​Protitumorsko delovanje genskega elektroprenosa plazmidne DNA z zapisom za kemokina CCL5 ali CCL17 in obsevanja na mišjih modelih karcinoma

Božič, Tim (avtor), Čemažar, Maja (mentor), Markelc, Boštjan (komentor)

Znanstveno izhodišče: V zadnjih letih se z razvojem novih tarčnih in genskih terapij spreminjajo tudi načini zdravljenja raka, vendar pa standardni pristopi, kot sta kemoterapija in radioterapija, v večini primerov še vedno veljajo za zlati standard. Posledično so osnova predkliničnih študij standardni pristopi, ki so kombinirani z drugimi terapijami, med njimi tudi genskimi, z namenom povečanja protitumorskega učinka. Med genske terapije spada tudi genski elektroprenos (GET), kjer z dovajanjem električnih pulzov omogočamo lokalno dostavljanje plazmidne DNA v celice različnih tkiv. Tako lahko dosežemo tudi vnos plazmidne DNA z zapisom za kemokina CCL5 in CCL17 v tumorje, ki se nato izražata v transfeciranih celicah in kot vnetna kemokina sprožita imunski odziv. Čeprav je bila genska terapija z GET različnih citokinov že opisana v literaturi, ostajata kemokina CCL5 in CCL17 z opisano metodo vnosa v tumorje še neraziskana. Številne študije namreč kažejo pomembno vlogo vnetnih kemokinov CCL5 in CCL17 pri usmerjanju celic T ubijalk (CD8+) in drugih imunskih celic v tumorsko mikrookolje. Povečana infiltracija imunskih celic v tumor sama po sebi ne vodi vedno v aktivacijo imunskega odziva in posledično protitumorskega učinka; genska terapija s kemokinoma je lahko zato učinkovita le v kombinaciji s terapijo, ki aktivira imunski odziv. Ena izmed takih terapij je radioterapija. Namen doktorske naloge je bil raziskati protitumorsko delovanje GET plazmidne DNA z zapisom za kemokina CCL5 in CCL17 na ravni tumorskih celic in tumorskih modelov karcinoma pri laboratorijskih miših. Raziskali smo neposredni učinek plazmidov z zapisom za kemokina CCL5 in CCL17 na tumorske celice, vpliv na izražanje različnih citokinov v tumorskih celicah in tumorjih po terapiji ter imunološki učinek terapij in vitro ter in vivo. Z navedenimi raziskavami smo želeli razširiti obstoječe znanje o mehanizmih delovanja kemokinov CCL5 in CCL17 po GET ter raziskati učinkovitost te terapije kot dopolnilne imunoterapije v kombinaciji z obsevanjem tumorjev. Metode: V prvem sklopu doktorske naloge smo določili vpliv in vitro lipofekcije celic s plazmidno DNA z zapisom za kemokina CCL5 ali CCL17 na viabilnost in izražanje genov izbranih citokinov pri mišjih celičnih linijah raka debelega črevesa CT26 in MC38, ter mišjih celičnih linijah raka dojke 4T1 in E0771. Viabilnost smo določili tako, da smo pritrjenim celicam 24 ur po nasaditvi dodali mešanico plazmidne DNA z zapisom za kemokin CCL5 ali CCL17 in lipofektamina, ki omogoči transfekcijo (oziroma lipofekcijo) celic. Po 48 urah smo učinek transfekcije preverjali s pomočjo testa viabilnosti PrestoBlue?. Vzporedno smo tretiranim celicam po 48 urah izolirali RNA in določili raven izražanja citokinov Ccl5, Ccl17, Cxcl9, Cxcl10, Il-1b, Il-6, Il-12?, Il-18, Ifn-?, Ifn-ß in Tnf-? z metodo kvantitativnega PCR v realnem času (qRT-PCR). Ker smo v nadaljevanju terapijo s kemokini kombinirali z obsevanjem, smo okarakterizirali tudi preživetje izbranih celičnih linij CT26 in 4T1 po lipofekciji s plazmidno DNA z zapisom za kemokin CCL5 ali CCL17 in različnimi dozami obsevanja s testom klonogenosti, ki je standardni test za določevanje radioobčutljivosti. Z linearno-kvadratnim modelom smo določili razmerje ?