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Project titleNew aproaches in immunotherapy of glioma multiforme based on immonugenecity of ferroptotic cell death.

Keywordsferroptosis, immunogenic cell death, immunogenicity, DAMPs, tumor prophylactic vaccination, anti-tumor therapy

Annotation
Glioblastoma multiforme (GBM, WHO grade 4) is the most frequently occurring malignant central nervous system tumor with a global incidence of 0.59–3.69 per 100000. It is by far the most common and malignant of all glial tumors (Wen P.et al. N Engl J Med. 2008; Pearson J. et al. Signal Transduct Target Ther. 2017). Under the current therapeutic regimen of maximum safe resection, chemotherapy with temozolomide, radiotherapy (and where applicable additional modalities), median overall survival of only 14.6–20.9 months is achievable (Erhart F. et al. NPG Vaccines. 2020). Due to the low effectiveness of current GBM treatment methods, there is an urgent need to develop breakthrough therapeutic approaches to achieve maximum tumor destruction and level out the risk of secondary tumors and metastases. Immunotherapy is seen as an independent antitumor therapeutic strategy in addition to surgery, radio, and chemotherapy (Goldberg M.S. et al. Nat Rev Cancer. 2019). For GBM various immunotherapeutic approaches have been tested but so far none of them has resulted in a real clinical breakthrough (Berghoff A. et al. Nat Rev Neurol. 2019). Of the immunotherapies currently available, remarkable efforts have been invested in the development of dendritic cell (DC)–based vaccines (Mastelic-Gavillet B. et a. Front Immunol. 2020). In a therapeutic sense, DC vaccines largely act by stimulating tumor-specific cytotoxic CD8+ T cells that recognize and eliminate malignant cells. Current anti-GBM DC immunotherapies mostly use specific antigen peptides/RNA for pulsing DCs or whole-glioma tumor cells killed via freeze/thawing (F/T)–based necrosis (Garg A. et al. Trends Immunol. 2017). Although the former methodology might exhibit low efficacy due to the high antigenic heterogeneity of GBM, the latter procedure is associated with poor immunogenic potential. Thus, to create the next-generation anti-GBM vaccines, the immunogenicity of the dying/dead cancer cells used to pulse the DCs and the ability to induce a superior T helper 1–mediated immunity needs to be improved. Research in recent years convincingly proves that the manifestation of immunogenicity in dying tumor cells is one of the most critical factors that increase anti-cancer therapy's effectiveness. The induction of immunogenic cell death leads to the emission of Damage-Associated Molecular Patterns (DAMPs), which act as danger signals and as adjuvants to activate the immune system. The released DAMPs induce attraction of antigen-presenting cells (e.g., DCs) to the tumor bed and promotes antigen cross-presentation by the main Histocompatibility Class I complex to cells of the adaptive immune system (CD8+T cells), which target and eliminate the remaining tumor cells (Kepp O. et al. Cancer Metastasis Rev. 2011, Krysko D.V. et al. Nat Rev Cancer. 2012, Galluzzi L. et al. J ImmunoTher Cancer. 2020). Currently, several types of cell death with proven immunogenic properties (apoptosis, necroptosis) are known, but for many cancers, including GBM, the risk of resistance persists, which determines the escape of tumor cells from immunological control and resistance to therapy. That is why the search continues for alternative forms of cell death with immunogenic properties, through the induction of which effective tumor destruction can be achieved. Ferroptosis, an iron-dependent form of regulated necrosis, is a recently recognized type of cell death that can play an essential role in cancer biology (Dixon S.J. et al. Cell. 2012, Angeli J.P.F. et al. Trends Pharmacol Sci. 2017). In a recent study, it has been demonstrated that vaccination with early ferroptotic cancer cells induces efficient antitumor immunity pointing to the immunogenic potential of ferroptotic cancer cells (Efimova I. et al. J Immunother Cancer. 2020). These results pave the way for the development of new therapeutic strategies for cancers based on induction of ferroptosis, and thus broadens the current concept of immunogenic cell death and open the door for the development of new strategies in cancer immunotherapy. This project proposes to evaluate the immunomodulatory role of glioma cells that undergo ferroptosis and to unravel the role of DAMPs (calreticulin, HMGB1) in implementing the immunogenic properties of ferroptotic glioma cells and to evaluate the effectiveness of prophylactic and therapeutic DC vaccines loaded with ferroptotic glioma cells in the experimental orthotopic glioma mouse models (Fig. 1).

