VISTA drives macrophages towards a pro-tumoral phenotype that promotes cancer cell phagocytosis yet down-regulates T cell responses.

BACKGROUND: VISTA is a well-known immune checkpoint in T cell biology, but its role in innate immunity is less established. Here, we investigated the role of VISTA on anticancer macrophage immunity, with a focus on phagocytosis, macrophage polarization and concomitant T cell activation. METHODS: Macrophages, differentiated from VISTA overexpressed THP-1 cells and cord blood CD34+ cell-derived monocytes, were used in phagocytosis assay using B lymphoma target cells opsonized with Rituximab. PBMC-derived macrophages were used to assess the correlation between phagocytosis and VISTA expression. qRT-PCR, flow cytometry, and enzyme-linked immunosorbent assay were performed to analyze the impact of VISTA on other checkpoints and M1/M2-like macrophage biology. Additionally, flow cytometry was used to assess the frequency of CD14+ monocytes expressing VISTA in PBMCs from 65 lymphoma patients and 37 healthy donors. RESULTS: Ectopic expression of VISTA in the monocytic model cell line THP-1 or in primary monocytes triggered differentiation towards the macrophage lineage, with a marked increase in M2-like macrophage-related gene expression and decrease in M1-like macrophage-related gene expression. VISTA expression in THP-1 and monocyte-derived macrophages strongly downregulated expression of SIRPα, a prominent 'don't eat me' signal, and augmented phagocytic activity of macrophages against cancer cells. Intriguingly, expression of VISTA's extracellular domain alone sufficed to trigger phagocytosis in ∼ 50% of cell lines, with those cell lines also directly binding to recombinant human VISTA, indicating ligand-dependent and -independent mechanisms. Endogenous VISTA expression was predominantly higher in M2-like macrophages compared to M0- or M1-like macrophages, with a positive correlation observed between VISTA expression in M2c macrophages and their phagocytic activity. VISTA-expressing macrophages demonstrated a unique cytokine profile, characterized by reduced IL-1ß and elevated IL-10 secretion. Furthermore, VISTA interacted with MHC-I and downregulated its surface expression, leading to diminished T cell activation. Notably, VISTA surface expression was identified in monocytes from all lymphoma patients but was less prevalent in healthy donors. CONCLUSIONS: Collectively, VISTA expression associates with and drives M2-like activation of macrophages with a high phagocytic capacity yet a decrease in antigen presentation capability to T cells. Therefore, VISTA is a negative immune checkpoint regulator in macrophage-mediated immune suppression.

Galectin-9 has non-apoptotic cytotoxic activity toward acute myeloid leukemia independent of cytarabine resistance.

Acute myeloid leukemia (AML) is a malignancy still associated with poor survival rates, among others, due to frequent occurrence of therapy-resistant relapse after standard-of-care treatment with cytarabine (AraC). AraC triggers apoptotic cell death, a type of cell death to which AML cells often become resistant. Therefore, therapeutic options that trigger an alternate type of cell death are of particular interest. We previously identified that the glycan-binding protein Galectin-9 (Gal-9) has tumor-selective and non-apoptotic cytotoxicity towards various types of cancer, which depended on autophagy inhibition. Thus, Gal-9 could be of therapeutic interest for (AraC-resistant) AML. In the current study, treatment with Gal-9 was cytotoxic for AML cells, including for CD34+ patient-derived AML stem cells, but not for healthy cord blood-derived CD34+ stem cells. This Gal-9-mediated cytotoxicity did not rely on apoptosis but was negatively associated with autophagic flux. Importantly, both AraC-sensitive and -resistant AML cell lines, as well as AML patient samples, were sensitive to single-agent treatment with Gal-9. Additionally, Gal-9 potentiated the cytotoxic effect of DNA demethylase inhibitor Azacytidine (Aza), a drug that is clinically used for patients that are not eligible for intensive AraC treatment. Thus, Gal-9 is a potential therapeutic agent for the treatment of AML, including AraC-resistant AML, by inducing caspase-independent cell death.

Galectin-9 Triggers Neutrophil-Mediated Anticancer Immunity.

In earlier studies, galectin-9 (Gal-9) was identified as a multifaceted player in both adaptive and innate immunity. Further, Gal-9 had direct cytotoxic and tumor-selective activity towards cancer cell lines of various origins. In the current study, we identified that treatment with Gal-9 triggered pronounced membrane alterations in cancer cells. Specifically, phosphatidyl serine (PS) was rapidly externalized, and the anti-phagocytic regulator, CD47, was downregulated within minutes. In line with this, treatment of mixed neutrophil/tumor cell cultures with Gal-9 triggered trogocytosis and augmented antibody-dependent cellular phagocytosis of cancer cells. Interestingly, this pro-trogocytic effect was also due to the Gal-9-mediated activation of neutrophils with upregulation of adhesion markers and mobilization of gelatinase, secretory, and specific granules. These activation events were accompanied by a decrease in cancer cell adhesion in mixed cultures of leukocytes and cancer cells. Further, prominent cytotoxicity was detected when leukocytes were mixed with pre-adhered cancer cells, which was abrogated when neutrophils were depleted. Taken together, Gal-9 treatment potently activated neutrophil-mediated anticancer immunity, resulting in the elimination of epithelial cancer cells.