/ß ter nato izračunali inhibitorne doze IC10, IC50, IC90. V naslednjem sklopu raziskave smo določevali kemotaktične lastnosti kemokinov CCL5 in CCL17. In vitro smo s testoma kemotakse, Boydenovo komoro (angl. Boyden chamber) in z inserti s štirimi vdolbinicami (angl. Culture-Inserts 4 Well), spremljali migracijo mišjih makrofagov RAW264.7 in mišjih celic T ubijalk CTLL-2 proti kemokinskemu gradientu, ki ga vzpostavijo tumorske celice po lipofekciji s plazmidno DNA z zapisom za kemokin CCL5 ali CCL17. Pri obeh testih kemotakse smo imunske celice predhodno označili s fluorescenčnim barvilom CellTrace? CFSE Dye in njihovo migracijo določevali z enkratnim ali večkratnim zajemom slik z napravo Cytation 1. In vivo smo ovrednotili ekstravazacijo imunskih celic po GET plazmidne DNA z zapisom za CCL5 ali CCL17 z uporabo modela dorzalnega okna na mišjem modelu raka debelega črevesa CT26 in raka dojke 4T1. Štiriindvajset ur po namestitvi dorzalnega okna smo tumorje inducirali s podkožnim injiciranjem celične suspenzije, ki je vsebovala 3x105 celic CT26-GFP (celice CT26, ki stabilno izražajo zeleni fluorescirajoči protein (GFP)) ali 4T1-GFP (celice 4T1, ki stabilno izražajo GFP). Terapijo smo izvedli, ko je premer tumorjev dosegel 4 mm. Po intratumorskem injiciranju 5 µL suspenzije plazmidne DNA (2 µg/µL) z zapisom za CCL5 ali CCL17 smo z generatorjem električnih pulzov ELECTRO Cell B10 dovedli električne pulze, ki se uporabljajo pri elektrokemoterapiji (ECT pulzi - 8 pulzov, 1300 V/cm, 100 µs, 1 Hz) s ploščatimi elektrodami z razmakom 4 mm med elektrodama. Po 48 urah smo mišim intravensko injicirali splenocite, izolirane iz vranice zdravih donorskih miši. Splenocite smo pred injiciranjem predhodno označili s fluorescenčnim barvilom CellTracker? CM-DiI Dye. S konfokalnim mikroskopom Zeiss LSM800 smo nato 24 in 48 ur po injiciranju fluorescenčno označenih splenocitov zajeli 3D slike tumorjev. Protitumorski učinek GET plazmidne DNA z zapisom za kemokina CCL5 ali CCL17 smo določili na tumorskima modeloma CT26 in 4T1 s spremljanjem zaostanka v rasti podkožnih tumorjev po terapiji. Tumorje smo inducirali s podkožnim injiciranjem 100 µL celične suspenzije, ki je vsebovala 3x105 celic CT26 ali 4T1. Ko so tumorji dosegli velikost 50 mm3 smo začeli s terapijo. Pet minut po intratumorskem injiciranju 25 µL plazmidne DNA (s koncentracijo 1 ali 2 µg/µL) z zapisom za CCL5 ali CCL17 smo z generatorjem električnih pulzov (Cliniporator) dovedli ECT električne pulze s ploščatimi elektrodami z razmakom 6 mm med elektrodama. Zaostanek v rasti smo spremljali z merjenjem treh pravokotnih premerov tumorjev s kljunastim merilom, s katerimi smo po formuli za elipsoid (a × b × c × ?/6; pri čemer a, b in c predstavljajo pravokotne premere) izračunali prostornino tumorjev. Odziv na terapijo smo dodatno ovrednotili z določevanjem infiltracije imunskih celic v tumor z imunofluorescenčnim označevanjem in določevanjem izražanja izbranih citokinov s qRT-PCR. V tem primeru smo mišim odvzeli tumorje tretji in sedmi dan po terapiji. Polovico posameznega tumorja smo uporabili za določitev izražanja Ccl5, Ccl17, Cxcl9, Cxcl10, Il-6, Il-12? in Ifn-? z metodo qRT-PCR, iz druge polovice pa smo pripravili serijo zaledenelih tumorskih rezin, ki smo jih imunofluorescenčno označili s primarnimi in sekundarnimi protitelesi proti površinskim označevalcem celic T pomagalk (CD4+), celic T ubijalk (CD8+), makrofagov (F4/80+) in endotelijskih celic (CD31+). S konfokalnim mikroskopom Zeiss LSM800 smo nato zajeli slike robov in notranjosti tumorjev ter določili število imunskih celic in površino žil. Kombinirana terapija GET plazmidne DNA z zapisom za kemokina CCL5 ali CCL17 in radioterapije