scholarly journals Circulating tumour cells in patients with urothelial tumours: Enrichment and in vitro culture

2014 ◽  
Vol 8 (9-10) ◽  
pp. 715 ◽  
Author(s):  
Vladimir Bobek ◽  
Martin Cegan ◽  
Katarina Kolostova
Keyword(s):  
Early Stage ◽  
Tumour Progression ◽  
Urinary Bladder Cancer ◽  
Tumour Cells ◽  
Circulating Tumour Cells ◽  
Cellular Analysis ◽  
Increased Risk ◽  
In Vitro Cell Cultures ◽  
Urothelial Tumours

Introduction: Results of clinical trials have demonstrated that circulating tumour cells (CTCs) are frequently detected in patients with urothelial tumours. The monitoring of CTCs has the potential to improve therapeutic management at an early stage and also to identify patients with increased risk of tumour progression or recurrence before the onset of clinically detected metastasis. In this study, we report a new effectively simplified methodology for a separation and in vitro culturing of viable CTCs from peripheral blood.Method: We include patients diagnosed with 3 types of urothelial tumours (prostate cancer, urinary bladder cancer, and kidney cancer). A size-based separation method for viable CTC - enrichment from unclothed peripheral blood has been introduced (MetaCell, Ostrava, Czech Republic). The enriched CTCs fraction was cultured directly on the separation membrane, or transferred from the membrane and cultured on any plastic surface or a microscopic slide.Results: We report a successful application of a CTCs isolation procedure in patients with urothelial cancers. The CTCs captured on the membrane are enriched with a remarkable proliferation potential. This has enabled us to set up in vitro cell cultures from the viable CTCs unaffected by any fixation buffers, antibodies or lysing solutions. Next, the CTCs were cultured in vitro for a minimum of 10 to 14 days to enable further downstream analysis (e.g., immunohistochemistry).Conclusion: We demonstrated an efficient CTCs capture platform, based on a cell size separation principle. Furthermore, we report an ability to culture the enriched cells – a critical requirement for post-isolation cellular analysis.

scholarly journals Pre-metastatic niche triggers SDF-1/CXCR4 axis and promotes organ colonisation by hepatocellular circulating tumour cells via downregulation of Prrx1

2019 ◽  
Vol 38 (1) ◽  
Author(s):  
Yujun Tang ◽  
Yishi Lu ◽  
Yuan Chen ◽  
Lei Luo ◽  
Lei Cai ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Line ◽  
Lentiviral Vector ◽  
Tumour Cells ◽  
Clinical Samples ◽  
Circulating Tumour Cells ◽  
Metastatic Niche ◽  
Metastatic Sites

Abstract Background Circulating tumour cells (CTCs), especially mesenchymal CTCs, are important determinants of metastasis, which leads to most recurrence and mortality in hepatocellular carcinoma (HCC). However, little is known about the underlying mechanisms of CTC colonisation in pre-metastatic niches. Methods Detection and classification of CTCs in patients were performed using the CanPatrol™ system. A lentiviral vector expressing Prrx1-targeting shRNA was constructed to generate a stable HCC cell line with low expression of Prrx1. The effect of Prrx1 knockdown on stemness, migration, and drug resistance of the cell line was assessed, including involvement of SDF-1/CXCR4 signalling. Promising clinical applications of an inhibitor of STAT3 tyrosine phosphorylation, C188–9, and specific blockade with CXCR4 antibody were explored. Results The number of mesenchymal CTCs in blood was closely associated with tumour recurrence or metastasis. Pre-metastatic niche-derived SDF-1 could downregulate Prrx1, which induced the stemness, drug resistance, and increased expression of CXCR4 in HCC cells through the STAT3 pathway in vitro. In vivo, mice bearing tumours of Prrx1 low-expressing cells had significantly shorter survival. In xenograft tumours and clinical samples, loss of Prrx1 was negatively correlated with increased expression of CXCR4 in lung metastatic sites compared with that in the primary foci. Conclusions These findings demonstrate that decreased expression of Prrx1 stimulates SDF-1/CXCR4 signalling and contributes to organ colonisation with blood CTCs in HCC. STAT3 inhibition and specific blockade of CXCR4 have clinical potential as therapeutics for eliminating organ metastasis in advanced HCC.

scholarly journals An Exosomal Urinary miRNA Signature for Early Diagnosis of Renal Fibrosis in Lupus Nephritis

Cells ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 773 ◽  
Author(s):  
Solé ◽  
Moliné ◽  
Vidal ◽  
Ordi-Ros ◽  
Cortés-Hernández
Keyword(s):  
Lupus Nephritis ◽  
Renal Fibrosis ◽  
Target Genes ◽  
Early Stage ◽  
High Sensitivity ◽  
Increased Risk ◽  
Chronicity Index ◽  
Stage Renal Disease ◽  
End Stage

