Eat-me Signals Research Articles

Loss of GABARAP mediates resistance to immunogenic chemotherapy in multiple myeloma

AbstractImmunogenic cell death (ICD) is a form of cell death by which cancer treatments can induce a clinically relevant antitumor immune response in a broad range of cancers. In multiple myeloma (MM), the proteasome inhibitor bortezomib is an ICD inducer and creates durable therapeutic responses in patients. However, eventual relapse and resistance to bortezomib appear inevitable. Here, by integrating patient transcriptomic data with an analysis of calreticulin (CRT) protein interactors, we found that GABA type A receptor–associated protein (GABARAP) is a key player whose loss prevented tumor cell death from being perceived as immunogenic after bortezomib treatment. GABARAP is located on chromosome 17p, which is commonly deleted in patients with high risk MM. GABARAP deletion impaired the exposure of the eat-me signal CRT on the surface of dying MM cells in vitro and in vivo, thus reducing tumor cell phagocytosis by dendritic cells and the subsequent antitumor T-cell response. Low GABARAP was independently associated with shorter survival in patients with MM and reduced tumor immune infiltration. Mechanistically, we found that GABARAP deletion blocked ICD signaling by decreasing autophagy and altering Golgi apparatus morphology, with consequent defects in the downstream vesicular transport of CRT. Conversely, upregulating autophagy using rapamycin restored Golgi morphology, CRT exposure, and ICD signaling in GABARAPKO cells undergoing bortezomib treatment. Therefore, coupling an ICD inducer, such as bortezomib, with an autophagy inducer, such as rapamycin, may improve patient outcomes in MM, in which low GABARAP in the form of del(17p) is common and leads to worse outcomes.

Cardiolipin-mediated temporal response to hydroquinone toxicity in human retinal pigmented epithelial cell line.

Hydroquinone, a potent toxic agent of cigarette smoke, damages retinal pigmented epithelial cells by triggering oxidative stress and mitochondrial dysfunction, two events causally related to the development and progression of retinal diseases. The inner mitochondrial membrane is enriched in cardiolipin, a phospholipid susceptible of oxidative modifications which determine cell-fate decision. Using ARPE-19 cell line as a model of retinal pigmented epithelium, we analyzed the potential involvement of cardiolipin in hydroquinone toxicity. Hydroquinone exposure caused an early concentration-dependent increase in mitochondrial reactive oxygen species, decrease in mitochondrial membrane potential, and rise in the rate of oxygen consumption not accompanied by changes in ATP levels. Despite mitochondrial impairment, cell viability was preserved. Hydroquinone induced cardiolipin translocation to the outer mitochondrial membrane, and an increase in the colocalization of the autophagosome adapter protein LC3 with mitochondria, indicating the induction of protective mitophagy. A prolonged hydroquinone treatment induced pyroptotic cell death by cardiolipin-mediated caspase-1 and gasdermin-D activation. Cardiolipin-specific antioxidants counteracted hydroquinone effects pointing out that cardiolipin can act as a mitochondrial "eat-me signal" or as a pyroptotic cell death trigger. Our results indicate that cardiolipin may act as a timer for the mitophagy to pyroptosis switch and propose cardiolipin-targeting compounds as promising approaches for the treatment of oxidative stress-related retinal diseases.

Defective Clearance of Neutrophils Causes Pathogenic Microenvironmental Interactions and Offers a Candidate Immunotherapy in the Myeloproliferative Neoplasms