na tumorskem modelu CT26 in 4T1 je bila izvedena enako kot zgoraj opisana monoterapija s kemokini, le da smo tumorje po GET dodatno obsevali. Pri tem smo uporabili dva obsevalna režima – obsevanje z enkratno dozo 10 Gy in obsevanje s frakcionirano dozo 3x 5 Gy. Pri obeh obsevalnih režimih so tumorji prejeli biološko ekvivalentno dozo ?22 Gy. Z namenom povečanja protitumorske učinkovitosti smo testirali tudi kombinacijo dvakratnega GET plazmidne DNA z zapisom za kemokina CCL5 ali CCL17 in obsevanja, pri čemer smo drugi GET izvedli 24 ur po zadnjem obsevanju. Pri vseh kombiniranih terapijah smo odziv na terapijo ovrednotili z določevanjem zaostanka v rasti tumorjev, določevanjem infiltracije imunskih celic v tumor z imunofluorescenčnim označevanjem in izražanjem izbranih citokinov s qRT-PCR. V primeru kombinirane terapije smo tumorje za lažjo primerjavo med skupinami odvzeli na tretji dan po zadnjem obsevanju. Raziskave na živalih so potekale v skladu z navodili in dovoljenjem Ministrstva za kmetijstvo, gozdarstvo in prehrano Republike Slovenije (št. dovoljenja U34401-1/2015/17 in U34401-3/2022/11). Za statistično obdelavo podatkov smo uporabili program GraphPad. Vse podatke smo testirali za normalno porazdelitev podatkov. Skupinam z normalno porazdelitvijo podatkov smo določili srednje vrednosti in standardno napako ter ugotavljali statistično značilne razlike med skupinami s testom enosmerne analize variance (One-Way ANOVA) ali t-testom. Podatke, ki niso imeli normalne porazdelitve, smo analizirali z neparametričnimi testi (Wilcoxonov test, Mann-Whitneyjev test in Kruskal-Wallis ANOVA). Rezultati: V prvem delovnem sklopu smo okarakterizirali mišji celični liniji raka debelega črevesa CT26 in MC38, ter raka dojke 4T1 in E0771 na ravni bazalnega izražanja vnetnih citokinov. Nato smo določili vpliv lipofekcije s plazmidno DNA z zapisom za kemokin CCL5 ali CCL17 ter kontrolnim plazmidom pDNA Ctrl na viabilnost celic. Lipofekcija ni vplivala na viabilnost celic, vnos posamezne plazmidne DNA pa je povzročil statistično značilno spremembo v izražanju vnesenih kemokinov kot tudi nekaterih vnetnih citokinov. Določevanje preživetja celic v odvisnosti od doze obsevanja po transfekciji s posamezno plazmidno DNA je pokazalo, da je celična linija CT26 občutljivejša na obsevanje v primerjavi s 4T1. S testi kemotakse in vitro smo določevali stopnjo migracije mišjih makrofagov RAW264.7 in celic T ubijalk CTLL-2 proti tumorskim celicam CT26 in 4T1 po lipofekciji s posamezno plazmidno DNA. Test z Boydenovo komoro je pokazal zmanjšano migracijo makrofagov in celic T ubijalk proti transfeciranim tumorskim celicam CT26 in 4T1 v primerjavi s kontrolo. Pri tem med tretiranimi skupinami nismo opazili statistično značilnih razlik, test kemotakse insertov s štirimi vdolbinicami pa je pokazal povečano migracijo makrofagov proti tumorskim celicam CT26 transfeciranim s plazmidno DNA z zapisom za CCL17 ali CCL5. Povišano migracijo makrofagov smo opazili tudi proti tumorskim celicam 4T1, transfeciranim s plazmidno DNA z zapisom za CCL17, medtem ko je bila migracija proti celicam 4T1, transfeciranim s kontrolno plazmidno DNA ali plazmidno DNA z zapisom za CCL5 precej nižja in primerljiva med skupinama. Kemotaktične lastnosti kemokinov CCL5 in CCL17 smo s spremljanjem ekstravazacije splenocitov določevali tudi in vivo na modelu dorzalnega okna, pri čemer smo po GET obeh kemokinov pokazali, da CCL5 in CCL17 povzročita infiltracijo splenocitov v tumorje CT26 in 4T1. Nato smo ovrednotili protitumorski učinek GET plazmidne DNA z zapisom za kemokina