RIPK3 dampens mitochondrial bioenergetics and lipid droplet dynamics in metabolic liver disease.

BACKGROUND AND AIMS: Receptor-interacting protein kinase 3 (RIPK3) mediates NAFLD progression, but its metabolic function is unclear. Here, we aimed to investigate the role of RIPK3 in modulating mitochondria function, coupled with lipid droplet (LD) architecture in NAFLD. APPROACH AND RESULTS: Functional studies evaluating mitochondria and LD biology were performed in wild-type (WT) and Ripk3-/- mice fed a choline-deficient, amino acid-defined (CDAA) diet for 32 and 66 weeks and in CRISPR-Cas9 Ripk3 -null fat-loaded immortalized hepatocytes. The association between hepatic perilipin (PLIN) 1 and 5, RIPK3, and disease severity was also addressed in a cohort of patients with NAFLD and in PLIN1 -associated familial partial lipodystrophy. Ripk3 deficiency rescued impairment in mitochondrial biogenesis, bioenergetics, and function in CDAA diet-fed mice and fat-loaded hepatocytes. Ripk3 deficiency was accompanied by a strong upregulation of antioxidant systems, leading to diminished oxidative stress upon fat loading both in vivo and in vitro. Strikingly, Ripk3-/- hepatocytes displayed smaller size LD in higher numbers than WT cells after incubation with free fatty acids. Ripk3 deficiency upregulated adipocyte and hepatic levels of LD-associated proteins PLIN1 and PLIN5. PLIN1 upregulation controlled LD structure and diminished mitochondrial stress upon free fatty acid overload in Ripk3-/- hepatocytes and was associated with diminished human NAFLD severity. Conversely, a pathogenic PLIN1 frameshift variant was associated with NAFLD and fibrosis, as well as with increased hepatic RIPK3 levels in familial partial lipodystrophy. CONCLUSIONS: Ripk3 deficiency restores mitochondria bioenergetics and impacts LD dynamics. RIPK3 inhibition is promising in ameliorating NAFLD.

Effect of Cisplatin and Its Cationic Analogues in the Phase Behavior and Permeability of Model Lipid Bilayers.

Increasing evidence suggests a critical role of lipids in both the mechanisms of toxicity and resistance of cells to platinum(II) complexes. In particular, cisplatin and other analogues were reported to interact with lipids and transiently promote lipid phase changes both in the bulk membranes and in specific membrane domains. However, these processes are complex and not fully understood. In this work, cisplatin and its cationic species formed at pH 7.4 in low chloride concentrations were tested for their ability to induce phase changes in model membranes with different lipid compositions. Fluorescent probes that partition to different lipid phases were used to report on the fluidity of the membrane, and a leakage assay was performed to evaluate the effect of cisplatin in the permeability of these vesicles. The results showed that platinum(II) complex effects on membrane fluidity depend on membrane lipid composition and properties, promoting a stronger decrease in the fluidity of membranes containing gel phase. Moreover, at high concentration, these complexes were prone to alter the permeability of lipid membranes without inducing their collapse or aggregation.

Meeting Report - The 2019 FEBS special meeting on sphingolipid biology: sphingolipids in physiology and pathology.

Sphingolipids are a fundamental class of molecules that are involved in structural, organizational and signaling properties of eukaryotic membranes. Defects in their production or disposal lead to acquired and inherited human diseases. A growing community of scientists has embraced the challenge to dissect different aspects of sphingolipid biology using a variety of approaches, and a substantial part of this community met last May in the beautiful town of Cascais in Portugal. Over 200 scientists from 26 countries animated the conference, held in a 15th century citadel, sharing their data and opinions on the current understanding and future challenges in sphingolipid research. Here, we report some of their contributions to provide the readers with a bird's-eye view of the themes discussed at the meeting.

Canonical and 1-Deoxy(methyl) Sphingoid Bases: Tackling the Effect of the Lipid Structure on Membrane Biophysical Properties.

