Annotation of the results obtained in 2020
The Project is devoted to study the mechanism of cell death induction by photodynamic therapy (PDT) of malignant brain cancer (glioma) and to analyze its immunogenicity. In the third stage of the Project, we studied immunogenic properties of photosensitizers developed in house from tetra(aryl)tetracyanoporphyrazines with different side radicals in the periphery of the macricycle. To confirm the universality of identified features of immunogenic cell death, we evaluated the effects of pz I – and pz III- based PDT on murine fibrosarcoma MCA cells, which is widely used in in vivo experimental studies on immunogenic cell death. Similar to glioma GL261 cells, the accumulation rate and localization of photosensitizers, and the type of cell death in PDT were determined for MCA205 cells. Dark toxicity and photodynamic activity (irradiation dose of 20 J/cm2) of pz I and pz III were also analyzed for MCA205 cells. An inhibitor analysis revealed that pz I-PDT induces apoptotic cell death, while pz III-PDT activates a mixed type of cell death with features of apoptosis and necroptosis. PDT based on pz I and pz III leads to the considerable increase in HMGB1 and ATP levels in the extracellular environment, which are typical features for immunogenic cell death. High phagocytic activity by primary dendritic cell cultures (DCs) towards MCA cells stimulated with pz I-PDT and pz III-PDT was shown. Moreover, tumor cells stimulated with PDT and co-cultured with DCs provided a phenotypic maturation of DCs characterized by increased expression of CD80 and CD86 markers. In the tumor prophylactic vaccination model based on pz I-PDT and pz III-PDT induced MCA205 cells, pronounced signs of activation of the adaptive immune system were observed, characterized by a significant decrease in the rate of the development of tumor growth. Vaccination with PDT-induced MCA205 cells of immunodeficient Nude mice confirmed a substantial contribution of the adaptive immune system in the induction of antitumor response. By the end of the 3rd year of the Project, an experimental article was published in the Journal for ImmunoTherapy of Cancer (IF WoS 10.252, Q1; top 6% of journals in «immunology» (10/159) and «oncology» (16/244)). It has been shown for the first time that early ferroptotic cancer cells are immunogenic in vitro and in vivo (Efimova Iu. et al. J ImmunoТher Cancer. 2020). This article has an Altmetric Attention Score of 37, which makes it the top 5% of all research outputs ever tracked by Altmetric (https://bmj.altmetric.com/details/94389453#score). Earlier on the second stage of the Project, it was shown that photosens-based PDT activates a mixed type of glioma GL261 cell death with features of apoptosis and ferroptosis, and revealed pronounced immunogenic effects (DAMPs release, phagocytosis by DCs and induction of DCs phenotypic maturation, significant inhibition of tumor growth in prophylactic vaccination model in vivo) (Turubanova et al. J ImmunoТher Cancer. 2019, 14 citations in WoS). This article has an Altmetric Attention Score of 130, which makes it the top 5% of all research outputs ever tracked by Altmetric (https://bmj.altmetric.com/details/73159302#score). Additional analysis of the literature and based on our obtained data allowed us to propose that ferroptotic cell death and photodynamic reactions can work synergistically; therefore, it is reasonable to assume that the induction of ferroptosis in PDT is a powerful alternative strategy to improve the effectiveness of anticancer therapy, in particular in tumors which are resistant to apoptosis and necroptosis. Our hypothesis will be presented to the scientific community in the Forum article by Mishchenko T.A., Balalaeva I.V., Vedunova M.V., Krysko D.V. New insights into effective anticancer strategy: a synergistic action of ferroptosis and photodynamic therapy in Trends in Cancer journal (IF WoS = 11.592, Q1; top 6% in «oncology» (13/244)). The manuscript is currently under reconsideration. A review by Alzeibak R., Mishchenko T.A., Shilyagina N.Yu., Balalaeva I.V., Vedunova M.V., Krysko D.V. Targeting immunogenic cell death by photodynamic therapy: past, present and future was also accepted for publication in the Journal for ImmunoTherapy of Cancer (Editor's decision from December 2, 2020; http://dx.doi.org/10.1136/jitc-2020-001926) (IF WoS 10.252, Q1; top 6% of journals in «immunology» (10/159) and «oncology» (16/244)). Experimental article based on the 3rd year project results Turubanova V., Mishchenko T., Balalaeva I., Efimova Iu., Peskova N., Klapshina L., Lermontova S., Bachert C., Krysko O., Vedunova M., Krysko D.V. Novel porphyrazine-based photodynamic anticancer therapy induces immunogenic cell death is currently under reconsideration in Scientific reports journal (IF WoS 4.576, Q1). These results were also presented at three international conferences, including in Switzerland and at three national conferences. Besides, three oral presentations by V. Turubanova at the all-Russian Conferences were awarded by the first-degree diploma. The results were also reported in numerous Russian and international media worldwide.

