Intracellular iron is essential for numerous biological processes, yet its redox activity makes it potentially cytotoxic. Because of this, a tight regulation of its cellular compartmentalization is required. Lysosomes and mitochondria play central roles in iron metabolism. Lysosomes are crucial for iron redistribution after its endocytosis, while mitochondria utilize it for heme and Fe-S cluster synthesis. Disruption of the functional crosstalk between these two organelles can lead to iron dyshomeostasis and ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation. Recent evidence highlights the importance of mitochondria-lysosome contact sites (MLCs) in mediating iron trafficking, particularly under pathological conditions. However, studying these nanoscopic, dynamic structures poses significant technical challenges. Here, we describe a novel live-cell imaging protocol combining super-resolution structured illumination microscopy (SIM) with organelle-specific dyes and a selective mitochondrial Fe(II) probe to visualize MLC formation and track iron transfer in real time. This approach enables the precise investigation of subcellular iron dynamics and their implications for ferroptosis and disease.

Lymphatic endothelial cells (LECs) and the lymphatic vasculature have evolved from being viewed as passive conduits for fluid drainage and metastatic dissemination to active, dynamic regulators of inflammation and tumor immunity. In solid tumors, both tumor-associated and lymph node (LN)–resident LECs engage in complex interactions with their environment to orchestrate immune processes, including antigen transport and presentation to T cells, leukocyte recruitment and trafficking via chemokine gradients, and local immune modulation through the expression of co-inhibitory ligands such as programmed death-ligand 1 (PD-L1). These multifaceted roles enable LECs to either amplify effector responses or induce tolerance, profoundly influencing the efficacy of cancer immunotherapies depending on their activation state, tissue context, and molecular programming. This minireview synthesizes and discusses recent advances in tumor lymphangiogenesis, the role of LECs and their intensive crosstalk with the immune compartments, in the coordination of anti-tumor immune responses, with particular focus on LEC-autophagy as a lipid metabolic checkpoint controlling lymph node T cell egress, and its far-reaching implications for optimizing immunotherapy outcomes in solid tumors.

Abstract Glioblastoma (GBM) cell states reflect spatial microenvironmental interactions. Here, using COMET spatial proteomics and RNAscope across multiregional human GBM tissue, spanning tumor cores with pseudopalisading regions, infiltrative margins and peripheral regions, together with multiplex spatial profiling of 202 specimens from 33 patients with matched primary and recurrent tumors, we identify full-length gasdermin E (GSDME-FL) as a macrophage-instructed regulator of malignant cell plasticity. Integration with single-cell transcriptomics and functional perturbation shows that GSDME-high tumor cells localize to macrophage-rich perivascular niches and are associated with delayed recurrence and longer patient survival. Mechanistically, macrophage-derived S100A4 activates EGFR-Sp1 signaling to induce GSDME-FL in neighboring GBM cells. GSDME-FL restrains hypoxia-associated mesenchymal plasticity and shapes macrophage-induced tumor state transitions independently of caspase activation. During immunogenic cell death (ICD), cleaved GSDME promotes pre-lytic swelling, early ATP efflux, and the release of canonical ICD-associated cytokines and chemokines. NLRP3 signaling further supports ATP release and licenses macrophage phagocytosis of dying GBM cells, whereas NINJ1-dependent membrane rupture enables terminal HMGB1 release. Vaccination with ICD-treated glioma cells elicits tumor rejection in a prophylactic intracranial challenge model. Notably, co-expression of GSDME and NINJ1 in macrophage-rich perivascular niches provides a spatial correlate of pathway convergence in patient tumors. Thus, our findings define how a spatial macrophage niche induces GSDME-FL, thereby coupling malignant cell state plasticity to the inflammatory properties of GBM cell death.

