The alpha-adrenergic antagonist prazosin promotes cytosolic siRNA delivery from lysosomal compartments.

The widespread use of small interfering RNA (siRNA) is limited by the multiple extra- and intracellular barriers upon in vivo administration. Hence, suitable delivery systems, based on siRNA encapsulation in nanoparticles or its conjugation to targeting ligands, have been developed. Nevertheless, at the intracellular level, these state-of-the-art delivery systems still suffer from a low endosomal escape efficiency. Consequently, the bulk of the endocytosed siRNA drug rapidly accumulates in the lysosomal compartment. We recently reported that a wide variety of cationic amphiphilic drugs (CADs) can promote small nucleic acid delivery from the endolysosomal compartment into the cytosol via transient induction of lysosomal membrane permeabilization. Here, we describe the identification of alternate siRNA delivery enhancers from the NIH Clinical Compound Collection that do not have the typical physicochemical properties of CADs. Additionally, we demonstrate improved endolysosomal escape of siRNA via a cholesterol conjugate and polymeric carriers with the α1-adrenergic antagonist prazosin, which was identified as the best performing delivery enhancer from the compound screen. A more detailed assessment of the mode-of-action of prazosin suggests that a different cellular phenotype compared to typical CAD adjuvants drives cytosolic siRNA delivery. As it has been described in the literature that prazosin also induces cancer cell apoptosis and promotes antigen cross-presentation in dendritic cells, the proof-of-concept data in this work provides opportunities for the repurposing of prazosin in an anti-cancer combination strategy with siRNA.

Mechanobiology of Ferroptotic Cancer Cells as a Novel "Eat-Me" Signal: Regulating Efferocytosis through Layer-by-Layer Coating.

The importance of the clearance of dead cells is shown to have a regulatory role for normal tissue homeostasis and for the modulation of immune responses. However, how mechanobiological properties of dead cells affect efferocytosis remains largely unknown. Here, it is reported that the Young's modulus of cancer cells undergoing ferroptosis is reduced. To modulate their Young's modulus a layer-by-layer (LbL) nanocoating is developed. Scanning electron and fluorescence microscopy confirm coating efficiency of ferroptotic cells while atomic force microscopy reveals encapsulation of the dead cells increases their Young's modulus dependent on the number of applied LbL layers which increases their efferocytosis by primary macrophages. This work demonstrates the crucial role of mechanobiology of dead cells in regulating their efferocytosis by macrophages which can be exploited for the development of novel therapeutic strategies for diseases where modulation of efferocytosis can be potentially beneficial and for the design of drug delivery systems for cancer therapy.

Ferroptosis: friend or foe in cancer immunotherapy?

Ferroptosis has gained interest due to it immunogenicity and the higher sensitivity of cancer cells to it. However, it was recently shown that ferroptosis in tumor-associated neutrophils leads to immunosuppression and negatively impacts therapy. Here, we discuss the potential implications of the two sides (friend versus foe) of ferroptosis in cancer immunotherapy.

DC vaccines loaded with glioma cells killed by photodynamic therapy induce Th17 anti-tumor immunity and provide a four-gene signature for glioma prognosis.

