métodos , Repetições de Microssatélites/genética , Animais , Biomarcadores/análise , Linhagem Celular , Impressões Digitais de DNA , Bases de Dados Factuais , Cães , Humanos , Camundongos , SoftwareRESUMO
The use of cell lines in research can be affected by cell line misidentification. Short tandem repeat (STR) analysis is an effective method, and the gold standard, for the identification of the genetic origin of a cell line, but methods that allow the discrimination between cell lines of the same genetic origin are lacking. Here, we use intact cell MALDI-ToF mass spectrometry analysis, routinely used for the identification of bacteria in clinical diagnostic procedures, for the authentication of a set of cell lines consisting of three parental neuroblastoma cell lines (IMR-5, IMR-32 and UKF-NB-3) and eleven drug-adapted sublines. Principal component analysis (PCA) of intact-cell MALDI-ToF mass spectrometry data revealed clear differences between most, but not all, of the investigated cell lines. Mass spectrometry whole-cell fingerprints enabled the separation of IMR-32 and its clonal subline IMR-5. Sublines that had been adapted to closely related drugs, for example, the cisplatin- and oxaliplatin-resistant UKF-NB-3 sublines and the vincristine- and vinblastine-adapted IMR-5 sublines, also displayed clearly distinctive patterns. In conclusion, intact whole-cell MALDI-ToF mass spectrometry has the potential to be further developed into an authentication method for mammalian cells of a common genetic origin.