Journal: Frontiers in Pharmacology

Article Title: IL6/CCL2 from M2-polarized microglia promotes breast cancer brain metastasis and the reversal effect of β-elemene

doi: 10.3389/fphar.2025.1547333

Figure Lengend Snippet: M2 microglia phenotype and IL-6 and CCL2 cytokines were significantly elevated in the BC-BM brain. (A) Breast cancer 4T1-luc and MDA-MB-231-luc cells brain metastasis established by intracardiac inoculation. (B) HE staining of brain tissue with BC-BM. IHC staining of brain tissue with IBA1 (C) , IL6 (D) , and CCL2 (E) . (F) Representative images of immunofluorescence staining, with DAPI (blue) for nuclei, Arg1 (red) for M2-marker, iNOS (red) for M1-marker (magnification: 20×). Upper image was from non-brain-metastatic lesion and lower image was brain-metastatic lesion of mice brain.

Article Snippet: Tocilizumab was purchased from PeproTech Inc. (Suzhou, China), IL6 was purchased from GLPbio (CA, United States), anti-mouse/human/rat CCL2 2H5 was purchased from Selleck Chemicals (Shanghai, China), temozolomide (TMZ) was purchased from Sigma-Aldrich (St. Louis, United States), and β-elemene was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

Techniques: Staining, Immunohistochemistry, Immunofluorescence, Marker

Journal: Frontiers in Pharmacology

Article Title: IL6/CCL2 from M2-polarized microglia promotes breast cancer brain metastasis and the reversal effect of β-elemene

doi: 10.3389/fphar.2025.1547333

Figure Lengend Snippet: Breast cancer cell CM promoted M2-type polarization, and IL-6 and CCL2 secretion of primary microglia. (A) Schematic diagram of in vitro CM-co-culture with primary microglia model. ELISA assays of murine-derived IL6 (B) and CCL2 (C) cytokines from primary microglia cells CM-co-culture model supernatants. Cell supernatants from primary microglia cells treated with CM-coculture medium for 0, 12, 24, and 48 h were collected and measured by IL6 (D) and CCL2 (E) ELISA assays. (F) Representative images of primary microglia cells treated with CM-co-culture medium immunofluorescence staining with DAPI (blue) for nuclei, Arg1 (red) for M2-marker (left image), iNOS (red) for M1-marker (right image) (magnification: 20×). Western blot (G) and corresponding gray value analysis (H) were used to investigate the expression Arg1, CD206, and iNOS in primary microglia cells treated with CM-coculture medium for 0, 12, 24, and 48 h. (I) Relative expression of murine-derived IL6, CCL2, M1-markers (iNOS), and M2-markers (Arg1) mRNA in primary microglia cells treated with CM-coculture medium for 48 h detected by quantitative RT-PCR. GAPDH was used to normalize gene expression. Data are mean ± SD (n = 3). Significant difference versus control group, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Tocilizumab was purchased from PeproTech Inc. (Suzhou, China), IL6 was purchased from GLPbio (CA, United States), anti-mouse/human/rat CCL2 2H5 was purchased from Selleck Chemicals (Shanghai, China), temozolomide (TMZ) was purchased from Sigma-Aldrich (St. Louis, United States), and β-elemene was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

Techniques: In Vitro, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Immunofluorescence, Staining, Marker, Western Blot, Expressing, Quantitative RT-PCR, Gene Expression, Control

Journal: Frontiers in Pharmacology

Article Title: IL6/CCL2 from M2-polarized microglia promotes breast cancer brain metastasis and the reversal effect of β-elemene

doi: 10.3389/fphar.2025.1547333

Figure Lengend Snippet: BC-BM cells revealed high BM rate and significantly recruited M-MDSC in vivo . In vivo and in situ fluorescence intensity of human breast cancer parent MDA-MB-231-luc and MDA-MB-231-BM2 cells (A) , and mouse breast cancer parent 4T1-luc and 4T1-BM2 cells (C) . ELISA assays of murine-derived IL6 and CCL2 cytokines from human breast cancer parent MDA-MB-231-luc and MDA-MB-231-BMs cell supernatants (B) , and mouse breast cancer parent 4T1-luc and 4T1-BM2 cell supernatants (D) . (E) Schematic diagram of immune cells separated from brain tissue and stained with cluster of differentiation markers. (F) M-MDSC and P-MDSC distributions in normal mice brain, 4T1-BM1 inoculation mice brain, and 4T1-BM2 inoculation mice brain were detected by flow cytometer. Data are mean ± SD (n = 3). Significant difference versus control group, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Tocilizumab was purchased from PeproTech Inc. (Suzhou, China), IL6 was purchased from GLPbio (CA, United States), anti-mouse/human/rat CCL2 2H5 was purchased from Selleck Chemicals (Shanghai, China), temozolomide (TMZ) was purchased from Sigma-Aldrich (St. Louis, United States), and β-elemene was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