Neutrophils are the most abundant leukocytes and essential components of the immune microenvironment. Myeloproliferative neoplasms (MPNs) are characterized by excessive myelopoiesis and serious complications, such as thrombosis, myelofibrosis and leukemic transformation. Neutrophils and hematopoietic stem cells (HSCs) carrying the most common MPN driver mutation (JAK2 V617F) are protected from apoptosis. Moreover, mutant neutrophils aggregate with platelets and interact with activated endothelium, increasing thrombosis risk in MPN (Blood 107:3676; Thromb Res 129:263; Sci Transl Med 10:eaan8292; Leukemia 33:2544). However, possible pathogenic interactions between heterogeneous bone marrow (BM) niches in MPN (Nat Cancer doi:10.1038/s43018-023-00607-x) and neutrophils are understudied. Circulating HSCs and leukocytes home daily to the BM (Nature 452:442), where apoptotic/senescent neutrophils are cleared by macrophages (Immunology 125:281; Cell 153:1025). We found that senescent neutrophils carrying JAK2 V617F or CALR del52 mutation evade normal clearance and accumulate in the BM of MPN mice. The homeostatic recognition and efferocytosis of neutrophils by macrophages rely on the balance of “eat-me” and “don't-eat-me” signals. Supervised analyses of two independent human JAK2 V617F+ MPN granulocyte RNAseq datasets (Blood 123:e123; Blood 134:199) showed specific upregulation of CD24 - reportedly a don't-eat-me signal in solid tumors (Nature 572:392). CD24 expression was high in granulocytes from MPN transformed into AML and in the JAK inhibitors ruxolitinib and fedratinib-resistant HEL cells (Nat Cancer 4:108). Unlike CD274 or CD47, which are extensively studied in myeloid malignancies, our data showed that CD24 expression was high in JAK2 V617F+ human or mouse neutrophils. Functional blockade in co-culture with macrophages or after in vivo adoptive transfer showed that CD24 serves as a don't-eat-me signal in neutrophils. Given that oncogenic driver mutations lead to the constitutive activation of cytokine-regulated JAK-STAT signaling, we investigated the effect of cytokines activating this pathway. We found that high GM-CSF in MPN BM induces CD24 expression and prevents neutrophil clearance via JAK-STAT5 signaling. Consequently, ruxolitinib normalized GM-CSF-induced CD24 expression and decreased human and mouse neutrophils in MPN. Our study showed that accumulation of mutant senescent neutrophils in the BM triggered abnormal interactions within the BM microenvironment. Mutated neutrophils were found closer to Megakaryocytes (Mk) and ~30% of Mks showed neutrophils transiently invading their demarcation membrane system through a process termed “emperipolesis”, which increases thrombopoiesis and intercellular exchange between neutrophils and Mks (Elife 8:e44031) and frequently occurs in diseases associated with myelofibrosis, such as MPN (Blood 104:3573) and grey platelet syndrome (Blood 124:3624). To examine whether mutated neutrophils affect platelet production, we transferred wild-type or JAK2 V617F+ senescent neutrophils and found that JAK2 V617F+ mutated (but not WT) senescent neutrophils increased Mk emperipolesis and circulating platelets. Supervised RNA-seq analysis of CD24 hi JAK2 V617F+ human neutrophils highlighted their pro-inflammatory signature. Furthermore, CD24 expression positively correlated with neutrophil degranulation signatures and proteolytic enzymes that activate latent pro-fibrotic TGF-β in Mk, subsequently triggering myelofibrosis. Taken together, these findings suggest that accumulation of senescent neutrophils might lead to their pathogenic interactions with Mks or platelets, which may increase thrombocytosis and myelofibrosis risk in MPN. To test this hypothesis and the therapeutic potential of CD24 inhibition, MPN mice were chronically treated with CD24 blocking antibody. This treatment improved thrombocytosis, decreased platelet-neutrophil complexes, restored normal clearance of mutated senescent neutrophils and reduced emperipolesis levels which, in turn, decreased active TGF-β, ultimately improving myelofibrosis. This study reveals defective neutrophil clearance as a cause of pathogenic microenvironmental interactions that may increase thrombosis and myelofibrosis risk, and postulate CD24 as a candidate target for innate immune checkpoint in myeloid malignancies.

TMIC-24. FERROPTOSIS INDUCES IMMUNOGENIC CELL DEATH AND SYNERGIZES WITH CD47 BLOCKADE TO REPROGRAM THE GLIOMA MICROENVIRONMENT

