Epithelial-mesenchymal transition (EMT) is a crucial process with significance in the metastasis of malignant tumors. It is through the acquisition of plasticity that cancer cells become more mobile and gain the ability to metastasize to other tissues. The mesenchymal-epithelial transition (MET) is the return to an epithelial state, which allows for the formation of secondary tumors. Both processes, EMT and MET, are regulated by different pathways and different mediators, which affects the sophistication of the overall tumorigenesis process. Not insignificant are also cancer stem cells and their participation in the angiogenesis, which occur very intensively within tumors. Difficulties in effectively treating cancer are primarily dependent on the potential of cancer cells to rapidly expand and occupy secondarily vital organs. Due to the ability of these cells to spread, the concept of the circulating tumor cell (CTC) has emerged. Interestingly, CTCs exhibit molecular diversity and stem-like and mesenchymal features, even when derived from primary tumor tissue from a single patient. While EMT is necessary for metastasis, MET is required for CTCs to establish a secondary site. A thorough understanding of the processes that govern the balance between EMT and MET in malignancy is crucial.
- Klíčová slova
- Cancer stem Cells, Circulating Tumor cell (CTC), Epithelial-mesenchymal Transition (EMT), Mesenchymal-epithelial Transition (MET),
- MeSH
- epitelo-mezenchymální tranzice * MeSH
- fenotyp MeSH
- kmenové buňky metabolismus cytologie patologie MeSH
- lidé MeSH
- nádorové cirkulující buňky * patologie metabolismus MeSH
- nádorové kmenové buňky * patologie metabolismus MeSH
- nádory patologie metabolismus MeSH
- patologická angiogeneze * patologie MeSH
- proliferace buněk genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Breast cancer metastases are the main reason for women´s highest cancer mortality. Even though tumor cell dissemination via circulating tumor cells (CTC) released from the primary site is a very ineffective process, distant metastases appear in 46% of triple-negative breast cancer (TNBC) patients corresponding to the disease aggressiveness. Laboratory models for functional testing which mimic the spread of metastatic cells are needed for efficient investigation of the underlying mechanisms and therapeutic intervention. Here, we describe novel isogenic variants LMC3 and CTC3 of human TNBC cell line MDA-MB-231 that were derived by repeated injection of tumor cells into the tail vein of immunodeficient mice and subsequent selection of metastatic cells from lung metastases. These variants have increased migration potential, altered expression profiles, and elevated tumorigenic potential. Moreover, cell line CTC3 readily produces metastases in the lungs and bone marrow and detectable viable circulating tumor cells in the blood. This model enables rapid and cost-efficient strategies for biomarker exploration and novel intervention approaches to limit the CTC presence in the blood and hence tumor dissemination.
- MeSH
- biologické markery MeSH
- lidé MeSH
- metastázy nádorů MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádorové cirkulující buňky * metabolismus MeSH
- nádory plic * sekundární MeSH
- triple-negativní karcinom prsu * genetika patologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- biologické markery MeSH
Cell immunocapture microsystems are a fast-emerging field with several potential medical diagnostic applications. Isolation and quantification of circulating rare cells (CRCs) show great importance in the early stages of disease diagnostics and prognostics. Here, we present a simple and robust stop-flow microsystem (fabricated by a combination of glass microblasting and 3D printing) based on a planar antibody-coated surface that is effective in the immunocapture of the model as well as naturally occurring rare cells. A chip with a planar immunocapture channel working in the so-called stop-flow dynamic regime was designed to enable monitoring the efficiency of the cell capture by fluorescence microscopy. Up to 90% immunocapture efficiency of MCF-7 cells spiked into whole blood on CD326 antibody-coated planar surfaces was achieved. We discuss the role of the planar surface modifications, the influence of the set stop-flow dynamic conditions, and medium complexity on the efficiency of cell immunocapture. The presented results could be further employed in the design of microsystems for cell-size-independent isolation and identification of rare cells from blood.
