Elena Catanzaro

Background Immunotherapy represents the future of clinical cancer treatment. The type of cancer cell death determines the antitumor immune response and thereby contributes to the efficacy of anticancer therapy and long-term survival of patients. Induction of immunogenic apoptosis or necroptosis in cancer cells does activate antitumor immunity, but resistance to these cell death modalities is common. Therefore, it is of great importance to find other ways to kill tumor cells. Recently, ferroptosis has been identified as a novel, iron-dependent form of regulated cell death but whether ferroptotic cancer cells are immunogenic is unknown. Methods Ferroptotic cell death in murine fibrosarcoma MCA205 or glioma GL261 cells was induced by RAS-selective lethal 3 and ferroptosis was analyzed by flow cytometry, atomic force and confocal microscopy. ATP and high-mobility group box 1 (HMGB1) release were detected by luminescence and ELISA assays, respectively. Immunogenicity in vitro was analyzed by coculturing of ferroptotic cancer cells with bone-marrow derived dendritic cells (BMDCs) and rate of phagocytosis and activation/maturation of BMDCs (CD11c + CD86 + , CD11c + CD40 + , CD11c + MHCII + , IL-6, RNAseq analysis). The tumor prophylactic vaccination model in immune-competent and immune compromised (Rag-2 −/− ) mice was used to analyze ferroptosis immunogenicity. Results Ferroptosis can be induced in cancer cells by inhibition of glutathione peroxidase 4, as evidenced by confocal and atomic force microscopy and inhibitors’ analysis. We demonstrate for the first time that ferroptosis is immunogenic in vitro and in vivo. Early, but not late, ferroptotic cells promote the phenotypic maturation of BMDCs and elicit a vaccination-like effect in immune-competent mice but not in Rag-2 −/− mice, suggesting that the mechanism of immunogenicity is very tightly regulated by the adaptive immune system and is time dependent. Also, ATP and HMGB1, the best-characterized damage-associated molecular patterns involved in immunogenic cell death, have proven to be passively released along the timeline of ferroptosis and act as immunogenic signal associated with the immunogenicity of early ferroptotic cancer cells. Conclusions 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 opens the door for the development of new strategies in cancer immunotherapy.

<h3>Background</h3> Anti-cancer therapy is more successful when it can also induce an immunogenic form of cancer cell death (ICD). Therefore, when developing new treatment strategies, it is extremely important to choose methods that induce ICD and thereby activate anti-tumor immune response leading to the most effective destruction of tumor cells. The aim of this work was to analyze whether the clinically widely used photosensitizers, photosens (PS) and photodithazine (PD), can induce ICD when used in photodynamic therapy (PDT). <h3>Methods</h3> Cell death in murine glioma GL261 or fibrosarcoma MCA205 cells was induced by PS- or PD-PDT and cell death was analyzed by MTT or flow cytometry. Intracellular distribution of PS and PD was studied by using the laser scanning microscope. Calreticulin exposure and HMGB1 and ATP release were detected by flow cytometry, ELISA and luminescence assay, respectively. Immunogenicity in vitro was analyzed by co-culturing of dying cancer cells with bone-marrow derived dendritic cells (BMDCs) and rate of phagocytosis and maturation (CD11c⁺CD86⁺, CD11c⁺CD40⁺) of BMDCs and production of IL-6 in the supernatant were measured. In vivo immunogenicity was analyzed in mouse tumor prophylactic vaccination model. <h3>Results</h3> We determined the optimal concentrations of the photosensitizers and found that at a light dose of 20 J/cm² (λex 615–635 nm) both PS and PD efficiently induced cell death in glioma GL261 and fibrosarcoma MCA205 cells. We demonstrate that PS localized predominantly in the lysosomes and that the cell death induced by PS-PDT was inhibited by zVAD-fmk (apoptosis inhibitor) and by ferrostatin-1 and DFO (ferroptosis inhibitors), but not by the necroptosis inhibitor necrostatin-1 s. By contrast, PD accumulated in the endoplasmic reticulum and Golgi apparatus, and the cell death induced by PD-PDT was inhibited only by z-VAD-fmk. Dying cancer cells induced by PS-PDT or PD-PDT emit calreticulin, HMGB1 and ATP and they were efficiently engulfed by BMDCs, which then matured, became activated and produced IL-6. Using dying cancer cells induced by PS-PDT or PD-PDT, we demonstrate the efficient vaccination potential of ICD in vivo. <h3>Conclusions</h3> Altogether, these results identify PS and PD as novel ICD inducers that could be effectively combined with PDT in cancer therapy.

Despite the huge investment into research and the significant effort and advances made in the search for new anticancer drugs in recent decades, cancer cure and treatment continue to be a formidable challenge. Many sources, including plants, animals, and minerals, have been explored in the oncological field because of the possibility of identifying novel molecular therapeutics. Marine sponges are a prolific source of secondary metabolites, a number of which showed intriguing tumor chemopreventive and chemotherapeutic properties. Recently, Food and Drug Administration-approved drugs derived from marine sponges have been shown to reduce metastatic breast cancer, malignant lymphoma, and Hodgkin's disease. The chemopreventive and potential anticancer activity of marine sponge-derived compounds could be explained by multiple cellular and molecular mechanisms, including DNA protection, cell-cycle modulation, apoptosis, and anti-inflammatory activities as well as their ability to chemosensitize cancer cells to traditional antiblastic chemotherapy. The present article aims to depict the multiple mechanisms involved in the chemopreventive and therapeutic effects of marine sponges and critically explore the limitations and challenges associated with the development of marine sponge-based anticancer strategy.

systems. However, the mechanisms at the basis of CAP-cell interaction are not yet completely understood. The aim of this study is to investigate CAP proapoptotic effect and identify some of the molecular mechanisms triggered by CAP in human T-lymphoblastoid leukemia cells. CAP treatment was performed by means of a wand electrode DBD source driven by nanosecond high-voltage pulses under different operating conditions. The biological endpoints were assessed through flow cytometry and real-time PCR. CAP caused apoptosis in Jurkat cells mediated by p53 upregulation. To test the involvement of intrinsic and/or extrinsic pathway, the expression of Bax/Bcl-2 and caspase-8 was analyzed. The activation of caspase-8 and the upregulation of Bax and Bcl-2 were observed. Moreover, CAP treatment increased ROS intracellular level. The situation reverts after a longer time of treatment. This is probably due to compensatory cellular mechanisms such as the posttranscriptional upregulation of SOD1, CAT, and GSR2. According to ROS increase, CAP induced a significant increase in DNA damage at all treatment conditions. In conclusion, our results provide a deeper understanding of CAP potential in the oncological field and pose the basis for the evaluation of its toxicological profile.