CCL5 ali CCL17 na mišjih tumorskih modelih CT26 in 4T1 z določevanjem izražanja vnetnih citokinov, stopnjo infiltriranih imunskih celic v tumorje in z določevanjem zaostanka v rasti tumorjev po terapiji. Čeprav je po terapiji prišlo do povišanega izražanja vnesenih kemokinov in spremenjenega izražanja nekaterih vnetnih citokinov, GET s kemokini ni povzročil daljših zaostankov v rasti tumorjev v primerjevi s tumorji po GET s pDNA Ctrl. Nobena izmed terapij tudi ni vodila do popolnih odgovorov. Prav tako pri nobenem tumorskem modelu po terapijah ni bilo značilnih sprememb v številu citotoksičnih CD8+ celic T ubijalk, CD4+ celic T pomagalk in F4/80+ makrofagov v primerjavi s kontrolo. Kombinirano terapijo GET plazmidne DNA z zapisom za kemokina CCL5 ali CCL17 in radioterapije smo na mišjih tumorskih modelih CT26 in 4T1 ovrednotili na enak način kot monoterapijo. Spremembe v izražanju vnetnih citokinov smo v tumorjih CT26 zaznali po vseh kombiniranih terapijah, tudi pri kombiniranih terapijah z GET pDNA Ctrl. V tumorjih 4T1 so kombinirane terapije, ne glede na uporabljeno plazmidno DNA, vodile predvsem do povišanega izražanja gena Ccl5. V primeru 2x GET s 25 µg plazmidne DNA kot monoterapije ali v kombinaciji s frakcioniranim obsevanjem 3x 5 Gy se raven izražanja vnetnih citokinov v obeh tumorskih modelih znatno ni spremenila, z izjemo povečanega izražanja vnesenih kemokinov. Imunofluorescenčno označevanje zaledenelih tumorskih rezin po kombiniranih terapijah je pokazalo spremembe v populacijah CD4+ in CD8+ celic T, pri čemer se je število CD8+ celic T pri obeh tumorskih modelih na robu tumorjev zmanjšalo že po samem obsevanju z enkratno ali frakcionirano dozo. Po kombiniranih terapijah pa je bilo zmanjšanje števila CD8+ celic T v nekaterih primerih tudi statistično značilno v primerjavi s kontrolo. Iz števila popolnih odgovorov in zaostanka v rasti tumorjev CT26, je razvidno, da GET s plazmidno DNA doprinese k protitumorskemu učinku obsevanja ne glede na uporabljeno plazmidno DNA. Nasprotno pa je v neimunogenih tumorjih 4T1 primerjava zaostankov v rasti tumorjev med skupinami pokazala statistično značilno večji protitumorski učinek obsevanja v kombinaciji z GET kemokinov CCL5 ali CCL17 glede na kombinacijo obsevanja in GET s kontrolno plazmidno DNA. Zaključki: V raziskavi smo dokazali, da povečano izražanje kemokinov CCL5 ali CCL17 v transfeciranih tumorskih celicah vpliva na izražanje ostalih vnetnih citokinov. Povečano izražanje je vodilo do migracije makrofagov v primeru testov kemotakse in vitro kot tudi do infiltracije splenocitov v tumorje na modelu dorzalnega okna in vivo. Genski elektroprenos kemokinov CCL5 ali CCL17 vodi do popolnih odgovorov le v kombinaciji z obsevanjem. GET kemokinov in obsevanje vodita do popolnih odgovorov pri imunogenih tumorjih CT26 ne glede na uporabljeno plazmidno DNA. Pri neimunogenih tumorjih 4T1 pa GET kemokinov in obsevanja vodi v značilno daljši zaostanek v rasti tumorjev, kar kaže na doprinos kemokinov k protitumorskemu učinku. Kemokina CCL5 in CCL17 tako predstavljata potencial v imunoterapiji raka, vendar je v primeru kombinirane terapije potrebna nadaljnja optimizacija terapevtskega režima, še posebej v povezavi z imunološkim statusom tumorja.

• Jezik: Slovenski jezik
• Ključne besede: Kemokini, CCL5, CCL17, genski elektroprenos, radioterapija, mišji modeli karcinomov
• Vrsta gradiva: Doktorsko delo/naloga
• Organizacija: MF - Medicinska fakulteta
• Leto izida: 2023
• PID: 20.500.12556/RUL-143943
• COBISS.SI-ID: 147767555