Expected results
The planned experiments in vitro and in vivo will allow to reveal the mechanisms of immunogenicity of ferroptotically dying glioma cells and to develop novel dendric cells’ vaccines based on the early ferroptotic cells. According to the literature, this is an innovative and timely project. In particular, the profile and level of emission of danger signals (DAMPs) in the case of ferroptotic glioma cells will be determined. In the project, original stable cell lines of glioma GL261 with a blocked expression of each of these DAMPs (Calreticulin, HMGB1) will be obtained. The role of each DAMPs in the manifestation of the immunogenic effect in the animal models will be established. We will also unravel the effectiveness of the use of prophylactic and therapeutic dendritic vaccines loaded with ferroptotic glioma cells in the orthotopic glioma mouse models and will evaluate the dynamics of tumor growth by magnetic resonance imaging technique and neurological deficit in animals and effects on T cells, macrophages, neutrophils, myeloid suppressor cells infiltration level in the tumor microenvironment. Based on the obtained results, 8 articles will be published in scientific journals indexed by Web of Science or Scopus databases. Implementation of the project will create a unique fundamental platform for developing a new strategy of antitumor immunotherapy for glioblastoma based on dendritic cells’ vaccines loaded with ferroptotic glioma cells. This approach may lead to a significant paradigm shift in cancer therapy and the development of pilot clinical trials in the future. In the project complex but complementary experimental techniques and methods in vitro and in vivo will be performed, which will thoroughly investigate ferroptotic immunogenicity mechanisms in dendritic cells’ vaccination in the orthotopic glioma mouse models. Applying these innovative methods will accelerate and facilitate the transfer of the necessary knowledge leading to the development of novel oncological experimental immunotherapies for glioblastoma based on ferroptosis induction.

Annotation of the results obtained in 2022
Glioblastoma multiforme (GBM) is the most common malignancy of the central nervous system. Due to the low effectiveness of current GBM treatment methods, there is an urgent need to develop breakthrough therapeutic approaches to achieve maximum tumor destruction and level out the risk of secondary tumors and metastases. Immunotherapy is seen as an independent antitumor therapeutic strategy in addition to surgery, radio, and chemotherapy. Development of dendritic cell (DC)-based vaccines, the effect of which is aimed at stimulating cytotoxic CD8+ T-cells that perform the function of recognition and destruction of tumor cells, is one of the intensively developing areas in GBM immunotherapy. Current anti-GBM DC immunotherapies mostly use specific antigen peptides/RNA for pulsing DCs or whole-glioma tumor cells killed via freeze/thawing (F/T)–based necrosis, but none of them has yet led to breakthrough results in the clinic. To create a new generation of anti-GBM vaccines with high efficacy, the immunogenic potential of the dying/dead glioma cells used to activate DCs and their ability to activate Th1-mediated immunity should be increased. The Project aims to study the role of damage-associated molecular patterns (DAMPs) in the immunogenicity of ferroptotic glioma cells and to investigate the efficacy of prophylactic and therapeutic DC vaccines loaded with ferroptotic glioma cells in an experimental orthotopic glioma model in vivo. At the first stage of the project, we optimized the CRISPR-Cas9 protocols to develop an in vitro model for studying the role of key DAMPs in the realization of cancer cell death through the ferroptosis pathway. We performed experiments to assess the role of the key DAMPs, calreticulin (CRT), in manifesting the immunogenicity of ferroptotic cancer cell death and found that CRT is an important participant in the immunogenic mechanisms that ensure the formation of an anti-tumor immune response and the effectiveness of preventive DC vaccination against glioma in vivo. We also analyzed the efficacy and specificity of glioma cell death through the ferroptosis pathway by using a selective inducer RSL3 and established its concentrations which leads to glioma cell death in IC50 and IC85 values. High specificity of ferroptotic glioma cell death induced by RSL3 was confirmed by an inhibitor analysis. An analysis of the key DAMPs (calreticulin, ATP, and HMGB1) exposure in RSL3 ferroptosis induction of glioma GL261 cells established a pattern of DAMPs characterized by early release of ATP followed by active calreticulin exposure from dying tumor cells and active release of HMGB1 in the late stages of ferroptosis. Active DAMPs release suggests immunogenic properties of glioma cell death through the ferroptosis pathway. Co-culturing of DCs with RSL3-induced glioma cells reveals a tendency to increase phagocytic activity and phenotypic maturation of antigen-presenting cells, and no increased content of proinflammatory cytokine IL6 in the cell supernatants. On the other hand, the first pilot experiments to identify the nature of macrophage polarization in the presence of RSL3-induced glioma cells revealed a pronounced tendency to polarize the immunosuppressive M2 phenotype into the antitumor M1 phenotype. Analysis of the specific response of antigen-presenting cells in the presence of early-stage ferroptosis glioma cells will be performed in the next stage of the project. We also developed protocol of orthotopic glioma model in vivo, which ensures active development of neoplastic processes in the brain tissue for 28 days after intracranial inoculation of viable GL261 glioma cells into the mouse brain. The model developed and verified by us will be used in the next stages of the project in terms of research on the effectiveness of prophylactic and therapeutic DC-based vaccination against glioma. By the end of the 1st year of Project one review article was published in Cells journal (Q1) indexed by WoS (IF=6.7) and Scopus (SJR = 1.45). The results were also presented at 2 international conferences, including 1 conference abroad (Belgium).