Compared to the canonical sphingoid backbone of sphingolipids (SLs), atypical long-chain bases (LCBs) lack C1-OH (1-deoxy-LCBs) or C1-CH2OH (1-deoxymethyl-LCBs). In addition, when unsaturated, they present a cis-double bond instead of the canonical  Δ4-5 trans-double bond. These atypical LCBs are directly correlated with the development and progression of hereditary sensory and autonomic neuropathy type 1 and diabetes type II through yet unknown mechanisms. Changes in membrane properties have been linked to the biological actions of SLs. However, little is known about the influence of the LCB structure, particularly 1-deoxy(methyl)-LCB, on lipid-lipid interactions and their effect on membrane properties. To address this question, we used complementary fluorescence-based methodologies to study membrane model systems containing POPC and the different LCBs of interest. Our results show that 1-deoxymethyl-LCBs have the highest ability to reduce the fluidity of the membrane, while the intermolecular interactions of 1-deoxy-LCBs were found to be weaker, leading to the formation of less-ordered domains compared to their canonical counterparts-sphinganine and sphingosine. Furthermore, while the presence of a trans-double bond at the Δ4-5 position of the LCB increased the fluidity of the membrane compared to a saturated LCB, a cis-double bond completely disrupted the ability of the LCB to segregate into ordered domains. In conclusion, even small changes on the structure of the LCB, as seen in 1-deoxy(methyl)-LCBs, strongly affects lipid-lipid interactions and membrane fluidity. These results provide evidence that altered balance between species with different LCBs affect membrane properties and may contribute to the pathobiological role of these lipids.

Ceramide Domains in Health and Disease: A Biophysical Perspective.

Ceramides are the central molecules in sphingolipid metabolism. In addition, they are recognized as important modulators of cell function, playing key roles in several cellular processes that range from cell proliferation to cell death. Moreover, ceramides were implicated in multiple diseases, including cancer, neurodegenerative and metabolic diseases, and also in infection by different pathogens. The mechanisms underlying the diverse biological and pathological actions of ceramides are yet to be fully elucidated. Several lines of evidence suggest that the structural features of ceramides, namely their high hydrophobicity and ability to establish strong H-bond network, are responsible for changes in the biophysical properties of biological membranes that can affect the activity of proteins and activate signaling pathways. Ceramide-induced alterations in membrane biophysical properties might also influence the internalization, trafficking and sorting of lipids, proteins, drugs and even pathogens contributing to cell pathophysiology. In this chapter, we critically discuss the ability of ceramides to form lipid domains with atypical biophysical properties and how these domains can be involved in those processes.

N,O-Iminoboronates: Reversible Iminoboronates with Improved Stability for Cancer Cells Targeted Delivery.

Herein a new class of iminoboronates obtained from 2-acetylbenzene boronic acids and aminophenols is presented. The N,O-ligand topology enabled the formation of an additional B-O bond that locks the boron center in a tetrahedral geometry. This molecular arrangement decisively contributes to improve the construct's stability in biocompatible conditions and retaining the iminoboronate reversibility in more acidic environments. 2-Acetylbenzene boronic acid was reacted with a fluorescent amino-coumarin to yield a stable and non-fluorescent N,O-iminoboronate. This mechanism was further used to assemble a folate receptor targeting conjugate that selectively delivered the fluorescent amino-coumarin to MDA-MB-231 human breast cancer cells.

Changes in membrane biophysical properties induced by the Budesonide/Hydroxypropyl-ß-cyclodextrin complex.

Budesonide (BUD), a poorly soluble anti-inflammatory drug, is used to treat patients suffering from asthma and COPD (Chronic Obstructive Pulmonary Disease). Hydroxypropyl-ß-cyclodextrin (HPßCD), a biocompatible cyclodextrin known to interact with cholesterol, is used as a drug-solubilizing agent in pharmaceutical formulations. Budesonide administered as an inclusion complex within HPßCD (BUD:HPßCD) required a quarter of the nominal dose of the suspension formulation and significantly reduced neutrophil-induced inflammation in a COPD mouse model exceeding the effect of each molecule administered individually. This suggests the role of lipid domains enriched in cholesterol for inflammatory signaling activation. In this context, we investigated the effect of BUD:HPßCD on the biophysical properties of membrane lipids. On cellular models (A549, lung epithelial cells), BUD:HPßCD extracted cholesterol similarly to HPßCD. On large unilamellar vesicles (LUVs), by using the fluorescent probes diphenylhexatriene (DPH) and calcein, we demonstrated an increase in membrane fluidity and permeability induced by BUD:HPßCD in vesicles containing cholesterol. On giant unilamellar vesicles (GUVs) and lipid monolayers, BUD:HPßCD induced the disruption of cholesterol-enriched raft-like liquid ordered domains as well as changes in lipid packing and lipid desorption from the cholesterol monolayers, respectively. Except for membrane fluidity, all these effects were enhanced when HPßCD was complexed with budesonide as compared with HPßCD. Since cholesterol-enriched domains have been linked to membrane signaling including pathways involved in inflammation processes, we hypothesized the effects of BUD:HPßCD could be partly mediated by changes in the biophysical properties of cholesterol-enriched domains.

