Vloga katepsina X pri protitumorskem imunskem odzivu

doktorska disertacija

Tanja Jakoš (Avtor), Janko Kos (Mentor), Anja Pišlar (Komentor)

Povzetek

Raziskovanje interakcij med tumorskimi in imunskimi celicami je spodbudilo razvoj novih terapevtskih strategij za modulacijo imunskega odziva. Te se osredotočajo predvsem na izboljšanje delovanja citotoksičnih celic in premostitev stanja tumorske imunske tolerance. Regulatorne mieloidne celice, med katerimi imajo vidnejšo vlogo mieloidne supresorske celice (MDSC), so močne zaviralke protitumorskega imunskega odziva. Podobno kot pri tumorskih celicah je tudi v njih povečana aktivnost cisteinskih katepsinov, peptidaz, ki zaradi svojih tumor-promotorskih vlog predstavljajo potencialne terapevtske tarče. Cisteinski katepsini so ključni tudi za normalno delovanje imunskega sistema v procesih antigenske predstavitve, regulacije citotoksičnosti, adhezije ipd., vendar je vpliv njihovih inhibitorjev na imunski odziv pri raku še vedno slabo raziskan. Farmakološka inhibicija predstavlja pomembno orodje za odkrivanje funkcije cisteinskih peptidaz. V okviru pretekle študije smo identificirali nov inhibitor katepsina X, Z9, ki je zaradi reverzibilnega mehanizma vezave in izboljšane selektivnosti bolj primeren kot obstoječa spojina AMS36. Z njegovo pomočjo smo preučili vpliv katepsina X na citotoksične in imunosupresivne celice. V ta namen smo najprej pripravili protokol za pridobivanje MDSC in vitro ter ga ustrezno validirali. Prvi smo pokazali, da lahko tumorska celična linija raka dojke spodbuja prehod monocitov v MDSC, ki močno zavirajo proliferacijo limfocitov T in vitro. Pri tem se v celicah poviša izražanje cisteinskih katepsinov, med drugim tudi katepsina L in X. Z dodatkom inhibitorja katepsina X v ko-kulture tumorskih in imunskih celic smo dosegli povišanje invazivnosti celic MDA-MB-231, pri čemer se citotoksičnost imunskih celic ni spremenila. Tudi inhibicija katepsina L s CLIK-148 je vplivala na recipročne interakcije med tumorskimi in imunskimi celicami, vendar smo ravno nasprotno opazili povečano citotoksičnost CD8+ celic T ob nespremenjeni invaziji tumorskih celic. Oba inhibitorja sta okrepila imunosupresivno funkcijo MDSC. Hkrati smo predpostavili, da je katepsin X pomemben za vzpostavitev imunske sinapse in citotoksičnosti, saj sodeluje pri uravnavanju afinitete integrinov z 2 podenoto, med katere sodi adhezijska molekula LFA-1. Eksperimenti so pokazali, da se katepsin X obnaša podobno kot katepsin W, saj se izloča ob degranulaciji citotoksičnih celic in ne vpliva na stabilnost imunske sinapse niti ne na citotoksičnost. Inhibicija aktivnosti katepsina X torej ne spremeni funkcije imunske sinapse, kar kaţe na to, da prevladajo drugi načini regulacije afinitete LFA-1. Celični modeli so nepogrešljivi za validacijo tarč in testiranje učinkovitosti zdravilnih učinkovin v predkliničnih testih. Z uporabo kompleksnega modela ko-kulture smo dokazali, da lahko inhibitorji cisteinskih katepsinov prek vpliva na recipročne interakcije med tumorskimi in imunskimi celicami regulirajo imunski odziv. Spremembe funkcionalnih lastnosti MDSC in citotoksičnih celic, o katerih poročamo, nudijo novo izhodišče za bodoče študije pomena cisteinskih katepsinov pri regulaciji protitumorskega imunskega odziva.

Ključne besede

DNA-topoizomeraza II;protirakave učinkovine;funkcija topoizmeraz;testiranje zaviralne aktivnosti;sinteza zaviralcev;sintezni postopki;

Podatki

Jezik:	Slovenski jezik
Leto izida:	2020
Tipologija:	2.08 - Doktorska disertacija
Organizacija:	UL FFA - Fakulteta za farmacijo
Založnik:	[T. Jakoš]
UDK:	577.152.34:616-097(043.3)
COBISS:	32564739
Št. ogledov:	148
Št. prenosov:	0
Ocena:	0 (0 glasov)
Metapodatki:

Ostali podatki

Sekundarni jezik:	Angleški jezik
Sekundarni naslov:	ǂThe ǂrole of cathepsin X in antitumor immune response
Sekundarni povzetek:	In the light of growing understanding of tumor-immune interactions, immune modulating agents are becoming integral part of improved therapeutic strategies for cancer patients. While the primary goal is to engage cytotoxic cells in tumor elimination, targeting immunosuppressive environment is another topmost priority. Immunosuppression is mediated by innate myeloid cells, in particular myeloid-derived suppressor cells (MDSC), which are known to over-express cysteine cathepsins. Increased activities of several cathepsins, including cathepsins L and X, were associated with cancer progression for many cancer types. Therefore, cancer patients could benefit from selective small-molecule inhibitors that would balance cathepsins' excessive activity. However, little is known on their concurrent impact on the immune system, where cathepsins are known to regulate several important functions such as antigen presentation, regulation of cytotoxicity, adhesion etc. Recently, we identified novel selective cathepsin X inhibitor, Z9, with potential for clinical applications due to its reversible mechanism of action. Z9, along with established irreversible inhibitor AMS36, was used to study the impact of cathepsin X inhibition on antitumor immune response. For this purpose, we established and validated protocol for in vitro generation of MDSC. We showed that MDA-MB-231 breast cancer cell line promoted transition of human monocytic cells towards MDSC. Such MDSC potently reduced T cell proliferation in in vitro assay. Moreover, we demonstrated increased expression of several cathepsins in MDSC, most notably cathepsin L and cathepsin X. Inhibition of cathepsin X resulted in increased invasion of MDA-MB-231 cells in co-culture with immune cells. On the other hand, cathepsin L inhibition by CLIK-148 improved cytotoxicity of CD8+ cells during tumor-immune cell interactions. Inhibition of both cathepsins, however, potentiated MDSC immunosuppressive activity. Additionally, we hypothesized that proteolytic processing of [beta]2 integrin LFA-1 by cathepsin X could impact the immunological synapse formation and cytotoxicity against target cells. Surprisingly, we discovered that cathepsin X was regulated similarly to cathepsin W, and it was found to be secreted after cytotoxic cell degranulation. Based on our experiments we conclude that cathepsin X does not play essential role in regulation of LFA-1 in immunological synapse. In this regard cathepsin X inhibitors would not exert negative impact on cytotoxicity. We have demonstrated advantage of co-culture systems over tumor cell mono-cultures for in vitro testing of cysteine cathepsin inhibitors, since they provide additional insights into reciprocal interactions between tumor and immune cells. By using human cells, we succeeded in bridging the inter-species differences and in supplementing the lack of knowledge with novel information about the role of cysteine cathepsins in MDSC. Also, by revealing the functional impact on MDSC and cytotoxic cells, we provided consequential data that should be taken into consideration when evaluating cathepsin X inhibitors in vivo.
Sekundarne ključne besede:	Imunski odziv;Katepsini;
Vrsta dela (COBISS):	Doktorska disertacija
Komentar na gradivo:	Univ. v Ljubljani, Fak. za farmacijo
Strani:	XVI, 139 str.
ID:	15504120