The antitumor effect of electrochemotherapy with cisplatin or oxaliplatin in combination with gene electrotransfer of plasmid encoding interleukin-12 in murine melanoma model

Znanstveno izhodišče: Za razliko od začetnih stadijev raka, zaradi agresivnosti in odpornosti na terapije uspešnost zdravljenja malignega melanoma še vedno ne dosega pričakovanj. Novi terapevtski pristopi temeljijo predvsem na kombiniranju že uveljavljenih terapij z novejšimi terapijami, ki vplivajo na komponente imunskega sistema. Medtem ko je elektrokemoterapija s cisplatinom že dobro uveljavljena v kliniki, elektrokemoterapija z oksaliplatinom do danes še ni bila raziskana. Oksaliplatin je primeren kandidat za vpeljavo v elektrokemoterapijo zaradi podobnosti s cisplatinom, opisanih manjših stranskih učinkov in predpostavljeno večjega imunomodulatornega vpliva. Čeprav v kliniki z elektrokemoterapijo pri 80 % tumorjev dosežemo lokalno tumorsko kontrolo, protitumorski učinek na oddaljene nezdravljene tumorje in/ali metastaze (t. i. »abscopal effect«) še ni bil opisan. Namen doktorske naloge je bil preučiti učinkovitost intratumoralne elektrokemoterapije z oksaliplatinom in jo primerjati z elektrokemoterapijo s cisplatinom na mišjem malignem melanomu B16F10. Preverili smo tudi, v kolikšni meri lahko peritumoralni genski elektroprenos plazmida z zapisom za interlevkin-12 (IL-12) poveča lokalno učinkovitost elektrokemoterapije in ali lahko spodbudi tudi sistemski odgovor, to je protitumorski učinek na oddaljene nezdravljene tumorje. Metode: Raziskave so potekale na celični liniji mišjega malignega melanoma B16F10. V prvem, in vitro delu smo raziskali, ali električni pulzi potencirajo privzem citostatika oksaliplatina v celice ter vezavo platine na DNA. Vsebnost platine v vzorcih smo izmerili s pomočjo masne spektrometrije z induktivno sklopljeno plazmo. Zanimala nas je tudi potenciacija v citotoksičnosti ob aplikaciji električnih pulzov in primerjava občutljivosti celic B16F10 na oksaliplatin in cisplatin. Dokazali smo jo s testom klonogenosti. Citometrično smo določili vrsto celične smrti oziroma delež celic, ki umirajo z apoptozo, nekrozo ali imunogeno celično smrtjo. Nadaljevali smo s poskusi na miših C57Bl/6. Tumorske celice mišjega melanoma B16F10 so po injiciranju v podkožje na boku miši tvorile čvrste tumorje. S terapijo smo začeli, ko so tumorji dosegli 35&#6148540 mm3. Preverili smo protitumorsko učinkovitost elektrokemoterapije z oksaliplatinom in jo primerjali z elektrokemoterapijo s cisplatinom. Kombinirano terapijo sta sestavljala peritumoralni genski elektroprenos plazmida z zapisom za IL-12 in intratumoralna elektrokemoterapija s cisplatinom ali oksaliplatinom. Najprej smo izvedli genski elektroprenos, in sicer tako, da smo v štiri mesta peritumoralno intradermalno injicirali plazmid (skupaj 50 µg). Nizkonapetostne električne pulze (12 pulzov, 170 V/cm, 150 ms, 0,4/1,39 Hz) smo dovedli z več-točkovno elektrodo (MEA) s krožno razporeditvijo šestih elektrodnih konic takoj po injiciranju plazmida. Pet minut po genski terapiji smo intratumoralno injicirali cisplatin ali oksaliplatin, po dveh minutah pa dovedli električne pulze s ploščatimi elektrodami (8 pulzov, 1300 V/cm, 100 µs, 1 Hz). Preverili smo, ali dodatne ponovitve genskega elektroprenosa IL-12 izboljšajo protitumorski učinek kombinirane terapije. Za določanje sistemskega vpliva kombinirane terapije oziroma vpliva na oddaljene nezdravljene tumorje smo uvedli model za sistemsko bolezen. Tumorske celice smo injicirali v oba boka živaliv desni bok 1 x 106 celic, v levi bok pa 0,7 x 106 celic. Med izvedbo elektrokemoterapije in genskega elektroprenosa so bile miši pod inhalacijsko anestezijo z izofluranom. Zdravili smo desni tumor, zaostanek v rasti pa spremljali na obeh tumorjih, zdravljenem in nezdravljenem. Protitumorski učinek oziroma zaostanek v rasti tumorjev smo spremljali s kljunastim merilom. Za določanje koncentracije IL-12 v serumu, vsebnosti platine v tumorjih in serumu ter histološko analizo smo po humani usmrtitvi živali odvzeli tumorje in kri. Imunohistokemijsko smo označili nekrozo in apoptozo (kaspaza 3), izražanje transgena (IL-12) in populacije imunskih celic v tumorju (grancim B, Foxp3). Rezultati: Citotoksičnost oksaliplatina v kombinaciji z električnimi pulzi je bila visoka in primerljiva s citotoksičnostjo cisplatina