IFN-gamma priming of adipose-derived stromal cells at "physiological" hypoxia.

Multipotent mesenchymal stromal cells (MSCs) are considered cue regulators of tissue remodeling. Their activity is strongly governed by local milieu, where O2 level is most important. The elevation of inflammatory mediators and acute O2 lowering may additionally modulate MSC activity. In present paper the priming effects of IFN-gamma on adipose tissue-derived MSCs (ASCs) at tissue-related O2 level (5%) and acute hypoxic stress (0.1% O2 ) were assessed as alterations of ASCs' CFU-F, proliferation, migration, osteo-commitment. IFN-gamma priming provoked ROS elevation, cell growth slowdown, attenuation of both spontaneous and induced osteodifferentiation of tissue O2 -adapted ASCs. The prominent changes in ASC cytoskeleton-related gene transcription was detected. IFN-gamma exposure shifted the ASC paracrine profile, suppressing the production of VEGF and IL-8, while MCP-1 and IL-6 were stimulated. Conditioned medium of IFN-gamma-primed ASCs did not activate vessel growth in the CAM assay, but induced endothelial cell migration in "wound closure." Short-term hypoxia suppressed CFU-F number, IFN-gamma-induced elevation of IL-6 and endothelial cell migration, while it abolished IFN-gamma-provoked VEGF inhibition. After N-acetyl cysteine treatment ROS level was partly abolished providing additional enhancement of IL-6 and suppression of IL-8 and VEGF production. These findings demonstrated that paracrine activity of ASCs in part may be governed by ROS level. Thus, this study first demonstrated that IFN-gamma priming itself and in combination with acute O2 deprivation could supply dual effects on ASC functions providing both stimulatory and hampering effects. The equilibrium of these factors is a substantial requirement for the execution of MSC remodeling functions.

Response of Adipose Tissue-Derived Stromal Cells in Tissue-Related O2 Microenvironment to Short-Term Hypoxic Stress.

A microenvironment low in O2 ('physiological' hypoxia) governs the functions of perivascular multipotent mesenchymal stromal cells, defining their involvement in tissue physiological homeostasis and regenerative remodelling. Acute hypoxic stress is considered as one of the important factors inducing tissue damage. Here, we evaluate the influence of short-term hypoxia (1% O2 for 24 h) on perivascular adipose tissue-derived cells (ASCs) permanently expanded in tissue-related O2 (5%) microenvironment. After hypoxic exposure, ASCs retained high viability, stromal cell morphology and mesenchymal phenotype (CD73+, CD90+, CD105+ and CD45-). Mild oxidative damage was unveiled as elevation of reactive oxygen species and thiobarbituric acid-active products, while no reduction in the activity of the antioxidant enzymes catalase and glutathione peroxidase and a 20% statistically significant increase in superoxide dismutase activity was detected. Expression of hypoxia-inducible factor (HIF)-1α and HIF-3α isoforms was differently regulated. HIF-1α displayed transient up-regulation, with maximum levels 30 min after acute hypoxic exposure, while HIF-3α was significantly up-regulated after 24 h. Up-regulation of ERK7, MEK1 and c-fos, and down-regulation of MKK6, p53, CCNA2, CCNB1 and CCNB2 were observed after 24 h of oxygen deprivation. Acute hypoxic exposure did not affect the gene expression of other mitogen-activated protein kinases (MAPKs) and MAPK kinases, MAPK/ERK kinase-interacting proteins, MAPK-activated transcription factors and scaffolding proteins. Significant stimulation of vascular endothelial growth factor α and interleukin-6 production was detected in ASC-conditioned medium. Thus, tissue O2-adapted ASCs are resistant to hypoxic stress, which can ensure their effective involvement in the regeneration of tissue damage under significant oxygen deprivation.

Botensilimab, an Fc-enhanced anti-CTLA-4 antibody, is effective against tumors poorly responsive to conventional immunotherapy.

Conventional immune checkpoint inhibitors (ICI) targeting CTLA-4 elicit durable survival, but primarily in patients with immune-inflamed tumors. Although the mechanisms underlying response to anti-CTLA-4 remain poorly understood, Fc-gamma receptor (FcγR) IIIA co-engagement appears critical for activity, potentially explaining the modest clinical benefits of approved anti-CTLA-4 antibodies. We demonstrate that anti-CTLA-4 engineered for enhanced FcγR affinity leverages FcγR-dependent mechanisms to potentiate T cell responsiveness, reduce intratumoral Tregs, and enhance antigen presenting cell activation. Fc-enhanced anti-CTLA-4 promoted superior efficacy in mouse models and remodeled innate and adaptive immunity versus conventional anti-CTLA-4. These findings extend to patients treated with botensilimab, an Fc-enhanced anti-CTLA-4 antibody, with clinical activity across multiple poorly immunogenic and ICI treatment-refractory cancers. Efficacy was independent of tumor neoantigen burden or FcγRIIIA genotype. However, FcγRIIA and FcγRIIIA expression emerged as potential response biomarkers. These data highlight the therapeutic potential of Fc-enhanced anti-CTLA-4 antibodies in cancers unresponsive to conventional ICI therapy.