For lupus nephritis (LN) management, it is very important to detect fibrosis at an early stage. Urinary exosomal miRNAs profiling can be used as a potential multi-marker phenotyping tool to identify early fibrosis. We isolated and characterised urinary exosomes and cellular pellets from patients with biopsy-proven LN (n = 45) and healthy controls (n = 20). LN chronicity index (CI) correlated with urinary exosomal miR-21, miR-150, and miR-29c (r = 0.565, 0.840, −0.559, respectively). This miRNA profile distinguished low CI from moderate-high CI in LN patients with a high sensitivity and specificity (94.4% and 99.8%). Furthermore, this multimarker panel predicted an increased risk of progression to end-stage renal disease (ESRD). Pathway analysis identified VEGFA and SP1 as common target genes for the three miRNAs. Immunohistochemistry in LN renal biopsies revealed a significant increase of COL1A1 and COL4A1 correlated with renal chronicity. SP1 decreased significantly in the high-CI group (p = 0.002). VEGFA levels showed no differences. In vitro experiments suggest that these miRNA combinations promote renal fibrosis by increasing profibrotic molecules through SP1 and Smad3/TGFβ pathways. In conclusion, a urinary exosomal multimarker panel composed of miR-21, miR-150, and miR-29c provides a non-invasive method to detect early renal fibrosis and predict disease progression in LN.

scholarly journals Analysis of Circulating Tumour Cells in Early-Stage Uveal Melanoma: Evaluation of Tumour Marker Expression to Increase Capture

Cancers ◽  
2021 ◽  
Vol 13 (23) ◽  
pp. 5990
Author(s):  
Aaron B. Beasley ◽  
Timothy W. Isaacs ◽  
Tersia Vermeulen ◽  
James Freeman ◽  
Jean-Louis DeSousa ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Lines ◽  
Uveal Melanoma ◽  
Early Stage ◽  
Tumour Cells ◽  
Stem Cell Markers ◽  
Circulating Tumour Cells ◽  
Marker Panel ◽  
Cell Markers ◽  
Molecular Features

(1) Background: The stratification of uveal melanoma (UM) patients into prognostic groups is critical for patient management and for directing patients towards clinical trials. Current classification is based on clinicopathological and molecular features of the tumour. Analysis of circulating tumour cells (CTCs) has been proposed as a tool to avoid invasive biopsy of the primary tumour. However, the clinical utility of such liquid biopsy depends on the detection rate of CTCs. (2) Methods: The expression of melanoma, melanocyte, and stem cell markers was tested in a primary tissue microarray (TMA) and UM cell lines. Markers found to be highly expressed in primary UM were used to either immunomagnetically isolate or immunostain UM CTCs prior to treatment of the primary lesion. (3) Results: TMA and cell lines had heterogeneous expression of common melanoma, melanocyte, and stem cell markers. A multi-marker panel of immunomagnetic beads enabled isolation of CTCs in 37/43 (86%) patients with UM. Detection of three or more CTCs using the multi-marker panel, but not MCSP alone, was a significant predictor of shorter progression free (p = 0.040) and overall (p = 0.022) survival. (4) Conclusions: The multi-marker immunomagnetic isolation protocol enabled the detection of CTCs in most primary UM patients. Overall, our results suggest that a multi-marker approach could be a powerful tool for CTC separation for non-invasive prognostication of UM.

scholarly journals IFN-λ4 may contribute to HCV persistence by increasing ER stress and enhancing IRF1 signaling

2020 ◽  
Author(s):  
Olusegun O. Onabajo ◽  
Fang Wang ◽  
Mei-Hsuan Lee ◽  
Oscar Florez-Vargas ◽  
Adeola Obajemu ◽  
...  
Keyword(s):  
Er Stress ◽  
Early Stage ◽  
Positive Regulation ◽  
Antiproliferative Effects ◽  
Functional Relationships ◽  
Hepatic Cells ◽  
Increased Risk ◽  
Chronic Hepatitis C Virus ◽  
Chronic Hcv

ABSTRACTChronic hepatitis C virus (HCV) infection and cirrhosis are major risk factors for developing hepatocellular carcinoma (HCC). Genetic polymorphisms in the IFNL3/IFNL4 locus have been associated both with impaired clearance of HCV and protection from liver fibrosis, an early stage of cirrhosis. Here, we aimed to address the genetic and functional relationships between IFNL3/IFNL4 polymorphisms, HCV-related cirrhosis, and HCC risk. We evaluated associations between IFNL4 genotype, defined as the presence of rs368234815-dG or rs12979860-T alleles, with cirrhosis and HCC risk in patients with chronic HCV - 2,931 from Taiwan and 3,566 from Japan. We detected associations between IFNL4 genotype and decreased risk of cirrhosis (OR=0.66, 95%CI=0.46-0.93, P=0.018, in Taiwan), but increased risk of HCC (OR=1.28, 95%CI=1.07-1.52, P=0.0058, in Japan). In-vitro, IFN-λ4 expression increased ER stress, and enhanced positive regulation of IFN responses via IRF1 induction, which mediated antiproliferative effects in hepatic cells. Our data present novel IFN-λ4-associated pathways that may be contributing to HCV persistence and development of HCC.