Publications

1. Krysko D.V., Demuynck R., Efimova Iu., Naessens F., Krysko O., Catanzaro E. In Vitro Veritas: From 2D Cultures to Organ-on-a-Chip Models to Study Immunogenic Cell Death in the Tumor Microenvironment Cells, 11(22), 3705 (year - 2022) https://doi.org/10.3390/cells11223705

2. Savyuk M.O., Turubanova V.D., Mishchenko T.A., Efimova Iu., Sleptsova E.E., Redkin T.S., Gorshkova E.N., Vedunova M.V., Krysko D.V., Krysko O.A. Analysis of the DAMPs release from ferroptotic glioma cells Fbstact book of BACR Annual meeting 2022: Tumour plasticity and heterogeneity during cancer progression, P. 1 (year - 2022)

3. Turubanova V.D., Saviuk M.O., Sleptsova E.E., Redkin T.S., Vedunova M.V., Krysko D.V. Анализ иммуногенного потенциала ферроптотически-индуцируемой клеточной гибели глиомы с использованием RSL3. IX Международная конференция молодых ученых: вирусологов, биотехнологов, биофизиков, молекулярных биологов и биоинформатиков — 2022: Cборник тезисов, АНО «Иннов. центр Кольцово». — Новосибирск : ИПЦ НГУ, 2022. С. 681 (year - 2022)

4. - Научные проекты Университета Лобачевского получили 17 грантов Российского научного фонда Независимое информационное агентство "Нижний Новгород" (НИА "Нижний Новгород"), 01.04.2022 (year - )

5. - 17 научных проектов Университета Лобачевского получили гранты РНФ ННГУ им. Н.И. Лобачевского (Новости ННГУ), 04.04.2022 (year - )

6. - 17 проектов нижегородцев получили гранты Российского научного фонда Нижегородская правда, 01.04.2022 (year - )