Practical computational toolkits for dendrimers and dendrons structure design.

Dendrimers and dendrons offer an excellent platform for developing novel drug delivery systems and medicines. The rational design and further development of these repetitively branched systems are restricted by difficulties in scalable synthesis and structural determination, which can be overcome by judicious use of molecular modelling and molecular simulations. A major difficulty to utilise in silico studies to design dendrimers lies in the laborious generation of their structures. Current modelling tools utilise automated assembly of simpler dendrimers or the inefficient manual assembly of monomer precursors to generate more complicated dendrimer structures. Herein we describe two novel graphical user interface toolkits written in Python that provide an improved degree of automation for rapid assembly of dendrimers and generation of their 2D and 3D structures. Our first toolkit uses the RDkit library, SMILES nomenclature of monomers and SMARTS reaction nomenclature to generate SMILES and mol files of dendrimers without 3D coordinates. These files are used for simple graphical representations and storing their structures in databases. The second toolkit assembles complex topology dendrimers from monomers to construct 3D dendrimer structures to be used as starting points for simulation using existing and widely available software and force fields. Both tools were validated for ease-of-use to prototype dendrimer structure and the second toolkit was especially relevant for dendrimers of high complexity and size.

Glucosylceramide Reorganizes Cholesterol-Containing Domains in a Fluid Phospholipid Membrane.

Glucosylceramide (GlcCer), one of the simplest glycosphingolipids, plays key roles in physiology and pathophysiology. It has been suggested that GlcCer modulates cellular events by forming specialized domains. In this study, we investigated the interplay between GlcCer and cholesterol (Chol), an important lipid involved in the formation of liquid-ordered (lo) phases. Using fluorescence microscopy and spectroscopy, and dynamic and electrophoretic light scattering, we characterized the interaction between these lipids in different pH environments. A quantitative description of the phase behavior of the ternary unsaturated phospholipid/Chol/GlcCer mixture is presented. The results demonstrate coexistence between lo and liquid-disordered (ld) phases. However, the extent of lo/ld phase separation is sparse, mainly due to the ability of GlcCer to segregate into tightly packed gel domains. As a result, the phase diagram of these mixtures is characterized by an extensive three-phase coexistence region of fluid (ld-phospholipid enriched)/lo (Chol enriched)/gel (GlcCer enriched). Moreover, the results show that upon acidification, GlcCer solubility in the lo phase is increased, leading to a larger lo/ld coexistence region. Quantitative analyses allowed us to determine the differences in the composition of the phases at neutral and acidic pH. These results predict the impact of GlcCer on domain formation and membrane organization in complex biological membranes, and provide a background for unraveling the relationship between the biophysical properties of GlcCer and its biological action.

A Three-Component Assembly Promoted by Boronic Acids Delivers a Modular Fluorophore Platform (BASHY Dyes).

The modular assembly of boronic acids with Schiff-base ligands enabled the construction of innovative fluorescent dyes [boronic acid salicylidenehydrazone (BASHY)] with suitable structural and photophysical properties for live cell bioimaging applications. This reaction enabled the straightforward synthesis (yields up to 99%) of structurally diverse and photostable dyes that exhibit a polarity-sensitive green-to-yellow emission with high quantum yields of up to 0.6 in nonpolar environments. These dyes displayed a high brightness (up to 54,000 M(-1) cm(-1)). The promising structural and fluorescence properties of BASHY dyes fostered the preparation of non-cytotoxic, stable, and highly fluorescent poly(lactide-co-glycolide) nanoparticles that were effectively internalized by dendritic cells. The dyes were also shown to selectively stain lipid droplets in HeLa cells, without inducing any appreciable cytotoxicity or competing plasma membrane labeling; this confirmed their potential as fluorescent stains.