Autophagy, the primary lysosomal degradation pathway, plays a key role in cell survival and homeostasis. In tumors, it is upregulated to support cancer cell plasticity, adaptation to the microenvironment, and therapy resistance, making its inhibition an attractive therapeutic strategy. However, since autophagy is essential in healthy tissues, selective inhibition in tumors is critical. To address this, we designed inhibitors of two autophagy initiation factors (ULK1/2 and VPS34) equipped with tumor-targeting vectors. Our most promising candidates combine a low-nanomolar ULK1/2 inhibitory scaffold with an RGR-sequence targeting peptide. These compounds were validated across in vitro, in cellulo, and in vivo models, demonstrating selective activity and preserved efficacy. As the first examples of tumor-targeted autophagy inhibitors, they open new avenues for developing tissue-specific modulators of autophagy, with potential applications in oncology and beyond.

Abstract Cellular polyamine depletion is a promising anticancer strategy, but compensatory polyamine uptake limits efficacy when synthesis is blocked by DFMO (difluoromethylornithine), a clinically approved inhibitor of ornithine decarboxylase. The transporter and feedback mechanism driving this adaptive response have remained unclear. Despite their similar biochemical properties, we identify ATP13A3, rather than ATP13A2, as the principal DFMO-responsive polyamine importer, suggesting that these isoforms regulate distinct polyamine fluxes. Mechanistically, the polyamine sensor antizyme not only restrains polyamine biosynthesis but also selectively inhibits ATP13A3-mediated uptake, a brake that is relieved upon DFMO treatment. This regulatory circuit exposes distinct polyamine-acquisition states across cancers, defining synthesis- and/or uptake-biased subtypes that can shift during disease progression. Melanoma metastasis and vemurafenib resistance evolve toward increased ATP13A3-dependent uptake. The polyamine uptake branch controlled by ATP13A3-antizyme regulation can be pharmacologically blocked by AMXT 1501, which directly inhibits ATP13A3. Together, our findings explain DFMO adaptation through ATP13A3–antizyme control and establish ATP13A3 as a targetable node for polyamine depletion strategies in multiple cancers, supporting ongoing clinical evaluation of combined DFMO/AMXT 1501 therapy.

Deficiency of adenosine deaminase 2 (DADA2) causes a complex phenotype of autoinflammation and immunodeficiency. Bone marrow failure is often refractory to treatment with tumour necrosis factor-alpha (TNF-alpha) inhibitors and additional treatment options are needed. However, the pathomechanisms underlying the disease remain incompletely understood. The aim of this study was to examine the viability and metabolic profile of ADA2-deficient cells and to characterise the activity of different cell death pathways to advance the mechanistic understanding of DADA2. By flow cytometry and western blot, we showed that ADA2-/- U-937 cells and PBMCs from DADA2 patients showed significantly elevated levels of cell death compared with cells expressing wild-type ADA2. Viability of ADA2-deficient cells was not improved by inhibitors of apoptosis, necroptosis, pyroptosis and ferroptosis. Blocking of TNF-alpha, type I interferon and STING signalling as well as reintroduction of wild-type ADA2 protein did not rescue the cell death phenotype in vitro. ADA2-deficient cells had an aberrant morphology with increased cell size and granularity and were impaired in their proliferative capacity. To identify the cause of the impaired viability, we performed 13C glucose tracer metabolomics experiments which revealed disturbances in the pentose phosphate pathway of ADA2-deficient cells. This tended to be associated with increased exposure to intracellular reactive oxygen species that was attenuated in the PBMCs of a DADA2 patient measured after successful hematopoietic stem cell transplantation. Collectively, our findings established increased levels of cell death as a possible pathomechanism of DADA2 and showed that the absence of ADA2 leads to an impairment of the pentose phosphate pathway which may account for the cellular vulnerability of ADA2-deficient cells.