 

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Annotation of the results obtained in 2018
The Project is devoted to study a mechanism of cell death under photodynamic treatment of malignant brain cancer (glioma) and to analyze its immunogenicity. The basis for developing of approaches in neurooncology where photodynamic therapy (PDT) as an alternative therapeutic method for malignant brain tumors will be used indicate recent experimental results and first clinical studies. These data show a significant increase in patient survival, increase of disease-free intervals, and decrease of the risks of severe neurological effects. It has been recently shown in this field that regulated malignant cell death programs can either suppress or increase their immunogenic potential during treatment. Therefore, it is extremely important to characterize not only photosensitizers’ toxicity under various modes of PDT and the type of cell death, but also the molecular and immunological aspects of tumor cell death. One of the key aims of the first part of the Project was to choose photodynamic agents that will induce effectively cell death in glioma cells after PDT and in the same time will have a low toxicity to normal brain cells. Thefore in this part the following compounds have been studied as photodynamic agents: (i) photosensitizers, which are currently used in Russian clinical practice (Photoditazine or Photosens), (ii) hypericin which is used as a positive control, for which we have previously shown the induction of immunogenic cell death, and iii) unique compounds developed in house from tetra(aryl)tetracyanoporphyrazines group with different side radicals in the periphery of the macricycle: phenanthrenyl (Pz I), biphenyl (Pz II), fluorobenzyl -O-phenyl (Pz III), and diethyl-N-phenyl (Pz IV). In total we have tested seven photodynamic agents. Absorption and fluorescence spectra were obtained for all these studied compounds. We have characterized the features of penetration rate and intracellular localization of these photosensitizers for a murine glioma cell line GL261. It was shown that significant accumulation of amount of all studied photosensitizers in glioma cells occurred after several hours of incubation. Pz I and hypericin have the highest rate of internalization among the tested original (home made) and commercial compounds. The lowest internalization rate was shown for Photosens, as the most hydrophilic compound. We found that these photosensitizers significantly differ in their intracellular localization (being either lysosomes, ER or the Golgi apparatus). For example, we have confirmed the previously reported data by the Project leader that hypericin localizes in the ER. Indeed it has been shown that the ability of photosensitizer to induce immunogenic cell death is dependent on its localization in the ER and its ability to induce ER stress. Therefore, it can be assumed that the studied photosensitizers differ in their potential ability to induce immunogenic cell death. This issue will be investigated in the next part of the Project. For all studied photosensitizers, we analysed the ability to induce cell death in murine GL261 cells during short-term incubation in the dark, as well as during irradiation at a dose of 20 J/cm2. Photosens incubation in the dark at concentrations up to 100uM did not cause negative effect on glioma cells. Concentrations more than 30 μM of Photoditazine and Pz I-III significantly decreased cultures viability. The ability to induce cell death for Pz IV and hypericin was apparent when concentration of these photosensitizers exceeded 10 mM. Irradiation at a dose of 20 J/cm2 resulted in cell death at photosensitizer concentrations not exceeding approximately 1 μM. IC50 values corresponding to a photosensitizer concentration causing cell death with 50% efficiency were calculated. In order to preliminarily characterize the type of cell death induced by PDT with a photosensitizer, we performed experiments with cell death inhibitors. Selective blockers of apoptosis (a pancaspase inhibitor zVAD-fmk), necroptosis (inhibitor of kinase RIP1 - necrostatin-1s), ferroptosis (lipid radicals trap - ferrostatin-1, and iron chelator deferoxamine (DFO)) were used. For all photosensitizers the effect of inhibitors was statistically not significant 2 hours after irradiation. However, when this incubation time was increased, some cell death inhibitors could significantly block cell death. Moreover, this response was depended on the type of used photosensitizer and cell death inhibitor, which confirms the assumption that the type of induced cell death can vary depending on the physical and chemical properties of the photodynamic agent and its localization in the cell. To evaluate possible side effects, which photodynamic agents can cause on normal (non-malignant) nervous cells, we have performed an analysis of delayed toxicity effects of the studied substances by using primary murine brain cells cultures. We have found that despite the similarity of chemical nature, all porphyrazines (Pz I-IV) have different levels of cytotoxicity. The least cytotoxic effect was shown for PzI and PzII. PzIII and commercial photosensitizer Photoditazin caused pronounced cell death. Photodynamic agents Hypericin and Photosens have low dark toxicity to normal brain cells. Analysis of penetration rate of studied photosensitizers revealed that commercial photosensitizer Hypericin and Photoditazin as well as all porphyrazines PzI, PzII, PzIII and PzIV actively accumulate in neurons bodies, neuronal outgrowths, and in glial cells several hours after starting of incubation. The lowest rate of penetration into nervous cells has Photosens. Based on the obtained results, photosensitizers for further study have been chosen. Since pronounced toxicity effects could limit the practical application of the photosensitizer, the main criterion for selection was a lack of pronounced toxicity of photosensitizers for normal brain cells. Thus, the following photosensitizers will be used in the next part of the Project: hypericin, photosens, PZI / II.

