Journal: American Journal of Cancer Research

Article Title: Macrophage-derived exosomes induce M2 microglial polarization to alleviate bone cancer pain

doi: 10.62347/BCLF6941

Figure Lengend Snippet: Characterization of exosomes derived from M2-polarized RAW264.7 macrophages. A. Transmission electron microscopy (TEM) image showing the morphology of the isolated exosomes with a typical double-layer membrane structure (× 30000). Scale bar: 50 nm . B. Nanoparticle tracking analysis (NTA) of exosome size distribution, with the majority of particles around 100 nm in diameter. C. The expression of exosome-specific markers CD63, CD81, TSG101, and the endoplasmic reticulum marker Calnexin was analyzed by Western blot (n=3). Note: Transmission electron microscopy (TEM), Nanoparticle tracking analysis (NTA), Cluster of differentiation 63 (CD63), Cluster of differentiation 81 (CD81), Tumor susceptibility gene 101 (TSG101), Endoplasmic reticulum marker Calnexin, Western blot (WB).

Article Snippet: The expression of M1-related proteins iNOS, CD80, and CD86 was significantly upregulated in the LPS-treated group, whereas their expression was markedly downregulated in the LPS + Exosome-treated group ( Figure 3B ), indicating that exosomes inhibited LPS-induced M1 polarization.

Techniques: Derivative Assay, Transmission Assay, Electron Microscopy, Isolation, Membrane, Expressing, Marker, Western Blot

Journal: American Journal of Cancer Research

Article Title: Macrophage-derived exosomes induce M2 microglial polarization to alleviate bone cancer pain

doi: 10.62347/BCLF6941

Figure Lengend Snippet: Exosome uptake by BV2 microglial cells and its effect on M1/M2 polarization. A. Immunofluorescence staining showing the uptake of PKH67-labeled exosomes (red) by BV2 microglial cells. The nuclei were stained with DAPI (blue). The merged image confirms the internalization of exosomes into the cells (× 400). B. Bar graph displaying the expression levels of M1-related proteins (iNOS, CD80, CD86) in the Control, LPS, and LPS + Exosome groups. LPS treatment significantly increased M1-associated protein expression, while exosome treatment reduced these levels. C. Bar graph showing the expression levels of M2-related proteins (CD206, Arg-1). Exosome treatment significantly upregulated M2-related protein expression compared to the Control and LPS groups. Note: BV2 (a microglial cell line), Inducible nitric oxide synthase (iNOS), Cluster of differentiation 80 (CD80), Cluster of differentiation 86 (CD86), Cluster of differentiation 206 (CD206), Arginase 1 (Arg-1), Lipopolysaccharide (LPS), Standard deviation (SD). Data are presented as mean ± SD. ****P < 0.0001, ***P < 0.001 vs. Control group (n=3).

Article Snippet: The expression of M1-related proteins iNOS, CD80, and CD86 was significantly upregulated in the LPS-treated group, whereas their expression was markedly downregulated in the LPS + Exosome-treated group ( Figure 3B ), indicating that exosomes inhibited LPS-induced M1 polarization.

Techniques: Immunofluorescence, Staining, Labeling, Expressing, Control, Standard Deviation

Journal: American Journal of Cancer Research

Article Title: Macrophage-derived exosomes induce M2 microglial polarization to alleviate bone cancer pain

doi: 10.62347/BCLF6941

Figure Lengend Snippet: Exosomes promoted BV2 microglial cell proliferation and survival, and modulated M1/M2-related gene expression. A. CCK-8 assay showing the proliferation of BV2 cells at 24 h, 48 h, and 72 h. The LPS + Exosome group exhibited significantly higher proliferation compared to the LPS group. B. Colony formation assay indicating the number of BV2 cells in each group. Exosome treatment improved the survival of BV2 cells, as shown by the increased number of colonies in the LPS + Exosome group compared to the LPS group. C. qRT-PCR analysis of M2-related genes (Arg-1, IL-10) showing significant upregulation in the LPS + Exosome group compared to the LPS group. D. qRT-PCR analysis of M1-related genes (iNOS, TNF-α) showing significant downregulation in the LPS + Exosome group. Note: Cell counting kit-8 (CCK-8), Quantitative reverse transcription polymerase chain reaction (qRT-PCR), M1 (classically activated microglia), M2 (alternatively activated microglia), Tumor necrosis factor-alpha (TNF-α), Interleukin-10 (IL-10). Data are presented as mean ± SD. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 vs. Control or LPS group (n=3).

Article Snippet: The expression of M1-related proteins iNOS, CD80, and CD86 was significantly upregulated in the LPS-treated group, whereas their expression was markedly downregulated in the LPS + Exosome-treated group ( Figure 3B ), indicating that exosomes inhibited LPS-induced M1 polarization.

