Spremljanje citotoksične aktivnosti naravnih celic ubijalk izbranih bolnikov z rakom prostate tekom eksperimentalne imunske terapije

Zidar, Anže (author), Jeras, Matjaž (mentor)

Naravne celice ubijalke (NK) so efektorske celice prirojenega imunskega sistema. Njihova ključna naloga je prepoznavanje in uničevanje rakavo spremenjenih ter z virusi in glivicami okuženih celic. Poleg tega so zgodnji vir provnetnih citokinov, zlasti interferona ? (IFN-?) in kemokinov, ki aktivirajo in privabljajo tudi druge celice imunskega sistema. V periferni krvi sta prisotni dve večji subpopulaciji celic NK, in sicer izrazito citotoksične CD56PdimP, ki so v večini ter regulatorne CD56PbrightP, ki jih je malo in so pomemben vir IFN-?. Aktivnost celic NK (NKA) lahko določamo tako z merjenjem sproščanja IFN-? in vitro kot s testom njihove neposredne citotoksičnosti zoper modelne tarčne celice. NKA je pri zdravih posameznikih skozi daljše časovno obdobje dokaj stabilna, prav tako pa se pri njih tudi razmerja med subpopulacijama celic NK ne spreminjajo bistveno. V literaturi večinoma poročajo o zmanjšanju vrednosti NKA z napredovanjem raka prostate, ob tem pa obstajajo tudi podatki o zmanjšanju deleža CD16P-PCD56Pbright Pcelic NK v periferni krvi. V okviru magistrske naloge smo določali NKA v vzorcih mononuklearnih celic iz periferne krvi, odvzetih šestim izbranim bolnikom z rakom prostate v različnih časovnih obdobjih, in sicer pred, med in po kliničnem preskušanju protitumorske imunske celične terapije. Odstotke citotoksičnosti celic NK v omenjenih vzorcih smo določali v pogojih in vitro, na osnovi rezultatov testa za ugotavljanje obsega neposredne celične citotoksičnosti, ki temelji na količini sproščenega znotrajceličnega encima laktatne dehidrogenaze (LDH) iz uničenih tarčnih celic K562. Za izvajanje testov smo na podlagi predhodnih poskusov izbrali mikrotitrske plošče s 96 vdolbinicami v obliki črke V. Pri dveh bolnikih smo za primerjavo vzporedno izvedli tudi test citotoksičnosti s pretočno citometrijo, pri katerem smo tarčne celice K562 označevali s fluorescenčnima barviloma CFSE in 7-AAD. Rezultati naših poskusov so v večini primerov pokazali precejšnja nihanja NKA tekom klinične študije, glede na kontrolne vrednosti odstotka citotoksičnosti, določene v vzorcih, odvzetih pred začetkom eksperimentalne imunske celične terapije, kar je najverjetneje posledica omenjenega zdravljenja. Izbrani bolniki so bili predstavniki treh skupin, ki so jih izvajalci klinične študije oblikovali glede na njihovo odzivnost na preskušano terapijo, ovrednoteno z definiranimi kliničnimi parametri. Povezave med klinično oceno njihove odzivnosti in NKA z našim omejenim številom poskusov nismo uspeli dokazati, vendar pa so se pokazali določeni trendi, ki nakazujejo na to, da bi nam tak dokaz uspel, če bi imeli na razpolago dovolj velik vzorec preiskovancev.

• Language: Slovenian
• Keywords: rak prostate, citotoksična aktivnost celic NK, imunska celična terapija, test citotoksičnosti LDH, pretočna citometrija
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-109225
• Publication date in RUL: 28.08.2019

Secondary language

• Language: English
• Title: Natural killer cell cytotoxic activity monitoring in selected prostate cancer patiens during the course of experimental immunotherapy

Abstract

Natural killer cells (NK) are effector cells of innate immune system. Their key role is to recognise and destroy cancerous, virus infected and fungus infected cells in humans. They are also an early source of proinflammatory cytokines, especially interferon ? (IFN-?) and chemokines that activate and attract other immune cells. There are two main subpopulations of NK cells in peripheral blood, distinctly cytotoxic subpopulation of CD56Pdim PNK cells and mostly regulatory CD56PbrightP NK cells that are an important source of early IFN-?. NK cell activity (NKA) can be determined by in vitro measuring of released IFN-? as well as with a direct cytotoxicity assay using model target cells. NKA in healthy individuals is relatively stable through longer periods of time and the ratio between NK subpopulations remains mostly unchanged. In clinical studies it is reported that in patients with prostate cancer, NKA decreases with advancing cancer stage. These studies are accompanied with reports of diminishing subpopulation of CD16P-PCD56Pbright Pin peripheral blood, which promotes a stronger immune response against cancer cells. In our work we measured cytotoxic NKA in samples of cells acquired from PBMC of six patients with prostate cancer in different time points during a clinical trial of an anticancer immune therapy. The percentages of cytotoxicity were measured in in vitro conditions on the basis of results from a direct cytotoxicity assay based on the amount of released intracellular enzyme lactate dehydrogenase (LDH) from killed target cells K562. We used a letter V shaped 96 well microtiter plates for performing the assay on the basis of preliminary testing. In two patients we also performed a cytotoxic assay using flow cytometry, where we dyed the target cells K562 with CFSE and 7-AAD dyes. Flow cytometry is a good alternative to LDH assay because we can determine the cytotoxicity on a single cell level with this technique. Our results mainly show large variations in NKA through the course of our clinical trial compared to our control values of samples taken before the first application of the trial substances, which is most likely a consequence of before mentioned therapy. Patients tested for NKA were representing three groups based on their responsiveness to antitumor therapy based on defined clinical parameters. We could not prove a correlation between NKA and clinical outcomes with our limited study subjects, but we observed trends which could in a large enough sample prove a correlation between the two. We would also need to design our clinical study for this purpose and take samples during a longer period of time, especially after the end of experimental treatment.

• Keywords: cytotoxic NK cell activity, prostate cancer, immune therapy, flow cytometry, LDH cytotoxic assay