scholarly journals Exosomes derived from Fusobacterium nucleatum-infected colorectal cancer cells facilitate tumour metastasis by selectively carrying miR-1246/92b-3p/27a-3p and CXCL16

Gut ◽  
2020 ◽  
pp. gutjnl-2020-321187
Author(s):  
Songhe Guo ◽  
Jun Chen ◽  
Fangfang Chen ◽  
Qiuyao Zeng ◽  
Wan-Li Liu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Tumour Progression ◽  
Proteome Analysis ◽  
Fusobacterium Nucleatum ◽  
Tumour Cells ◽  
Migration Ability ◽  
Tumour Metastasis ◽  
Infected Cells

ObjectiveExosomes released from tumour cells are packed with unique RNA and protein cargo, and they are emerging as an important mediator in the communication network that promotes tumour progression. The facultative intracellular bacterium Fusobacterium nucleatum (Fn) is an important colorectal cancer (CRC)-associated bacterium. To date, the function of exosomes from Fn-infected CRC cells has not been explored.DesignExosomes were isolated by sequential differential centrifugation and verified by transmission electron microscopy, NanoSight analysis and Western blotting. Given that exosomes have been shown to transport miRNAs and proteins to alter cellular functions, we performed miRNA sequencing and proteome analysis of exosomes from Fn-infected and non-infected cells. The biological role and mechanism of exosomes from Fn-infected cells in CRC tumour growth and liver metastasis were determined in vitro and in vivo.ResultsWe demonstrated that exosomes delivered miR-1246/92b-3p/27a-3p and CXCL16/RhoA/IL-8 from Fn-infected cells into non-infected cells to increase cell migration ability in vitro and promote tumour metastasis in vivo. Finally, both circulating exosomal miR-1246/92b-3p/27a-3p and CXCL16 levels were closely associated with Fn abundance and tumour stage in patients with CRC.ConclusionThis study suggests that Fn infection may stimulate tumour cells to generate miR-1246/92b-3p/27a-3p-rich and CXCL16/RhoA/IL-8 exosomes that are delivered to uninfected cells to promote prometastatic behaviours.

scholarly journals Circulating biomarkers in patients with glioblastoma

2019 ◽  
Vol 122 (3) ◽  
pp. 295-305 ◽  
Author(s):  
Juliana Müller Bark ◽  
Arutha Kulasinghe ◽  
Benjamin Chua ◽  
Bryan W. Day ◽  
Chamindie Punyadeera
Keyword(s):  
Extracellular Vesicles ◽  
Tumour Progression ◽  
Imaging Techniques ◽  
Therapy Response ◽  
Invasive Procedure ◽  
Tumour Cells ◽  
Circulating Tumour Cells ◽  
Circulating Biomarkers ◽  
Liquid Biopsies ◽  
The Central Nervous System

Abstract Gliomas are the most common tumours of the central nervous system and the most aggressive form is glioblastoma (GBM). Despite advances in treatment, patient survival remains low. GBM diagnosis typically relies on imaging techniques and postoperative pathological diagnosis; however, both procedures have their inherent limitations. Imaging modalities cannot differentiate tumour progression from treatment-related changes that mimic progression, known as pseudoprogression, which might lead to misinterpretation of therapy response and delay clinical interventions. In addition to imaging limitations, tissue biopsies are invasive and most of the time cannot be performed over the course of treatment to evaluate ‘real-time’ tumour dynamics. In an attempt to address these limitations, liquid biopsies have been proposed in the field. Blood sampling is a minimally invasive procedure for a patient to endure and could provide tumoural information to guide therapy. Tumours shed tumoural content, such as circulating tumour cells, cell-free nucleic acids, proteins and extracellular vesicles, into the circulation, and these biomarkers are reported to cross the blood–brain barrier. The use of liquid biopsies is emerging in the field of GBM. In this review, we aim to summarise the current literature on circulating biomarkers, namely circulating tumour cells, circulating tumour DNA and extracellular vesicles as potential non-invasively sampled biomarkers to manage the treatment of patients with GBM.