na celicah mišjega melanoma B16F10 in vitro. Primerljiva citotoksičnost je bila posledica primerljive potenciacije v privzemu platine v celice in vezavi platine na DNA. Na melanomskem modelu in vivo smo pokazali, da je elektrokemoterapija z oksaliplatinom enako učinkovita kot elektrokemoterapija s cisplatinom, če dozo oksaliplatina dvignemo za faktor 1,6. Izpostavitev melanomskih celic elektrokemoterapiji z oksaliplatinom ali cisplatinom je vodila tudi v imunogeno celično smrt, slednje pa je koreliralo s povišano infiltracijo imunskih celic v tumor po terapiji. Po elektrokemoterapiji z nizkimi dozami oksaliplatina ali cisplatina smo opazili le zaostanek v rasti primarnih tumorjev, ne pa tudi popolnih odgovorov. Pokazali smo, da peritumoralni genski elektroprenos plazmida z zapisom za IL-12 potencira protitumorski učinek intratumoralne elektrokemoterapije s cisplatinom ali oksaliplatinom na mišjem malignem melanomu B16F10. Ob enako učinkoviti elektrokemoterapiji s cisplatinom ali oksaliplatinom je bil prispevek genskega elektroprenosa IL-12 večji pri elektrokemoterapiji s cisplatinom, dosegli smo namreč 38 % popolnih odgovorov. Po kombinirani terapiji z oksaliplatinom smo opazili le značilen zaostanek v rasti tumorjev. Dodatno ponavljanje genskega elektroprenosa plazmida z zapisom za IL-12 ni izboljšalo terapevtskega učinka, povzročilo je tudi infiltracijo peritumoralnega prostora s celicami T zaviralkami. Najpomembneje, kombinirana terapija, pri kateri smo za elektrokemoterapijo uporabili cisplatin, je imela poleg lokalnega tudi sistemski učinek, dvodnevni zaostanek v rasti oddaljenih nezdravljenih tumorjev. Za uspešnost opisane kombinirane terapije je najverjetneje odgovorno lokalno in sistemsko spodbujanje imunskega odziva. Po kombinirani terapiji s cisplatinom v elektrokemoterapiji smo namreč v zdravljenih in nezdravljenih tumorjih opazili imunske celice, pozitivne za grancim B. Zaključki: Genski elektroprenos plazmida z zapisom za IL-12 je učinkovit pri spodbujanju lokalnega protitumorskega učinka elektrokemoterapije. Poleg tega ima kombinirana terapija, pri kateri smo za elektrokemoterapijo uporabili cisplatin, tudi sistemski protitumorski učinek. Rezultati doktorske naloge bodo v pomoč pri vpeljevanju oksaliplatina v elektrokemoterapijo, kot samostojno terapijo, v kliniko. Rezultati v sklopu testiranja kombinirane terapije pa bodo osnova za prehod na naslednjo stopnjo v verigi translacijskih raziskav, prijavo klinične študije.Scientific background: Treatment of malignant melanoma remains a challenge due to its aggressiveness and resistance to treatment. Combining established local ablative therapies with recently discovered immunotherapies is a promising approach for treating malignant melanoma. While electrochemotherapy with cisplatin is already a well-established treatment, oxaliplatin has not yet been tested for use with electrochemotherapy. Based on its similar characteristics to cisplatin, described lesser side-effect and its presumably more pronounced immunomodulatory effect, oxaliplatin is an eligible candidate for electrochemotherapy. Although local effectiveness of electrochemotherapy is up to 80 % of local tumor control, noticeable effects on distant non-treated metastases (abscopal effect) have yet to be described. With the aim to increase the armamentarium of drugs for electrochemotherapy, oxaliplatin was compared to the already well-established electrochemotherapy with cisplatin in murine malignant melanoma B16F10. In addition, we also investigated if peritumoral gene electrotransfer of a plasmid encoding interleukin-12 (IL-12) as an adjuvant immunotherapy enhances the responses seen with electrochemotherapy on a local and systemic level (abscopal effect). Methods: Murine B16F10 melanoma cells with a high metastatic potential were used. Firstly, we determined the potentiation in the uptake of platinum after applying electric pulses and platinum-based drugs oxaliplatin or cisplatin in vitro. Platinum content in cells and the amount of platinum bound to DNA were measured by inductively coupled plasma mass spectrometry. Using the clonogenic assay we determined the cellular reproductive potential following electroporation with oxaliplatin or cisplatin. We also determined the type of cell death after electroporation with oxaliplatin or cisplatin. More