Iron is indispensable for cellular metabolism yet potentially cytotoxic, making its intracellular handling a fundamental determinant of cell fate decisions. The endo-lysosomal system has recently emerged as a central iron rheostat that integrates transferrin uptake, ferritinophagy, and lysosomal iron export to control iron bioavailability for mitochondria and other iron-dependent pathways. Growing studies further show that lysosomal iron is not merely permissive for ferroptosis but can directly initiate lipid damage through localized iron activation, lysosomal lipid peroxidation, and lysosomal membrane permeabilization. At the same time, emerging studies on organelle contact sites reveal that ferroptosis arises from the failure of a coordinated multi-organellar communication system, in which lysosomes, the endoplasmic reticulum, and mitochondria exchange iron, lipids, and redox signals in an effort to metabolically adapt to stress. This perspective is particularly relevant to drug-tolerant persisters and mesenchymal cancer cell states, which rely on rewired lysosomal iron trafficking to sustain plasticity while becoming highly susceptible to ferroptosis. In this minireview, we discuss emerging insights into the spatial organization of iron metabolism and propose a model in which ferroptosis sensitivity depends on the intracellular routing, chemical reactivity, and release dynamics of iron, highlighting lysosomal iron handling as a key therapeutic vulnerability in minimal residual disease.

A major obstacle in treating glioblastoma is the remarkable resistance of glioblastoma stem cells to standard-of-care therapies such as temozolomide and irradiation. A new study now reveals that dismantling protective structures called stress granules can make these cells more susceptible to these treatment regimens by unleashing ferroptosis.

High endothelial venules (HEVs) play a crucial role in adaptive immune responses in secondary lymphoid organs (SLOs). They are equipped with high amounts of peripheral node addressin (PNAd), harboring carbohydrate structures that serve as L-selectin ligands to efficiently facilitate lymphocyte homing. During inflammation, the HEV network expands in SLOs, increasing lymphocyte infiltration, but the underlying mechanisms that maintain HEVs remain underexplored. Here, we report that autophagy is essential for HEV function and expansion. Using single-cell transcriptomics, unbiased proteomics, intravital imaging, and an inducible HEV tracer system in mice, we demonstrate that autophagy deficiency compromises lymphotoxin beta receptor (LTβR) signaling and the unfolded protein response in HEVs, leading to disrupted PNAd production, dedifferentiation, and reduced lymphocyte homing. Autophagy deficiency and LTβR blockade impair HEV function and reduce skin inflammation in psoriasis-affected mice by limiting immune infiltration and cytokine release. Our work reveals that autophagy safeguards HEV identity and function during inflammation.

Macroautophagy/autophagy is the main lysosomal pathway for the degradation and recycling of cytoplasmic cargo, with emerging roles in endothelial cell (EC) biology. While autophagy has been extensively studied in blood ECs, its function in lymphatic ECs (LECs) remains unexplored. Given the central role of the lymphatic system in antitumor immunity and metastatic spread, we investigated how LEC autophagy affects metastatic lung colonization. In line with previous reports showing that autophagy regulates the availability of the egress signal sphingosine 1 phosphate (S1P) in secondary lymphoid organs (SLOs), lungs of non-tumor-bearing mice with LEC-specific genetic deletion of Atg5 (ATG5 LEC-KO mice) exhibited reduced lymphocyte infiltration. Remarkably, in tumor-bearing mice, either genetic loss of LEC-autophagy or pharmacological blockade of S1P lyase by 4-deoxypyridoxine (DOP) suppressed lung metastasis. Pulmonary immune profiling revealed that while DOP enhanced effector T cell activity despite lower numbers, LEC-autophagy-deficient mice markedly increased the B and CD8 T Cell abundance coupled with profound reduction of VEGFR3 expression in the lung lymphatic vasculature. Together, these findings uncover an autophagy-dependent remodeling of the lymphatic routes and immune niches that fosters metastatic seeding and growth in the lung.

The peroxidation of membrane phospholipids (PLs) is a hallmark of ferroptosis. The endoplasmic reticulum and mitochondria have been implicated in ferroptosis, but whether intracellular PL peroxidation ensues at their contact sites (endoplasmic reticulum-mitochondria contact sites, EMCSs) is unknown. Using super-resolution live imaging, we charted the spatiotemporal events triggered by ferroptosis at the interorganelle level. Here we show that EMCSs expand minutes after localized PL peroxides are formed and secondarily spread to mitochondria, promoting mitochondrial reactive oxygen species and fission. Oxidative lipidomics unravels that EMCSs host distinct proferroptotic polyunsaturated-PLs, including doubly proferroptotic polyunsaturated-acylated PLs, demonstrating their high propensity to undergo PL peroxidation. Endoplasmic reticulum-mitochondria untethering blunts PL peroxidation and ferroptosis, while EMCS stabilization enhances them. Consistently, distancing EMCSs protects the ferroptosis-susceptible triple-negative breast cancer subtype, harbouring high EMCS-related gene expression and basal PL peroxide levels. Conversely, in insensitive triple-negative breast cancer subtypes, bolstering EMCSs sensitizes them to ferroptosis. Our data unveil endoplasmic reticulum-mitochondria appositions as initial hubs of PL peroxide formation and posit that empowering EMCSs endorses ferroptosis in cancer cells.

Mitochondria and lysosomes regulate a multitude of biological processes that are essential for the maintenance of nutrient and metabolic homeostasis and overall cell viability. Recent evidence reveals that these pivotal organelles, similarly to others previously studied, communicate through specialized membrane contact sites (MCSs), hereafter referred to as mitochondria-lysosome contacts (or MLCs), which promote their dynamic interaction without involving membrane fusion. Signal integration through MLCs is implicated in key processes, including mitochondrial fission and dynamics, and the exchange of calcium, cholesterol, and amino acids. Impairments in the formation and function of MLCs are increasingly associated with age-related diseases, specifically neurodegenerative disorders and lysosomal storage diseases. However, MLCs may play roles in other pathological contexts where lysosomes and mitochondria are crucial. In this review, we introduce the methodologies used to study MLCs and discuss known molecular players and key factors involved in their regulation in mammalian cells. We also argue other potential regulatory mechanisms depending on the acidic lysosomal pH and their impact on MLC's function. Finally, we explore the emerging implications of dysfunctional mitochondria-lysosome interactions in disease, highlighting their potential as therapeutic targets in cancer.

The peroxidation of membrane phospholipids (PLs) is a hallmark of ferroptosis. The endoplasmic reticulum and mitochondria have been implicated in ferroptosis, but whether intracellular PL peroxidation ensues at their contact sites (endoplasmic reticulum-mitochondria contact sites, EMCSs) is unknown. Using super-resolution live imaging, we charted the spatiotemporal events triggered by ferroptosis at the interorganelle level. Here we show that EMCSs expand minutes after localized PL peroxides are formed and secondarily spread to mitochondria, promoting mitochondrial reactive oxygen species and fission. Oxidative lipidomics unravels that EMCSs host distinct proferroptotic polyunsaturated-PLs, including doubly proferroptotic polyunsaturated-acylated PLs, demonstrating their high propensity to undergo PL peroxidation. Endoplasmic reticulum-mitochondria untethering blunts PL peroxidation and ferroptosis, while EMCS stabilization enhances them. Consistently, distancing EMCSs protects the ferroptosis-susceptible triple-negative breast cancer subtype, harbouring high EMCS-related gene expression and basal PL peroxide levels. Conversely, in insensitive triple-negative breast cancer subtypes, bolstering EMCSs sensitizes them to ferroptosis. Our data unveil endoplasmic reticulum-mitochondria appositions as initial hubs of PL peroxide formation and posit that empowering EMCSs endorses ferroptosis in cancer cells.

Ferroptosis, a regulated form of cell death driven by lipid peroxidation, holds promise for targeting treatment-resistant cancer cells. Using a panel of melanoma cell lines, we uncover variability in the timing of ferroptosis onset upon exposure to iron and polyunsaturated fatty acids (PUFAs). This heterogeneity is linked to differences in PUFA sequestration into lipid droplets (LDs) and their subcellular distribution, particularly near lipid-metabolizing organelles such as mitochondria. In late-onset models, ferroptosis is delayed by peripheral LD retention and triggered by nutrient deprivation and AMP-activated protein kinase (AMPK) activation, which promotes LD trafficking toward mitochondria. Early responders bypass this mechanism. Our findings identify nutrient status and LD dynamics as key modulators of PUFA- and iron-induced ferroptosis, offering insights for therapeutic exploitation in cancer.

