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.

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.

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.

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.

Laurdan in live cell imaging: Effect of acquisition settings, cell culture conditions and data analysis on generalized polarization measurements.

Cell function is highly dependent on membrane structure, organization, and fluidity. Therefore, methods to probe the biophysical properties of biological membranes are required. Determination of generalized polarization (GP) values using Laurdan in fluorescence microscopy studies is one of the most widely-used methods to investigate changes in membrane fluidity in vitro and in vivo. In the last couple of decades, there has been a major increase in the number of studies using Laurdan GP, where several different methodological approaches are used. Such differences interfere with data interpretation inasmuch as it is difficult to validate if Laurdan GP variations actually reflect changes in membrane organization or arise from biased experimental approaches. To address this, we evaluated the influence of different methodological details of experimental data acquisition and analysis on Laurdan GP. Our results showed that absolute GP values are highly dependent on several of the parameters analyzed, showing that incorrect data can result from technical and methodological inconsistencies. Considering these differences, we further analyzed the impact of cell variability on GP determination, focusing on basic cell culture conditions, such as cell confluency, number of passages and media composition. Our results show that GP values can report alterations in the biophysical properties of cell membranes caused by cellular adaptation to the culture conditions. In summary, this study provides thorough analysis of the factors that can lead to Laurdan GP variability and suggests approaches to improve data quality, which would generate more precise interpretation and comparison within individual studies and among the literature on Laurdan GP.

The long chain base unsaturation has a stronger impact on 1-deoxy(methyl)-sphingolipids biophysical properties than the structure of its C1 functional group.

1-deoxy-sphingolipids, also known as atypical sphingolipids, are directly implicated in the development and progression of hereditary sensory and autonomic neuropathy type 1 and diabetes type 2. The mechanisms underlying their patho-physiological actions are yet to be elucidated. Accumulating evidence suggests that the biological actions of canonical sphingolipids are triggered by changes promoted on membrane organization and biophysical properties. However, little is known regarding the biophysical implications of atypical sphingolipids. In this study, we performed a comprehensive characterization of the effects of the naturally occurring 1-deoxy-dihydroceramide, 1-deoxy-ceramideΔ14Z and 1-deoxymethyl-ceramideΔ3E in the properties of a fluid membrane. In addition, to better define which structural features determine sphingolipid ability to form ordered domains, the synthetic 1-O-methyl-ceramideΔ4E and 1-deoxy-ceramideΔ4E were also studied. Our results show that natural and synthetic 1-deoxy(methyl)-sphingolipids fail to laterally segregate into ordered domains as efficiently as the canonical C16-ceramide. The impaired ability of atypical sphingolipids to form ordered domains was more dependent on the presence, position, and configuration of the sphingoid base double bond than on the structure of its C1 functional group, due to packing constraints introduced by an unsaturated backbone. Nonetheless, absence of a hydrogen bond donor and acceptor group at the C1 position strongly reduced the capacity of atypical sphingolipids to form gel domains. Altogether, the results showed that 1-deoxy(methyl)-sphingolipids induce unique changes on the biophysical properties of the membranes, suggesting that these alterations might, in part, trigger the patho-biological actions of these lipids.

Biophysical impact of sphingosine and other abnormal lipid accumulation in Niemann-Pick disease type C cell models.

Niemann-Pick disease type C (NPC) is a complex and rare pathology, which is mainly associated to mutations in the NPC1 gene. This disease is phenotypically characterized by the abnormal accumulation of multiple lipid species in the acidic compartments of the cell. Due to the complexity of stored material, a clear molecular mechanism explaining NPC pathophysiology is still not established. Abnormal sphingosine accumulation was suggested as the primary factor involved in the development of NPC, followed by the accumulation of other lipid species. To provide additional mechanistic insight into the role of sphingosine in NPC development, fluorescence spectroscopy and microscopy were used to study the biophysical properties of biological membranes using different cellular models of NPC. Addition of sphingosine to healthy CHO-K1 cells, in conditions where other lipid species are not yet accumulated, caused a rapid decrease in plasma membrane and lysosome membrane fluidity, suggesting a direct effect of sphingosine rather than a downstream event. Changes in membrane fluidity caused by addition of sphingosine were partially sustained upon impaired trafficking and metabolization of cholesterol in these cells, and could recapitulate the decrease in membrane fluidity observed in NPC1 null Chinese Hamster Ovary (CHO) cells (CHO-M12) and in cells with pharmacologically induced NPC phenotype (treated with U18666A). In summary, these results show for the first time that the fluidity of the membranes is altered in models of NPC and that these changes are in part caused by sphingosine, supporting the role of this lipid in the pathophysiology of NPC.

Biophysical Analysis of Lipid Domains by Fluorescence Microscopy.

The study of the structure and dynamics of membrane domains in vivo is a challenging task. However, major advances could be achieved through the application of microscopic and spectroscopic techniques coupled with the use of model membranes, where the relations between lipid composition and the type, amount and properties of the domains present can be quantitatively studied.This chapter provides protocols to study membrane organization and visualize membrane domains by fluorescence microscopy both in artificial membrane and living cell models of Gaucher Disease (GD ). We describe a bottom-up multiprobe methodology, which enables understanding how the specific lipid interactions established by glucosylceramide, the lipid that accumulates in GD , affect the biophysical properties of model and cell membranes, focusing on its ability to influence the formation, properties and organization of lipid raft domains. In this context, we address the preparation of (1) raft-mimicking giant unilamellar vesicles labeled with a combination of fluorophores that allow for the visualization and comprehensive characterization of those membrane domains and (2) human fibroblasts exhibiting GD phenotype to assess the biophysical properties of biological membrane in living cells using fluorescence microscopy.

Biophysical Analysis of Lipid Domains in Mammalian and Yeast Membranes by Fluorescence Spectroscopy.

The use of steady-state and time-resolved fluorescence spectroscopy to study sterol and sphingolipid-enriched lipid domains as diverse as the ones found in mammalian and fungal membranes is herein described. We first address how to prepare liposomes that mimic raft-containing membranes of mammalian cells and how to use fluorescence spectroscopy to characterize the biophysical properties of these membrane model systems. We further illustrate the application of Förster resonance energy transfer (FRET) to study nanodomain reorganization upon interaction with small bioactive molecules, phenolic acids, an important group of phytochemical compounds. This methodology overcomes the resolution limits of conventional fluorescence microscopy allowing for the identification and characterization of lipid domains at the nanoscale.We continue by showing how to use fluorescence spectroscopy in the biophysical analysis of more complex biological systems, namely the plasma membrane of Saccharomyces cerevisiae yeast cells and the necessary adaptations to the filamentous fungus Neurospora crassa , evaluating the global order of the membrane, sphingolipid-enriched domains rigidity and abundance, and ergosterol-dependent properties.