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.

Department of Laboratory Genetics University Hospital Kralovske Vinohrady Prague Czech Republic;

Department of Laboratory Genetics University Hospital Kralovske Vinohrady Prague Czech Republic; ; Department of Histology and Embryology Wroclaw Medical University Wroclaw Poland

Department of Pathology Masaryk's hospital in Usti nad Labem Krajska zdravotni Usti nad Labem Czech Republic;

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Msaouel P, Koutsilieris M. Diagnostic value of circulating tumor cell detection in bladder and urothelial cancer: Systematic review and meta-analysis. BMC Cancer. 2011;4:336. doi: 10.1186/1471-2407-11-336. PubMed DOI PMC

de Bono JS, Scher HI, Montgomery RB, et al. Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer. Clin Cancer Res. 2008;14:6302–9. doi: 10.1158/1078-0432.CCR-08-0872. PubMed DOI

Rink M, Chun FK, Minner S, et al. Detection of circulating tumour cells in peripheral blood of patients with advanced non-metastatic bladder cancer. BJU Int. 2011;107:1668–75. doi: 10.1111/j.1464-410X.2010.09562.x. PubMed DOI

Fidler IJ, Kripke ML. Metastasis results from preexisting variant cells within a malignant tumor. Science. 1977;197:893–5. PubMed

Fidler IJ. Metastasis: Guantitative analysis of distribution and fate of tumor emboli labeled with 125 I-5-iodo-2’-deoxyuridine. J. Natl. Cancer Inst. 1970;45:773–82. PubMed

Liotta LA, Saidel MG, Kleinerman J. The significance of hematogenous tumor cell clumps in the metastatic process. Cancer Res. 1976;36:889–94. PubMed

Friedlander TW, Fong L. The end of the beginning: Circulating tumor cells as a biomarker in castration-resistant prostate cancer. J Clin Oncol. 2014;32:1104–6. doi: 10.1200/JCO.2013.54.7307. . Epub 2014 Mar 10. PubMed DOI

Danila DC, Fleisher M, Scher HI. Circulating tumor cells as biomarkers in prostate cancer. Clin Cancer Res. 2011;17:3903–12. doi: 10.1158/1078-0432.CCR-10-2650. PubMed DOI PMC

Shaffer DR, Leversha MA, Danila DC, et al. Circulating tumor cell analysis in patients with progressive castration-resistant prostate cancer. Clin Cancer Res. 2007;13:2023–9. PubMed

Danila DC, Heller G, Gignac GA, et al. Circulating tumor cell number and prognosis in progressive castration-resistant prostate cancer. Clin Cancer Res. 2007;13:7053–8. PubMed

Goodman OB, Jr, Symanowski JT, Loudyi A, et al. Circulating tumor cells as a predictive biomarker in patients with hormone-sensitive prostate cancer. Clin Genitourin Cancer. 2011;9:31–8. PubMed

Goldkorn A, Ely B, Quinn DI, et al. Circulating tumor cell counts are prognostic of overall survival in Southwest Oncology Group trial S0421: A phase III trial of docetaxel with or without atrasentan for meta-static castration-resistant prostate cancer. J Clin Oncol. 2014;32:1136–42. doi: 10.1200/JCO.2013.51.7417. PubMed DOI PMC

Scher HI, Jia X, de Bono JS, et al. Circulating tumour cells as prognostic markers in progressive, castration-resistant prostate cancer: A reanalysis of IMMC38 trial data. Lancet Oncol. 2009;10:233–9. doi: 10.1016/S1470-2045(08)70340-1. PubMed DOI PMC

Xu T, Lu B, Tai YC, et al. A cancer detection platform which measures telomerase activity from live circulating tumor cells captured on a microfilter. Cancer Res. 2010;70:6420–6. PubMed PMC

Lin HK, Zheng S, Wiliams AJ, et al. Portable filter-based microdevice for detection and characterization of circulating tumor cells. Clin Cancer Res. 2010;16:1–8. PubMed PMC

Danila DC, Anand A, Sung CC, et al. TMPRSS2-ERG status in circulating tumor cells as a predictive biomarker of sensitivity in castrationresistantprostate cancer patients treated with abiraterone acetate. Eur Urol. 2011;60:897–904. PubMed PMC

Jiang Y, Palma JF, Agus DB, et al. Detection of androgen receptor mutations in circulating tumor cells in castration-resistant prostate cancer. Clin Chem. 2010;56:1492–5. doi: 10.1373/clinchem.2010.143297. PubMed DOI

Scher HI, Heller G, Molina A, et al. Evaluation of circulating tumor cell (CTC) enumeration as an efficacy response biomarker of overall survival (OS) in metastatic castration-resistant prostate cancer (mCRPC): Planned final analysis (FA) of COU-AA-301, a randomized double-blind, placebo-controlled phase III study of abiraterone acetate (AA) plus low-dose prednisone (P) post docetaxel. J Clin Oncol. 2011;29:293s.

Prentice RL. Surrogate endpoints in clinical trials: definition and operational criteria. Stat Med. 1989;8:431–40. PubMed

Allard WJ, Matera J, Miller MC, et al. Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin Cancer Res. 2004;10:6897–904. PubMed

Pantel K, Den´eve E, Nocca D, et al. Circulating epitelial cells in patients with benign colon diseases. Clin Chem. 2012;58:936–40. PubMed

Mikolajczyk L, Millar S, Tsinberg P, et al. Detection of EpCAM-negative and cytokeratin-negative circulating tumor cells in peripheral blood. J Oncol. 2011;252361 doi: 10.1155/2011/252361. . Epub 2011 Apr 19. PubMed DOI PMC

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