Techniques: Gene Expression, CCK-8 Assay, Colony Assay, Quantitative RT-PCR, Cell Counting, Reverse Transcription, Polymerase Chain Reaction, Control

Journal: American Journal of Cancer Research

Article Title: Macrophage-derived exosomes induce M2 microglial polarization to alleviate bone cancer pain

doi: 10.62347/BCLF6941

Figure Lengend Snippet: Exosomes suppressed TLR4/NF-κB signaling pathway related protein expression in BV2 microglial cells. A. Western blot analysis of TLR4, p-p65, p65, p-IκBα, and IκBα protein expression in BV2 microglial cells. LPS treatment significantly increased the expression of TLR4, p-p65, and p-IκBα, indicating activation of the TLR4/NF-κB pathway. Exosome treatment in the LPS + Exosome group reduced the expression of these proteins. β-actin was used as an internal control. B. ELISA analysis of inflammatory cytokines (IL-6, TNF-α, MCP-1) in the culture supernatants of BV2 cells. LPS treatment significantly increased the levels of these cytokines, while exosome treatment reduced their expression. Note: Toll-like receptor 4 (TLR4), Phosphorylated p65 (p-p65), p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (p65), Phosphorylated inhibitor of κB alpha (p-IκBα), Inhibitor of κB alpha (IκBα), Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), Enzyme-linked immunosorbent assay (ELISA). Data are presented as mean ± SD. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 vs. Control or LPS group (n=3).

Article Snippet: The expression of M1-related proteins iNOS, CD80, and CD86 was significantly upregulated in the LPS-treated group, whereas their expression was markedly downregulated in the LPS + Exosome-treated group ( Figure 3B ), indicating that exosomes inhibited LPS-induced M1 polarization.

Techniques: Expressing, Western Blot, Activation Assay, Control, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Cancer Research

Article Title: Macrophage-derived exosomes induce M2 microglial polarization to alleviate bone cancer pain

doi: 10.62347/BCLF6941

Figure Lengend Snippet: Exosomes alleviated bone cancer pain and modulated M1/M2 polarization in the spinal cord of rats. A. Paw withdrawal threshold (PWT) test showing that BCP rats exhibited significantly lower PWT values, indicating heightened mechanical pain sensitivity. Exosome treatment significantly increased PWT values, reducing pain sensitivity. B. WB of the M1 marker CD86 in the spinal cord, showing significantly higher expression in the BCP group compared to the Sham group, while exosome treatment reduced CD86 expression. C. WB of the M2 marker CD206, showing lower expression in the BCP group and significantly increased expression after exosome treatment. Note: Paw withdrawal threshold (PWT), Bone cancer pain model (BNT), Control group (Sham), Western blot (WB). Data are presented as mean ± SD. ***P < 0.001, *P < 0.05 vs. Sham or BCP group (n=3).

Article Snippet: The expression of M1-related proteins iNOS, CD80, and CD86 was significantly upregulated in the LPS-treated group, whereas their expression was markedly downregulated in the LPS + Exosome-treated group ( Figure 3B ), indicating that exosomes inhibited LPS-induced M1 polarization.

Techniques: Marker, Expressing, Control, Western Blot

Journal: American Journal of Cancer Research

Article Title: Macrophage-derived exosomes induce M2 microglial polarization to alleviate bone cancer pain

doi: 10.62347/BCLF6941

Figure Lengend Snippet: Exosomes modulated F4/80 and Iba-1 expression in the spinal cord of rats with bone cancer pain. A. Protein band of F4/80, Iba-1 in spinal cord sections of Sham, BCP and BCP + Exo groups. B. Quantified result of protein levels of F4/80 and Iba-1. Note: Macrophage marker F4/80 (F4/80), Ionized calcium-binding adaptor molecule 1 (Iba-1), Western blot (WB). Data are presented as mean ± SD. ***P < 0.001,**P < 0.01, vs. Sham or BCP group (n=3).

Article Snippet: The expression of M1-related proteins iNOS, CD80, and CD86 was significantly upregulated in the LPS-treated group, whereas their expression was markedly downregulated in the LPS + Exosome-treated group ( Figure 3B ), indicating that exosomes inhibited LPS-induced M1 polarization.