• Datum objave v RUL: 21.01.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Antitumor effectiveness of gene electrotransfer of plasmid DNA encoding chemokines CCL5 or CCL17 and irradiation in murine carcinoma models

Izvleček

Scientific background: In recent years, the development of new targeted therapies and gene therapies has redefined the ways of cancer treatment. However, classical approaches such as chemotherapy and radiotherapy remain a gold standard. Therefore, preclinical studies are focusing on therapies that combine classical approaches with novel strategies, which include gene therapies to increase overall antitumor efficacy. Gene electrotransfer (GET) as a gene therapy method uses electric pulses to achieve enhanced local delivery of plasmid DNA into cells of various tissues. The use of GET also enables the delivery of plasmid DNA encoding the chemokines CCL5 and CCL17 into tumors, where they are expressed by transfected cells and act as proinflammatory chemokines to elicit an immune response. Although gene therapy utilizing GET of cytokines has been described in the literature, GET of chemokines CCL5 and CCL17 has never been studied in tumors. Several studies describe the important role of proinflammatory chemokines CCL5 or CCL17 in recruiting and directing cytotoxic T cells (CD8+) and other immune cells into the tumor microenvironment [1]. However, increased infiltration of immune cells into tumors does not necessarily lead to immune activation and antitumor activity [2]. Therefore, gene therapy with chemokines can be effective only in combination with a therapy capable of eliciting an immune response. One of these therapies is radiotherapy [3,4]. The aim of this doctoral dissertation was to investigate the antitumor effect of GET of plasmid DNA encoding the chemokines CCL5 or CCL17 on tumor cells and tumor carcinoma models in laboratory mice. We determined the direct effect of plasmids encoding chemokines CCL5 or CCL17 on tumor cells, their effect on the expression of several cytokines in tumor cells and tumors after therapy, and the immunological effect in vitro and in vivo. With the above studies, we aimed to expand the existing knowledge on the mechanisms of action of the chemokines CCL5 and CCL17 after GET and to investigate the efficacy of this therapy as a complementary immunotherapy in combination with tumor irradiation. Methods: In the first section of the doctoral dissertation, we determined the effect of in vitro lipofection of cells with plasmid DNA encoding chemokines CCL5 or CCL17 on the viability and gene expression of selected cytokines in murine colon cancer cell lines CT26 and MC38, and murine breast cancer cell lines 4T1 and E0771. Viability was determined on attached cells 24 hours after seeding, by adding a mixture of plasmid DNA encoding CCL5 or CCL17 and lipofectamine, which enables transfection (or lipofection) of cells. After 48 hours, the effect of transfection was assessed using the PrestoBlue〢 viability assay. In parallel, RNA of treated cells was isolated 48 hours after lipofection and the expression levels of cytokines Ccl5, Ccl17, Cxcl9, Cxcl10, Il-1b, Il-6, Il-12α, Il-18, Ifn-γ, Ifn-β and Tnf-α were determined by quantitative real-time PCR (qRT-PCR). Because we subsequently combined chemokine therapy with irradiation, we also characterized the survival of selected cell lines CT26 and 4T1 after lipofection with plasmid DNA encoding chemokine CCL5 or CCL17 and different doses of irradiation using the clonogenic assay, which is a standard test for determining radiosensitivity. Using the linear-quadratic model, we determined