Enzalutamide, an Androgen Receptor Antagonist, Enhances Myeloid Cell-Mediated Immune Suppression and Tumor Progression.

Androgen receptor (AR) antagonism increases overall survival in prostate cancer; however, treatment failure leads to tumor progression and patient mortality. The effect of AR modulation on AR+ nontumor cells that participate in the resistance to AR antagonism is poorly understood. Tumor-infiltrating myeloid cells, including macrophages and myeloid-derived suppressor cells (MDSC), express AR and promote prostate cancer progression. We investigated how AR antagonism affects myeloid cell function and metabolism in an AR-independent murine colon tumor model. Systemic blockade of AR with enzalutamide resulted in increased MC-38 tumor growth in vivo even when AR was knocked out of MC-38 tumor cells. MC-38 tumor growth was also increased when immunocompetent, but not immunodeficient, mice were coinjected with tumor cells and MDSCs treated with enzalutamide or lacking AR, suggesting that AR regulated the ability of MDSCs to suppress adaptive immunity. Myeloid AR-knockout male mice also displayed increased growth of TRAMP C2 prostate tumors when compared with wild type. Inhibition of AR signaling suppressed mitochondrial respiration in myeloid cells via MPC/AMPK signaling pathways; suppression of mitochondrial respiration increased MDSC tumor-promoting functions. Our work showed that AR regulates a tumor-promoting myeloid cell phenotype and influences myeloid cell metabolism. These findings suggest that tumor resistance to AR antagonism is due, in part, to changes in myeloid cell function and metabolism.

Low-dose photodynamic therapy promotes angiogenic potential and increases immunogenicity of human mesenchymal stromal cells.

Photodynamic therapy (PDT) is a non-invasive FDA and EMA-approved anticancer treatment modality. Initially developed for elimination of malignant cells, PDT affects all cells in the tumor bed including stromal cells. Stroma represents not only an important component of tumor microenvironment, but has a significant impact on tumor susceptibility to PDT and other anticancer therapies. However, the effects of PDT on stromal cells are poorly investigated. During PDT the tumor stroma can receive low-dose irradiation as a result of chosen regimen or limited depth of light penetration. Here, we characterized response of human mesenchymal stromal cells (MSCs) to low-dose PDT. In an in vitro model we demonstrated that low-dose PDT resulted in activation of Erk1/2 and inhibition of GSK-3 signaling in MSCs. PDT-mediated induction of intracellular reactive oxygen species (ROS) resulted in reorganization of MSC cytoskeleton and decreased cell motility. More importantly, low-dose PDT dramatically upregulated secretion of various proangiogenic factors (VEGF-A, IL-8, PAI-1, MMP-9, etc.) by MSCs and improved MSC ability to promote angiogenesis suggesting an increase in the pro-tumorigenic potential of MSCs. In contrast, co-cultivation of PDT-treated MSCs with lymphocytes resulted in significant decrease of MSC viability and potential increase in MSC immunogenicity, which may lead to increased anti-tumor immunity. Low-dose PDT in MSCs significantly inhibited secretion of CCL2 (MCP-1) potentially limiting infiltration of pro-tumorigenic macrophages. Altogether, our findings demonstrate that low-dose PDT significantly modifies functional properties of MSCs improving their pro-tumorigenic potential while simultaneously increasing potential immune stimulation suggesting possible mechanisms of stromal cell contribution to PDT efficacy.

KLF9-dependent ROS regulate melanoma progression in stage-specific manner.