In-situ polymerized polypyrrole nanoparticles immobilized poly(ε-caprolactone) electrospun conductive scaffolds for bone tissue engineering.

Despite intensive attempts to fabricate polypyrrole nanoparticles (PPy-NPs) incorporated nanofibrous scaffolds, a low-cost facile strategy is still demanded. Herein, we developed a novel strategy- in-situ polymerization of PPy-NPs and immobilized them into the PCL polymeric matrix in a single step. For the in-situ polymerization of PPy-NPs, ferric chloride hexahydrate (FeCl3.6H2O) was introduced as an oxidant into the blended solution of PCL and pyrrole monomers. Due to the chemical oxidative polymerization process, the clear solution changed into a black PCL/PPy solution. After electrospinning the solution, PCL/PPy composite nanofibers were fabricated. The immobilization of PPy-NPs into PCL matrix was clearly revealed by Bio-TEM images. The Field emission scanning electron microscopy (FESEM) results exhibited that the PCL/PPy scaffolds showed significantly decreased fiber diameter. The atomic force microscopy (AFM) study showed increased surface roughness in the PCL/PPy scaffolds. The mechanical strength test of PCL/PPy scaffolds showed improved Young's Modulus (YM = 2 to 4-folds) and tensile strength (TS = 3 to 4-folds). As well as the YM and TS were gradually increased with increased concentration of PPy-NPs in composite scaffolds. The conductivity measurement conducted on polymeric solution and electrospun scaffolds showed an increasing trend of conductive property in the PCL/PPy solution and scaffolds too. The surface wettability test exhibited decreased water contact angle measurement from 126° for pure PCL to 93° for the PCL/PPy-200 composite scaffold. The biomineralization test conducted by simulated body fluid (SBF) incubation showed enhanced calcium-phosphate crystal deposition on the PCL/PPy scaffolds. The CCK-8 assay and confocal laser scanning microscopic (CLSM) imaging conducted without and with electrical stimulation (ES) displayed enhanced cell adhesion, growth, and proliferation of MC3T3-E1 cells on the PCL/PPy conductive scaffolds. Furthermore, ALP and ARS staining assays showed significant enhancement of the calcium-phosphate deposition on the PCL/PPy scaffolds after ES treatment. Hence, the current study provides a novel strategy for the fabrication of PCL/PPy conductive scaffolds with enhanced bioactivity, biocompatibility, and osteogenic differentiation under electrical stimulation confirmed its promising application towards bone tissue engineering.

Biocompatible superparamagnetic sub-micron vaterite particles for thermo-chemotherapy: From controlled design to in vitro anticancer synergism.

A promising candidate for the development of controlled and targeted nanoscale drug delivery system but less studied so far is calcium carbonate (CaCO3) in the form of porous polycrystalline vaterite spheres. Vaterite has been shown to exhibit various beneficial properties such as excellent biocompatibility, high drug loading capacity, and pH-sensitive decomposition under mild conditions. However, fabricating vaterite particles with improved porosity, high surface area and loading a payload into the common synthesis method is still a challenge. Here we report on the synthesis of a highly porous, spherical superparamagnetic vaterite particles (PMVP) of size ∼800 nm encapsulating Iron oxide nanoparticles (IONPs) in a one-step reaction and loaded with DOX molecules through electrostatic attractions and physisorption for cancer thermo-chemotherapy application. The main advantage of the PMVP-DOX is that it can be magnetically targeted into the tumor region and once exposed to the tumor tissues characteristic acidic pH, the PMVP nanoparticle dissociates, releasing the DOX and intelligently converts the pH-triggered drug release into a tumor triggered drug release. Simultaneous application of alternating magnetic field (AMF) generates localized heat of the tumor tissues due to the hyperthermic capability of the IONPs in the PMVP and results in the synergistic tumoricidal activities.

Drug release and kinetic models of anticancer drug (BTZ) from a pH-responsive alginate polydopamine hydrogel: Towards cancer chemotherapy.

A pH-sensitive polymeric carrier was developed in this study for local delivery of anticancer drug bortezomib (BTZ) to cancer cells. Our strategy is based on the conjugation of BTZ to polymeric carriers containing catechol groups, which are considered to release BTZ selectively in cancer cells. In this study we used alginate-conjugated polydopamine as a building block polymer. The catechol moiety of polydopamine binds to the boronic acid group of BTZ drug and release the drug molecules in a pH-dependent method. Cancer tissue has acidic environment where BTZ dissociate from the catechol group of polydopamine to control the release of the free drug. Mathematical equation models were used to clarify the mechanism of drug release. The release profile fitted first order with correlation coefficient (R2 = 0.98), the release mechanism was studied using Korsmeyer-Peppas, Higuchi, Hixson-Crowell, and Kopcha models. We revealed the release mechanism follows non-fickian and diffusion was the dominant mechanism while small portion contributed to erosion. The pH-sensitive mechanism controls the release of BTZ in targeted cancer cells, hence developing a novel idea that is applicable in future towards other boronic acid-containing drugs to treat various kinds of health challenges.