Abstract Immunotherapy has thus far proven ineffective in glioblastoma, in part due to the immune cold tumor microenvironment. Here we demonstrate that ferroptosis specifically targets a quiescent, immunosuppressive astrocyte-like glioma population and can therefore be used as a novel inducer of immunogenic cell death to repolarize the immune microenvironment by promoting tumor cell phagocytosis and antigen presentation. First, we characterized the immunogenic effects of ferroptosis by using a glioma cell-myeloid cell co-culture system and patient-derived organotypic slice cultures and performed phagocytosis assays, flow-cytometry, immunohistochemistry, cytokine arrays and single cell RNAseq. GPX4-dependent ferroptosis induced translocation of the pro-phagocytic eat-me signal calreticulin in both murine glioma cells and human tissue samples. Ferroptotic-driven tumor cell death reprogramed local myeloid populations to promote glioma phagocytosis, MHCII-driven antigen presentation, and paracrine cytokine signaling involved in T-cell recruitment. Local delivery of RSL3, a GPX4 inhibitor, in a murine glioma model polarized myeloid cells to a pro-phagocytic phenotype and significantly increased intra-tumoral levels of CD4+ and CD8+ T-cells. Astrocytic-like tumor cells upregulate CD47 as a mechanism to evade phagocytosis. Therefore, we combined ferroptosis with an anti-CD47 monoclonal antibody (aCD47) to enhance reprogramming of the immune cold microenvironment. In an orthotopic murine glioma model, adding aCD47 to RSL3 led to significantly increased tumor phagocytosis, antigen presentation and prolonged survival. Thus, we demonstrate that local delivery of ferroptosis-inducing agents in conjunction with CD47 blockade is an effective multi-modality therapeutic regimen capable of reversing the immunosuppressive glioma microenvironment.

The Drosophila chemokine–like Orion bridges phosphatidylserine and Draper in phagocytosis of neurons

Phagocytic clearance of degenerating neurons is triggered by "eat-me" signals exposed on the neuronal surface. The conserved neuronal eat-me signal phosphatidylserine (PS) and the engulfment receptor Draper (Drpr) mediate phagocytosis of degenerating neurons in Drosophila. However, how PS is recognized by Drpr-expressing phagocytes invivo remains poorly understood. Using multiple models of dendrite degeneration, we show that the Drosophila chemokine-like protein Orion can bind to PS and is responsible for detecting PS exposure on neurons; it is supplied cell-non-autonomously to coat PS-exposing dendrites and to mediate interactions between PS and Drpr, thus enabling phagocytosis. As a result, the accumulation of Orion on neurons and on phagocytes produces opposite outcomes by potentiating and suppressing phagocytosis, respectively. Moreover, the Orion dosage is a key determinant of the sensitivity of phagocytes to PS exposed on neurons. Lastly, mutagenesis analyses show that the sequence motifs shared between Orion and human immunomodulatory proteins are important for Orion function. Thus, our results uncover a missing link in PS-mediated phagocytosis in Drosophila and imply conserved mechanisms of phagocytosis of neurons.

Apoptosis and Phagocytosis as Antiviral Mechanisms.

Viruses are infectious entities that make use of the replication machinery of their hosts to produce more progenies, causing disease and sometimes death. To counter viral infection, metazoan hosts are equipped with various defense mechanisms, from the rapid-evoking innate immune responses to the most advanced adaptive immune responses. Previous research demonstrated that cells in fruit flies and mice infected with Drosophila C virus and influenza, respectively, undergo apoptosis, which triggers the engulfment of apoptotic virus-infected cells by phagocytes. This process involves the recognition of eat-me signals on the surface of virus-infected cells by receptors of specialized phagocytes, such as macrophages and neutrophils in mice and hemocytes in fruit flies, to facilitate the phagocytic elimination of virus-infected cells. Inhibition of phagocytosis led to severe pathologies and death in both species, indicating that apoptosis-dependent phagocytosis of virus-infected cells is a conserved antiviral mechanism in multicellular organisms. Indeed, our understanding of the mechanisms underlying apoptosis-dependent phagocytosis of virus-infected cells has shed a new perspective on how hosts defend themselves against viral infection. This chapter explores the mechanisms of this process and its potential for developing new treatments for viral diseases.

Alzheimer's disease-associated R47H TREM2 increases, but wild-type TREM2 decreases, microglial phagocytosis of synaptosomes and neuronal loss.