Although antioxidants promote melanoma metastasis, the role of reactive oxygen species (ROS) in other stages of melanoma progression is controversial. Moreover, genes regulating ROS have not been functionally characterized throughout the entire tumor progression in mouse models of cancer. To address this question, we crossed mice-bearing knock-out of Klf9, an ubiquitous transcriptional regulator of oxidative stress, with two conditional melanocytic mouse models: BrafCA mice, where BrafV600E causes premalignant melanocytic hyperplasia, and BrafCA/Pten-/- mice, where BrafV600E and loss of Pten induce primary melanomas and metastases. Klf9 deficiency inhibited premalignant melanocytic hyperplasia in BrafCA mice but did not affect formation and growth of BrafCA/Pten-/- primary melanomas. It also, as expected, promoted BrafCA/Pten-/- metastasis. Treatment with antioxidant N-acetyl cysteine phenocopied loss of Klf9 including suppression of melanocytic hyperplasia. We were interested in a different role of Klf9 in regulation of cell proliferation in BrafCA and BrafCA/Pten-/- melanocytic cells. Mechanistically, we demonstrated that BRAFV600E signaling transcriptionally upregulated KLF9 and that KLF9-dependent ROS were required for full-scale activation of ERK1/2 and induction of cell proliferation by BRAFV600E. PTEN depletion in BRAFV600E-melanocytes did not further activate ERK1/2 and cell proliferation, but rendered these phenotypes insensitive to KLF9 and ROS. Our data identified an essential role of KLF9-dependent ROS in BRAFV600E signaling in premalignant melanocytes, offered an explanation to variable role of ROS in premalignant and transformed melanocytic cells and suggested a novel mechanism for suppression of premalignant growth by topical antioxidants.

Macroporous modified poly (vinyl alcohol) hydrogels with charged groups for tissue engineering: Preparation and in vitro evaluation.

Poly(vinyl alcohol) (PVA) hydrogels are widely employed for various biomedical applications, including tissue engineering, due to their biocompatibility, high water solubility, low protein adsorption, and chemical stability. However, non-charged surface of PVA-based hydrogels is not optimal for cell adhesion and spreading. Here, cross-linked macroporous hydrogels based on low molecular weight acrylated PVA (Acr-PVA) was synthesized by modification of the pendant alcohol groups on the PVA with glycidyl methacrylate (GMA). To enhance cell affinity, charged groups were introduced to the hydrogel composition. For this purpose, Acr-PVA was copolymerized with either negatively charged acrylic acid (AA) or positively charged 2-(diethylamino) ethyl methacrylate (DEAEMA) monomers. A surface charge of the obtained hydrogels was found to be in function of the co-monomer type and content. Confocal microscopy observations confirmed that adhesion and spreading of both mouse fibroblasts (L929) and human mesenchymal stem cells (hMSC) on the modified Acr-PVA-AA and Acr-PVA-DEAEMA hydrogels were better than those on the non-modified Acr-PVA hydrogel. The increase of DEAEMA monomer content from 5 to 15mol% resulted in the enhancement of cell viability which was 1.5-fold higher for Acr-PVA-DEAEMA-15 hydrogel than that of the non-modified Acr-PVA hydrogel sample.

Acute Hypoxic Stress Affects Migration Machinery of Tissue O2-Adapted Adipose Stromal Cells.

The ability of mesenchymal stromal (stem) cells (MSCs) to be mobilised from their local depot towards sites of injury and to participate in tissue repair makes these cells promising candidates for cell therapy. Physiological O2 tension in an MSC niche in vivo is about 4-7%. However, most in vitro studies of MSC functional activity are performed at 20% O2. Therefore, this study focused on the effects of short-term hypoxic stress (0.1% O2, 24 h) on adipose tissue-derived MSC motility at tissue-related O2 level. No significant changes in integrin expression were detected after short-term hypoxic stress. However, O2 deprivation provoked vimentin disassembly and actin polymerisation and increased cell stiffness. In addition, hypoxic stress induced the downregulation of ACTR3, DSTN, MACF1, MID1, MYPT1, NCK1, ROCK1, TIAM1, and WASF1 expression, the products of which are known to be involved in leading edge formation and cell translocation. These changes were accompanied by the attenuation of targeted and nontargeted migration of MSCs after short-term hypoxic exposure, as demonstrated in scratch and transwell migration assays. These results indicate that acute hypoxic stress can modulate MSC function in their native milieu, preventing their mobilisation from sites of injury.

Polyelectrolyte microcapsules with entrapped multicellular tumor spheroids as a novel tool to study the effects of photodynamic therapy.

In the current study, semi-permeable alginate-oligochitosan microcapsules for multicellular tumor spheroids (MTS) generation were elaborated and tested, to estimate a response of the microencapsulated MTS (MMTS) to photodynamic therapy (PDT). The microcapsules (mean diameter 600 µm) with entrapped human breast adenocarcinoma MCF-7 cells were obtained using an electrostatic bead generator, and MMTS were generated by in vitro long-term cell cultivation. The formed MMTS were incubated in Chlorin e6 photosensitizer solution and then irradiated using 650-nm laser light. The cell viability was measured by MTT-assay in 24 h after irradiation, and histological analysis was performed. The proposed MTS-based model was found to be more resistant to the PDT than the two-dimensional monolayer cell culture model. Thus, MMTS could be considered as a promising three-dimesional in vitro model to estimate the doses of drugs or parameters for PDT in vitro before carrying out preclinical tests.