In a cell-based non-invasive prenatal test (cbNIPT), intact circulating trophoblasts (CTs) are isolated from maternal blood for subsequent genetic analysis. Enrichment of these CTs from maternal blood is the most challenging step in the cbNIPT workflow. This study aims to assess the suitability of the filtration-based Metacell® technology to enrich CTs from maternal blood at week 10 to 13 of gestation. The Metacell® technology is a novel size-based enrichment technology that combines blood filtration through 8 μm pores with an in vitro culture method. Three protocols were evaluated. First, 8 mL or 16 mL of maternal blood was filtered and subsequently cultured in vitro on the separation membrane for 3 days in RPMI 1640. In addition, 16 mL of maternal blood was filtered, and immediately processed without further culturing. Y-chromosome-specific qPCR or STR analysis was performed to evaluate the enrichment of CTs. A total of 44 samples from pregnant women, out of which 26 were carrying a male fetus, were processed. Although five enriched male fetus samples show detectable male DNA quantities, it cannot be excluded that the obtained positive signal is caused by cell-free fetal DNA sticking to the Metacell® separation membrane. In conclusion, the Metacell® technology, tested as described, is not suitable for consistent enrichment of CTs.

• MeSH
• DNA MeSH
• lidé MeSH
• plod MeSH
• prenatální diagnóza * metody   MeSH
• technologie MeSH
• těhotenství MeSH
• trofoblasty * MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

OBJECTIVE: Approximately one third of patients diagnosed with muscle-invasive urinary bladder cancer (UBC) have undetected metastases at the time of treatment of the primary tumor. Currently there are no reliable specific serum markers for monitoring and evaluating risk profiles of urothelial cancers. Several studies suggest that detection of circulating tumor cells (CTCs) may correlate with the disease status and prognosis at baseline and early in the treatment of cancers. In this study a new way of isolation and in vitro cultivation of CTCs of urinary bladder cancer was introduced. MATERIALS AND METHODS: Peripheral blood (PB) samples from 53 patients who had undergone urological procedure were evaluated using the MetaCell device (MetaCell s.r.o., Ostrava, Czech Republic). The patients enrolled in the study were both oncological patients with UBC and non-oncological patients with inflammation (14 patients). The sensitivity and quantification of CTCs were evaluated. The separated CTCs were cultured in vitro. RESULTS: 39 patients with confirmed UBC were enrolled in the study. CTCs were detected in 25 (64%) patients, and most of these patients had between 6 and 10 cells. The separated CTCs were successfully cultured in vitro. CONCLUSION: CTCs were detected in a higher percentage of patients than in other studies. This paper describes the first successful culturing of human UBC cells. The MetaCell approach used in this study enabled the capture of viable intact virgin CTCs (virgin CTC) suitable for next in vitro culturing, single cell analysis or drug testing.

• MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• nádorové cirkulující buňky patologie   MeSH
• nádory močového měchýře krev   patologie   MeSH
• papilární karcinom krev   patologie   MeSH
• proliferace buněk MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• separace buněk metody   MeSH
• staging nádorů MeSH
• studie případů a kontrol MeSH
• stupeň nádoru MeSH
• viabilita buněk MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Monitoring of circulating tumor cells' (CTCs) presence has the potential to improve therapeutic management of oncological diseases at an early stage and also to identify patients with increased risk of tumor progression or recurrence before the onset of clinically detected metastasis. Here we describe a new simplified efficient methodology for the separation and in vitro culturing of viable CTCs from peripheral blood by size-based filtration (MetaCell®). The isolation protocol yields preferentially cells bigger than 8 μm enabling further cytomorphological and molecular analysis.

