Circulating tumor cells (CTCs) are rare cells that can be found in the peripheral blood of cancer patients. They have been demonstrated to be useful prognostic markers in many cancer types. Within the last decade various methods have been developed to detect rare cells within a liquid biopsy from a cancer patient. These methods have revealed the phenotypic diversity of CTCs and how they can represent the complement of cells that are found in a tumor. Single-cell proteogenomics has emerged as an all-encompassing next-generation technological approach for CTC research. This allows for the deconstruction of cellular heterogeneity, dynamics of metastatic initiation and progression, and response or resistance to therapeutics in the clinical settings. We take advantage of this opportunity to investigate CTC heterogeneity and understand their full potential in precision medicine.The high-definition single-cell analysis (HD-SCA) workflow combines detection of the entire population of CTCs and rare cancer related cells with single-cell genomic analysis and may therefore provide insight into their subpopulations based on molecular as well as morphological data. In this chapter we describe in detail the protocols from isolation of a candidate cell from a microscopy slide, through whole-genome amplification and library preparation, to CNV analysis of identified cells from the HD-SCA workflow. This process may also be applicable to any platform starting with a standard microscopy slide or isolated cell of interest.

• MeSH
• analýza jednotlivých buněk metody   MeSH
• individualizovaná medicína MeSH
• lidé MeSH
• mutace MeSH
• nádorové cirkulující buňky MeSH
• nádory diagnóza   genetika   MeSH
• proteogenomika metody   MeSH
• variabilita počtu kopií segmentů DNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Transcriptome analysis of circulating tumor cells (CTCs) holds great promise to unravel the biology of cancer cell dissemination and identify expressed genes and signaling pathways relevant to therapeutic interventions. METHODS: CTCs were enriched based on their EpCAM expression (CellSearch(®)) or by size and deformability (Parsortix(TM)), identified by EpCAM and/or pan-keratin-specific antibodies, and isolated for single cell multiplex RNA profiling. RESULTS: Distinct breast and prostate CTC expression signatures could be discriminated from RNA profiles of leukocytes. Some CTCs positive for epithelial transcripts (EpCAM and KRT19) also coexpressed leukocyte/mesenchymal associated markers (PTPRC and VIM). Additional subsets of CTCs within individual patients were characterized by divergent expression of genes involved in epithelial-mesenchymal transition (e.g., CDH2, MMPs, VIM, or ZEB1 and 2), DNA repair (RAD51), resistance to cancer therapy (e.g., AR, AR-V7, ERBB2, EGFR), cancer stemness (e.g., CD24 and CD44), activated signaling pathways involved in tumor progression (e.g., PIK3CA and MTOR) or cross talks between tumors and immune cells (e.g., CCL4, CXCL2, CXCL9, IL15, IL1B, or IL8). CONCLUSIONS: Multimarker RNA profiling of single CTCs reveals distinct CTC subsets and provides important insights into gene regulatory networks relevant for cancer progression and therapy.

• MeSH
• epitelo-mezenchymální tranzice genetika   MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• nádorové cirkulující buňky metabolismus   patologie   MeSH
• RNA nádorová genetika   MeSH
• stanovení celkové genové exprese * MeSH
• transkriptom genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články 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

The reason why a few myeloma cells egress from the bone marrow (BM) into peripheral blood (PB) remains unknown. Here, we investigated molecular hallmarks of circulating tumor cells (CTCs) to identify the events leading to myeloma trafficking into the bloodstream. After using next-generation flow to isolate matched CTCs and BM tumor cells from 32 patients, we found high correlation in gene expression at single-cell and bulk levels (r ≥ 0.94, P = 10-16), with only 55 genes differentially expressed between CTCs and BM tumor cells. CTCs overexpressed genes involved in inflammation, hypoxia, or epithelial-mesenchymal transition, whereas genes related with proliferation were downregulated in CTCs. The cancer stem cell marker CD44 was overexpressed in CTCs, and its knockdown significantly reduced migration of MM cells towards SDF1-α and their adhesion to fibronectin. Approximately half (29/55) of genes differentially expressed in CTCs were prognostic in patients with newly-diagnosed myeloma (n = 553; CoMMpass). In a multivariate analysis including the R-ISS, overexpression of CENPF and LGALS1 was significantly associated with inferior survival. Altogether, these results help understanding the presence of CTCs in PB and suggest that hypoxic BM niches together with a pro-inflammatory microenvironment induce an arrest in proliferation, forcing tumor cells to circulate in PB and seek other BM niches to continue growing.