Triggering receptor on myeloid cells 2 (TREM2) is an innate immune receptor, upregulated on the surface of microglia associated with amyloid plaques in Alzheimer's disease (AD). Individuals heterozygous for the R47H variant of TREM2 have greatly increased risk of developing AD. We examined the effects of wild-type (WT), R47H and knock-out (KO) of human TREM2 expression in three microglial cell systems. Addition of mouse BV-2 microglia expressing R47H TREM2 to primary mouse neuronal cultures caused neuronal loss, not observed with WT TREM2. Neuronal loss was prevented by using annexin V to block exposed phosphatidylserine, an eat-me signal and ligand of TREM2, suggesting loss was mediated by microglial phagocytosis of neurons exposing phosphatidylserine. Addition of human CHME-3 microglia expressing R47H TREM2 to LUHMES neuronal-like cells also caused loss compared to WT TREM2. Expression of R47H TREM2 in BV-2 and CHME-3 microglia increased their uptake of phosphatidylserine-beads and synaptosomes versus WT TREM2. Human iPSC-derived microglia with heterozygous R47H TREM2 had increased phagocytosis of synaptosomes vs common-variant TREM2. Additionally, phosphatidylserine liposomes increased activation of human iPSC-derived microglia expressing homozygous R47H TREM2 versus common-variant TREM2. Finally, overexpression of TREM2 in CHME-3 microglia caused increased expression of cystatin F, a cysteine protease inhibitor, and knock-down of cystatin F increased CHME-3 uptake of phosphatidylserine-beads. Together, these data suggest that R47H TREM2 may increase AD risk by increasing phagocytosis of synapses and neurons via greater activation by phosphatidylserine and that WT TREM2 may decrease microglial phagocytosis of synapses and neurons via cystatin F.

Combined In Vitro Toxicity and Immunogenicity of Cold Plasma and Pulsed Electric Fields.

In modern oncology, therapies are based on combining monotherapies to overcome treatment resistance and increase therapy precision. The application of microsecond-pulsed electric fields (PEF) is approved to enhance local chemotherapeutic drug uptake within combination electrochemotherapy regimens. Reactive oxygen species (ROS) have been implicated in anticancer effects, and cold physical plasma produces vast amounts of ROS, which have recently been shown to benefit head and neck cancer patients. PEF and cold plasma technology have been linked to immunogenic cell death (ICD) induction, a regulated cell death accompanied by sterile inflammation that promotes antitumor immunity. To this end, we investigated the combined effect of both treatments regarding their intracellular ROS accumulation, toxicity, ICD-related marker expression, and optimal exposure sequence in a leukemia model cell line. The combination treatment substantially increased ROS and intracellular glutathione levels, leading to additive cytotoxic effects accompanied by a significantly increased expression of ICD markers, such as the eat-me signal calreticulin (CRT). Preconditioned treatment with cold plasma followed by PEF exposure was the most potent treatment sequence. The results indicate additive effects of cold plasma and PEF, motivating further studies in skin and breast tumor models for the future improvement of ECT in such patients.

PARP-inhibition reprograms macrophages toward an anti-tumor phenotype.

Poly(ADP)ribosylation inhibitors (PARPis) are toxic to cancer cells with homologous recombination (HR) deficiency but not to HR-proficient cells in the tumor microenvironment (TME), including tumor-associated macrophages (TAMs). As TAMs can promote or inhibit tumor growth, we set out to examine the effects of PARP inhibition on TAMs in BRCA1-related breast cancer (BC). The PARPi olaparib causes reprogramming of TAMs toward higher cytotoxicity and phagocytosis. A PARPi-related surge in NAD+ increases glycolysis, blunts oxidative phosphorylation, and induces reverse mitochondrial electron transport (RET) with an increase in reactive oxygen species (ROS) and transcriptional reprogramming. This reprogramming occurs in the absence or presence of PARP1 or PARP2 and is partially recapitulated by addition of NAD derivative methyl-nicotinamide (MNA). Invivo and exvivo, the effect of olaparib on TAMs contributes to the anti-tumor efficacy of the PARPi. Invivo blockade of the "don't-eat-me signal" with CD47 antibodies in combination with olaparib improves outcomes in a BRCA1-related BC model.

Membrane-localized calreticulin induced by ER stress in cytotrophoblast-like BeWo cells acts as an eat-me signal.

Membrane-localized calreticulin induced by ER stress in cytotrophoblast-like BeWo cells acts as an eat-me signal.