Integration of mRNA and microRNA profiles as prognostic and predictive markers in lung adenocarcinoma

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 7522-7522
Author(s):  
J. H. Strickler ◽  
W. Mostertz ◽  
W. Kim ◽  
K. Walters ◽  
M. Stevenson ◽  
...  
Keyword(s):  
High Risk ◽  
Lung Adenocarcinoma ◽  
Cell Lines ◽  
Cisplatin Resistance ◽  
Early Stage ◽  
Gene Signature ◽  
Discovery Cohort ◽  
Risk Of Recurrence ◽  
Increased Risk

7522 Background: Lung adenocarcinoma (ADC) is a distinct biologic entity with unique gene amplifications (Weir B, Nature 2008). Yet, comprehensive transcriptomic analysis, including microRNAs, specific to lung ADC are lacking. Methods: Using mRNA expression data from a discovery cohort of 154 patients with histologically proven early stage (I and II) lung ADC, signatures of oncogenic pathway and tumor microenvironment status were applied and further organized by hierarchical clustering to develop a metagene model. Further, using in vitro assays in a large cohort of lung ADC cell lines (n = 42) with corresponding mRNA and microRNA data, novel microRNAs associated with a poor prognosis and their relationship to cisplatin resistance was elucidated. Results: In the discovery cohort of 154 patients with early stage disease, activation of oncogenic pathways associated with wound healing (angiogenesis), chromosomal instability, and STAT signaling were associated with an increased risk of recurrence (p<0.001). Utilizing the extremes of survival to identify cohorts of patients as high and low risk phenotypes, using bayesian regression, a 100 gene signature (‘metagene') that captured the diversity of signaling pathways unique to patients at increased risk of recurrence was identified and validated in an independent cohort (n = 364) of lung ADC samples with 78.3% accuracy. Kaplan Meier survival analysis and multivariate analysis further confirmed the independent prognostic value of the 100 gene signature (p= 0.007). Using in vitro cell proliferation assays, predicted high risk lung ADC cell lines were identified as being more resistant to cisplatin therapy than those predicted to be low risk (p=0.001). In a novel manner, we also identified several microRNAs (miR-215, miR-98, miR- 643, let-7b, miR-665, miR-629) associated with a high risk of recurrence and more importantly cisplatin resistance. Conclusions: mRNA and microRNA profiles reflect unique aspects of individual tumors and may characterize histology-specific tumor heterogeneity in lung ADC, providing an opportunity to better characterize the oncogenic process and refine therapeutic options. No significant financial relationships to disclose.

scholarly journals Detection of circulating tumour cells in the breast cancer using CytoTrack system

2019 ◽  
Vol 65 (4) ◽  
pp. 31-36
Author(s):  
Anna Bogacz ◽  
Marlena Wolek ◽  
Aleksandra Górska ◽  
Ewa Leporowska ◽  
Danuta Procyk ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Metastatic Cancer ◽  
Tumour Cells ◽  
Circulating Tumour Cells ◽  
Scanning Microscope ◽  
Blood Samples ◽  
Flow Sorting ◽  
Method Of Assessment ◽  
Mcf 7

SummaryIntroduction: Plants are a rich source of healing substances. Cancer is a leading cause of death worldwide while breast cancer is the most common cancer among women. Circulating tumour cells (CTCs) are potential founder cells for metastasis. Therefore, their assessment may be used for monitoring of treatment as well as detecting cancer metastatis. Hence, it is suggested that the number of CTCs may be a valuable tumour biomarker during therapy.Objective: The purpose of this study was to detect CTCs in breast cancer and to validate the method of assessment of CTC count using CytoTrack CT11 technology.Methods: MCF-7 cells were sorted by a FACSARIA flow cytometer from blood samples derived from patients who have not been diagnosed with cancer. Identification and quantitative assessment of MCF-7 cells in blood samples were determined by flow sorting. Then, blood samples containing MCF-7 cells or without MCF-7 were scanned with the use of an automated fluorescence scanning microscope.Results: In in vitro model analysing the glass CytoDisc™ with stained MCF-7 cells, we noted the correlation between the amount of observed tumour cells and expected number of tumour cells. Moreover, coefficient of variation in case of the recovery rate of the assumed number of MCF-7 cells was 30%, 17%, 18% and 15%, respectively.Conclusion: Our study suggest that CTCs could be predictive factor in patients with metastatic cancer especially in breast cancer.

scholarly journals Identification of circulating tumour cells in early stage breast cancer patients using multi marker immunobead RT-PCR

2009 ◽  
Vol 2 (1) ◽  
pp. 24 ◽  
Author(s):  
Michael P Raynor ◽  
Sally-Anne Stephenson ◽  
Kenneth B Pittman ◽  
David CA Walsh ◽  
Michael A Henderson ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Patients ◽  
Early Stage ◽  
Tumour Cells ◽  
Early Stage Breast Cancer ◽  
Circulating Tumour Cells ◽  
Breast Cancer Patients ◽  
Rt Pcr
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