• MeSH
• epitelo-mezenchymální tranzice genetika   MeSH
• exprese genu genetika   MeSH
• genetická transkripce genetika   MeSH
• hypoxie genetika   patologie   MeSH
• kostní dřeň patologie   MeSH
• lidé MeSH
• mnohočetný myelom genetika   patologie   MeSH
• nádorové buněčné linie MeSH
• nádorové cirkulující buňky patologie   MeSH
• nádorové kmenové buňky patologie   MeSH
• nádorové mikroprostředí genetika   MeSH
• pohyb buněk genetika   MeSH
• prognóza MeSH
• proliferace buněk genetika   MeSH
• zánět genetika   patologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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

Circulating tumor cells (CTCs) represent one of the promising diagnostic targets in monitoring of lung adenocarcinoma progression and malignant transformation. Here we propose a microfluidic chip (MLC Chip) which will enable to isolate (count) population of CTCs from peripheral blood sample of histologically proved adenocarcinoma stage IIb-IV patients and to sort them on their respective phenotype (epithelial/mesenchymal) on the level of the individual cells with the possibility of subsequent cell release for cultivation and/or gene expression profiling. The MLC Chip with filtration and single-cell sorting unit will be presented along with the novel types of binding proteins for EpCAM and N-Cadherin markers for CTCs on-chip capture. The overall goal is to evaluate the clinical relevance of different CTC phenotypes on early diagnostics of lung adenocarcinoma, correlate their occurrence with the carcinoma stage and progression, divide a population of patients in high/low risk groups, predict diagnosis and finally to adjust a medical treatment for individual patients.

Cirkulující nádorové buňky (CTC) jsou jedním z potenciálně významných diagnostických markerů pro monitorování metastatické progrese adenokarcinomu plic. Projekt je zaměřen na vývoj mikrofluidního čipu (MLC čip), který umožní izolovat (kvantifikovat) populace CTC buněk ve vzorku periferní krve pacientů s histologicky prokázaným adenokarcinomem plic fáze IIb-IV a rozdělit je dle jejich fenotypového projevu (epiteliální/mesenchymální) na úrovni jednotlivých buněk, s možností jejich následného uvolnění z čipu pro in vitro kultivaci, popř. analýzu genové exprese. MLC čip se bude skládat z filtrační a buněčné sortovací jednotky s imobilizovanými vazebnými proteiny pro zachycení buněk pomocí povrchových membránových markerů EpCAM a N-cadherinu. Hlavním cílem projektu je zhodnocení klinické relevance různých fenotypů CTC buněk na včasnou diagnostiku adenokarcinomu plic, korelovat je se stádiem onemocnění, rozdělit pacienty na high/low risk skupiny, predikovat diagnózu a ve výsledku uzpůsobit léčbu z hlediska individuálních potřeb pacienta.

• MeSH
• adenokarcinom plic diagnóza   MeSH
• adhezní molekula epiteliálních buněk MeSH
• fenotyp MeSH
• kadheriny MeSH
• laboratoř na čipu MeSH
• lidé MeSH
• mikročipová analýza MeSH
• nádorové biomarkery MeSH
• nádorové cirkulující buňky MeSH
• ribozomy MeSH
• Check Tag
• lidé MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• onkologie
• pneumologie a ftizeologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

As cancer care is transitioning to personalized therapies with necessary complementary or companion biomarkers there is significant interest in determining to what extent non-invasive liquid biopsies reflect the gold standard solid biopsy. We have established an approach for measuring patient-specific circulating and solid cell concordance by introducing tumor touch preparations to the High-Definition Single Cell Analysis workflow for high-resolution cytomorphometric characterization of metastatic colorectal cancer (mCRC). Subgroups of cells based on size, shape and protein expression were identified in both liquid and solid biopsies, which overall displayed high inter- and intra- patient pleomorphism at the single-cell level of analysis. Concordance of liquid and solid biopsies was patient-dependent and between 0.1-0.9. Morphometric variables displayed particularly high correlation, suggesting that circulating cells do not represent distinct subpopulations from the solid tumor. This was further substantiated by significant decrease in concentration of circulating cells after mCRC resection. Combined with the association of circulating cells with tumor burden and necrosis of hepatic lesions, our overall findings demonstrate that liquid biopsy cells can be informative biomarkers in the mCRC setting. Patient-specific level of concordance can readily be measured to establish the utility of circulating cells as biomarkers and define biosignatures for liquid biopsy assays.

