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Uvod: Serozni rak jajčnika visokega visokega gradusa (ang. high grade serous ovarian cancer, HGSC) je najpogostejši in najbolj agresiven histološki tip raka jajčnika. Običajno ga odkrijemo v napredovalem stadiju bolezni z izlivi v telesne votline, ki so pogosto prvi znak bolezni. Kljub napredku v diagnostiki in zdravljenju, se preživetje bolnic v zadnjih letih ni izboljšalo. Bioloških označevalcev s katerimi bi lahko zanesljivo napovedali potek bolezni in odgovor na zdravljenje zaenkrat še nimamo. V doktorski nalogi smo želeli preučiti tumorske sferoide ter imunske celice (ki sestavljajo tumorsko mikrookolje v izlivih bolnic s HGSC) kot potencialne napovedne dejavnike poteka bolezni, ovrednotiti izražanje PD-1 (receptor celične smrti 1) in PD-L1 (ligand PD-1) na imunskih in tumorskih celicah ter določiti morfološke, imunofenotipske in molekularne značilnosti tumorskih celic za takojšno in zanesljivo citopatološko diagnostiko. Cilji: V sklopu raziskave smo zastavili tri glavne cilje: 1) kvantificirati različne imunske celice v izlivih bolnic s HGSC in izražanje PD-1 in PD-L1 ter opredeliti njihovo napovedno vrednost preživetja 2) opisati tumorske sferoide v izlivih, s poudarkom na njihovih morfoloških značilnostih, sestavi celic (tumorskih, imunskih in endotelijskih celic) ter izražanju PD-1 in PD-L1 3) oceniti zanesljivost citopatologije za določanje imunofenotipskih značilnosti tumorskih celic in mutacij BRCA1/2 iz izlivov HGSC. Metode: V raziskavo smo vključili bolnice s histološko potrjenim HGSC in malignim izlivom. S pretočno citometrijo smo določili prisotnost (delež) različnih imunskih celic v izlivih: limfociti T (CD3+ in njihovih podskupin CD4+, CD8+), regulatorni limfociti T (Treg), NK T celice, CD103+ limfociti T, limfociti B, NK celice, makrofagi in dendritične celice ter izražanje PD-1 in PD-L1. Nizek in visok delež imunskih celic smo opredelili na osnovi mediane. Poleg tega smo v ramazih pobarvanih po Giemsi ocenili velikost in gostoto tumorskih sferoidov, določili mejno vrednost za razlikovanje bolnic z nizko in visoko gostoto tumorskih sferoidov, ki lahko napove boljše oz. slabše preživetje. Na citoblokih smo z imunocitokemijo (ICK) določili celično sestavo sferoidov. Poleg tega smo opazovali tudi izražanje PD-1 in PD-L1. Rezultate pretočno-citometrične analize imunskih celic in ICK analiz sferoidov smo uporabili za izračun morebitne korelacije s preživetjem brez napredovanja bolezni (ang. progression-free survival, PFS) ter celotnim preživetjem (ang. overall survival, OS). Na citospinih in citoblokih smo izvedli ICK barvanje za označevalce, značilne za HGSC histotip: PAX8, WT1, P53, P16 in Ki-67. S sekvenciranjem nove generacije (ang. next-generation sequencing, NGS) smo določili prisotnost zarodnih ali somatskih mutacij BRCA1/2 v izlivih. Rezultate ICK in NGS smo primerjali z rezultati imunohistokemije (IHK) in NGS iz vzorcev tkiva primarnega tumorja ter vzorcev krvi. Rezultati: Rezultati pretočne citometrije so pokazali, da so limfociti T prevladujoče imunske celice v izlivih (mediana 51%), medtem ko je drugih imunskih celic bistveno manj (mediana ?10%). PD-1 je bil izražen predvsem na limfocitih T (mediana 20%), manjše izražanje pa smo opazili tudi na drugih imunskih celicah. Izražanje PD-L1 nismo zaznani. Bolnice z visokim deležem CD103+ T limfocitov, NK celic, dendritičnih celic in PD-1+ Treg celic so imele statistično značilno boljši PFS in OS kot bolnice z nizkim deležem imunskih celic. Bolnice z visokim deležem CD8+ limfocitov T, makrofagov in PD1+ NK celic ter nizkim deležem CD4+ limfocitov T pa so imele le statistično značilno boljši OS. Poleg tega smo v razmazih pobarvanih po Giemsi opazili majhne, srednje velike in velike sferoide, vendar njihova velikost ni bila povezana s preživetjem. Določili smo 10% mejno vrednost gostote sferoidov, ki je bila povezna s statistično značilno boljšim oz. slabšim PFS in OS. Preučevali smo tudi sferoide v citoblokih, vendar le pri vzorcih, ki so vsebovali velike in številne sferoide. Rezultati ICK so potrdili, da