Techniques: In Vivo, In Situ, Fluorescence, Enzyme-linked Immunosorbent Assay, Derivative Assay, Staining, Flow Cytometry, Control

Journal: Frontiers in Pharmacology

Article Title: IL6/CCL2 from M2-polarized microglia promotes breast cancer brain metastasis and the reversal effect of β-elemene

doi: 10.3389/fphar.2025.1547333

Figure Lengend Snippet: M2-type microglia secrete cytokines that promote the MET process of tumor cells and recruit M-MDSCs. (A) E-cadherin (magnification: 400×) and vimentin (magnification: 200×) expression of 4T1-luc cells cultured with different conditions presented by immunofluorescence staining. Blue signal represents DAPI-stained nuclei. Control: normal 4T1-luc cells; TGF-β1: 4T1-luc cells treated with TGF-β1 (2.5 ng/mL) for 24 h; TGF-β1 and CC-CM: 4T1-luc cells treated with TGF-β1 (2.5 ng/mL) and incubated with co-culture CM for 24 h; CC-CM: 4T1-luc cells treated with co-culture CM for 24 h. (B) Western blot and corresponding gray value analysis of E-cadherin and vimentin in 4T1-luc cells cultured with different conditions. (C) Western blot and corresponding gray value analysis of p-JAK2, JAK2, p-STAT3, and STAT3 in 4T1-luc cells treated with Tocilizumab (2.5 μg/mL), IL6 (200 ng/mL) alone, or IL6-supplemented with tocilizumab. (D) Flowchart of in vitro MDSC recruitment experiment, flow cytometer detection results, and data statistics diagram. (E) Flowchart of in vitro MDSC recruitment with anti-mouse/human/rat CCL2 2H5 experiment and flow cytometer detection results. Data are mean ± SD (n = 3). Significant difference versus control group, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Tocilizumab was purchased from PeproTech Inc. (Suzhou, China), IL6 was purchased from GLPbio (CA, United States), anti-mouse/human/rat CCL2 2H5 was purchased from Selleck Chemicals (Shanghai, China), temozolomide (TMZ) was purchased from Sigma-Aldrich (St. Louis, United States), and β-elemene was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

Techniques: Expressing, Cell Culture, Immunofluorescence, Staining, Control, Incubation, Co-Culture Assay, Western Blot, In Vitro, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: IL6/CCL2 from M2-polarized microglia promotes breast cancer brain metastasis and the reversal effect of β-elemene

doi: 10.3389/fphar.2025.1547333

Figure Lengend Snippet: β-elemene regulated IL6/STAT3 signaling pathway and inhibited M-MDSC recruitment. (A) Representative images of brain HE staining treated with TMZ and β-elemene for 14 days depicted. (B) Western blot analysis of p-JAK2, JAK2, p-STAT3, and STAT3 in mice brain tissue treated with TMZ and β-elemene for 14 days. Relative expressions of IL6 (C) and CCL2 (D) in each group’s mice brain tissue were detected by quantitative RT-PCR. GAPDH was used to normalize gene expression. (E) M-MDSC and P-MDSC distributions in normal mice brain, 4T1-luc inoculation mice brain, and 4T1-luc inoculation treated with TMZ and β-elemene mice brain detected by flow cytometer. Data are mean ± SD. significant difference versus control group, ***, P < 0.001; significant difference versus model group, #, p < 0.05, ##, p < 0.01, and ###, p < 0.001.

Article Snippet: Tocilizumab was purchased from PeproTech Inc. (Suzhou, China), IL6 was purchased from GLPbio (CA, United States), anti-mouse/human/rat CCL2 2H5 was purchased from Selleck Chemicals (Shanghai, China), temozolomide (TMZ) was purchased from Sigma-Aldrich (St. Louis, United States), and β-elemene was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

Techniques: Staining, Western Blot, Quantitative RT-PCR, Gene Expression, Flow Cytometry, Control