• 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

Východiska: Maligní lymfomy představují vysoce heterogenní skupinu nádorů s rozmanitým klinickým chováním – od indolentních až po velmi agresivní formy s přežitím v rámci měsíců. Tato onemocnění jsou od počátku považována za systémová, často se vyskytující na několika místech současně. Nicméně diagnóza a přesná klasifikace obvykle probíhá z biopsie jediné patologické uzliny či infiltrátu. Klinické zkušenosti přitom ukazují, že biologické chování lymfomu nemusí být v různých jeho lokalizacích zcela shodné. Ve snaze vyřešit tento problém, ale také zlepšit diagnostiku z obtížně dostupných kompartmentů, je poslední dobou intenzivně zkoumána tzv. volná cirkulující DNA (cfDNA), jejíž součástí je také DNA uvolněná z nádorových buněk – cirkulující nádorová DNA (ctDNA). Tuto DNA lze snadno získat z tekutých biopsií, jako je krev, případně jiné tělní tekutiny. Cíl: Tento článek shrnuje dosavadní poznatky o cfDNA a ctDNA, zejména pak právě v kontextu maligních lymfomů, a nastiňuje potenciální směry jejího budoucího praktického využití. Závěr: Detekce a analýza ctDNA představuje novou modalitu, která může v budoucnu vést ke zkvalitnění všech fází léčby maligních lymfomů od diagnostiky až po sledování tzv. minimální zbytkové choroby.

Background: Malignant lymphomas represent a highly heterogeneous group of tumors with varied clinical behavior – from indolent to very aggressive forms with survival in the order of months. From the very beginning, these diseases are considered systemic, often occurring in several anatomical locations simultaneously. However, diagnosis and exact classification are usually inferred from a biopsy of a single pathological lymph node or infiltrate, even though clinical experience shows that the biological behavior of lymphoma is not necessarily identical across anatomical locations. In an effort to address this issue as well as the problem of biopsy of not easily accessible compartments, circulating free DNA (cfDNA), which contains circulating tumor DNA (ctDNA) released from dead tumor cells, has been extensively studied in recent years. This DNA is easily accessible from liquid biopsies such as blood or other patient‘s bodily fluids. Purpose: This article summarizes current scientific knowledge on cfDNA and ctDNA, particularly in the context of malignant lymphoma, and foreshadows its potential future uses. Conclusion: Detection and analysis of cfDNA represents a new approach that can lead to future improvements in all phases of lymphoma treatment from diagnostics to minimal residual disease monitoring.

• MeSH
• cirkulující nádorová DNA analýza   MeSH
• difúzní velkobuněčný B-lymfom diagnóza   genetika   MeSH
• lidé MeSH
• lymfom diagnóza   genetika   klasifikace   MeSH
• progrese nemoci MeSH
• reziduální nádor diagnóza   genetika   MeSH
• tekutá biopsie metody   MeSH
• volné cirkulující nukleové kyseliny * analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• přehledy MeSH

Molecular analysis of circulating and disseminated tumor cells (CTCs/DTCs) has great potential as a means for continuous evaluation of prognosis and treatment efficacy in near-real time through minimally invasive liquid biopsies. To realize this potential, however, methods for molecular analysis of these rare cells must be developed and validated. Here, we describe the integration of imaging mass cytometry (IMC) using metal-labeled antibodies as implemented on the Fluidigm Hyperion Imaging System into the workflow of the previously established High Definition Single Cell Analysis (HD-SCA) assay for liquid biopsies, along with methods for image analysis and signal normalization. Using liquid biopsies from a metastatic prostate cancer case, we demonstrate that IMC can extend the reach of CTC characterization to include dozens of protein biomarkers, with the potential to understand a range of biological properties that could affect therapeutic response, metastasis and immune surveillance when coupled with simultaneous phenotyping of thousands of leukocytes.

• Publikační typ
• časopisecké články MeSH