so poleg tumorskih celic v sferoidih tudi limfociti, makrofagi in endotelijske celice. PD-1 je bil v sferoidih izražen predvsem na limfocitih, medtem ko smo opazili izražanje PD-L1 tako na tumorskih celicah kot tudi na imunskih celicah. Korelacije s preživetjem bolnic nismo uspeli pokazati. Rezultati ICC/IHC za PAX8, WT1, P53 in P16 so pokazali zelo dobro korelacijo med citospini, citobloki in tkivnimi bloki (? > 0,75), vendar korelacija za Ki-67 je bila slabša (? < 0,26 p < 0,001). Dodatno smo potrdili 100% korelacijo zarodnih mutacij BRCA1/2, ki so bile določene iz izlivov in primarnega tumorja, vendar to ni bilo opaženo pri somatskih mutacijah. Zaključek: Rezultati doktorske disertacije so pokazali, da imajo vzorci izlivov bolnic s HGSC velik potencial za preučevanje in razvoj novih prognostičnih označevalcev ter so enakovredni tkivnim vzorcem primarnega tumorja za postavitev zanesljive diagnoze HGSC. Introduction: High-grade serous ovarian cancer (HGSC) is the most common and aggressive histotype of ovarian cancer, often associated with effusions at presentation. Despite advancements in diagnosis and treatment, survival rates for HGSC patients have not improved in recent years. Currently, there is a need for new biological markers that can reliably predict the course of the disease and response to treatment. One potential source of such markers is the effusion that often accompanies HGSC. HGSC effusions contain tumor spheroids, dissociated tumor cells, and immune cells, and by studying the features and prognostic significance of these components, we may be able to identify new markers that can aid in the diagnosis and treatment of HGSC. However, there is a lack of quantitative data about the immune cells in HGSC effusions and data on their clinical importance. Furthermore, no study has yet investigated the presence of immune cells and endothelial cells in tumor spheroids and the expression of PD-1 and PD-L1. In general, cytopathology has also been underused as a modality to identify immunophenotypic and molecular features of HGSC. Objective: Three main objectives were established: 1) to quantify different immune cells in HGSC effusions, the expression of programmed death receptor-1 (PD-1) and its ligand (PD-L1) in both tumor and immune cells, and to calculate their correlation with patient progression-free survival (PFS) and overall survival (OS) 2) to study tumor spheroids within effusions with an emphasis on their morphological features, cell composition (tumor cells, immune cells, and endothelial cells), and PD-1 and PD-L1 expression, and 3) to evaluate the accuracy of cytopathology to identify immunophenotypic features of tumor cells and BRCA1/2 mutations from HGSC effusions. Methods: Patients with histologically confirmed HGSC and malignant effusions were included in the study. Flow cytometry analysis was performed to detect percentages of different immune cells in the effusions: T lymphocytes (CD3+ and their subsets CD4+, CD8+, regulatory T cells (Tregs), NK T (NKT) cells, and CD103+ T lymphocytes), B lymphocytes, NK cells, macrophages, and dendritic cells (DCs), as well as their expression of PD-1 and PD-L1. The cut-off of low and high percentages of different immune cells was determined by the median of the variables. Giemsa smears were used to evaluate the size and density of tumor spheroids in effusions, and a cut-off value for low and high density of tumor spheroids that could predict better/poorer survival outcomes was determined. Cytoblock immunocytochemistry (ICC) was used to describe the cell composition of the spheroids to establish if immune cells (T and B lymphocytes, and macrophages) and endothelial cells composed tumor spheroids together with the tumor cells, as well as PD-1 and PD-L1 expression within the spheroids. Flow-cytometric results on immune cells and morphological and immunophenotypic features of spheroids were used to calculate if there is any correlation with PFS and OS. ICC staining for PAX8, WT1, P53, P16, and Ki-67 was performed on cytospins and cytoblocks and compared with the results in tissue blocks of the primary tumor. Next-generation sequencing (NGS) was used to detect germline/somatic BRCA1/2 mutations in the effusions. Both ICC and NGS results were compared with immunohistochemistry (IHC) and NGS results from tissue blocks of the primary tumor and blood samples. Results: Flow-cytometric results showed that T lymphocytes were the predominant cells in the effusions (median 51%), while the presence of other immune cells was much lower (median 䁤10%). PD-1 was mainly expressed on T lymphocytes (median 20%), and lower expression was observed on other immune cells. PD-L1 expression was not detected in immune cells at all. Patients with high percentages of CD103+ T lymphocytes, NK cells, DCs, and PD-1+ Tregs were significantly associated with better PFS and OS than patients with low percentages of those immune cells, while high percentages of CD8+ T subsets, macrophages, and PD1+ NK cells, and low percentages of CD4+ T subsets associated with significantly better OS. The presence of small, medium, and large spheroids was observed in Giemsa smears but the size of the spheroids did not correlate with patients’ survival. A 10% cut-off for spheroid density in Giemsa smears was established, which significantly predicted the PFS and OS of the patients. The presence of spheroids was also observed in cytoblocks, but only in cases of big-sized and high-density spheroids (25/43 cytoblocks). ICC results confirmed the presence of spheroid-associated lymphocytes, macrophages, and endothelial cells in the spheroids. PD-1 was mainly expressed in spheroid-associated lymphocytes, while PD-L1 expression was seen in both spheroid-associated tumor cells and immune cells. No correlation with patient survival was observed. ICC/IHC results for PAX8, WT1, P53, and P16 showed good reliability between cytospins, cytoblocks, and tissue blocks (α > 0.75), whereas poor reliability and significant differences were observed for Ki-67 between effusions and tissue blocks (α < 0.26 p < 0.001). A 100% concordance was confirmed for the germline BRCA1/2 mutations between effusions and tissue blocks, but only 14% concordance for the somatic mutations. Conclusions: The results from the doctoral thesis highlight the potential of HGSC effusions to describe tumor and immune cells, explore their prognostic significance and the possibility of evolving new prognostic markers, as well as the importance of cytopathology to confirm HGSC histotype and BRCA1/2 mutations from effusions as reliable as from the primary tumor tissue.

Bisfenoli (BP) so skupina industrijskih kemikalij, ki se uporabljajo pri proizvodnji plastike in epoksi smol. Iz njih izdelujejo najrazličnejše izdelke kot so obloge za konzerve, stekleničke za dojenčke, igrače, papir za račune in mnoge druge, ki se uporabljajo v našem vsakdanjem življenju in jih posledično zasledimo v okolju. Zaradi kopičenja podatkov o nevarnih lastnostih in škodljivih vplivih najpogostejšega izmed BP-jev, bisfenola A (BPA), predvsem motenj endokrinega sistema, je bila njegova uporaba v Evropski uniji postopoma omejena oziroma celo prepovedana, da bi zaščitili zdravje ljudi in okolje. Prepoved uporabe BPA je spodbudila razvoj domnevno varnejših analogov, za katere pa v literaturi ni veliko toksikoloških podatkov. Tako smo v magistrski nalogi proučevali citotoksično in genotoksično aktivnost BPA in njegovih analogov: bisfenol AF (BPAF), bisfenol AP (BPAP), bisfenol C (BPC), bisfenol FL (BPFL), bisfenol S (BPS) na jetrnem in vitro 3D celičnem modelu. Sferoide smo pripravili s prisilno plavajočo metodo, vpliv na živost celic sferoidov po izpostavitvi BP-jem smo merili s testom MTS. S pomočjo mikroskopije, smo spremljali površino, obliko in kompaktnost sferoidov, s testom komet pa smo preučevali genotoksičnost v časovnih izpostavitvah 24 in 96 ur. Statistično značilen vpliv na živost celic v sferoidu po 24 urah so imeli BPFL > BPAF = BPAP, po 96 urah pa BPFL > BPC = BPAP > BPA = BPAF. Mikroskopske analize so pokazale, da sta imela največji vpliv na rast in spremembo površine ter kompaktnosti sferoidov BPC in BPFL po 96-urni izpostavitvi. S testom komet smo pokazali, da sta bila po 24 urah izpostavitve najbolj genotoksična BPAF in BPS (⡥ 10 μM), sledil je BPAP ( ⡥ 40 μM). Po 96 urah izpostavitve je BPAP povzročil poškodbe DNA pri koncentraciji ⡥ 0,1 μM, pri vseh ostalih BP-jih smo poškodbe DNA zaznali pri višjih koncentracijah. Povzamemo lahko, da so za analoge BPA potrebne dodatne študije mehanizmov delovanja za pripravo ustrezne ocene tveganja, saj je toksična aktivnost za posamezne analoge, kljub podobni kemijski strukturi, različna. Bisphenols (BPs) are a group of industrial chemicals used in the manufacture of plastics and epoxy resin. They are found in various products, including the inner linings of cans, baby bottles, toys, thermal paper, and others used in our daily life. Therefore, BPs are present in the environment. The most widely used BP is bisphenol A (BPA), on which most studies have been conducted showing its adverse effects on human health, especially its endocrine disrupting activity. Therefore, the European Union began to restrict and eventually ban the use of BPA to protect human health and the environment. The ban has led to the development of supposedly safer BPA analogues, for which few toxicological data are available. In the master thesis, we investigated the cytotoxic and genotoxic activity of BPA and its analogues (BPAF, BPAP, BPC, BPFL, BPS) in a liver in vitro 3D cell model. In this study, we prepared spheroids by the forced floating method and measured cell viability after exposure to BPs using the MTS assay. We measured the surface area and observed the shape and compactness of the spheroids with microscopy and studied the genotoxic effects with the comet assay at 24 and 96-hour exposure. After 24-hour exposure to BPs, BPFL > BPAF = BPAP statistically significantly decreased the viability of HepG2 and after 96 hours, we observed a similar effect on cell viability in BPFL > BPC = BPAP > BPA = BPAF. BPC and BPFL had the greatest effect on morphological changes in growth, surface area, compactness, and shape of the spheroids that were evident at 96-hour exposure. Of the six BPs tested by the comet assay, BPAF and BPS (⡥ 10 μM) and BPAP (⡥ 40 μM) exhibited stronger genotoxic potential than BPA, BPFL, and BPC after 24 hours of exposure. After 96 hours of exposure, BPAP was the most genotoxic causing/inducing DNA damage at ⡥1 µM. All others BPs studied statistically significantly increased DNA damage at higher concentrations. To summarize, despite their similar chemical structure, the toxic activity of the analogues is not the same, and therefore further studies on the BPA analogues and their molecular activity are needed to provide/make accurate risk assessment.

Cilj istraživanja: Odrediti zastupljenost autofagije u neurosferama dobivenih iz glioblastoma te utvrditi ima li ulogu vremenski period uzgoja neurosfera na pojavnost autofagije. Nacrt studije: Rad je koncipiran kao in vitro studija (kontrolirani pokus). Materijal i metode: Stanična linija D54 nakon uzgoja i kultivacije u inkubatoru nasađena je na mikrotitarsku pločicu sa tzv. V dnom kako bi se omogućilo formiranje sfera. Sferoidi su tako uzgajani kroz 5, 10 i 15 dana. Po isteku vremena stanice su tretirane tripsinom kako bi se razdvojile na pojedinačne stanice koje će se detektirati protočnim citometrom. Za detekciju autofagije obojane su acridin orange fluorescentnom bojom. Izmjerena fluorescencija proporcionalna je razini autofagije u stanicama. Uzorci su analizirani u duplikatu na BD FACScanto II protočnom citometru te su rezultati obrađeni programom FlowJo. Rezultati: Duljim uzgojem sferoida razina autofagije opada. Dokazana je statistički značajna razlika međusobnim uspoređivanjem sferoida formiranih tijekom 5, 10 i 15 dana. Zaključak: Regulatori autofagije su raspodjela hranjivih tvari i kisika koja u 3D staničnoj kulturi više sliči onoj u ljudskom organizmu nego što je slučaj u 2D staničnoj kulturi. Razina autofagije smanjuje se od sferoida starih 5 dana prema sferoidima starim 15 dana jer rastom sferoida raste i zona mrtvih stanica.

Določitev genotoksične aktivnosti kemikalij je pomemben del ocene tveganja za ljudi. Za testiranje genotoksičnosti se najpogosteje uporabljajo in vitro jetrne celične linije, saj imajo ohranjeno določeno stopnjo presnovne aktivnosti ksenobiotikov. Vendar imajo tradicionalni dvodimenzionalni (2D) in vitro celični modeli številne pomanjkljivosti, zato v zadnjih letih raziskovalci razvijajo številne in vitro tridimenzionalne (3D) celične modele, ki zaradi svoje večje kompleksnosti bolje posnemajo in vivo pogoje. Prednosti 3D celičnih modelov pred 2D celičnimi modeli so povrnitev številnih bioloških funkcij, kot so medcelične povezave in povezave celica ¬– zunajcelični matriks, kar vodi do boljše diferenciacije celic. V magistrskem delu smo želeli ovrednotiti občutljivost in specifičnost novorazvitega jetrnega 3D in vitro celičnega modela. V prvem delu smo okarakterizirali in optimizirali pogoje za razvoj in vitro 3D celičnega modela (sferoidi), ki smo ga pripravili iz celične linije humanega hepatocelularnega karcinoma (HepG2 celice). Sferoidi, ki smo jih pripravili s prisilno plavajočo metodo, so bili kompaktni ter enakomerni v obliki, velikosti in rasti. V drugem delu smo sferoide pri starosti 72 ur izpostavili različnim koncentracijam dveh posredno delujočih genotoksičnih spojin, in sicer benzo(a)pirenu (BaP) in 2-amino-1-metil-6-fenilimidazo(4,5-b)piridinu (PhIP) za 24 in 72 ur. Proliferacijo celic, celični cikel ter nastanek dvoverižnih prelomov DNA smo spremljali s pretočno citometrijo. Rezultati kažejo, da spojina BaP vpliva tako na celični cikel kot na proliferacijo celic, medtem ko pri spojini PhIP nismo zaznali razlik. Z analizo prisotnosti dvoverižnih prelomov DNA smo pokazali, da BaP povzroči poškodbe DNA tako po 24-urni kot po 72-urni izpostavitvi že pri koncentraciji 䁥 1 μM. PhIP po 24-urni izpostavitvi povzroči poškodbe DNA pri koncentraciji 200 μM, po 72-urni izpostavitvi pa že pri koncentraciji 䁥 25 μM. Genotoksične učinke BaP in PhIP (z izjemo kratkotrajne izpostavitve sferoidov spojini PhIP) smo zaznali pri nižjih koncentracijah, kot so bili ti zaznani v študijah na in vitro 2D celičnem modelu. Zaključimo lahko, da jetrni in vitro 3D celični model kaže izboljšano občutljivost za zaznavanje citotoksičnega in genotoksičnega delovanja posredno delujočih genotoksičnih spojin. Z našo raziskavo smo tako korak bližje k premostitvi vrzeli med in vivo raziskavami in testi, ki temeljijo na in vitro 2D celičnih modelih. Determination of genotoxic activity of chemicals is an essential part of human risk assessment. For genotoxicity assessment, in vitro hepatic cell lines are most commonly used as they have retained certain level of xenobiotic metabolic activity. However, the traditional two-dimensional (2D) in vitro cell models have numerous limitations. Therefore scientists have in the last few years developed many in vitro three-dimensional (3D) cell models, which due to their higher complexity better imitate in vivo conditions. The advantage of 3D cell models compared to 2D monolayer models is the recovery of several biological functions such as intercellular interactions and cell – extracellular matrix interactions, which lead to better cell differentiation. The objective of this master's thesis was to evaluate sensitivity and specificity of newly developed 3D in vitro cell model. In the first part we optimized the conditions for development of in vitro 3D cell model (spheroids) which was formed from human hepatocellular carcinoma (HepG2) cell line. The spheroids were formed by the forced floating method. They were compact and of uniform shape, size and growth. In the second part, 72 hours old spheroids were exposed to graded concentrations of two indirect acting genotoxic compounds, benzo(a)pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) for 24 and 72 hours. Cell proliferation, cell cycle and induction of DNA double-strand breaks were asessed by flow citometry. The results showed that BaP compound affected both cell cycle and cell proliferation, although no differences were noticed for PhIP. By analysing the presence of DNA double-strand breaks it was shown that BaP caused DNA damage after 24- and 72-hour exposure at concentration 䁥 1 μM. PhIP caused DNA damage at 200 μM after 24-hour exposure and at 䁥 25 μM after 72-hour exposure. Genotoxic effects of BaP and PhIP were (with the exception of short term exposure of spheroids to PhIP) detected at lower concentrations as we reported for in vitro 2D monolayer model. We can conclude that hepatic 3D cell model showed improved sensitivity for detection of cytotoxic and genotoxic activity of indirect acting genotoxic compounds. This research presents a step forward to bridge the gap between in vivo research and tests that are based on 2D monolayer models.

Za vnos plazmidne DNA (pDNA) v celice ali tkiva lahko uporabimo različne dostavne metode. Ena izmed teh je elektroporacija, oz. genski elektroprenos (GEP) oz. elektrogenska terapija. Pri GEP celice izpostavimo zunanjemu električnemu polju z določeno jakostjo in trajanjem ter pri tem vplivamo na prepustnost membrane. Pri zdravljenju rakavih sprememb se pri elektrogenski terapiji uporabljajo predvsem geni, ki vplivajo na imunski odgovor ali angiogenezo tumorja. Pri predhodnjih poskusih smo na mišjem modelu z GEP uspeli regresijo tumorja doseči tudi pri kontrolni skupini, kjer smo uporabili le plazmidno DNA, brez terapevtskega gena. Upočasnjeno rast in regresijo tumorja po GEP plazmidne DNA brez terapevskega gena so opazili tudi v drugih raziskavah. Ker plazmidna DNA brez terapevtskega gena ne nosi zapisa za terapevtski protein, do regresije tumorja ni prišlo zaradi terapevtskega učinka. Predvidevamo, da plazmidna DNA z GEP vstopi v celico z endocitozo ali skozi pore v membrani, ki nastanejo po izpostavitvi celice električnim pulzom. Celice citosolno DNA zaznajo s pomočjo citosolnih senzorjev DNA v celici. Po vstopu pDNA v celico se ob njeni vezavi na senzorje DNA sproži kaskada reakcij znotrajceličnega signaliziranja, ki vodi do prepisa genov, ki kodirajo vnetne citokine in interferone. Poleg prepisovanja genov lahko pride tudi do sprožitve različnih oblik celične smrti in sicer piroptoze, nekroptoze in apotoze. Prisotnost senzorjev DNA so dokazali v imunskih in v nekaterih neimunskih celicah. For the entry of pDNA into the cells and tissues we can use different delivery methodes. One of them is electroporation, eg. gene electrotransfer (GET), eg. electrogene therapy in which the cells are exposed to electric pulses with specific intensities and durations in order to increase the permeability of the cell membrane. In cancer electrogene therapy, genes that affect the immune system or angioginesis of the tumor are used. In our laboratory, we noticed a regression of tumors after electrogene therapy of mice in control groups, where we used only pDNA without any therapeutic gene. In other laboratories tumor growth delay and regression of tumors after GET of pDNA was also determined. Because pDNA has no terapeutic genes, regression is not due to therapeutic effect. It was predicted that after GET, pDNA enters the cell with endocytosis or through pores, that are established in the membrane after exposure to the electric field. After the entery of pDNA into the cells, the DNA sensors can bind to it and this leads to dfferent signal pathways, resulting in transcription of genes coding proimmlamatory cytokines, interferons and can also lead to different triggered cell deaths, such as piroptosis and necroptosis. The presence of DNA sensors was shown in immune cells and some of the non-immune cells.