Annotation of the results obtained in 2023
Glioblastoma multiforme (GBM) is the most common malignancy of the central nervous system. Due to the low effectiveness of current GBM treatment methods, there is an urgent need to develop breakthrough therapeutic approaches to achieve maximum tumor destruction and level out the risk of secondary tumors and metastases. Immunotherapy is seen as an intriguing independent antitumor therapeutic strategy in addition to surgery, radio, and chemotherapy. Development of dendritic cell (DC)-based vaccines, the effect of which is aimed at stimulating cytotoxic CD8+ T-cells that perform the function of recognition and destruction of tumor cells, is one of the intensively developing areas in GBM immunotherapy. Current anti-GBM DC immunotherapies mostly use specific antigen peptides/RNA for pulsing DCs or whole-glioma tumor cells killed via freeze/thawing (F/T)–based necrosis, but none of them has yet led to breakthrough results in the clinic. The project aims to study the role of damage-associated molecular patterns (DAMPs) in the immunogenicity of glioma cells undergoing regulated cell death and study the efficacy of prophylactic and therapeutic DC vaccines loaded with dying/dead glioma cells in an experimental orthotopic glioma model in vivo. At the second stage of the project, genetic transformation of glioma GL261 cells was performed to obtain persistent cell line GL261-bCRT with gene blockade of one of the key DAMPs, i.e. calreticulin (CRT). Viable GL261-bCRT monoclonal cultures were obtained by single-cell optical sorting, and their efficiency was analyzed by PCR and sequencing. At the third stage of the project, the selected monoclones are planned to test the efficiency of CRT gene knockout by assessing the level of protein expression. The phenotypic maturation profile of dendritic cells (DCs) in the presence of glioma GL261 cells at early (3 h) and late (24 h) stages of regulated cell death and their lysates were characterized. According to the results of cytokine profile analysis in the supernatants obtained by co-culturing DCs with dead GL261 glioma cells at ratios of 1:5 and 1:10 a significant increase of proinflammatory cytokine IL-6 was found when DCs were co-cultured with glioma cells at the early stage of regulated cell death in both studied ratios. The data suggest the potential participation of activated antigen-presenting cells in the differentiation and activation of T cells in the process of antigen-presentation with the subsequent formation of a T-cell immune response. Approbation and selection of optimal protocols for DCs vaccination based on lysates of glioma cells undergoing regulated cell death in prophylactic and therapeutic regimens were performed. The efficacy of prophylactic DCs vaccination based on lysates of dead glioma cells under blockade of CRT was evaluated. It has been shown that CRT is an important link of immunogenic mechanisms ensuring the formation of antitumor immune response and the efficacy of prophylactic DC vaccination against glioma based on lysates of glioma cells undergoing regulated cell death. By the end of the 2nd year of the project we published protocols for evaluating immunogenic cell death in vitro and in vivo, including ferroptosis, using photodynamic therapy as an example of one therapeutic strategy against glioma – Mishchenko T. et al. Methods in Сell Biology (impact factor WoS 1.83, Scopus). One review was published in the Vaccines journal (impact factor WoS 7.8, Scopus, Q1). We have proposed a concept for creating effective DCs-based vaccines against glioma and published it in the Trends in Cancer journal (impact factor WoS 18.4, Scopus, Q1). A review article uncovering possible strategies to overcome the immunosuppressive glioma microenvironment, the importance of identifying the immunological profile for each patient in order to develop a personalized immunotherapeutic strategy, has been accepted for publication in the Frontiers in Immunology journal (impact factor WoS 7.3, Scopus, Q1). The results were also presented at regional and international conferences.

Publications

1. Mishchenko T.A., Turubanova V.D., Gorshkova E.N., Krysko O., Vedunova M.V., Krysko D.V. Targeting immunogenic cell death for glioma immunotherapy Trends in Cancer, S2405-8033(23)00216-9 (year - 2023) https://doi.org/10.1016/j.trecan.2023.10.005

2. Mishchenko T.A., Turubanova V.D., Gorshkova E.N., Krysko O., Vedunova M.V., Krysko D.V. Glioma: Bridging the Tumor Microenvironment, Patient Immune Profiles for Novel Personalized Immunotherapy Frontiers in Immunology, - (year - 2023)

3. Perenkov A.D., Sergeeva A.D., Vedunova M.V., Krysko D.V. In Vitro Transcribed RNA-Based Platform Vaccines: Past, Present, and Future Vaccines, 11(10), 1600 (year - 2023) https://doi.org/10.3390/vaccines11101600

4. Mishchenko T.A., Balalaeva I.V., Turubanova V.D., Saviuk M.O., Shylyagina N.Yu., Krysko O., Vedunova M.V., Krysko D.V. Gold standard assessment of immunogenic cell death induced by photodynamic therapy: From in vitro to tumor mouse models and anti-cancer vaccination strategies Methods in Cell Biology, Academic Press, Амстердам, P. 1-62 (year - 2023) https://doi.org/10.1016/bs.mcb.2023.05.003