P06.03.A Combination of EGFRvIII CAR T cell therapy and paracrine GAM modulation for the treatment of GBM

Abstract Background The glioblastoma (GBM) immune microenvironment mainly consists of protumoral glioma-associated microglia and macrophages (GAMs). Previously, we showed that blockade of the don't-eat-me signal CD47, overexpressed by GBM cells, rescued GAMs' phagocytic function in mice. However, CD47 blockade monotherapy has been ineffective in treating human solid tumors to date. Thus, we propose a combinatorial approach of local chimeric antigen receptor (CAR) T cell therapy with paracrine GAM modulation for a synergistic elimination of GBM. Material and Methods We lentivirally transduced healthy donor human T cells to generate humanized EGFRvIII-directed CAR T that constitutively secrete a SIRPγ-related protein (SGRP) with high affinity to CD47. Killing assays were performed with endogenous EGFRvIII-expressing BS153 or EGFRvIII-overexpressed U251 GBM cells and assessed by Incucyte time-lapse imaging analysis. CAR T cell activation was confirmed by flow cytometry (FC) and IFNγ was detected from co-culture supernatants or mouse plasma by ELISA. The CAR T cell secretome was analyzed by liquid chromatography-mass spectrometry (LC-MS) to confirm the secretion of SGRP. NSG mice were orthotopically implanted with either EGFRvIII+ BS153 or U251 cells and treated intratumorally with one or two CAR T cell infusions. Results EGFRvIII and EGFRvIII-SGRP CAR T proliferated and killed tumor cells in vitro in a dose-dependent manner within 72h with complete cytotoxicity at E:T 1:1 compared to CD19 CAR T. CAR T cells specifically co-expressed CD25 and CD107a and secreted IFNγ in the presence of tumor antigen (24h CD25/CD107a co-expression: EGFRvIII=59.3±3.00%, EGFRvIII-SGRP=52.6±1.42%, CD19=0.1±0.07%; 24h IFNγ secretion: EGFRvIII=173.6±2.50%, EGFRvIII-SGRP=113.8±6.42%, CD19=4.5±1.49%). Differential expression analysis of the CAR T cell secretome identified SGRP in EGFRvIII-SGRP CAR T supernatants (-Log10qValue/Log2fold-change=13.72/6.62). Consistent with studies of systemic EGFRvIII CAR T cell therapy, our data suggest that intratumoral EGFRvIII CAR T were insufficient to eliminate BS153 tumors with endogenous EGFRvIII (Overall survival; EGFRvIII: 20%, CD19: 0%, n=5/group). Conclusion Here, we show that EGFRvIII CAR T specifically targeted and killed EGFRvIII+ GBM cells in vitro, but failed to control tumor growth in vivo without GAM modulation. EGFRvIII-SGRP CAR T secretome analysis identified SGRP from the supernatants of unstimulated monocultures. SGRP impaired the binding of SIRPα-Fc to CD47-upregulated GBM cells in vitro, but did not elicit macrophage-mediated phagocytosis of GBM cells in our current in vitro experimental setup. Future work will focus on the functional characterization of SGRP and on further investigating the additive effect of CAR T cell therapy and GAM modulation using translational in vivo and ex vivo models.

Phospholipid Scramblases: Role in Cancer Progression and Anticancer Therapeutics.

Phospholipid scramblases (PLSCRs) that catalyze rapid mixing of plasma membrane lipids result in surface exposure of phosphatidyl serine (PS), a lipid normally residing to the inner plasma membrane leaflet. PS exposure provides a chemotactic eat-me signal for phagocytes resulting in non-inflammatory clearance of apoptotic cells by efferocytosis. However, metastatic tumor cells escape efferocytosis through alteration of tumor microenvironment and apoptotic signaling. Tumor cells exhibit altered membrane features, high constitutive PS exposure, low drug permeability and increased multidrug resistance through clonal evolution. PLSCRs are transcriptionally up-regulated in tumor cells leading to plasma membrane remodeling and aberrant PS exposure on cell surface. In addition, PLSCRs interact with multiple cellular components to modulate cancer progression and survival. While PLSCRs and PS exposed on tumor cells are novel drug targets, many exogenous molecules that catalyze lipid scrambling on tumor plasma membrane are potent anticancer therapeutic molecules. In this review, we provide a comprehensive analysis of scramblase mediated signaling events, membrane alteration specific to tumor development and possible therapeutic implications of scramblases and PS exposure.

Statins boost the macrophage eat-me signal to keep atherosclerosis at bay.

Statins boost the macrophage eat-me signal to keep atherosclerosis at bay.