Iron sustains cancer cell plasticity, yet it also sensitizes the mesenchymal, drug-tolerant phenotype to ferroptosis. This posits that iron compartmentalization must be tightly regulated. However, the molecular machinery governing organelle Fe(II) compartmentalization remains elusive. Here, we show that BDH2 is a key effector of inter-organelle Fe(II) redistribution and ferroptosis vulnerability during melanoma transition from a melanocytic (MEL) to a mesenchymal-like (MES) phenotype. In MEL cells, BDH2 localizes at the mitochondria–lysosome contacts (MLCs) to generate the siderophore 2,5-dihydroxybenzoic acid (2,5-DHBA), which ferries iron into the mitochondria. Fe(II) transfer by BDH2 supports mitochondrial bioenergetics, which is required to maintain lysosomal acidification and MLC formation. Loss of BDH2 alters lysosomal pH and MLC tethering dynamics, causing lysosomal iron sequestration, which primes MES cells for ferroptosis. Rescuing BDH2 expression, or supplementing 2,5-DHBA, rectifies lysosomal pH and MLCs, protecting MES cells from ferroptosis and enhancing their ability to metastasize. Thus, we unveil a BDH2-dependent mechanism that orchestrates inter-organelle Fe(II) transfer, linking metabolic regulation of lysosomal pH to the ferroptosis vulnerability of the mesenchymal, drug-tolerant cancer cells. Rizzollo et al. show that BDH2 participates in iron distribution between cellular compartments, which sets the threshold for the ferroptosis vulnerability of the melanoma cell phenotypes, ultimately affecting their metastatic capacity

Reprogramming T cell metabolism can improve intratumoural fitness. By performing a CRISPR/Cas9 metabolic survey in CD8+ T cells, we identified 83 targets and we applied single-cell RNA sequencing to disclose transcriptome changes associated with each metabolic perturbation in the context of pancreatic cancer. This revealed elongation of very long-chain fatty acids protein 1 (Elovl1) as a metabolic target to sustain effector functions and memory phenotypes in CD8+ T cells. Accordingly, Elovl1 inactivation in adoptively transferred T cells combined with anti-PD-1 showed therapeutic efficacy in resistant pancreatic and melanoma tumours. The accumulation of saturated long-chain fatty acids in Elovl1-deficient T cells destabilized INSIG1, leading to SREBP2 activation, increased plasma membrane cholesterol and stronger T cell receptor signalling. Elovl1-deficient T cells increased mitochondrial fitness and fatty acid oxidation, thus withstanding the metabolic stress imposed by the tumour microenvironment. Finally, ELOVL1 in CD8+ T cells correlated with anti-PD-1 response in patients with melanoma. Altogether, Elovl1 targeting synergizes with anti-PD-1 to promote effective T cell responses.

Ferroptosis, driven by uncontrolled peroxidation of membrane phospholipids, is distinct from other cell death modalities because it lacks an initiating signal and is surveilled by endogenous antioxidant defenses. Glutathione peroxidase 4 (GPX4) is the guardian of ferroptosis, although its membrane-protective function remains poorly understood. Here, structural and functional analyses of a missense mutation in GPX4 (p.R152H), which causes early-onset neurodegeneration, revealed that this variant disrupts membrane anchoring without considerably impairing its catalytic activity. Spatiotemporal Gpx4 deletion or neuron-specific GPX4R152H expression in mice induced degeneration of cortical and cerebellar neurons, accompanied by progressive neuroinflammation. Patient induced pluripotent stem cell (iPSC)-derived cortical neurons and forebrain organoids displayed increased ferroptotic vulnerability, mirroring key pathological features, and were sensitive to ferroptosis inhibition. Neuroproteomics revealed Alzheimer's-like signatures in affected brains. These findings highlight the necessity of proper GPX4 membrane anchoring, establish ferroptosis as a key driver of neurodegeneration, and provide the rationale for targeting ferroptosis as a therapeutic strategy in neurodegenerative disease.

