Metode za izolaciju cirkulirajućih tumorskih stanica

master's thesis

• Title (english): Methods for isolation circulating tumor cells
• Author: Antonija Hanžek
• Mentor: Jozsef Petrik (mentor)
• Committee member: Jozsef Petrik (predsjednik povjerenstva)
• Committee member: Karmela Barišić (član povjerenstva)
• Committee member: Sandra Šupraha Goreta (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2019-09-02, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Područje primjene cirkulirajućih tumorskih stanica (CTC) raste s pojavom novih tehnologija koje nude različite pristupe izolacije i
karakterizacije CTC-a iz krvi i kliničkom potvrdom CTC kao tumorskog markera. CTC su stanice porijeklom iz primarnog tumora
prisutne u perifernoj cirkulaciji te se koriste kao multifunkcionalan biomarker za odabir terapije, praćenje maligne bolesti i otkrivanje rezistencije na terapiju. CTC su vrlo rijetke stanice, stoga je potrebna osjetljiva metoda koja mora razlikovati CTC od ostalih stanica krvi. Cilj ovog diplomskog rada je prikazati metode za izolaciju cirkulirajućih tumorskih stanica, naglasiti prednosti i nedostatke pojedinih tehnologija te prikazati rezultate izolacije MDA-MB-231 tumorskih stanica karcinoma dojke dobivenih pomoću ScreenCell sustava. Referentna metoda izolacije i detekcije CTC-a je CellSearch sustav, imunomagnetska tehnologija koja se koristi za praćenje metastatskog karcinoma dojke, kolona i prostate. Temelji se na detekciji epitelnih biljega, stoga je primjenjiva za tumore epitelnog porijekla. Danas se razvijaju nove tehnologije koje ne zahtjevaju predanalitičku obradu uzoraka krvi, kao što su Magsweeper ili mikrofluidni uređaji, a omogućuju izolaciju CTC-a visoke specifičnosti i osjetljivosti. Interes raste za metode izolacije koje su neovisne o ekspresiji epitelnih biljega na CTC te uporabi antitijela, već se temelje na fizikalnim obilježjima stanica. Takva metoda je ScreenCell tehnologija koja omogućuje izolaciju CTC-a iz pune krvi na temelju mikrofiltracije. To je jednostavan uređaj koji omogućuje brzu i učinkovitu izolaciju, ali postoji rizik od začepljenja sustava. Pilot istraživanje s ciljem evaluacije ScreenCell sustava je pokušaj izolacije tumorskih stanica MDA-MB-231 u različitim koncentracijama iz pune krvi. Na temelju zapažanja i rezultata, možemo zaključiti da je kvaliteta uzorka i vrijeme od uzorkovanja do analize CTC-a ključan predananalitički kriterij za uspješnu izolaciju CTC-a iz pune krvi pomoću ScreenCell sustava. Poboljšanje postojećih tehnologija i novi inovativni pristupi potvrđuju klinički značaj CTC-a te ubrzavaju uvođenje tekuće biopsije i analize CTC-a u kliničku praksu.

Abstract (english)

The field of circulating tumor cells (CTCs) is expanding with the development of new technologies, all offering different approaches for isolation and characterization of CTCs from blood. CTCs are tumor-derived cells present in the peripheral circulation. They are used as multifunctional biomarker for selection of therapy, monitoring and follow up of patients and detection of resistance to therapy. CTCs are very rare cells, so detection requires a sensitive method able to differentiate CTCs from hematopoetic cells. The aim of this master thesis is to provide an overview of current methods for CTC isolation, discuss the advantages and challenges of each technology and present the results of isolation of MDA-MB-231 breast cancer cells from whole blood by using ScreenCell device. CellSearch® system, immunomagnetic technology is the reference method for the CTC isolation and detection. It is the only CTC technology approved by the FDA for monitoring patients with metastatic breast, prostate and colorectal cancer. This method uses specific epithelial biomarkers expressed on the cell surface to capture cells and is therefore suitable for tumors of epithelial origin. New technologies, such as Magsweeper or microfluidic devices, do not require pre-analytical processing of blood samples, but they
offer CTC isolation of high specificity and sensitivity. Strategies for CTC isolation based on biophysical properties have gained increasing popularity because they are “label free”. One example is ScreenCell®, a simple and innovative non-invasive technology for isolating CTCs from whole blood based on the microfiltration. It is a simple and inexpensive device that provides fast and efficient isolation, but as size-based technology, have risk of device clogging. A pilot study was conducted to evaluate the ScreenCell® system. The goal is to isolate the MDA-MB-231 breast cancer cells in different concentrations from whole blood. Based on the observations and results, the conclusion is that the key preanalyticals requirements for the successful CTC isolation from whole blood using ScreenCell system are the good quality of the blood sample and the time from sampling to CTC analysis. Improvements in existing technologies and a new innovative approaches are confirming the clinical utility of CTCs and accelerating the implementation of liquid biopsy and CTC analysis in clinical practice in oncology.

• Keywords: cirkulirajuće tumorske stanice; CTC; izolacija CTC; metode analize CTC; tekuća biopsija
• Keywords (english): circulating tumor cells; CTCs; CTC isolation; methods of CTC analysis; liquid biopsy
• Language: croatian
• URN:NBN: urn:nbn:hr:163:824878
• Study programme: Title: Medical biochemistry; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra medicinske biokemije (magistar/magistra medicinske biokemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2020-02-21 14:09:43