the α/β ratio and then calculated the inhibitory doses IC10, IC50, IC90. In the next part of the study, we determined the chemotactic properties of chemokines CCL5 and CCL17. In vitro, we used chemotaxis assays, Boyden chamber and a four-well insert assay (Culture-Inserts 4 Well), and observed migration of mouse macrophages RAW264.7 and mouse killer T cells CTLL-2 against a chemokine gradient, which is established by tumor cells 48 hours after lipofection with plasmid DNA encoding chemokine CCL5 or CCL17. In both chemotaxis assays, immune cells were first labeled with the fluorescent dye CellTrace〢 CFSE and their migration was determined by single or multiple acquisition of images with Cytation 1 multimodal imaging reader. In vivo, we evaluated immune cell extravasation after GET of plasmid DNA encoding CCL5 or CCL17 to CT26 colon and 4T1 breast tumors grown in dorsal window chamber model. Twenty-four hours after surgical implantation of the dorsal window chamber, tumors were induced by subcutaneous injection of a cell suspension containing 3x105 CT26-GFP cells (CT26 cells stably expressing green fluorescent protein (GFP)) or 4T1-GFP (4T1 cells stably expressing GFP). Therapy was performed when the diameter of the tumors reached 4 mm. After intratumoral injection of 5 µL of plasmid DNA (2 µg/µL) encoding CCL5 or CCL17, electrical pulses tipically used in electrochemotherapy [5] (ECT pulses - 8 pulses, 1300 V/cm, 100 µs, 1 Hz) were delivered using plate electrodes with a distance of 4 mm between the electrodes. After 48 hours, the mice were intravenously injected with splenocytes isolated from the spleen of healthy donor mice. Splenocytes were pre-labeled with the fluorescent dye CellTracker〢 CM-DiI Dye prior to injection. Then, 24 and 48 hours after the injection of fluorescently labeled splenocytes, 3D images of the tumors were acquired using a Zeiss LSM800 confocal microscope. The antitumor effect of GET of plasmid DNA encoding chemokine CCL5 or CCL17 was determined on tumor models CT26 and 4T1 by measuring the delay in growth of subcutaneous tumors after therapy. Tumors were induced by subcutaneous injection of 100 µL of a cell suspension containing 3x105 CT26 or 4T1 cells. When the tumor volume reached 50 mm3, we started the therapy. Five minutes after intratumoral injection of 25 µL of plasmid DNA (with a concentration of 1 or 2 µg/µL) encoding CCL5 or CCL17, ECT electric pulses were delivered delivered using plate electrodes with a distance of 6 mm between the electrodes. Tumor growth delay was deteremined by measuring the three rectangular diameters of the tumors with a caliper tool. Measured diameters were used to calculate the volume of the tumors according to the formula for ellipsoid (a × b × c × π/6; where a, b and c are perpendicular tumor diameters). The response to the therapy was additionally assessed by determining the infiltration of immune cells into the tumor by immunofluorescence labeling and determining the expression of selected cytokines by qRT-PCR. In this case, tumors were collected on day three and day seven after therapy. Half of each tumor was used to determine the expression of Ccl5, Ccl17, Cxcl9, Cxcl10, Il-6, Il-12α and Ifn-γ by the qRT-PCR, while the other half was used to prepare a series of frozen tumor sections, which were immunofluorescently labeled with primary and secondary antibodies against surface markers of helper T cells (CD4+), cytotoxic T cells (CD8+), macrophages (F4/80+), and endothelial cells (CD31+). We then used a Zeiss LSM800 confocal microscope to capture images