- Klíčová slova
- Cell immunocapture, Circulating endothelial cells, Circulating tumor cells, Microsystems, Rare cell population,
- MeSH
- biosenzitivní techniky * MeSH
- lidé MeSH
- mikrofluidní analytické techniky * metody MeSH
- nádorové buněčné linie MeSH
- nádorové cirkulující buňky * metabolismus MeSH
- protilátky MeSH
- separace buněk metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- protilátky MeSH
BACKGROUND: Dendritic cell (DC) therapy counts to the promising strategies how to weaken and eradicate cancer disease. We aimed to develop a good manufacturing practice (GMP) protocol for monocyte-derived DC (Mo-DC) maturation using circulating tumor cells lysates with subsequent experimental T-cell priming in vitro. METHODS: DC differentiation was induced from a population of immunomagnetically enriched CD14 + monocytes out of the leukapheresis samples (n = 6). The separation was provided automatically, in a closed bag system, using CliniMACS Prodigy® separation protocols (Miltenyi Biotec). For differentiation and maturation of CD14 + cells, DendriMACs® growing medium with supplements (GM-CSF, IL-4, IL-6, IL-1B, TNFa, PGE) was used. Immature Mo-DCs were loaded with autologous circulating tumor cell (CTCs) lysates. Autologous CTCs were sorted out by size-based filtration (MetaCell®) of the leukapheresis CD14-negative fraction. A mixture of mature Mo-DCs and autologous non-target blood cells (NTBCs) was co-cultured and the activation effect of mature Mo-DCs on T-cell activation was monitored by means of multimarker gene expression profiling. RESULTS: New protocols for mMo-DC production using automatization and CTC lysates were introduced including a feasible in vitro assay for mMo-DC efficacy evaluation. Gene expression analysis revealed elevation for following genes in NTBC (T cells) subset primed by mMo-DCs: CD8A, CD4, MKI67, MIF, TNFA, CD86, and CD80 (p ≤ 0.01). CONCLUSION: Summarizing the presented data, we might conclude mMo-DCs were generated using CliniMACS Prodigy® machine and CTC lysates in a homogenous manner showing a potential to generate NTBC activation in co-cultures. Identification of the activation signals in T-cell population by simple multimarker-qPCRs could fasten the process of effective mMo-DC production.
- Klíčová slova
- Circulating tumor cells, Dendritic cells, Immunotherapy, MetaCell, Personalized medicine, T cells,
- MeSH
- dendritické buňky * metabolismus MeSH
- faktor stimulující granulocyto-makrofágové kolonie farmakologie MeSH
- interleukin-4 farmakologie MeSH
- interleukin-6 farmakologie MeSH
- lidé MeSH
- monocyty * metabolismus MeSH
- nádorové cirkulující buňky * metabolismus MeSH
- prostaglandiny E farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- faktor stimulující granulocyto-makrofágové kolonie MeSH
- interleukin-4 MeSH
- interleukin-6 MeSH
- prostaglandiny E MeSH
New non-invasive approaches have developed for diagnosis and treatment of malignant diseases. Cells shed from the primary tumor circulating in the bloodstream with metastasis potential are called Circulating Tumor Cells (CTCs). These cells are easily acquired from the peripheral blood of patients, while several enrichment and isolation methods are available nowadays with different benefits and positive detection rates. A brief characterization of three major categories of detection is described (nucleic acid-based, physical properties-based, antibody-based). In this review we concentrate on gynecological malignancies and how CTCs could be used in the diagnosis of cancer, treatment management and its effective prognosis and early recurrence detection. Presence of CTCs in endometrial cancer patients show worse overall survival, while gene analysis could identify patients in need of systemic therapy after surgical treatment to prevent metastasis and recurrence. Based on the influence of human papillomavirus (HPV) in the etiology of cervical cancer, viral oncogene transcripts could be used as an ideal marker for cervical cancer cells detection. In ovarian cancer, CTCs could help in the differentiation from benign adnexal masses and show a high independence from other biomarkers such as CA125 and HE4. While detection of CTC after complete cytoreductive surgery could indicate invisible lesions, combination of tumor associated genes rises the specificity of CTC detection.