Techniques: Expressing, Marker, Binding Assay, Western Blot

Journal: American Journal of Cancer Research

Article Title: Macrophage-derived exosomes induce M2 microglial polarization to alleviate bone cancer pain

doi: 10.62347/BCLF6941

Figure Lengend Snippet: Exosomes regulated M1/M2 polarization and the TLR4/NF-κB signaling pathway in the spinal cord of rats with bone cancer pain. A. Immunofluorescence staining showing the localization and internalization of red PKH67-labeled exosomes (red) in microglia and macrophages in the gray matter region of the spinal cord. Nuclei were stained with DAPI (blue), and merged images confirm exosome uptake in the BCP + Exo group (× 600). B. Western blot analysis of M1 polarization markers (iNOS, CD86, CD68) in the Sham, BCP , and BCP + Exo groups. M1 markers were significantly upregulated in the BCP group, while exosome treatment reduced their expression. C. Western blot analysis of M2 polarization markers (CD206, Arg-1). M2 markers were downregulated in the BCP group, and exosome treatment significantly upregulated their expression. D. Western blot analysis of TLR4, p-p65, and p-IκBα. The TLR4/NF-κB pathway was activated in the BCP group, and exosome treatment inhibited the activation of this pathway. Note: Inducible nitric oxide synthase (iNOS), Cluster of differentiation 86 (CD86), Cluster of differentiation 206 (CD206), Arginase 1 (Arg-1), Phosphorylated p65 (p-p65), Phosphorylated inhibitor of κB alpha (p-IκBα), Lipopolysaccharide (LPS), Western blot (WB). Data are presented as mean ± SD. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 vs. Sham or BCP group (n=3).

Article Snippet: The expression of M1-related proteins iNOS, CD80, and CD86 was significantly upregulated in the LPS-treated group, whereas their expression was markedly downregulated in the LPS + Exosome-treated group ( Figure 3B ), indicating that exosomes inhibited LPS-induced M1 polarization.

Techniques: Immunofluorescence, Staining, Labeling, Western Blot, Expressing, Activation Assay

Journal: iScience

Article Title: Leveraging Vγ9Vδ2 T cells against prostate cancer through a VHH-based PSMA-Vδ2 bispecific T cell engager

doi: 10.1016/j.isci.2024.111289

Figure Lengend Snippet: Cross-presentation of virus-specific and tumor-associated antigens by PSMA-Vδ2 bsTCE activated Vγ9Vδ2 T cells (A) Expression of CD83, HLA-A2 and HLA-DR (MFI) by non-expanded Vγ9Vδ2 T cells ( n = 3–4) after 24 h incubation with 10 pM PSMA-Vδ2 bsTCE or medium on 1 μg/mL PSMA-coated plates. (B–F) Vγ9Vδ2 T cells (expanded or non-expanded) were pre-incubated for 24 h with 10 pM PSMA-Vδ2 hu-bsTCE or medium with or without 1 μM M1, MART-1, or pp65 LPs on 1 μg/mL PSMA-coated plates and were subsequently incubated with M1, MART-1, or pp65-specific CD8 + T cells. (B) Expression of CD69, CD25, CD107a, or IFN-γ (%) by M1-specific CD8 + T cells incubated for 16–24 h with expanded Vγ9Vδ2 T cells as described. (C) Expression of CD107a and IFN-γ (%) by MART-1 specific CD8 + T cells incubated 16–24 h with expanded Vγ9Vδ2 T cells as described. (D) Expression of CD69 and CD107a (%) by pp65-specific CD8 + T cells incubated 16–24 h with non-expanded Vγ9Vδ2 T cells as described. (E) Percentage of pp65-specific CD8 + T cells within CD8 + T cell population (CD8 + cells of CMV + healthy donor were used) incubated for 7 days with expanded Vγ9Vδ2 T cells as described. (F) Lysis of THP-1 tumor cells unloaded (left) or pre-loaded with 1 μM pp65 SP (right) after 24 h co-culture with CD8 + T cell fractions that were variably enriched for pp65-specific cells during the 7 days co-culture with expanded Vγ9Vδ2 T cells as shown in (E). Live target cells were determined as % of 7AAD − THP-1 cells. Data were all generated using flow cytometry, circles represent individual data-points and mean ± S.E.M. is shown. MFI = mean fluorescent intensity. Paired t test (A), two-way ANOVA with Tukey multiple comparisons test (B–E). p < 0.05: ∗, p < 0.01: ∗∗ and p < 0.0001: ∗∗∗∗.

Article Snippet: Expanded or non-expanded Vγ9Vδ2 T cells were pre-incubated for 24h on PSMA-coated wells with 0 or 10 p.m. PSMA-Vδ2 bsTCE in the presence or absence of 1 μM long peptides (LP) M1 (30-mer, influenza-derived antigen, LKTRPILSPLTKGILGFVFTLTVPSERGLQ), MART-1 (25-mer, tumor-associated antigen, GHGHSYTTAEELAGIGILTVILGVL) or pp65 (30-mer, CMV-derived antigen, TWPPWQAGILARNLVPMVATVQGQNLKYQE) which were custom made by ProImmune.