of the tumor edges and tumor center and determined the number of immune cells and the surface area of the vessels. Combined therapy of GET plasmid DNA encoding chemokine CCL5 or CCL17 and radiotherapy on CT26 and 4T1 tumor models was performed in the same manner as the chemokine monotherapy described above, except that the tumors were additionally irradiated after GET. We used two irradiation regimens - irradiation with a single dose of 10 Gy and irradiation with a fractionated dose of 3x 5 Gy. After both irradiation regimens, tumors received a biologically equivalent dose of 䁈22 Gy. To increase anti-tumor efficacy, we also tested the combination of a double GET of plasmid DNA encoding chemokine CCL5 or CCL17 and irradiation, with the second GET performed 24 h after the last irradiation. For all combination therapies, the response to the therapy was assessed by determining the tumor growth delay, the immune cell infiltration into the tumor by immunofluorescence labeling, and the expression of selected cytokines by qRT-PCR. In the case of combined therapy, tumors were removed on day 3 after the last irradiation to allow comparison between groups. Animal experiments were performed in accordance with the instructions and approval of the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia (permission no. U34401-1/2015/17 and U34401-3/2022/11). The GraphPad program was used for statistical analysis. All data were tested for normal distribution. Means and standard errors were determined for groups with normally distributed data, and statistically significant differences between groups were determined using the One-Way ANOVA or the t-test. Data that were not normally distributed were analyzed with nonparametric tests (Wilcoxon test, Mann-Whitney test, and Kruskal-Wallis ANOVA). Results: First, basal expression of inflammatory cytokines was determined in murine colon cancer cell lines CT26 and MC38 and breast cancer cell lines 4T1 and E0771. We then determined the effect of lipofection with plasmid DNA encoding CCL5 or CCL17 and the control plasmid pDNA Ctrl on cell viability. Lipofection did not affect cell viability, and introduction of single plasmid DNA resulted in a statistically significant change in the expression of the introduced chemokines as well as some inflammatory cytokines. Because we subsequently combined chemokine therapy with radiation, we also determined a dose-response of cell survival after transfection with individual plasmid DNA. The data were analyzed using a linear quadratic model, and we found that the CT26 cell line was more sensitive to radiation compared to 4T1. In vitro chemotaxis assays were used to determine the extent of migration of mouse macrophages RAW264.7 and cytotoxic T cells CTLL-2 toward tumor cells CT26 and 4T1 after lipofection with individual plasmid DNA. Boyden chamber chemotaxis assay showed reduced migration of macrophages and killer T cells towards transfected tumor cells CT26 and 4T1 compared to control. However, the difference between treatment groups was negligible. A four-well insert (Culture-Inserts 4 Well) chemotaxis assay showed an increased macrophage migration toward CT26 tumor cells transfected with plasmid DNA enocoding CCL17 or CCL5. Increased migration of macrophages was also observed against 4T1 tumor cells transfected with plasmid DNA encoding CCL17, whereas migration against 4T1 cells transfected with control plasmid DNA or plasmid DNA encoding CCL5 was much lower and comparable between groups. The chemotactic properties of the chemokines CCL5 and CCL17 were