Pathological levels of glucosylceramide change the biophysical properties of artificial and cell membranes.

Glucosylceramide (GlcCer) plays an active role in the regulation of various cellular events. Moreover, GlcCer is also a key modulator of membrane biophysical properties, which might be linked to the mechanism of its biological action. In order to understand the biophysical implications of GlcCer on membranes of living cells, we first studied the effect of GlcCer on artificial membranes containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin (SM) and cholesterol (Chol). Using an array of biophysical methods, we demonstrate that at lower GlcCer/Chol ratios, GlcCer stabilizes SM/Chol-enriched liquid-ordered domains. However, upon decreasing the Chol content, GlcCer significantly increased membrane order through the formation of gel domains. Changes in pH disturbed the packing properties of GlcCer-containing membranes, leading to an increase in membrane fluidity and reduced membrane electronegativity. To address the biophysical impact of GlcCer in biological membranes, studies were performed in wild type and in fibroblasts treated with conduritol-B-epoxide (CBE), which causes intracellular GlcCer accumulation, and in fibroblasts from patients with type I Gaucher disease (GD). Decreased membrane fluidity was observed in cells containing higher levels of GlcCer, such as in CBE-treated and GD cells. Together, we demonstrate that elevated GlcCer levels change the biophysical properties of cellular membranes, which might compromise membrane-associated cellular events and be of relevance for understanding the pathology of diseases, such as GD, in which GlcCer accumulates at high levels.

Regulatory aspects on nanomedicines.

Nanomedicines have been in the forefront of pharmaceutical research in the last decades, creating new challenges for research community, industry, and regulators. There is a strong demand for the fast development of scientific and technological tools to address unmet medical needs, thus improving human health care and life quality. Tremendous advances in the biomaterials and nanotechnology fields have prompted their use as promising tools to overcome important drawbacks, mostly associated to the non-specific effects of conventional therapeutic approaches. However, the wide range of application of nanomedicines demands a profound knowledge and characterization of these complex products. Their properties need to be extensively understood to avoid unpredicted effects on patients, such as potential immune reactivity. Research policy and alliances have been bringing together scientists, regulators, industry, and, more frequently in recent years, patient representatives and patient advocacy institutions. In order to successfully enhance the development of new technologies, improved strategies for research-based corporate organizations, more integrated research tools dealing with appropriate translational requirements aiming at clinical development, and proactive regulatory policies are essential in the near future. This review focuses on the most important aspects currently recognized as key factors for the regulation of nanomedicines, discussing the efforts under development by industry and regulatory agencies to promote their translation into the market. Regulatory Science aspects driving a faster and safer development of nanomedicines will be a central issue for the next years.

Tackling the biophysical properties of sphingolipids to decipher their biological roles.

From the most simple sphingoid bases to their complex glycosylated derivatives, several sphingolipid species were shown to have a role in fundamental cellular events and/or disease. Increasing evidence places lipid-lipid interactions and membrane structural alterations as central mechanisms underlying the action of these lipids. Understanding how these molecules exert their biological roles by studying their impact in the physical properties and organization of membranes is currently one of the main challenges in sphingolipid research. Herein, we review the progress in the state-of-the-art on the biophysical properties of sphingolipid-containing membranes, focusing on sphingosine, ceramides, and glycosphingolipids.

Changes in membrane biophysical properties induced by sphingomyelinase depend on the sphingolipid N-acyl chain.

Ceramide (Cer) is involved in the regulation of several cellular processes by mechanisms that depend on Cer-induced changes on membrane biophysical properties. Accumulating evidence shows that Cers with different N-acyl chain composition differentially impact cell physiology, which may in part be due to specific alterations in membrane biophysical properties. We now address how the sphingolipid (SL) N-acyl chain affects membrane properties in cultured human embryonic kidney cells by overexpressing different Cer synthases (CerSs). Our results show an increase in the order of cellular membranes in CerS2-transfected cells caused by the enrichment in very long acyl chain SLs. Formation of Cer upon treatment of cells with bacterial sphingomyelinase promoted sequential changes in the properties of the membranes: after an initial increase in the order of the fluid plasma membrane, reorganization into domains with gel-like properties whose characteristics are dependent on the acyl chain structure of the Cer was observed. Moreover, the extent of alterations of membrane properties correlates with the amount of Cer formed. These data reinforce the significance of Cer-induced changes on membrane biophysical properties as a likely molecular mechanism by which different acyl chain Cers exert their specific biological actions.

