SiO2 Fibers of Two Lengths and Their Effect on Cellular Responses of Macrophage-like Cells.

The immunoreactivity or/and stress response can be induced by nanomaterials' different properties, such as size, shape, etc. These effects are, however, not yet fully understood. This study aimed to clarify the effects of SiO2 nanofibers (SiO2NFs) on the cellular responses of THP-1-derived macrophage-like cells. The effects of SiO2NFs with different lengths on reactive oxygen species (ROS) and pro-inflammatory cytokines TNF-α and IL-1ß in THP-1 cells were evaluated. From the two tested lengths, it was only the L-SiO2NFs with a length ≈ 44 ± 22 µm that could induce ROS. Compared to this, only S-SiO2NFs with a length ≈ 14 ± 17 µm could enhance TNF-α and IL-1ß expression. Our results suggested that L-SiO2NFs disassembled by THP-1 cells produced ROS and that the inflammatory reaction was induced by the uptake of S-SiO2NFs by THP-1 cells. The F-actin staining results indicated that SiO2NFs induced cell motility and phagocytosis. There was no difference in cytotoxicity between L- and S-SiO2NFs. However, our results suggested that the lengths of SiO2NFs induced different cellular responses.

In vitro Assessment of Efferocytic Capacity of Human Macrophages Using Flow Cytometry.

Clearance of dying cells, named efferocytosis, is a pivotal function of professional phagocytes that impedes the accumulation of cell debris. Efferocytosis can be experimentally assessed by differentially tagging the target cells and professional phagocytes and analyzing by cell imaging or flow cytometry. Here, we describe an assay to evaluate the uptake of apoptotic cells (ACs) by human macrophages in vitro by labeling the different cells with commercially available dyes and analysis by flow cytometry. We detail the methods to prepare and label human macrophages and apoptotic lymphocytes and the in vitro approach to determine AC uptake. This protocol is based on previously published literature and allows for in vitro modeling of the efficiency of AC engulfment during continual efferocytosis process. Also, it can be modified to evaluate the clearance of different cell types by diverse professional phagocytes.

Homogeneous polyporus polysaccharide inhibits bladder cancer by polarizing macrophages to M1 subtype in tumor microenvironment.

BACKGROUND: Polyporus polysaccharide (PPS), an active ingredient of traditional Chinese medicinal Polyporus umbellatus, has multiple biological functions, such as anti-cancer, immune-regulating and hepatoprotective activities. The purpose of this study was to investigate the mechanism of homogeneous polyporus polysaccharide (HPP) activated macrophages in the treatment of bladder cancer. METHODS: 100 ng/mL Phorbol myristate acetate (PMA) was used to induce THP-1 human leukemic cells as a macrophage model. Then macrophages derived from THP-1 were treated with different concentrations of HPP (1, 10 and 100 µg/mL). Flow cytometry and RT-PCR were used to detected the expression of CD16, CD23, CD86, CD40 and interleukin (IL)-Iß, iNOS mRNA. ELISA was used to test the change of IL-1ß and TNF-α in macrophage after the treatment with HPP. The conditioned medium from HPP-polarized macrophages was used to detect the effect of activated macrophages on bladder cancer. MTT assay, 5-ethynyl-2'-deoxyuridine assay, flow cytometry, Transwell assay, and Western blot analysis were used to detect the effects of polarized macrophages on the viability, proliferation, apoptosis, and migration of bladder cancer cells. Western blot was also used to analysis the change of JAK2/NF-κB pathway protein. RESULTS: HPP promoted the expression of pro-inflammatory factors, such as IL-Iß, TNF-α and iNOS, and surface molecules CD86, CD16, CD23, and CD40 in macrophages and then polarized macrophages to M1 type. Results demonstrated that activated macrophages inhibited the proliferation of bladder cancer cells, regulated their apoptosis, and inhibited migration and epithelial-mesenchymal transformation (EMT). JAK2/NF-κB pathways were downregulated in the anti-bladder cancer process of activated macrophages. CONCLUSION: The findings indicated that HPP inhibited the proliferation and progression of bladder cancer by the polarization of macrophages to M1 type, and JAK2/NF-κB pathway was downregulated in the process of anti-bladder cancer.

Co-culturing polarized M2 Thp-1-derived macrophages enhance stemness of lung adenocarcinoma A549 cells.

BACKGROUND: The tumor microenvironment (TME) is highly associated with cancer stem cells, and affects tumor initiation, progression, and metastasis. This study aimed to explore the underlying molecular mechanism of induction of A549 cancer cell stemness by THP-1-derived macrophages. METHOD: The Hedgehog inhibitor (Vismodegib), Notch inhibitor Gamma Secretase Inhibitor (GSI), and Signal Transducer and Activator of Transcription 3 (STAT3) inhibitor Cucurbitacin I (JSI-124) were added separately into the co-culture system of A549 cancer cell with THP-1-derived macrophages. Cell Counting Kit-8 (CCK-8) assay and the Cell-IQ continuous surveillance system were used to examine the cell growth and morphological changes of A549 cells. The messenger ribonucleic acid (mRNA) and protein expression levels of stem cell markers were respectively analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, and the activity of Acetaldehyde dehydrogenase (ALDH) enzyme was assessed by flow cytometry analysis. Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR assays were performed to evaluate the activation and differentiation of macrophages. RESULTS: Results showed that the proliferation and stemness of A549 cells were significantly enhanced by co-culturing with THP-1-derived macrophages. The expression levels of Transforming growth factor-ß (TGF-ß) and Interleukin-6 (IL-6) in macrophages were notably increased after co-culturing with A549 cells. Meanwhile, co-culturing with A549 cells induced the polarization of macrophages towards the M2 phenotype. Moreover, the inhibitors could reduce the proliferation and stemness of the co-culture system, and decrease the expression of TGF-ß and IL-6. CONCLUSIONS: These results suggested that co-culturing A549 cells with THP-1-derived macrophages could induce the stemness of A549 cells via multiple pro-tumorigenic pathways. Thus, inhibition of the interaction between macrophages and lung cancer stem cells may be a viable target for lung cancer treatment in the future.