also determined in vivo by examining the extravasation of splenocytes in the dorsal window chamber model. In this setting GET of both chemokines showed that CCL5 and CCL17 induce the infiltration of splenocytes in CT26 and 4T1 tumors. We further evaluated the anti-tumor activity of GET plasmid DNA encoding CCL5 or CCL17 on tumor models CT26 and 4T1 by determining the expression of inflammatory cytokines, analyzing infiltrated immune cells in the tumor, and determining the tumor growth delay after therapy. Although increased expression of administered chemokines and altered expression of some inflammatory cytokines were observed after therapy, none of the therapies resulted in a complete response. The number of infiltrated helper CD4+ T cells, cytotoxic CD8+ T cells and F4/80+ macrophages did not change significantly after therapy in any tumor model. Combined therapy of GET plasmid DNA encoding CCL5 or CCL17 and radiotherapy was evaluated in the same manner as monotherapy in the CT26 and 4T1 mouse tumor models. Changes in the expression were detected in CT26 tumors after all combined therapies, even in combined therapies with GET pDNA Ctrl. In 4T1 tumors, combined therapies resulted mainly in increased expression of Ccl5 gene, regardless of the plasmid DNA used. In the case of 2x GET with 25 µg of plasmid DNA as monotherapy or in combination with fractionated irradiation of 3x 5 Gy, the expression level of inflammatory cytokines in both tumor models does not change significantly, with the exception of increased expression of introduced chemokines. Immunofluorescence labeling of frozen tumor sections after combined therapies showed changes in CD4+ and CD8+ T cell populations. Namely, the number of CD8+ T cells in the tumor edge of both tumor models decreased already after single or fractionated irradiation alone. After combined therapies, the decrease in the number of CD8+ T cells compared with controls was in some cases also statistically significant. From the number of complete responses and the tumor growth delay of CT26 tumors, it can be seen that GET of plasmid DNA contributes to the antitumor effect of irradiation regardless of the plasmid DNA used. In contrast, in non-immunogenic 4T1 tumors, comparison of tumor growth delay between groups showed a statistically significant antitumor effect of irradiation in combination with GET of chemokines CCL5 or CCL17 compared with irradiation combined with GET of control plasmid DNA. Conclusions: In this study, we demonstrated that increased expression of the chemokines CCL5 or CCL17 in transfected tumor cells affects the expression of other inflammatory cytokines. Moreover, both chemokines show chemotactic properties in both in vitro chemotaxis assays and in vivo dorsal window chamber model. Gene electrotransfer of plasmid DNA encoding chemokines CCL5 or CCL17 leads to complete responses only in combination with irradiation. In immunogenic CT26 tumors, GET of chemokines and irradiation leads to complete responses regardless of the plasmid DNA used. In non-immunogenic 4T1 tumors, GET of chemokines and irradiation leads to a significantly longer tumor growth dealy, suggesting a contribution of chemokines to the antitumor effect. Therefore, chemokines CCL5 and CCL17 represent a potential in cancer immunotherapy, however in the case of combined therapy further optimization of the therapeutic regimen is required, especially in relation to the immunological status of the tumor.

• Ključne besede: Chemokines, CCL5, CCL17, gene electrotransfer, radiotherapy, murine carcinoma models