THE EXPRESSION OF GENES OF PROTEASES IN MESENCHYMAL STEM CELL AND GLIOBLASTOMA CELL CO-CULTURES

Glioblastom multiforme (GBM) je najbolj maligen primarni možganski tumor. Zaradi difuzne infiltracije posameznih celic v zdravo možgansko tkivo popolna kirurška odstranitev ni mogoča. Heterogenost tumorja in heterogene GBM celice same pa dodatno povzročajo odpornosti tumorja na radioterapijo in kemoterapijo. Posledično je ponovni pojav bolezni navkljub zdravljenju pričakovan in bolnik umre približno 15 mesecev po postavljeni diagnozi. Mezenhimske matične celice pridobljene iz kostnega mozga (angl. bone marrow derived mesenchymal stem cells – MSC) izražajo tropizem k celicam GBM. MSC uravnavajo imunski sistem in v tumorskem okolju tvorijo strukturni in funkcionalni sincicij s celicami GBM, zaradi česar predstavljajo obetaven transportni sistem za ciljano dostavo proti-tumorskih učinkovin v tumor. Proteaze so ključni encimi, ki prispevajo k nastanku in napredovanju GBM. Ker se migracija in invazija celic GBM ob so-gojenju z MSC spremeni, nas je zanimalo, kakšno vlogo imajo proteaze v tem procesu. Namen raziskave je bil tako preučiti vpliv medsebojne interakcije med celicami GBM in MSC. V magistrskem delu smo ovrednotili medsebojni vpliv MSC in GBM celic U373 na izražanje proteaz: kalapina 1, kalpaina 2, katepsina B, katepsina K in katepsina L. Dokazali smo, da so izbrane protezae višje izražene v MSC kot v celicah U373 in da se izražanje kalpaina 1 in katepsina B v celicah U373 zviša, kadar le-te rastejo v direktni so-kulturi z MSC. Nasprotno pa je izražanje kalpaina 1 in katepsina B v MSC, ki rastejo v direktni so-kulturi s celicami U373, zmanjšano. Na podlagi teh rezultatov sklepamo, da celice tumorskega okolja znatno vplivajo ena na drugo in v so-kulturah, v primejavi z monokulturami, spremenijo svojo invazivnost. Posledično bi z nadaljnimi raziskavami medsebojnega vpliva med stromalnimi celicami, kot so MSC in celicami GBM, pridobili bolj natančne in za posamezno vrsto celic bolj specifične podatke o njihovem delovanju v tumoskem mikrookolju. Proteazno poznavanje tumorskega mikrookolja bi lahko vodilo k bolj naprednemu celičnemu zdravljenju GBM.Glioblastoma multiforme (GBM) is by far the most common and most malignant of primary brain tumors. Total surgical resection is not possible, due to diffuse single cell infiltration into surrounding healthy brain parenchyma. Additionaly, heterogeneity of tumor and heterogeneity of GBM cells itself makes GBM resistant to conventional cancer therapy. Consequently, GBM relapse is inevitable and patients die within 15 months after being diagnosed. Mesenchymal stem cells of bone marrow origin (MSC) exert tropism to GBM, and have the ability to modulate immune system and form functional and structural syncytium with GBM cells within GBM microenvironment. That is why MSC represent an interesting new transport vehicle for drug delivery into the tumor. The aim of this research was to evaluate the effect of co-culturing of MSC and U373 GBM cells on protease expression of calpain 1, calpain 2, cathepsin B, cathepsin K, cathepsin L in both cell types. We proved that the expression of the selected proteases was higher in MSC than in U373 cells, and that calpain 1 and cathepsin B were up-regulated in U373 cells when they were grown in the co-culture with MSC. In contrast, the expression of calpain 1 and cathepsin B was decreased in MSC, wich were co-cultured with U373 cells. Based on these results, we conclude that the cells in the tumor microenvironment significantly influence each other and cell invasiveness is changed in co-cultures, when compared to monocultures. Aditional research on interactions between stromal cells, such as MSC, and GBM cells is needed to aquire a more precise information about behaviour of individual cell type in the tumor microenvironment. The knowledge of proteases in tumour microenvironment can lead to an advanced cell therapy of GBM