 

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Annotation of the results obtained in 2019
The Project is devoted to study a mechanism of cell death induction by a photodynamic therapy (PDT) of malignant brain cancer (glioma) and to analyze its immunogenicity. It has been previously shown that PDT can be an alternative therapeutic method for malignant brain tumors. These data showed a significant increase in patient survival, increase of disease-free intervals, and decrease of the risks of severe neurological side effects. It has been recently shown in this field that regulated malignant cell death programs can either suppress or increase their immunogenic potential during treatment. Therefore, it is extremely important to characterize the molecular and immunological aspects of tumor cell death. In the second part of the Project, we performed a detailed analysis of immunogenicity (i.e. immunogenic cell death, ICD) of murine glioma GL261 cells, exposed to PDT. For this we have used commercially available photosensitizers such as photosens (PS) and photoditazine (PD). As PS and PD are now widely used in clinical practice, understanding of their capacity to induce ICD in future perspective will allow to modify therapeutic strategies and increase the effectiveness of PDT anticancer therapy. In order to confirm these data on another cancer cell line, the effects of PDT-PS and PDT-PD were evaluated on murine fibrosarcoma MCA205 cells, which are most commonly used in experimental studies of ICD. We characterized immunogenicity of the tumor cells stimulated by PDT based on PS and PD. For this the cells were first induced by both PDT-PS or PDT-PD and we found that this treatment cause emission of damage-associated molecular patterns (DAMPs), which are well-known markers of ICD. A significant increase in surface-exposure of calreticulin (CRT) and we detected release of ATP and HMGB1. This fact points to immunogenic nature of tumor cell death. Next, we evaluated a phagocytic activity and phenotypic changes of antigen-presenting cells in the presence of PDT-induced tumor cells. It was found that PDT-induced tumor cells are potent inducers of phagocytic activity of bone-marrow derived dendritic cells and provide their phenotypic maturation (increase of CD40, CD86 and MHC-II expression). Pronounced signs of activation of an adaptive immune system were observed in a prophylactic tumor vaccination model in vivo. We showed a significant decrease frequency of tumors after the injection of vaccine based on PDT-induced tumor cells. Thus, in vitro and in vivo studies demonstrate that induction of death in cancer cells by PDT based on photosens and photodithazine activates an adaptive immune response which is one of the important properties of ICD. In this second stage of the Project, two articles (1 of which in Q1 journal) were published in journals indexed by WoS and Scopus. Moreover, one experimental article was accepted for publication in the Journal for ImmunoTherapy of Cancer (impact factor WoS = 8.728, Q1), which is included in the top 10% of journals in “immunology” (12/158) and “oncology” (19/230). The results were also presented at four international including Belgium and USA conferences and two national conferences. Oral report by V. Turubanova at the 72nd All-Russian with international participation School-Conference of Young Scientists "Biosystems: Organization, Behaviour, Management" was awarded the first-degree diploma.

 

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