A combined fluorescence spectroscopy, confocal and 2-photon microscopy approach to re-evaluate the properties of sphingolipid domains.

The aim of this study is to provide further insight about the interplay between important signaling lipids and to characterize the properties of the lipid domains formed by those lipids in membranes containing distinct composition. To this end, we have used a combination of fluorescence spectroscopy, confocal and two-photon microscopy and a stepwise approach to re-evaluate the biophysical properties of sphingolipid domains, particularly lipid rafts and ceramide (Cer)-platforms. By using this strategy we were able to show that, in binary mixtures, sphingolipids (Cer and sphingomyelin, SM) form more tightly packed gel domains than those formed by phospholipids with similar acyl chain length. In more complex lipid mixtures, the interaction between the different lipids is intricate and is strongly dictated by the Cer-to-Chol ratio. The results show that in quaternary phospholipid/SM/Chol/Cer mixtures, Cer forms gel domains that become less packed as Chol is increased. Moreover, the extent of gel phase formation is strongly reduced in these mixtures, even though Cer molar fraction is increased. These results suggest that in biological membranes, lipid domains such as rafts and ceramide platforms, might display distinctive biophysical properties depending on the local lipid composition at the site of the membrane where they are formed, further highlighting the potential role of membrane biophysical properties as an underlying mechanism for mediating specific biological processes.

Effect of glucosylceramide on the biophysical properties of fluid membranes.

Glucosylceramide (GlcCer), a relevant intermediate in the pathways of glycosphingolipid metabolism, plays key roles in the regulation of cell physiology. The molecular mechanisms by which GlcCer regulates cellular processes are unknown, but might involve changes in membrane biophysical properties and formation of lipid domains. In the present study, fluorescence spectroscopy, confocal microscopy and surface pressure-area (π-A) measurements were used to characterize the effect of GlcCer on the biophysical properties of model membranes. We show that C16:0-GlcCer has a high tendency to segregate into highly ordered gel domains and to increase the order of the fluid phase. Monolayer studies support the aggregation propensity of C16:0-GlcCer. π-A isotherms of single C16:0-GlcCer indicate that bilayer domains, or crystal-like structures, coexist within monolayer domains at the air-water interface. Mixtures with POPC exhibit partial miscibility with expansion of the mean molecular areas relative to the additive behavior of the components. Moreover, C16:0-GlcCer promotes morphological alterations in lipid vesicles leading to formation of flexible tubule-like structures that protrude from the fluid region of the bilayer. These results support the hypothesis that alterations in membrane biophysical properties induced by GlcCer might be involved in its mechanism of action.

Influence of intracellular membrane pH on sphingolipid organization and membrane biophysical properties.

Glucosylceramide (GlcCer) is a signaling lipid involved in the regulation of several cellular processes. It is present in different organelles, including the plasma membrane, Golgi apparatus, endoplasmic reticulum, and lysosomes. Accordingly, GlcCer is exposed to different pH environments in each organelle, which may lead to alterations in its properties and lateral organization and subsequent biological outcome. In this study, we addressed the effect of pH on the biophysical behavior of this lipid and other structurally related sphingolipids (SLs). Membranes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and C16-GlcCer, sphingomyelin, and different acyl chain ceramides were characterized by fluorescence spectroscopy, confocal microscopy, and surface pressure-area measurements under neutral and acidic conditions. The results show that changing the pH from 7.4 to 5.5 has a larger impact on C16-GlcCer-containing membranes compared to other SLs. In addition, acidification mainly affects the organization and packing properties of the GlcCer-enriched gel phase, suggesting that the interactions established by the glucose moiety, in the GlcCer molecule, are those most affected by the increase in the acidity. These results further highlight the role of GlcCer as a modulator of membrane biophysical properties and will possibly contribute to the understanding of its biological function in different organelles.

Methylation of glycosylated sphingolipid modulates membrane lipid topography and pathogenicity of Cryptococcus neoformans.

