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Title:Razvoj humanega sferoidnega 3-dimenzionalnega celičnega modela za testiranje učinkovin za zdravljenje raka glave in vratu na principu zaviranja matičnih celic raka
Authors:ID Goričan, Larisa (Author)
ID Potočnik, Uroš (Mentor) More about this mentor... New window
ID Gole, Boris (Comentor)
Files:.pdf DOK_Gorican_Larisa_2020.pdf (3,81 MB)
MD5: EFF20C50BD7DA38FC8FD6283E07716C5
PID: 20.500.12556/dkum/bc3e33c8-bfe0-4c4b-9014-3e32a44a6293
 
Language:Slovenian
Work type:Doctoral dissertation
Typology:2.08 - Doctoral Dissertation
Organization:MF - Faculty of Medicine
Abstract:Za ponovitve tumorjev in razvoj oddaljenih metastaz so odgovorne na primarno zdravljenje odporne celice raka z izvornimi lastnostmi, ti. matične celice raka (MCR). MCR predstavljajo kritično podskupino rakastih celic, ki imajo sposobnost samoobnavljanja in diferenciacije ter ohranjajo tumor tudi po tem, ko primarno zdravljenje uniči večino preostalih celic tumorja. Omenjena spoznanja so vodila v iskanje terapij, ciljanih na MCR. Zaradi potrebe po terapijah ciljanih na MCR in njihove redkosti v primarnih tkivih je narasla potreba po modelu za obogatitev MCR, ki bi omogočil obogatitev MCR ter hkrati dopuščal proučevanje učinkovin z aktivnostjo usmerjeno proti MCR z analizami visokozmogljivih presejalnih testov (VPT). V ta namen smo v sklopu doktorske naloge iz komercialne epitelne celične linije PCKGV razvili humani sferoidni tri-dimenzionalni (3D) celični model (SM) obogaten za MCR, primeren za testiranje aktivnih učinkovin za zdravljenje PCKGV. Omenjeni sferoidni model združuje prednosti in zaobide omejitve dveh trenutno najpogosteje uporabljanih tehnik nasajanja sferoidov, kulture prosto-plavajočih sfer (oz. model tumorske sfere) in modela večceličnega tumorskega sferoida (MVTS). Podobno kot v modelu prostoplavajočih sfer, smo v SM uporabili medij za spodbujanje in ohranjanje matičnosti. Slednji za razliko od običajnega medija ne vsebuje seruma ploda goveda (»fetal bovine serum« - FBS), znanega sprožilca diferenciacije, ter je dopolnjen z več dejavniki, ki spodbujajo rast matičnih celic (osnovni fibroblastni rastni faktor, epidermalni rastni faktor, sestavljen komercialni pripravek, im. B-27). Za razvoj SM smo uporabili komercialno dostopno, stabilno celično linijo, ki za razliko od primarnih celičnih kultur, omogoča izvedbo dalj časa trajajočih eksperimentov, ponovitve posameznih eksperimentov, ne zahteva invazivnih metod pridobivanja, prav tako pa omogoča uporaba takšnih linij večjo primerljivost rezultatov med različnimi raziskovalnimi skupinami / eksperimenti. Podobno kot v MVTS smo za nasaditev SM uporabili mikrotitrsko ploščico z zaobljenim dnom in neoprijemljivo podlago, ter večjo koncentracijo celic nasadili na posamezno jamico in ploščico po nasaditvi zavrteli. Omenjeno je omogočilo generacijo celične mase in kasneje kompaktnega sferoida. Razviti SM smo primerjali z adherentno celično kulturo in MVTS, pri čemer je SM pokazal večjo obogatitev markerjev matičnosti, tako na transkripcijskem, kot proteinskem nivoju. Nadalje smo s pomočjo tretiranja sferoidov z diferenciacijskim medijem z dodano retinojsko kislino (»all-trans retionic acid« - ATRA) uspeli izbrati nabor specifičnih kazalcev za zaznavo MCR-diferenciacijskega učinka. Eksperiment z ATRA potrjuje občutljivost SM na delovanje učinkovin in njegovo uporabnost za detekcijo aktivnosti učinkovin z delovanjem proti MCR na treh različnih nivojih, tj. transkripcijskem, proteinskem in na nivoju celičnega cikla. V končnem eksperimentu s peptidnimi frakcijami kostnega mozga smo na SM uspeli identificirati šest frakcij s statističnim trendom zvišanja/ znižanja ekspresije enega ali več markerjev matičnosti. V doktorski nalogi razviti SM za obogatitev matičnih celic raka predstavlja kompromis med kompleksnostjo tumorja in preprostostjo protokola. Je primer preprostega modela sferoidov, ki ga tvori samo ena vrsta celic. Čeprav ne zajame kompleksnosti pravega tumorja, zagotavlja visoko obogatitev MCR ter zaradi enostavne vzpostavitve modela, uniformnosti, kratkega časa gojenja in prostorske ločitve sferoidov, ki omogoča dodajanje ene same aktivne sestavine posameznemu sferoidu, omogoča identifikacijo učinkovin z aktivnostjo proti MCR v analizah VPT. Takšen preprost model omogoča cenejšo identifikacijo prvih spojin in zožitev izbora učinkovin za nadaljnjo validacijo v bolj kompleksnih in dragih sferoidnih ali živalskih modelih.
Keywords:Ploščatocelični karcinom glave in vratu, matične celice raka, 3D / sferoidna kultura / model, visokozmogljivi presejalni testi, učinkovine z delovanjem proti matičnim celicam raka, retinojska kislina, markerji matičnosti
Place of publishing:Maribor
Year of publishing:2020
PID:20.500.12556/DKUM-76783 New window
COBISS.SI-ID:39755011 New window
NUK URN:URN:SI:UM:DK:80HPQG3C
Publication date in DKUM:27.11.2020
Views:1543
Downloads:167
Metadata:XML DC-XML DC-RDF
Categories:MF
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Licences