- MeSH
- lidé MeSH
- nádorové cirkulující buňky metabolismus MeSH
- nádory ženských pohlavních orgánů metabolismus MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The combination of liquid biomarkers from a single blood tube can provide more comprehensive information on tumor development and progression in cancer patients compared to single analysis. Here, we evaluated whether a combined analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and circulating cell-free microRNA (miRNA) in total plasma and extracellular vesicles (EV) from the same blood sample is feasible and how the results are influenced by the choice of different blood tubes. Peripheral blood from 20 stage IV melanoma patients and five healthy donors (HD) was collected in EDTA, Streck, and Transfix tubes. Peripheral blood mononuclear cell fraction was used for CTC analysis, whereas plasma and EV fractions were used for ctDNA mutation and miRNA analysis. Mutations in cell-free circulating DNA were detected in 67% of patients, with no significant difference between the tubes. CTC was detected in only EDTA blood and only in 15% of patients. miRNA NGS (next-generation sequencing) results were highly influenced by the collection tubes and could only be performed from EDTA and Streck tubes due to hemolysis in Transfix tubes. No overlap of significantly differentially expressed miRNA (patients versus HD) could be found between the tubes in total plasma, whereas eight miRNA were commonly differentially regulated in the EV fraction. In summary, high-quality CTCs, ctDNA, and miRNA data from a single blood tube can be obtained. However, the choice of blood collection tubes is a critical pre-analytical variable.
- Klíčová slova
- CTC, EV, ctDNA, liquid biopsy, melanoma, miRNA,
- MeSH
- cirkulující nádorová DNA krev MeSH
- extracelulární vezikuly genetika metabolismus ultrastruktura MeSH
- lidé MeSH
- melanom krev patologie MeSH
- mikro RNA krev genetika MeSH
- mutace MeSH
- nádorové biomarkery krev genetika MeSH
- nádorové buněčné linie MeSH
- nádorové cirkulující buňky metabolismus MeSH
- senioři MeSH
- staging nádorů MeSH
- tekutá biopsie přístrojové vybavení metody MeSH
- transmisní elektronová mikroskopie MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cirkulující nádorová DNA MeSH
- mikro RNA MeSH
- nádorové biomarkery MeSH
Circulating tumor cells (CTCs), detached from the primary tumor or metastases and shed in the patient's bloodstream, represent a relatively easily obtainable sample of the cancer tissue that can indicate the actual state of cancer, and their evaluation can be repeated many times during the course of treatment. As part of liquid biopsy, evaluation of CTCs provides a lot of clinically relevant information, which reflects the actual, real-time conditions of the disease. CTCs can be used in cancer diagnosis or screening, real-time long-term disease monitoring and even therapy guidance. Their analysis can include their number, morphology, and biological features by using immunocytochemistry and all "-omic" technologies. This review describes methods of CTC isolation and potential clinical utilization in lung cancer.
- Klíčová slova
- Circulating tumor cells, biomarker, culturing, liquid biopsy, lung cancer, review,
- MeSH
- časná detekce nádoru MeSH
- diagnostické techniky molekulární MeSH
- lidé MeSH
- nádorové biomarkery MeSH
- nádorové cirkulující buňky metabolismus patologie MeSH
- nádory plic diagnóza etiologie terapie MeSH
- tekutá biopsie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- nádorové biomarkery MeSH
:Background: Current diagnosis and staging of advanced epithelial ovarian cancer (aEOC) has important limitations and better biomarkers are needed. We investigate the performance of non-haematopoietic circulating cells (CCs) at the time of disease presentation and relapse. Methods: Venous blood was collected prospectively from 37 aEOC patients and 39 volunteers. CCs were evaluated using ImageStream TechnologyTM and specific antibodies to differentiate epithelial cells from haematopoetic cells. qRT-PCR from whole blood of relapsed aEOC patients was carried out for biomarker discovery. Results: Significant numbers of CCs (CK+/WT1+/CD45-) were identified, quantified and characterised from aEOC patients compared to volunteers. CCs are abundant in women with newly diagnosed aEOC, prior to any treatment. Evaluation of RNA from the CCs in relapsed aEOC patients (n = 5) against a 79-gene panel revealed several differentially expressed genes compared to volunteers (n = 14). Size differentiation of CCs versus CD45+ haematopoietic cells was not reliable. Conclusion: CCs of non-haematopoetic origin are prevalent, particularly in patients with newly diagnosed aEOC. Exploiting a CC-rich population in aEOC patients offers insights into a part of the circulating microenvironment.