In previous studies we showed that the replication of Cryptococcus neoformans in the lung environment is controlled by the glucosylceramide (GlcCer) synthase gene (GCS1), which synthesizes the membrane sphingolipid GlcCer from the C9-methyl ceramide. Here, we studied the effect of the mutation of the sphingolipid C9 methyltransferase gene (SMT1), which adds a methyl group to position 9 of the sphingosine backbone of ceramide. The C. neoformans Δsmt1 mutant does not make C9-methyl ceramide and, thus, any methylated GlcCer. However, it accumulates demethylated ceramide and demethylated GlcCer. The Δsmt1 mutant loses more than 80% of its virulence compared with the wild type and the reconstituted strain. Interestingly, growth of C. neoformans Δsmt1 in the lung was decreased and C. neoformans cells were contained in lung granulomas, which significantly reduced the rate of their dissemination to the brain reducing the onset of meningoencephalitis. Thus, using fluorescent spectroscopy and atomic force microscopy we compared the wild type and Δsmt1 mutant and found that the altered membrane composition and GlcCer structure affects fungal membrane rigidity, suggesting that specific sphingolipid structures are required for proper fungal membrane organization and integrity. Therefore, we propose that the physical structure of the plasma membrane imparted by specific classes of sphingolipids represents a critical factor for the ability of the fungus to establish virulence.

Cell lipid droplet heterogeneity and altered biophysical properties induced by cell stress and metabolic imbalance.

Lipid droplets (LD) are important regulators of lipid metabolism and are implicated in several diseases. However, the mechanisms underlying the roles of LD in cell pathophysiology remain elusive. Hence, new approaches that enable better characterization of LD are essential. This study establishes that Laurdan, a widely used fluorescent probe, can be used to label, quantify, and characterize changes in cell LD properties. Using lipid mixtures containing artificial LD we show that Laurdan GP depends on LD composition. Accordingly, enrichment in cholesterol esters (CE) shifts Laurdan GP from ∼0.60 to ∼0.70. Moreover, live-cell confocal microscopy shows that cells present multiple LD populations with distinctive biophysical features. The hydrophobicity and fraction of each LD population are cell type dependent and change differently in response to nutrient imbalance, cell density, and upon inhibition of LD biogenesis. The results show that cellular stress caused by increased cell density and nutrient overload increased the number of LD and their hydrophobicity and contributed to the formation of LD with very high GP values, likely enriched in CE. In contrast, nutrient deprivation was accompanied by decreased LD hydrophobicity and alterations in cell plasma membrane properties. In addition, we show that cancer cells present highly hydrophobic LD, compatible with a CE enrichment of these organelles. The distinct biophysical properties of LD contribute to the diversity of these organelles, suggesting that the specific alterations in their properties might be one of the mechanisms triggering LD pathophysiological actions and/or be related to the different mechanisms underlying LD metabolism.

Polyoxazoline-Based Nanovaccine Synergizes with Tumor-Associated Macrophage Targeting and Anti-PD-1 Immunotherapy against Solid Tumors.

Immune checkpoint blockade reaches remarkable clinical responses. However, even in the most favorable cases, half of these patients do not benefit from these therapies in the long term. It is hypothesized that the activation of host immunity by co-delivering peptide antigens, adjuvants, and regulators of the transforming growth factor (TGF)-ß expression using a polyoxazoline (POx)-poly(lactic-co-glycolic) acid (PLGA) nanovaccine, while modulating the tumor-associated macrophages (TAM) function within the tumor microenvironment (TME) and blocking the anti-programmed cell death protein 1 (PD-1) can constitute an alternative approach for cancer immunotherapy. POx-Mannose (Man) nanovaccines generate antigen-specific T-cell responses that control tumor growth to a higher extent than poly(ethylene glycol) (PEG)-Man nanovaccines. This anti-tumor effect induced by the POx-Man nanovaccines is mediated by a CD8+ -T cell-dependent mechanism, in contrast to the PEG-Man nanovaccines. POx-Man nanovaccine combines with pexidartinib, a modulator of the TAM function, restricts the MC38 tumor growth, and synergizes with PD-1 blockade, controlling MC38 and CT26 tumor growth and survival. This data is further validated in the highly aggressive and poorly immunogenic B16F10 melanoma mouse model. Therefore, the synergistic anti-tumor effect induced by the combination of nanovaccines with the inhibition of both TAM- and PD-1-inducing immunosuppression, holds great potential for improving immunotherapy outcomes in solid cancer patients.