License:CC BY-NC-ND 4.0, Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
Link:http://creativecommons.org/licenses/by-nc-nd/4.0/
Description:The most restrictive Creative Commons license. This only allows people to download and share the work for no commercial gain and for no other purposes.
Licensing start date:06.07.2020

Secondary language

Language:English
Title:Development of a human spheroid 3-dimensional cell model for screening head and neck cancer active compounds with anti-cancer stem cell activity
Abstract:Responsible for tumor recurrences and the development of distant metastases are, primary treatment-resistant, the so-called cancer stem cells (CSCs). CSCs represent a critical subset of cancer cells that can self-renew and differentiate, preserving a tumor even after primary treatment destroyed most of the remaining tumor cells. These findings have led to the search for CSC-targeted therapies. Due to the need for CSC-targeted therapies and their rarity in primary tissues, there is a growing need for a CSC-enrichment model, allowing MCR enrichment while also allowing the study of active ingredients with anti-CSC activity through high-throughput screening (HTS) analyzes. To this end, we developed a human spheroid three-dimensional (3D) cell model (SM) enriched for CSCs, suitable for testing CSC-active ingredients for the treatment of HNSCC using the commercial epithelial HNSCC cell line. The developed SM combines the advantages and circumvents the limitations of the two currently most commonly used spheroid forming techniques, the culture of free-floating spheres (or tumorsphere model), and the multicellular tumor spheroid model (MCTS model). Similar to the culture of free-floating spheres, we used a stemness promoting medium. The latter, unlike the usual culture medium, is devoid of fetal bovine serum (FBS), a known differentiation trigger, and supplemented by several stem cell-promoting factors (basic fibroblast growth factor, epidermal growth factor, commercial B-27 supplement). We used a commercially available, stable cell line for the development of SM. Unlike primary cell cultures, it allows long-term and repeated experiments, does not require invasive obtaining methods, and also allows for greater comparability of results between different research groups/experiments. Similar to the MCTS model, we used an ultra-low attachment round-bottom microtiter plate and seeded a high concentration of cells per individual well, with subsequent centrifugation step after the seeding of the cells. This enabled the generation of cell mass and a compact spheroid afterward. When compared to the adherent cell line and MCTS model, the SM model showed higher stem markers enrichment, both at the transcriptional and protein level. Furthermore, by treating the spheroids with a differentiation medium with added all-trans retinoic acid (ATRA), we were able to select a set of specific CSC-differentiation markers. The experiment with ATRA confirmed the sensitivity of SM to the active substances effect and its usefulness in detecting anti-CSC substances activity at three different levels, i.e. transcriptional, protein, and at the cell cycle level. In a final experiment using bone marrow peptide fractions, we were able to identify six bone marrow peptide fractions with a statistical trend of increasing / decreasing gene expression of one or more stem markers in SM. In the doctoral thesis developed CSC-enriched SM represents a compromise between tumor complexity and protocol simplicity. It is an example of a simple spheroid model formed by only one type of cell. Although it does not capture the complexity of an actual tumor, it provides high CSC-enrichment. The developed SM is, due to easy setup, uniformity, short culture time, and spatial separation of spheroids, allowing the addition of a single active ingredient to a single spheroid, suitable for the identification of anti-CSC substances, using HTS analyzes. Such simple models, like ours, allow cheaper identification of the first compounds and narrow the selection of active ingredients for further validations in more complex and expensive spheroidal or animal models.
Keywords:Head and neck squamous cell carcinoma, Cancer stem cells, 3D / Spheroid culture/model, High-throughput screening, Anti-cancer stem cell compounds, ATRA, Stem cell marker


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