TGFβ + small extracellular vesicles from head and neck squamous cell carcinoma cells reprogram macrophages towards a pro‐angiogenic phenotype

Transforming growth factor β (TGFβ) is a major component of tumor-derived small extracellular vesicles (TEX) in cancer patients. Mechanisms utilized by TGFβ+ TEX to promote tumor growth and pro-tumor activities in the tumor microenvironment (TME) are largely unknown. TEX produced by head and neck squamous cell carcinoma (HNSCC) cell lines carried TGFβ and angiogenesis-promoting proteins. TGFβ+ TEX stimulated macrophage chemotaxis without a notable M1/M2 phenotype shift and reprogrammed primary human macrophages to a pro-angiogenic phenotype characterized by the upregulation of pro-angiogenic factors and functions. In a murine basement membrane extract plug model, TGFβ+ TEX promoted macrophage infiltration and vascularization (p < 0.001), which was blocked by using the TGFβ ligand trap mRER (p < 0.001). TGFβ+ TEX injected into mice undergoing the 4-nitroquinoline-1-oxide (4-NQO)-driven oral carcinogenesis promoted tumor angiogenesis (p < 0.05), infiltration of M2-like macrophages in the TME (p < 0.05) and ultimately tumor progression (p < 0.05). Inhibition of TGFβ signaling in TEX with mRER ameliorated these pro-tumor activities. Silencing of TGFβ emerges as a critical step in suppressing pro-angiogenic functions of TEX in HNSCC

Arginase-1+ Exosomes from Reprogrammed Macrophages Promote Glioblastoma Progression

Interactions between tumor cells and tumor-associated macrophages (TAMs) are critical for glioblastoma progression. The TAMs represent up to 30% of the glioblastoma mass. The role of TAMs in tumor progression and in the mechanisms underlying tumor growth remain unclear. Using an in vitro model resembling the crosstalk between macrophages and glioblastoma cells, we show that glioblastoma-derived exosomes (GBex) reprogram M1 (mediate pro-inflammatory function) and M2 (mediate anti-inflammatory function) macrophages, converting M1 into TAMs and augmenting pro-tumor functions of M2 macrophages. In turn, these GBex-reprogrammed TAMs, produce exosomes decorated by immunosuppressive and tumor-growth promoting proteins. TAM-derived exosomes disseminate these proteins in the tumor microenvironment (TME) promoting tumor cell migration and proliferation. Mechanisms underlying the promotion of glioblastoma growth involved Arginase-1+ exosomes produced by the reprogrammed TAMs. A selective Arginase-1 inhibitor, nor-NOHA reversed growth-promoting effects of Arginase-1 carried by TAM-derived exosomes. The data suggest that GBex-reprogrammed Arginase-1+ TAMs emerge as a major source of exosomes promoting tumor growth and as a potential therapeutic target in glioblastoma

Novel TGFβ Inhibitors Ameliorate Oral Squamous Cell Carcinoma Progression and Improve the Antitumor Immune Response of Anti–PD-L1 Immunotherapy

TGF beta is a key regulator of oral squamous cell carcinoma (OSCC) progression, and its potential role as a therapeutic target has been investigated with a limited success. This study evaluates two novel TGF beta inhibitors as mono or combinatorial therapy with anti-PD-L1 antibodies (alpha-PD-L1 Ab) in a murine OSCC model. Immunocompetent C57BL/6 mice bearing malignant oral lesions induced by 4-nitroquinoline 1-oxide (4-NQO) were treated for 4 weeks with TGF beta inhibitors mRER (i.p., 50 mu g/d) or mmTGF beta 2-7m (10 mg/d delivered by osmotic pumps) alone or in combination with alpha-PD-L1 Abs (7x i.p. of 100 mu g/72 h). Tumor progression and body weight were monitored. Levels of bioactive TGF beta in serum were quantified using a TGF beta bioassay. Tissues were analyzed by immunohistology and flow cytometry. Therapy with mRER or mmTGF beta 2-7m reduced tumor burden (P < 0.05) and decreased body weight loss compared with controls. In inhibitor-treated mice, levels of TGF beta in tumor tissue and serum were reduced (P < 0.05), whereas they increased with tumor progression in controls. Both inhibitors enhanced CD8(+) T-cell infiltration into tumors and mRER reduced levels of myeloid-derived suppressor cells (P < 0.001). In combination with alpha-PD-L1 Abs, tumor burden was not further reduced; however, mmTGF beta 2-7m further reduced weight loss (P < 0.05). The collagen-rich stroma was reduced by using combinatorial TGF beta/PD-L1 therapies (P < 0.05), enabling an accelerated lymphocyte infiltration into tumor tissues. The blockade of TGF beta signaling by mRER or mmTGF beta 2-7m ameliorated in vivo progression of established murine OSCC. The inhibitors promoted antitumor immune responses, alone and in combination with alpha-PD-L1 Abs