High endothelial venules (HEVs) play a crucial role in adaptive immune responses in secondary and tertiary lymphoid organs. They are uniquely equipped with high levels of peripheral node addressins (PNAd), harboring carbohydrate structures that serve as L-Selectin ligands to efficiently facilitate lymphocyte homing. During inflammation, the HEV network expands in SLOs, increasing lymphocyte infiltration, but the underlying mechanisms maintaining HEVs remain underexplored. Here, we report that autophagy is essential for HEV function and expansion. Using single-cell transcriptomics, intravital imaging, and an inducible HEV tracer system in mice, we demonstrate that autophagy deficiency compromises LTβR-signaling and the Unfolded Protein Response in HEVs, leading to disrupted PNAd production, dedifferentiation, and reduced lymphocyte homing. Autophagy deficiency and LTβR blockade impaired HEV function and reduced skin inflammation in psoriasis-bearing mice by limiting immune infiltration and cytokine release. Our work uncovers an unprecedented role of autophagy in safeguarding HEV identity and function during inflammation. Highlights ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND:Deficiency of adenosine deaminase 2 (DADA2) is an inborn error of immunity causing vasculitis and bone marrow failure. Bone marrow failure is mostly unresponsive to TNF-α inhibitors. The limited understanding of the pathomechanisms driving the disease impedes the development of new treatment options. Unlike cellular model systems expressing pathogenic ADA2 variants, primary monocytes from patients with DADA2 lack ADA2 protein expression. OBJECTIVES:This study aimed to analyze the role of protein degradation in the pathogenesis of DADA2 and the therapeutic potential of the lysosomotropic drug hydroxychloroquine in the treatment of patients with DADA2. METHODS:ADA2 protein expression in CD14+ monocytes from healthy controls (n = 8) and patients with DADA2 (n = 11) was determined by Western blot after inhibition of lysosomal and proteasomal degradation, as well as after hydroxychloroquine treatment in vivo in 1 patient with DADA2. Lipidation of microtubule associated protein 1 light chain 3 beta (LC3B) was analyzed as a measure of autophagic activity. Clinical and laboratory data were recorded in cytopenic patients with DADA2 treated with hydroxychloroquine, 200 mg per day. RESULTS:We demonstrated that inhibition of lysosomal degradation restores ADA2 protein expression in DADA2 monocytes in vitro. DADA2 monocytes exhibited increased autophagic activity. We observed clinical improvement in 2 cytopenic patients with DADA2 who were treated with hydroxychloroquine, and we showed a concomitant increase in ADA2 protein levels in monocytes from one of these patients in vivo. CONCLUSION:We identified lysosomal protein degradation of ADA2 as a pathomechanism of DADA2 and introduced hydroxychloroquine as a potential treatment option in patients with DADA2 with refractory cytopenia.

Smac mimetics that deplete cellular inhibitor of apoptosis (cIAP) proteins have been shown to activate Nuclear Factor-kappa B (NF-κB). Here, we report that Smac mimetic-mediated activation of NF-κB contributes to the rescue of cancer cells from tunicamycin (TM)-triggered apoptosis. The prototypic Smac mimetic BV6 activates non-canonical and canonical NF-κB pathways, while TM has little effect on NF-κB signaling. Importantly, ectopic expression of dominant-negative IκBα superrepressor (IκBα-SR), which inhibits canonical and non-canonical NF-κB activation, significantly reversed this BV6-imposed protection against TM. Similarly, transient or stable knockdown of NF-κB-inducing kinase, which accumulated upon exposure to BV6 alone and in combination with TM, significantly counteracted BV6-mediated inhibition of TM-induced apoptosis. Interestingly, while cIAP2 was initially degraded upon BV6 treatment, it was subsequently upregulated in an NF-κB-dependent manner, as this restoration of cIAP2 expression was abolished in IκBα-SR-overexpressing cells. Interestingly, upon exposure to TM/BV6 apoptosis was significantly increased in cIAP2 knockdown cells. Furthermore, NF-κB inhibition partially prevented BV6-stimulated expression of Mcl-1 upon TM treatment. Consistently, Mcl-1 silencing significantly inhibited BV6-mediated protection from TM-induced apoptosis. Thus, NF-κB activation by Smac mimetic contributes to Smac mimetic-mediated protection against TM-induced apoptosis.