• MeSH
• buněčné kultury MeSH
• DNA nádorová genetika   izolace a purifikace   MeSH
• lidé MeSH
• mutační analýza DNA metody   MeSH
• nádorové cirkulující buňky metabolismus   patologie   MeSH
• separace buněk * metody   MeSH
• stanovení celkové genové exprese metody   MeSH
• urologické nádory diagnóza   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The most promising near-term application of circulating tumor cells (CTCs) monitoring relates to the development of targeted cancer therapies, and the need to tailor such treatments to individual tumor characteristics. A high number of new innovative technologies to improve methods for detecting CTCs, with extraordinarily high sensitivity, have recently been presented. The identification and characterization of CTCs require extremely sensitive and specific methods that are able to isolate CTCs with the possibility of cultivation and downstream analysis of in vitro culture of separated CTCs. In this original research paper, we demonstrate that it is possible to isolate human CTCs from a patient with prostate cancer, with subsequent cultivation and proliferation in vitro. We show that the use of a filtration device implemented by MetaCell® can fulfil all the requirements mentioned above. Fifty-five patients with localized prostate cancer have so far been enrolled into the study. CTCs were detected in the blood samples of 28 (52%) out of the 55 patients. We report successful isolation of CTCs from patients with prostate cancer, capturing cells with a proliferative capacity in 18 (64.3%) out of the 28 CTC-positive patients. Direct correlation with Gleason score and T stage was not proven. The cells, captured by a size-based filtration approach, remain in a good state, unaffected by any antibodies or lysing solutions. During the filtration process, no interactions occurred between antibodies and antigens on the surface of CTCs. This biological interaction is specific for immunomagnetic methods. The MetaCell device provides the possibility of reaching virgin CTCs suitable for subsequent cultivation or single-cell analysis. This aspect will have an important impact on the future design of clinical trials testing new drugs against targets expressed on metastatic cancer cells. In addition to measurement of CTC counts, future trials with targeted therapies should also include the assessment of the specific therapeutic target on CTCs.

• MeSH
• cytologické techniky metody   MeSH
• kohortové studie MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• nádorové cirkulující buňky patologie   MeSH
• nádory prostaty krev   patologie   MeSH
• senioři MeSH
• staging nádorů MeSH
• stupeň nádoru MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In the present study, we demonstrate an animal model and recently introduced size-based exclusion method for circulating tumor cells (CTCs) isolation. The methodology enables subsequent in vitro CTC-culture and characterization. Human lung cancer cell line H460, expressing red fluorescent protein (H460-RFP), was orthotopically implanted in nude mice. CTCs were isolated by a size-based filtration method and successfully cultured in vitro on the separating membrane (MetaCell®), analyzed by means of time-lapse imaging. The cultured CTCs were heterogeneous in size and morphology even though they originated from a single tumor. The outer CTC-membranes were blebbing in general. Abnormal mitosis resulting in three daughter cells was frequently observed. The expression of RFP ensured that the CTCs originated from lung tumor. These readily isolatable, identifiable and cultivable CTCs can be used to characterize individual patient cancers and for screening of more effective treatment.

• MeSH
• lidé MeSH
• luminescentní proteiny biosyntéza   MeSH
• modely nemocí na zvířatech * MeSH
• myši nahé MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• nádorové cirkulující buňky patologie   MeSH
• nádory plic patologie   MeSH
• poréznost MeSH
• povrchové vlastnosti MeSH
• separace buněk MeSH
• transplantace nádorů patologie   MeSH
• velikost částic MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND/AIM: The presence of circulating tumor cells (CTCs) in the peripheral blood of patients with solid tumors is associated with a poor prognosis. However, there are limited data concerning the detection of CTCs in endometrial cancer (EC). The aim of this study was to evaluate the presence of CTCs in the peripheral blood of patients with EC. MATERIALS AND METHODS: Peripheral blood samples from 92 patients who underwent a surgical procedure were evaluated using MetaCell® separation technology for CTCs. RESULTS: CTCs were detected in 69 (75%) patients with EC. CONCLUSION: CTCs were detected in a higher percentage of patients than in other studies. The results showed that the technology applied in this study can efficiently capture viable tumor cells in the blood that can be cultured while maintaining their original phenotype. This paper discusses the first successful culturing of human circulating endometrial cancer cells for further downstream functional and molecular characterization.

• MeSH
• lidé MeSH
• nádorové cirkulující buňky patologie   MeSH
• nádory endometria krev   patologie   MeSH
• staging nádorů MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

The main focus of the study was to detect circulating tumor cells (CTCs) in ovarian cancer (OC) patients using a new methodological approach (MetaCell(TM)) which is based on size-dependent separation of CTCs and subsequent cytomorphological evaluation. Cytomorphological evaluation using vital fluorescence microscopy approach enables to use the captured cells for further RNA/DNA analysis. The cytomorphological analysis is then completed by gene expression analysis (GEA). GEA showed that relative expression of EPCAM is elevated in CTC-enriched fractions in comparison to the whole peripheral blood sample and that the expression grows with in vitro cultivation time. Comparison of the relative gene expression level in the group of peripheral blood samples and CTC-fraction samples confirmed a statistically significant difference for the following genes (p < 0.02): KRT7, WT1, EPCAM, MUC16, MUC1, KRT18 and KRT19. Thus, we suggest that the combination of the above listed genes could confirm CTCs presence in OC patients with higher specificity than when GEA tests are performed for one marker only. The GEA revealed two separate clusters identifying patients with or without CTCs.