Gliomas, the most frequent type of primary tumor of the central nervous system in adults, results in significant morbidity and mortality. Despite the development of novel, complex, multidisciplinary, and targeted therapies, glioma therapy has not progressed much over the last decades. Therefore, there is an urgent need to develop novel patient-adjusted immunotherapies that actively stimulate antitumor T cells, generate long-term memory, and result in significant clinical benefits. This work aimed to investigate the efficacy and molecular mechanism of dendritic cell (DC) vaccines loaded with glioma cells undergoing immunogenic cell death (ICD) induced by photosens-based photodynamic therapy (PS-PDT) and to identify reliable prognostic gene signatures for predicting the overall survival of patients. Analysis of the transcriptional program of the ICD-based DC vaccine led to the identification of robust induction of Th17 signature when used as a vaccine. These DCs demonstrate retinoic acid receptor-related orphan receptor-γt dependent efficacy in an orthotopic mouse model. Moreover, comparative analysis of the transcriptome program of the ICD-based DC vaccine with transcriptome data from the TCGA-LGG dataset identified a four-gene signature (CFH, GALNT3, SMC4, VAV3) associated with overall survival of glioma patients. This model was validated on overall survival of CGGA-LGG, TCGA-GBM, and CGGA-GBM datasets to determine whether it has a similar prognostic value. To that end, the sensitivity and specificity of the prognostic model for predicting overall survival were evaluated by calculating the area under the curve of the time-dependent receiver operating characteristic curve. The values of area under the curve for TCGA-LGG, CGGA-LGG, TCGA-GBM, and CGGA-GBM for predicting five-year survival rates were, respectively, 0.75, 0.73, 0.9, and 0.69. These data open attractive prospects for improving glioma therapy by employing ICD and PS-PDT-based DC vaccines to induce Th17 immunity and to use this prognostic model to predict the overall survival of glioma patients.

In Vitro Veritas: From 2D Cultures to Organ-on-a-Chip Models to Study Immunogenic Cell Death in the Tumor Microenvironment.

Immunogenic cell death (ICD) is a functionally unique form of cell death that promotes a T-cell-dependent anti-tumor immune response specific to antigens originating from dying cancer cells. Many anticancer agents and strategies induce ICD, but despite their robust effects in vitro and in vivo on mice, translation into the clinic remains challenging. A major hindrance in antitumor research is the poor predictive ability of classic 2D in vitro models, which do not consider tumor biological complexity, such as the contribution of the tumor microenvironment (TME), which plays a crucial role in immunosuppression and cancer evasion. In this review, we describe different tumor models, from 2D cultures to organ-on-a-chip technology, as well as spheroids and perfusion bioreactors, all of which mimic the different degrees of the TME complexity. Next, we discuss how 3D cell cultures can be applied to study ICD and how to increase the translational potential of the ICD inducers. Finally, novel research directions are provided regarding ICD in the 3D cellular context which may lead to novel immunotherapies for cancer.

Immunogenic ferroptosis and where to find it?

Ferroptosis is a recently discovered form of regulated cell death that is morphologically, genetically, and biochemically distinct from apoptosis and necroptosis, and its potential use in anticancer therapy is emerging. The strong immunogenicity of (early) ferroptotic cancer cells broadens the current concept of immunogenic cell death and opens up new possibilities for cancer treatment. In particular, induction of immunogenic ferroptosis could be beneficial for patients with cancers resistant to apoptosis and necroptosis. However, ferroptotic cancer cells may be a rich source of oxidized lipids, which contribute to decreased phagocytosis and antigen cross-presentation by dendritic cells and thus may favor tumor evasion. This could explain the non-immunogenicity of late ferroptotic cells. Besides the presence of lactate in the tumor microenvironment, acidification and hypoxia are essential factors promoting ferroptosis resistance and affecting its immunogenicity. Here, we critically discuss the crucial mediators controlling the immunogenicity of ferroptosis that modulate the induction of antitumor immunity. We emphasize that it will be necessary to also identify the tolerogenic (ie, immunosuppressive) nature of ferroptosis, which can lead to tumor evasion.

Novel porphyrazine-based photodynamic anti-cancer therapy induces immunogenic cell death.

