Multimodal treatment approaches, such as radio-immunotherapy, necessitate regimen optimization and the investigation of the interactions of different modalities. The aim of this study was two-fold. ...Firstly, to select the most effective combination of irradiation and the previously developed tumor cell-based vaccine and then to provide insight into the immune response to the selected combinatorial treatment. The study was performed in immunologically different murine tumor models: B16F10 melanoma and CT26 colorectal carcinoma. The most effective combinatorial treatment was selected by comparing three different IR regimens and three different vaccination regimens. We determined the local immune response by investigating immune cell infiltration at the vaccination site and in tumors. Lastly, we determined the systemic immune response by investigating the amount of tumor-specific effector lymphocytes in draining lymph nodes. The selected most effective combinatorial treatment was 5× 5 Gy in combination with concomitant single-dose vaccination (B16F10) or with concomitant multi-dose vaccination (CT26). The combinatorial treatment successfully elicited a local immune response at the vaccination site and in tumors in both tumor models. It also resulted in the highest amount of tumor-specific effector lymphocytes in draining lymph nodes in the B16F10, but not in the CT26 tumor-bearing mice. However, the amount of tumor-specific effector lymphocytes was intrinsically higher in the CT26 than in the B16F10 tumor model. Upon the selection of the most effective combinatorial treatment, we demonstrated that the vaccine elicits an immune response and contributes to the antitumor efficacy of tumor irradiation. However, this interaction is multi-faceted and appears to be dependent on the tumor immunogenicity.
Background Malignant mesothelioma (MM) is a rare aggressive tumour of mesothelium caused by asbestos exposure. It has been suggested that the genetic variability of proteins involved in DNA repair ...mechanisms affects the risk of MM. This study investigated the influence of functional polymorphisms in ERCC1 and XRCC1 genes, the interactions between these polymorphisms as well as the interactions between these polymorphisms and asbestos exposure on MM risk. Patients and methods In total, 237 cases with MM and 193 controls with no asbestos-related disease were genotyped for ERCC1 and XRCC1 polymorphisms. Results ERCC1 rs3212986 polymorphism was significantly associated with a decreased risk of MM (odds ratio OR = 0.61; 95% confidence interval CI = 0.41-0.91; p = 0.014). No associations were observed between other genetic polymorphisms and MM risk. Interactions between polymorphisms did not significantly influence MM risk. Interaction between ERCC1 rs11615 and asbestos exposure significantly influenced MM risk (OR = 3.61; 95% CI = 1.12-11.66; p = 0.032). Carriers of polymorphic ERCC1 rs11615 allele who were exposed to low level of asbestos had a decreased risk of MM (OR = 0.40; 95% CI = 0.19-0.84; p = 0.016). Interactions between other polymorphisms and asbestos exposure did not significantly influence MM risk. Conclusions Our findings suggest that the genetic variability of DNA repair mechanisms could contribute to the risk of developing MM.