- Klíčová slova
- advanced epithelial ovarian cancer, circulating endothelial cells, circulating tumour cells,
- MeSH
- antigeny CD45 metabolismus MeSH
- karcinom krev MeSH
- lidé středního věku MeSH
- lidé MeSH
- nádorové biomarkery metabolismus MeSH
- nádorové buněčné linie MeSH
- nádorové cirkulující buňky metabolismus patologie MeSH
- nádory vaječníků krev MeSH
- proteiny WT1 metabolismus MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antigeny CD45 MeSH
- nádorové biomarkery MeSH
- proteiny WT1 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.
- Klíčová slova
- Bladder cancer, CTCs, Circulating tumor cells, Cultivation, Gene expression, In vitro, Prostate cancer, Renal,
- 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
- Názvy látek
- DNA nádorová MeSH
BACKGROUND: Molecular characterization of tumors could be a key to therapeutic decision-making with regards to targeted therapies in castration-resistant prostate cancer (CRPC). A convenient solution may be non-invasive liquid biopsy testing of circulating tumor cells (CTCs). For this reason, CTC-enriched samples obtained by immunomagnetic separation (AdnaTest®) were studied as a source material for high-throughput gene expression analysis using BioMark™. PATIENTS AND METHODS: CTC-enriched samples from 41 CRPC patients previously determined to be CTC positive using the AdnaTest® were retrospectively re-analysed for androgen receptor (AR) messenger RNA (mRNA), using the updated AdnaTest®. Blood samples were drawn two times from each patient: at the time of CRPC diagnosis and after the third docetaxel cycle. A gene expression panel of 27 genes related to CRPC therapeutic decision-making, including AR full length (ARFL) and splice variant 7 (ARV7), was retrospectively analyzed on a BioMark™ platform in 29 of 41 patients. RESULTS: The AdnaTest® detected AR mRNA in three-quarters of CTC-positive samples taken at the time of CRPC diagnosis and after the third docetaxel cycle. AR detection was associated with a shorter disease-specific survival (45.0 vs. 20.4 months) at the time of CRPC diagnosis. ARFL expression at the time of CRPC diagnosis, measured on the BioMark™ platform, was associated with a lower decrease of serum level of prostate-specific antigen (sPSA) (p = 0.029), i.e., worse therapy response. ARV7 was found in 38% of the ARFL--positive samples at both analyzed timepoints. CONCLUSION: Detection of AR expression by AdnaTest® in CTC-enriched samples may help predict patients' survival. These AdnaTest® CTC-enriched samples can be used in a high-throughput quantitative polymerase chain reaction (qPCR) analysis of gene expression, provided that the specificity of the assay for each individual gene is properly validated. The BioMark™ platform can be used for the simultaneous detection of ARFL and ARV7 and other genes in CTC-enriched samples from CRPC patients.
- MeSH
- genetické testování metody MeSH
- imunomagnetická separace MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé středního věku MeSH
- lidé MeSH
- nádorové biomarkery * MeSH
- nádorové cirkulující buňky metabolismus patologie MeSH
- nádory prostaty rezistentní na kastraci diagnóza genetika MeSH
- následné studie MeSH
- rychlé screeningové testy MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- stanovení celkové genové exprese * metody MeSH
- transkriptom MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- nádorové biomarkery * MeSH