Externalized phosphatidylinositides on apoptotic cells are eat-me signals recognized by CD14

Apoptotic cells are rapidly engulfed and removed by phagocytes after displaying cell surface eat-me signals. Among many phospholipids, only phosphatidylserine (PS) is known to act as an eat-me signal on apoptotic cells. Using unbiased proteomics, we identified externalized phosphatidylinositides (PIPs) as apoptotic eat-me signals recognized by CD14+ phagocytes. Exofacial PIPs on the surfaces of early and late-apoptotic cells were observed in patches and blebs using anti-PI(3,4,5)P3 antibody, AKT- and PLCδ PH-domains, and CD14 protein. Phagocytosis of apoptotic cells was blocked either by masking exofacial PIPs or by CD14 knockout in phagocytes. We further confirmed that exofacial PIP+ thymocytes increased dramatically after in vivo irradiation and that exofacial PIP+ cells represented more significant populations in tissues of Cd14−/− than WT mice, especially after induction of apoptosis. Our findings reveal exofacial PIPs to be previously unknown cell death signals recognized by CD14+ phagocytes.

Cyclophilin A Impairs Efferocytosis and Accelerates Atherosclerosis by Overexpressing CD 47 and Down-Regulating Calreticulin.

Impairment of efferocytosis in apoptotic macrophages is a known determinant of the severity of atherosclerosis and the vulnerability of plaques to rupture. The precise mechanisms involved in impaired efferocytosis are unclear. Given the well-recognized role of the inflammatory cytokine cyclophilin A (Cyp A) in modulating several atherogenic mechanisms in high-glucose primed monocytes, we investigated the role of Cyp A in macrophage efferocytosis. The efficiency of efferocytosis in RAW 264.7 macrophages grown in vitro and primed with cyclophilin A was assessed using flow cytometry and confocal assays. Cholesterol content in cells was measured using cell-based cholesterol efflux assay. Proteomic analysis and bioinformatics tools were employed to decipher the link between cyclophilin A and the known ligand receptors involved in efferocytosis. Cyclophilin A was found to impair efferocytosis in apoptotic macrophages by reducing ABCA1-mediated cholesterol efflux in foam cells derived from macrophages. Cyclophilin A-primed macrophages showed an increase in expression of the don’t-eat-me signal CD 47 and a decrease in the expression of the eat-me signal, calreticulin. Phagocytosis was restored upon silencing of cyclophilin A. New Zealand white rabbits were fed a high-fat diet, and lesions in their aortae were analyzed histologically for evidence of atherosclerosis and the expression of Cyp A, CD 47 and calreticulin, the ligand receptor involved in efferocytosis. Gene and protein expressions in aortae and macrophages were analyzed by real-time PCR and Western blotting. Cyclophilin A, via its effects on the expression of CD 47 and calreticulin, impairs efferocytosis in apoptotic macrophages. Together with its impact on cholesterol efflux from macrophages, these effects can amplify other mechanisms of Cyp A in accelerating the progression of atherosclerosis.

Precise Delivery of Nanomedicines to M2 Macrophages by Combining "Eat Me/Don't Eat Me" Signals and Its Anticancer Application.

Targeted delivery of nanomedicines to M2 tumor-associated macrophages (TAMs) has been proposed to reduce tumor promotion and enhance the efficacy of anticancer therapy. However, upregulated receptors on M2 TAMs are also expressed on M1 TAMs and other macrophages in normal tissues. Therefore, improving targeting specificity remains a key challenge. Here, we developed a precise M2 TAM-targeted delivery system using "eat-me" and "don't-eat-me" signals. A CD47-derived self-peptide ligand (don't-eat-me signal) and galactose ligand (eat-me signal) were introduced on liposomes. Cleavable phospholipid-polyethylene glycol was covered on the surface and could combine with the self-peptide to inhibit macrophage recognition even after immunoglobulin M adsorption and protect galactose from hepatic clearance to prolong the circulation time and promote the accumulation of liposomes in tumors. This detachable polymer can be removed by the redox microenvironment upon transcytosis through the tumor endothelium and re-expose the self-peptide and galactose. The self-peptide highly reduced M1 macrophage phagocytosis, and the galactose ligand enhanced the interaction between the liposomes and M2 macrophages. Thus, the modified liposomes enabled specific recognition of M1/M2 TAMs. In vitro evidence revealed reduced endocytosis of the liposomes by M1 macrophages. Moreover, in vivo studies demonstrated that doxorubicin-loaded liposomes efficiently eliminated M2 TAMs but did not affect M1 TAMs, enhancing the potency of the antitumor therapy. Collectively, our results demonstrate the potential of combining active escape and active targeting for precisely delivering a drug of interest to M2 macrophages and suggest its application in anticancer therapy.