• Publikační typ
• časopisecké články MeSH

The presence of circulating tumor cells (CTCs) in patients with solid tumors is associated with poor prognosis. However, there are limited data concerning the detection of CTCs in renal cell cancer (RCC). The aim of this study is to evaluate the presence of CTCs in peripheral blood of patients with RCC undergoing surgery (n = 186). CTCs were tested before and after surgery as well as during the follow-up period afterwards. In total 495 CTC testing in duplicates were provided. To enrich CTCs, a size-based separation protocol and tube MetaCell® was used. CTCs presence was evaluated by single cell cytomorphology based on vital fluorescence microscopy. Additionally, to standardly applied fluorescence stains, CTCs viability was controlled by mitochondrial activity. CTCs were detected independently on the sampling order in up to 86.7% of the tested blood samples in patients undergoing RCC surgery. There is higher probability of CTC detection with growing tumor size, especially in clear cell renal cell cancer (ccRCC) cases. Similarly, the tumor size corresponds with metastasis presence and lymph node positivity and CTC detection. This paper describes for the first-time successful analysis of viable CTCs and their mitochondria as a part of the functional characterization of CTCs in RCC.

• Publikační typ
• časopisecké články MeSH

UNLABELLED: Malignant pleural mesothelioma (MPM) is an aggressive disease with very poor prognosis which tends to affect older patients. Progress in the management of this group of patients has been limited by the rarity of the disease and hence, difficulty in conducting randomized trials. The vast majority of cancer deaths occur due to metastasis of the primary tumor to distant sites via circulating tumor cells (CTCs) in the circulation. CTCs are extremely rare and limits in technology used to capture these cells hamper our complete understanding over the metastatic process. In the present study we present a new method for detection and cultivation of CTCs isolated from peripheral blood of MPM patients. PATIENTS AND METHODS: Patients with diagnosed MPM were enrolled into this study. RESULTS: A size-based separation method for viable CTC enrichment from unclothed peripheral blood has been introduced; MetaCell. The size-based enrichment process was based on filtration of peripheral blood (PB) through porous polycarbonate membrane. The separated CTCs are cultured on the membrane in vitro under standard cancer cell culture conditions and observed by an inverted microscope. CONCLUSION: The reported methodology allows for quick and easy enrichment of CTCs and their cultivation. The cultivated cells can be used for next specification of gene expression and histological/biological specificity of concrete mesothelioma.

• MeSH
• lidé středního věku MeSH
• lidé MeSH
• mezoteliom krev   patologie   MeSH
• nádorové cirkulující buňky patologie   MeSH
• nádory pleury krev   patologie   MeSH
• nádory plic krev   patologie   MeSH
• senioři MeSH
• separace buněk metody   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

OBJECTIVES: Endometriosis is a common disorder amongst women of reproductive age. Despite extensive research, no reliable blood tests currently exist for the diagnosis of endometriosis STUDY DESIGN: We report several new approaches enabling study of cell specific characteristic of endometrial cells, introducing enrichment and culturing of viable circulating endometrial cells (CECs) isolated from peripheral blood (PB) and peritoneal endometrial cells (PECs) from peritoneal washing (PW). Size-based enrichment method (MetaCell(®), Czech Republic) has been used for the filtration of PB and PW in patients with diagnosed endometriosis. RESULTS: The PECs were found in the PW in all of the tested patients (n=17), but CECs) only in 23.5% (4/17) cases. Their endometrial origin has been proved by immunohistochemistry. PECs were successfully cultured in vitro directly on the separating membrane (9/17) exhibiting both endometrial cell phenotypes: stromal and glandular within the culture. CECs were successfully cultured in the two of the four positive cases, but in none of them confluence has been reached. The occurrence in CECs in PB is clear and very specific evidence of an active endometrial disease. CONCLUSIONS: We demonstrated efficient, quick and user friendly endometrial cells capture platform based on a cell size. Furthermore, we demonstrated an ability to culture the captured cells, a critical requirement for post-isolation cellular analysis directed to better understanding of endometriosis pathogenesis.

• MeSH
• ascitická tekutina cytologie   MeSH
• buněčné kultury MeSH
• dospělí MeSH
• endometrióza krev   diagnóza   MeSH
• endometrium * MeSH
• imunohistochemie MeSH
• kultivované buňky chemie   cytologie   MeSH
• lidé MeSH
• peritoneální výplach MeSH
• separace buněk MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH