Učinki selektivnih inhibitorjev metaloproteaz MMP-2 in MMP-9 na invazijo celic glioblastoma

Osolin, Manca (author), Breznik, Barbara (mentor)

Glioblastom je najpogostejši in najagresivnejši primarni možganski tumor. Zdravljenje poteka s kirurško odstranitvijo tumorja, radioterapijo in kemoterapijo s temozolomidom. Kljub agresivnemu zdravljenju mediana preživetja bolnikov z glioblastomom znaša zgolj 15 mesecev po diagnozi, v vseh primerih pa pride do ponovnega pojava bolezni. Tumorsko tkivo glioblastoma je sestavljeno iz heterogene populacije celic v različnih stopnjah diferenciacije. Nizka učinkovitost zdravljenja glioblastoma je predvsem posledica invazije glioblastomskih celic v parenhim možganov, zaradi česar je popolna kirurška odstranitev rakavih celic nemogoča. Pomembno vlogo pri invaziji glioblastomskih celic imata metaloproteazi MMP-2 in MMP-9, ki razgrajujeta komponente zunajceličnega matriksa možganov. Namen magistrskega dela je bil analizirati izražanje in izločanje MMP-2 in MMP-9 v različnih celičnih in organoidnih modelih glioblastoma ter ugotoviti njuno vlogo v invaziji glioblastomskih celic z uporabo selektivnih inhibitorjev MMP. Vse z namenom postaviti in vitro modele za preučevanje vloge MMP v glioblastomu ter testiranje aktivnostnih sond in inhibitorjev MMP-2 in MMP-9. Ugotovili smo, da celični liniji diferenciranih glioblastomskih celic ter glioblastomski organoidi izražajo metaloproteazi MMP-2 in MMP-9, medtem ko celični liniji glioblastomskih matičnih celic ne izražata MMP-2 in MMP-9. Dokazali smo, da je v lizatu organoidov NIB309 prisotne precej manj MMP-9 kot v supernatantu, medtem ko MMP-2 ni prisotna ne v lizatu ne v supernatantu organoidov NIB309. Z metodo želatinske cimografije smo ugotovili, da sta v celičnih linijah diferenciranih glioblastomskih celic U87 in NIB140 prisotni pro- in aktivna oblika MMP-2 in MMP-9, medtem ko sta v organoidih NIB295 in NIB298 prisotni zgolj pro-obliki MMP-2 in MMP-9. S pomočjo testa invazije iz sferoidov v matrigelu smo pokazali, da širokospektralni inhibitor MMP batimastat zmanjša invazijo glioblastomskih celic iz sferoidov U87 ter ne vpliva na invazijo glioblastomskih celic iz sferoidov NIB140. Ugotovili smo tudi, da selektivna inhibitorja MMP-2 in MMP-9, SB-3CT in RXP500.1, ne vplivata na invazijo glioblastomskih celic iz sferoidov U87 in NIB140. Učinkovitosti aktivnostnih sond NASA in BBCH, ki selektivno označita aktivno obliko MMP-2 in MMP-9, nismo uspeli dokazati na glioblastomski celični liniji U87. Pokazali smo, da glioblastomske celične linije in organoidi v različnih nivojih izražajo in izločajo MMP-2 in MMP-9. V diferenciranih glioblastomskih celicah smo dokazali prisotnost aktivnih MMP-2 in MMP-9. Če v teh celicah inhibiramo MMP-2 in MMP-9, najverjetneje pride do kompenzacije njihove aktivnosti. Na primeru MMP-2 in MMP-9 smo dokazali, da je za glioblastom značilna intertumorska heterogenost, zato je zelo pomembno personalizirano zdravljenje. Poleg tega smo postavili in karakterizirali in vitro celične modele, na katerih lahko testiramo nove inhibitorje in sonde za detekcijo MMP-2 in MMP-9.

• Language: Slovenian
• Keywords: glioblastom, MMP-2, MMP-9, invazija, selektivni inhibitorji
• Work type: Master's thesis/paper
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2024
• PID: 20.500.12556/RUL-158680
• Publication date in RUL: 19.06.2024

Secondary language

• Language: English
• Title: Effects of selective inhibitors of MMP-2 and MMP-9 metalloproteases on glioblastoma cell invasion

Abstract

Glioblastoma is the most common and most aggressive primary brain tumour. Treatment includes surgical removal of the tumour, radiotherapy and chemotherapy with temozolomide. Despite aggressive treatment, the median survival of patients with glioblastoma is only 15 months after diagnosis, and in all cases the disease recurs. Glioblastoma tumour tissue is composed of a heterogeneous population of cells in different stages of differentiation. The low efficacy of glioblastoma treatment is mainly due to the invasion of glioblastoma cells into the brain parenchyma, which makes complete surgical removal of the cancer cells impossible. The MMP-2 and MMP-9 metalloproteases, which degrade components of the extracellular matrix of the brain, play an important role in glioblastoma cell invasion. The aim of this MSc thesis was to analyse the expression and secretion of MMP-2 and MMP-9 in different glioblastoma cell and organoid models and to determine their role in glioblastoma cell invasion using selective MMP inhibitors. All with the aim to establish in vitro models to study the role of MMP in glioblastoma and test the activity-based probes and inhibitors of MMP-2 and MMP-9. We found that the two cell lines of differentiated glioblastoma cells and glioblastoma organoids express the metalloproteases MMP-2 and MMP-9, while the cell lines of glioblastoma stem cells do not express MMP-2 and MMP-9. We have shown that MMP-9 is present at a much lower level in the lysate of NIB309 organoids than in the supernatant, whereas MMP-2 is not present in either the lysate or the supernatant of NIB309 organoids. Using gelatin zymography, we found that the pro- and active forms of MMP-2 and MMP-9 are present in the cell lines of the differentiated glioblastoma cells U87 and NIB140, whereas only the pro-forms of MMP-2 and MMP-9 are present in the NIB295 and NIB298 organoids. Using a matrigel spheroid invasion assay, we showed that the broad-spectrum MMP inhibitor batimastat reduces invasion of glioblastoma cells from U87 spheroids and has no effect on invasion of glioblastoma cells from NIB140 spheroids. We also found that the selective inhibitors of MMP-2 and MMP-9, SB-3CT and RXP500.1, do not affect the invasion of glioblastoma cells from U87 and NIB140 spheroids. We were not able to demonstrate the efficacy of activity-based NASA and BBCH probes, which selectively label the active form of MMP-2 and MMP-9, in the U87 glioblastoma cell line. We have shown that glioblastoma cell lines and organoids express and secrete MMP-2 and MMP-9 at different levels. We have demonstrated the presence of active MMP-2 and MMP-9 in differentiated glioblastoma cells. If MMP-2 and MMP-9 are inhibited in these cells, their activity is likely to be compensated. Using MMP-2 and MMP-9, we have shown that glioblastoma is characterised by intertumor heterogeneity and that personalised treatment is therefore very important. In addition, we have established and characterised in vitro cell models in which new inhibitors and probes for the detection of MMP-2 and MMP-9 can be tested.

• Keywords: glioblastoma, MMP-2, MMP-9, invasion, selective inhibitors