Abstract
Human papillomavirus (HPV) positive pharyngeal squamous cell carcinoma (PSCC) appears to differ from HPV negative PSCC regarding several different characteristics due to the expression of ...HPV viral oncoproteins E6 and E7. Clinical studies suggest that HPV positive PSCC are more radiosensitive than HPV negative ones, however the exact mechanisms are still not known. It was previously shown that after X-ray radiation DNA accumulates in the cytosol of survived cells and can be sensed by specific pattern recognition receptors, i.e. cytosolic DNA sensors, which can modulate immune response and thus radiosensitivity of tumors. Therefore, the aim of our study was first to investigate the radiosensitivity and involvement of DNA sensing pathways in response to radiation in HPV positive and HPV negative PSCC in vitro. For determination of the radiosensitivity, HPV negative (FaDu and UM-SCC-6) and HPV positive (2A3 and UPCI:SCC090) PSCC cell lines were plated in 96 well plates and irradiated with different single doses (0, 2, 4, 6, 8 and 10 Gy). After 4 cell’s division PrestoBlue™ Cell Viability Reagent was used to determine the survival of cells taking into account different growth rate of the cells. For determination of DNA sensors and cytokines mRNA levels, PSCC cell lines were plated and irradiated with different single doses (0, 4 and 8 Gy). After 24 and 48 hours RNA was isolated using peqGOLD Total RNA Kit (VWR, West Chester, PA, USA) and reverse transcribed into cDNA using the SuperScript VILO cDNA Synthesis Kit (Invitrogen, Thermo Fisher Scientific). qRT-PCR was used to determine viral load (expression of HPV oncoproteins E6 and E7 mRNAs), expression of DNA sensors (cGAS, STING, RIG-I, DAI, IFI16, DDX60) and cytokines (IFNb, TNFa, IL1b). The expression of cytosolic DNA sensors and cytokines was dose- and time-dependent in both types of cells. The expression of DNA sensors DAI and RIG-I after irradiation was upregulated in HPV negative cell lines but not in HPV positive ones. The expression of cytokines IFN β and IL 1B was also upregulated only in HPV negative cell lines. Between the two HPV negative cell lines FaDu and SCC6, the expression profile did not differ, while the expression of DNA sensors and cytokines was higher in HPV positive cell line 2A3 compared to UM-SCC-6 cell lines, which has a higher level of viral proteins E6 and E7. In conclusion, our results indicate that on the in vitro level, DNA sensors are differentially expressed in response to irradiation in PSCC cell lines based on HPV infection but does not affect the radiosensitivity of the cells. Further in vivo studies are needed to elucidate the involvement of immune system in the response.
Citation Format: Kristina Levpuscek, Tanja Jesenko, Primoz Strojan, Gregor Sersa, Maja Cemazar. Irradiation induced expression of cytosolic DNA sensors and cytokines is associated with HPV status of pharyngeal squamous cell carcinoma cells abstract. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-013.
Genska terapija postaja čedalje bolj zanimiva tudi v onkologiji. Med aplikacijami je morda najzanimivejša imunostimulacija. Pripravimo lahko plazmidno DNA, ki nosi zapis za različne imunostimulatorne ...molekule, ki jih vnesemo v celice tumorjev ali normalnih tkiv. Ta tkiva postanejo proizvajalci teh molekul, ki lahko delujejo lokalno ali pa se izločajo tudi sistemsko v krvni obtok. Ker plazmidna DNA ne prehaja celične membrane, so potrebni dostavni sistemi, virusni ali nevirusni. V naših študijah uporabljamo predvsem nevirusni dostavni sistem – elektroporacijo. Interlevkin 12 (IL-12) je eden od zanimivih citokinov, za katerega je znano protitumorsko delovanje s spodbujanjem imunskega odziva in antiangiogenim delovanjem. Namen projekta SmartGene.si je bil pripraviti plazmid z zapisom za interlevkin 12 (plazmid phIL12) in pripraviti vse potrebno za njegovo klinično testiranje za zdravljenje kožnih tumorjev. V konzorciju smo združili moči s partnerji z akademskega in industrijskega področja. Treba je bilo pripraviti plazmid za uporabo v humani onkologiji po zahtevah Evropske agencije za zdravila (EMA). Za prijavo klinične študije na Javno agencijo za zdravila in medicinske pripomočke (JAZMP) smo morali izvesti tudi vse neklinične raziskave o varnosti in učinkovitosti zdravila. Nato je bilo treba razviti postopek priprave zdravila, zagotoviti primerne prostore za pripravo in izvedbo postopka priprave zdravila. V treh letih smo dosegli vse te zastavljene cilje in dobili dovoljenje za izvajanje klinične študije na kožnih tumorjih, ki ga je izdala JAZMP na osnovi pozitivnega mnenja Komisije Republike Slovenije za medicinsko etiko. Zdaj poteka klinična študija faze I preizkušanja plazmida phIL12 na kožnih tumorjih glave in vratu z namenom preveriti varnost in sprejemljivost genskega elektroprenosa plazmida v tumorje. Cilj študije je prav tako določiti primeren odmerek zdravila, ki bi ga v nadaljnji klinični študiji uporabili kot adjuvantno zdravljenje k ablativnim terapijam, kot sta radioterapija ali elektrokemoterapija.
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