specifically, we determined the amount of cells which died via apoptosis, necrosis or immunogenic cell death. Secondly, in vivo experiments were performed in C57Bl/6 mice. Solid tumors were grown on the right flank of mice after a subcutaneous inoculation of tumor cells. The therapy was performed when tumors reached 35&#6148540 mm3 in volume. The antitumor effect of electrochemotherapy was compared to electrochemotherapy with cisplatin. Thereafter, the antitumor effect of the combined treatment consisting of intratumoral electrochemotherapy with cisplatin or oxaliplatin and peritumoral gene electrotransfer of plasmid encoding IL-12 was researched. For gene electrotransfer mice received four intradermal injections of plasmid DNA encoding IL-12 (50 µg) around the tumor. Immediately after low voltage electric pulses (12 pulses, 170 V/cm, 150 ms, 0.4/1.39 Hz) were applied with a multi-electrode array (MEA). Five minutes after gene electrotransfer, oxaliplatin or cisplatin were injected intratumorally and after two minutes electric pulses (8 pulses, 1300 V/cm, 100 µs, 1 Hz) were applied with plate electrodes. The antitumor effectiveness of the combined treatment with multiple repetitions of gene electrotransfer was also tested. A dual flank model mimicking systemic disease was used to determine the systemic antitumor effect of the therapy. It was established by subcutaneous injections of tumor cells on both flanks of mice, 1 x 106 cells on the right and 0.7 x 106 on the left flank. During the treatment, animals were under inhalational anesthesia with isoflurane. Tumor growth was followed by measuring three mutually orthogonal tumor diameters with a Vernier caliper. The right tumor was regarded as a primary tumor and was treatedthe left tumor was left untreated. Blood samples were collected for the detection of IL-12 in mice sera and tumor samples were collected for histological analysis. Immunohistochemical analysis was performed to detect necrosis, apoptosis (caspase 3), transgene expression (IL-12) and infiltration of immune cells into tumors (granzyme B, Foxp3). Results: Electroporation potentiated the cytotoxicity of oxaliplatin in vitro on B16F10 melanoma cells and no differences in cytotoxicity between cisplatin and oxaliplatin were observed. Cytotoxicity correlated with cisplatin or oxaliplatin uptake into cells and drug binding to DNA. Based on in vivo results pertaining to effectiveness and drug uptake in tumors, electrochemotherapy with oxaliplatin is as effective as electrochemotherapy with cisplatin when the dose of oxaliplatin is increased 1.6-fold. Electrochemotherapy induced immunogenic cell death when either oxaliplatin or cisplatin were used, which correlated with a comparable increase in lymphocyte infiltration into tumors after electrochemotherapy with either oxaliplatin or cisplatin. We observed a delay in tumor growth after electrochemotherapy with low doses of oxaliplatin or cisplatin, with no complete responses. We showed that peritumoral gene electrotransfer of plasmid encoding IL-12 potentiated the antitumor effect of electrochemotherapy with oxaliplatin or cisplatin in B16F10 melanoma on a local level. The most pronounced potentiation was with electrochemotherapy using cisplatin, resulting in 38% of complete responses. However, the additional repetitions of gene electrotransfer did not improve the therapeutic effectiveness. Moreover, we also observed an abscopal effect after the combination treatment with electrochemotherapy with cisplatin. Average tumor growth delay of the distant untreated tumors in the latter group was two days. We ascribe the success of this novel therapy to the induction of an immune response on a local and systemic level. Namely, infiltration of granzyme B positive immune cells after the combination therapy with electrochemotherapy with cisplatin was observed in both primary and distant tumors. Conclusion: We showed that gene electrotransfer of IL-12 potentiates the effect of electrochemotherapy on a local level. Additionally, an abscopal effect was detected after the combination therapy in which cisplatin was used for electrochemotherapy. Our findings highlight the potential clinical use of intratumoral oxaliplatin in electrochemotherapy. Most importantly, the preclinical data will serve as the basis for a clinical phase study application