The immunogenicity of dying cancer cells determines the efficacy of anti-cancer therapy. Photodynamic therapy (PDT) can induce immunogenic cell death (ICD), which is characterized by the emission of damage-associated molecular patterns (DAMPs) from dying cells. This emission can trigger effective anti-tumor immunity. Only a few photosensitizers are known to induce ICD and, therefore, there is a need for development of new photosensitizers that can induce ICD. The purpose of this work was to analyze whether photosensitizers developed in-house from porphyrazines (pz I and pz III) can induce ICD in vitro and in vivo when used in PDT. We indetified the optimal concentrations of the photosensitizers and found that, at a light dose of 20 J/cm2 (λex 615-635 nm), both pz I and pz III efficiently induced cell death in cancer cells. We demonstrate that pz I localized predominantly in the Golgi apparatus and lysosomes while pz III in the endoplasmic reticulum and lysosomes. The cell death induced by pz I-PDT was inhibited by zVAD-fmk (apoptosis inhibitor) but not by ferrostatin-1 and DFO (ferroptosis inhibitors) or by necrostatin-1 s (necroptosis inhibitor). By contrast, the cell death induced by pz III-PDT was inhibited by z-VAD-fmk and by the necroptosis inhibitor, necrostatin-1 s. Cancer cells induced by pz I-PDT or pz III-PDT released HMGB1 and ATP and were engulfed by bone marrow-derived dendritic cells, which then matured and became activated in vitro. We demonstrate that cancer cells, after induction of cell death by pz I-PDT or pz III-PDT, are protective when used in the mouse model of prophylactic tumor vaccination. By vaccinating immunodeficient mice, we prove the role of the adaptive immune system in protecting against tumours. All together, we have shown that two novel porphyrazines developed in-house are potent ICD inducers that could be effectively applied in PDT of cancer.

Vaccination with early ferroptotic cancer cells induces efficient antitumor immunity.

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.

A 3D Cell Death Assay to Quantitatively Determine Ferroptosis in Spheroids.

The failure of drug efficacy in clinical trials remains a big issue in cancer research. This is largely due to the limitations of two-dimensional (2D) cell cultures, the most used tool in drug screening. Nowadays, three-dimensional (3D) cultures, including spheroids, are acknowledged to be a better model of the in vivo environment, but detailed cell death assays for 3D cultures (including those for ferroptosis) are scarce. In this work, we show that a new cell death analysis method, named 3D Cell Death Assay (3DELTA), can efficiently determine different cell death types including ferroptosis and quantitatively assess cell death in tumour spheroids. Our method uses Sytox dyes as a cell death marker and Triton X-100, which efficiently permeabilizes all cells in spheroids, was used to establish 100% cell death. After optimization of Sytox concentration, Triton X-100 concentration and timing, we showed that the 3DELTA method was able to detect signals from all cells without the need to disaggregate spheroids. Moreover, in this work we demonstrated that 2D experiments cannot be extrapolated to 3D cultures as 3D cultures are less sensitive to cell death induction. In conclusion, 3DELTA is a more cost-effective way to identify and measure cell death type in 3D cultures, including spheroids.

Biochemical thresholds for pathological presentation of ATP synthase deficiencies.

Mitochondrial ATP synthase is responsible for production of the majority of cellular ATP. Disorders of ATP synthase in humans can be caused by numerous mutations in both structural subunits and specific assembly factors. They are associated with variable pathogenicity and clinical phenotypes ranging from mild to the most severe mitochondrial diseases. To shed light on primary/pivotal functional consequences of ATP synthase deficiency, we explored human HEK 293 cells with a varying content of fully assembled ATP synthase, selectively downregulated to 15-80% of controls by the knockdown of F1 subunits γ, δ and ε. Examination of cellular respiration and glycolytic flux revealed that enhanced glycolysis compensates for insufficient mitochondrial ATP production while reduced dissipation of mitochondrial membrane potential leads to elevated ROS production. Both insufficient energy provision and increased oxidative stress contribute to the resulting pathological phenotype. The threshold for manifestation of the ATP synthase defect and subsequent metabolic remodelling equals to 10-30% of residual ATP synthase activity. The metabolic adaptations are not able to sustain proliferation in a galactose medium, although sufficient under glucose-rich conditions. As metabolic alterations occur when the content of ATP synthase drops below 30%, some milder ATP synthase defects may not necessarily manifest with a mitochondrial disease phenotype, as long as the threshold level is not exceeded.

Immunogenic cell death induced by a new photodynamic therapy based on photosens and photodithazine.

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: Altogether, these results identify PS and PD as novel ICD inducers that could be effectively combined with PDT in cancer therapy.