Development and characterization of CD73-siRNA-loaded nanoemulsion: effect on C6 glioma cells and primary astrocytes

Glioblastoma (GB) is the most common malignant brain tumor and is characterized by high invasiveness, poor prognosis, and limited therapeutic options. Silencing gene expression, through the use of small interfering RNA (siRNA), has been proposed as an alternative to conventional cancer therapy. Here, we evaluated the potential of CD73 as a new therapeutic target, since it is overexpressed in solid tumors and has emerged as a promising target to control GB progression. Methods: A cationic nanoemulsion (NE) as an intravenous siRNA-CD73 delivery system was developed and its effect on C6 glioma cell viability was determined. Results: The nanostructured system was effective in complexing oligonucleotides for delivery to target cells. In addition, we observed that the NE-siRNA-CD73 complex was effective in reducing CD73 protein levels and AMPase activity, which were related to decreased C6 glioma cell viability. Conclusions: These findings indicate the potential of siRNA-CD73-loaded cationic NE as a therapeutic alternative for glioma treatment254408415CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DO RIO GRANDE DO SUL - FAPERGS422298/2016-6; 312187/2018-1sem informação12/2011-7; 16/2551-0000265-7; 16/2551-0000473-

Synthesis and biological evaluation of benzothiazin-4-ones: a possible new class of acetylcholinesterase inhibitors

A series of nineteen benzothiazin-4-ones from N-(3-aminopropyl) piperidine, 4-(2-aminoethyl)morpholine or 1-(2-aminoethyl)piperidine, aliphatic or aromatic aldehyde and thiosalicylic acid, were synthesized in good yields by multicomponent one-pot reactions. The solvent was toluene and this efficient procedure afforded the desired heterocycles in 5 h. Identification and characterization were achieved by NMR and GC–MS techniques. In vitro AChE activities of all compounds were evaluated in cerebral cortex and hippocampus of rats and in general, the results in cortex were more promising than hippocampus. The benzothiazinone 5Bd showed the best AChE inhibition activity IC50 8.48 μM (cortex) and IC50 39.80 μM (hippocampus). The cytotoxicity of seven compounds in MCR-5 human fibroblast cell by SRB test in 24 h were evaluated and 5Bd suggest preliminary safety, showing no cytotoxicity at 100 µM. Finally, these important findings could be a starting point for the development of new AChE inhibitors agents and will provide the basis for new studies

Pneumococcal Extracellular Vesicles Modulate Host Immunity

Extracellular vesicles (EVs) have recently garnered attention for their participation in host-microbe interactions in pneumococcal infections. However, the effect of EVs on the host immune system remain poorly understood. Our studies focus on EVs produced by Streptococcus pneumoniae (pEVs), and reveal that pEVs are internalized by macrophages, T cells, and epithelial cells. In vitro, pEVs induce NF -KB activation in a dosage-dependent manner and polarize human macrophages to an alternative (M2) phenotype. In addition, pEV pretreatment conditions macrophages to increase bacteria uptake and such macrophages may act as a reservoir for pneumococcal cells by increasing survival of the phagocytosed bacteria. When administered systemically in mice, pEVs induce cytokine release; when immobilized locally, they recruit lymphocytes and macrophages. Taken together, pEVs emerge as critical contributors to inflammatory responses and tissue damage in mammalian hosts. IMPORTANCE Over the last decade, pathogen-derived extracellular vesicles (EVs) have emerged as important players in several human diseases. Therefore, a thorough understanding of EV-mediated mechanisms could provide novel insights into vaccine/therapeutic development. A critical question in the field is: do pathogen-derived EVs help the pathogen evade the harsh environment in the host or do they help the host to mount a robust immune response against the pathogen? This study is a step towards answering this critical question for the Gram-positive pathogen, Streptococcus pneumoniae. Our study shows that while S. pneumoniae EVs (pEVs) induce inflammatory response both in vitro and in vivo, they may also condition the host macrophages to serve as a reservoir for the bacteria