Combined Blockade of CD47-Sirpa Interaction By 5F9 (Magrolimab) and Azacitidine/Venetoclax Therapy Facilitates Macrophage-Mediated Anti-Leukemia Efficacy in AML Pre-Clinical Models

Combined Blockade of CD47-Sirpa Interaction By 5F9 (Magrolimab) and Azacitidine/Venetoclax Therapy Facilitates Macrophage-Mediated Anti-Leukemia Efficacy in AML Pre-Clinical Models

Dimethyl sulfide mediates microbial predator-prey interactions between zooplankton and algae in the ocean.

Phytoplankton are key components of the oceanic carbon and sulfur cycles1. During bloom events, some species can emit large amounts of the organosulfur volatile dimethyl sulfide (DMS) into the ocean and consequently the atmosphere, where it can modulate aerosol formation and affect climate2,3. In aquatic environments, DMS plays an important role as a chemical signal mediating diverse trophic interactions. Yet, its role in microbial predator-prey interactions remains elusive with contradicting evidence for its role in either algal chemical defence or in the chemo-attraction of grazers to prey cells4,5. Here we investigated the signalling role of DMS during zooplankton-algae interactions by genetic and biochemical manipulation of the algal DMS-generating enzyme dimethylsulfoniopropionate lyase (DL) in the bloom-forming alga Emiliania huxleyi6. We inhibited DL activity in E. huxleyi cells in vivo using the selective DL-inhibitor 2-bromo-3-(dimethylsulfonio)-propionate7 and overexpressed the DL-encoding gene in the model diatom Thalassiosira pseudonana. We showed that algal DL activity did not serve as an anti-grazing chemical defence but paradoxically enhanced predation by the grazer Oxyrrhis marina and other microzooplankton and mesozooplankton, including ciliates and copepods. Consumption of algal prey with induced DL activity also promoted O. marina growth. Overall, our results demonstrate that DMS-mediated grazing may be ecologically important and prevalent during prey-predator dynamics in aquatic ecosystems. The role of algal DMS revealed here, acting as an eat-me signal for grazers, raises fundamental questions regarding the retention of its biosynthetic enzyme through the evolution of dominant bloom-forming phytoplankton in the ocean.

The Phagocytic Code Regulating Phagocytosis of Mammalian Cells.

Mammalian phagocytes can phagocytose (i.e. eat) other mammalian cells in the body if they display certain signals, and this phagocytosis plays fundamental roles in development, cell turnover, tissue homeostasis and disease prevention. To phagocytose the correct cells, phagocytes must discriminate which cells to eat using a ‘phagocytic code’ - a set of over 50 known phagocytic signals determining whether a cell is eaten or not - comprising find-me signals, eat-me signals, don’t-eat-me signals and opsonins. Most opsonins require binding to eat-me signals – for example, the opsonins galectin-3, calreticulin and C1q bind asialoglycan eat-me signals on target cells - to induce phagocytosis. Some proteins act as ‘self-opsonins’, while others are ‘negative opsonins’ or ‘phagocyte suppressants’, inhibiting phagocytosis. We review known phagocytic signals here, both established and novel, and how they integrate to regulate phagocytosis of several mammalian targets - including excess cells in development, senescent and aged cells, infected cells, cancer cells, dead or dying cells, cell debris and neuronal synapses. Understanding the phagocytic code, and how it goes wrong, may enable novel therapies for multiple pathologies with too much or too little phagocytosis, such as: infectious disease, cancer, neurodegeneration, psychiatric disease, cardiovascular disease, ageing and auto-immune disease.

Putting the brakes on phagocytosis: "don't-eat-me" signaling in physiology and disease.

Timely removal of dying or pathogenic cells by phagocytes is essential to maintaining host homeostasis. Phagocytes execute the clearance process with high fidelity while sparing healthy neighboring cells, and this process is at least partially regulated by the balance of "eat-me" and "don't-eat-me" signals expressed on the surface of host cells. Upon contact, eat-me signals activate "pro-phagocytic" receptors expressed on the phagocyte membrane and signal to promote phagocytosis. Conversely, don't-eat-me signals engage "anti-phagocytic" receptors to suppress phagocytosis. We review the current knowledge of don't-eat-me signaling in normal physiology and disease contexts where aberrant don't-eat-me signaling contributes to pathology.