Techniques: Virus, Expressing, Incubation, Lysis, Co-Culture Assay, Generated, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Antibodies Targeting Human or Mouse VSIG4 Repolarize Tumor-Associated Macrophages Providing the Potential of Potent and Specific Clinical Anti-Tumor Response Induced across Multiple Cancer Types

doi: 10.3390/ijms25116160

Figure Lengend Snippet: siRNA knockdown of VSIG4 attenuates the M2c phenotype, and 12A12c repolarizes M2c-macrophages toward an M1-like phenotype ( A ) siRNA knockdown of VSIG4 in M2c macrophages transfected with 25 nM VSIG4 siRNA LNPs on days 1 and 3 during the differentiation and polarization period. VSIG4 mRNA and protein levels were measured on day 8 by branched chain DNA analysis and flow cytometry, respectively ( n = 4 donors). Green represents M1 macrophages, black represents M2 macrophages, red represents M2 macrophages transfected with VSIG4 siRNA. ( B ) The fold change of M2c markers CD16, CD163, and CD206 as determined by flow cytometry in M2c macrophages treated with siRNA LNPs relative to those treated with luciferase siRNA. ( C ) Flow cytometry determined the fold change of the M2c marker CD163 based on the % CD163 + of CD11b + CD3 − CD45 + M2c macrophages treated with 12A12 for 24 h followed by 0.1 ng/mL LPS for 24 h. ( D ) The fold change based on pg/mL secreted proteins measured by a 25-plex Luminex assay from human monocyte-derived M2c macrophages treated with 12A12c or isotype control for 30 min at 37 °C followed by 0.1 ng/mL LPS for 24 h. Fold changes were calculated with respect to the human IgG4 isotype control antibody for each donor and each concentration. Black lines represent individual donors ( n = 5). Purple lines represent the mean fold change across all donors.

Article Snippet: On day 6 of the assay, differentiated M2 cells were polarized to M2c macrophages with 1 mL of fresh media containing 50 ng/mL M-CSF and 10 ng/mL of interleukin-10 (IL-10, Cat #574004, Biolegend, San Diego, CA, USA) while M1 cells wells were polarized with 100 ng/mL LPS (Cat #tlrl-eblps, Invivogen, San Diego, CA, USA) and 20 ng/mL IFNg (Cat #570204, Biolegend, San Diego, CA, USA).

Techniques: Knockdown, Transfection, Flow Cytometry, Luciferase, Marker, Luminex, Derivative Assay, Control, Concentration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Carbon Dioxide Sensing by Immune Cells Occurs through Carbonic Anhydrase 2-Dependent Changes in Intracellular pH.

doi: 10.4049/jimmunol.2100665

Figure Lengend Snippet: FIGURE 2. High CO2 attenuates macrophage activation. (AE) Bone marrowderived macrophages (BMDMs) were polarized into proinflammatory (M1) macrophages utilizing LPSs (100 ng/ml). Absolute mRNA levels were assessed by a metabolic and inflammatory gene panel. (A) Venn diagram depicting the number of differentially expressed genes under unbuffered (no BM) and/or buffered conditions (BM). Further in-depth analysis included gene set analysis (B), single pathway score analysis of inflammatory signaling pathways (C), heat map analysis of proinflammatory genes involved in NF-kB signaling (D), and absolute mRNA count analysis (E) regarding the effects of CO2 on gene expression profiles in LPS-polarized M1 macrophages. (F) Quantitative RT-PCR analysis of the M1 marker TNF-a (Tnfa) in LPS-polarized M1 macrophages. (G) NF-kB activity was determined in transfected THP-1 monocytes upon different levels of CO2 and LPS stimulation. (H) Upon M1 polarization, THP-1 macrophages were treated with 5 or 10% CO2. ELISA-based analysis of CCL2 protein levels within cell culture medium of 5% CO2- or 10% CO2-treated macrophages under unbuffered (left graph) or buffered conditions (right graph). Graphs in (A)(H) depict pooled data from three biological replicates. Statistical significance was analyzed by one-way ANOVA and an appropriate post hoc test (in F and G), and a paired Student t test in (H). For statistical testing of large scale gene expression analysis, refer to Materials and Methods.

Article Snippet: After 6 h of CO2 treatment, macrophages were polarized into proinflammatory (M1) macrophages using LPS (100 ng/ml; InvivoGen, San Diego, CA, tlrl-3pelps) for an additional 18 h. At the end of experiments cells were harvested for RNA isolation to analyze mRNA levels of M1 markers.

Techniques: Activation Assay, Protein-Protein interactions, Gene Expression, Quantitative RT-PCR, Marker, Activity Assay, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture