Journal: Breast Cancer Research : BCR

Article Title: AXL promotes inflammatory breast cancer progression by regulating immunosuppressive macrophage polarization

doi: 10.1186/s13058-025-02015-8

Figure Lengend Snippet: AXL inhibition suppresses the polarization of immunosuppressive M2 macrophages. A AXL was knocked out in human THP-1 monocytes using the CRISPR/Cas9 system. These cells and control cells were subsequently induced into M2 macrophages, and AXL depletion was confirmed in AXL-KO M2 macrophages using Western blotting. M2 macrophages polarized from AXL-KO THP-1 cells had lower expression of AXL than M2 macrophages polarized from control cells. B The expression of M2 macrophage markers and secreted cytokines was measured by qRT-PCR. AXL KO reduced the expression of CD163 , CD206 , CCL17 , and CCL18 in M2 macrophages. C Flow cytometric analysis was conducted to compare the CD206 + macrophage population polarized from AXL-KO versus control THP-1 cells. AXL KO reduced the population of CD206 + macrophages polarized from THP-1 cells. D Treatment with TP-0903 inhibited the expression of AXL mRNA (left panel) and protein (right panel) in THP-1–derived M2 macrophages as determined using qRT-PCR and Western blotting, respectively. E TP-0903–treated M2 macrophages showed reduced mRNA level of CD163 , CD206 , CCL17 , and CCL18 compared to vehicle-treated M2 macrophages. F Flow cytometry and Western blotting showed that TP-0903 reduced the population of CD206 + cells (left panel) and CD206 protein expression (right panel) in THP-1–derived M2 macrophages. All experiments were repeated at least three times. Data were summarized as means ± SD. A 2-tailed Student t test ( B and 1-way analysis of variance followed by Dunnett’s multiple comparison test ( D – F ) were used to calculate P values. * P < 0.01

Article Snippet: Protein levels of human CCL20, CCL26, EREG, CXCL9, and CXCL10 in lysates or CM from AXL-KO or control M2 macrophages were quantified using a DuoSet ELISA kit, following the manufacturer’s instructions (R&D Systems).

Techniques: Inhibition, CRISPR, Control, Western Blot, Expressing, Quantitative RT-PCR, Derivative Assay, Flow Cytometry, Comparison

Journal: Breast Cancer Research : BCR

Article Title: AXL promotes inflammatory breast cancer progression by regulating immunosuppressive macrophage polarization

doi: 10.1186/s13058-025-02015-8

Figure Lengend Snippet: AXL depletion reduces the impact of M2 macrophages on IBC cell growth and migration. A SUM149 and BCX010 cells were co-cultured with 100% CM collected after culturing vehicle- or TP-0903–treated M2 macrophages for 48 h, and cell numbers after 3 days were measured by CTB assay. CM from TP-0903–treated M2 macrophages reduced the growth of SUM149 and BCX010 IBC cells. B The migration of human IBC cells induced by 100% CM from TP-0903– or vehicle-treated M2 macrophages after 48 h of culture was examined using transwell migration assay. CM from TP-0903–treated M2 macrophages inhibited the migration of SUM149 and BCX010 cells ( C ). D 100% CM from AXL-KO M2 macrophages after 48 h of culture reduced the growth C and migration D of SUM149 and BCX010 cells. All experiments were repeated at least three times. Data were summarized as means ± SD. One-way analysis of variance followed by Dunnett’s multiple comparison test ( A and B ) and 2-tailed Student t test ( C and D ) were used to calculate P values. * P < 0.05; ** P < 0.01

Article Snippet: Protein levels of human CCL20, CCL26, EREG, CXCL9, and CXCL10 in lysates or CM from AXL-KO or control M2 macrophages were quantified using a DuoSet ELISA kit, following the manufacturer’s instructions (R&D Systems).

Techniques: Migration, Cell Culture, CtB Assay, Transwell Migration Assay, Comparison

Journal: Breast Cancer Research : BCR

Article Title: AXL promotes inflammatory breast cancer progression by regulating immunosuppressive macrophage polarization

doi: 10.1186/s13058-025-02015-8

Figure Lengend Snippet: AXL suppression inhibits the polarization of immunosuppressive M2 macrophages via STAT6. A Treatment with TP-0903 reduced AXL, phospho-STAT6, and STAT6 protein expression as determined by Western blotting. B M2 macrophages polarized from AXL-KO THP-1 cells had lower phospho-AXL, AXL, phospho-STAT6, and STAT6 protein expression than those polarized from control THP-1 cells, as determined using Western blotting. C–E STAT6 was knocked down in THP-1 cells using siRNAs, and then THP-1 cells were induced to M2 macrophages. Knockdown of STAT6 in THP-1–polarized M2 macrophages C decreased the CD163 + CD206 + macrophage population as determined by flow cytometry D and decreased the expression of the CD163 and CD206 genes as determined using qRT-PCR E . F STAT6 was overexpressed in M2 macrophages polarized from AXL-KO THP-1 cells as tested using qRT-PCR. G STAT6 overexpression mitigated the inhibitory effect of AXL KO on the expression of M2 macrophage markers and cytokines, including CD163 , CD206 , CCL17 , and CCL18 . H The CM from control, AXL-KO, and AXL-KO + STAT6–overexpressing M2 macrophages and fresh media were used as attractants plated in the bottom chamber of transwells to test the migration of SUM149 cells. Migration of SUM149 cells was greater with CM from AXL-KO + STAT6–overexpressing M2 macrophages than with CM from AXL-KO M2 macrophages. All experiments were repeated at least three times. Data were summarized as means ± SD. One-way analysis of variance followed by Dunnett’s multiple comparison test was used to calculate P values. * P < 0.05; ** P < 0.01

Article Snippet: Protein levels of human CCL20, CCL26, EREG, CXCL9, and CXCL10 in lysates or CM from AXL-KO or control M2 macrophages were quantified using a DuoSet ELISA kit, following the manufacturer’s instructions (R&D Systems).

Techniques: Expressing, Western Blot, Control, Knockdown, Flow Cytometry, Quantitative RT-PCR, Over Expression, Migration, Comparison

Journal: Breast Cancer Research : BCR

Article Title: AXL promotes inflammatory breast cancer progression by regulating immunosuppressive macrophage polarization

doi: 10.1186/s13058-025-02015-8

Figure Lengend Snippet: AXL regulates the expression of cytokines via STAT6 in M2 macrophages. A – C mRNA was collected from M2 macrophages polarized from AXL-KO THP-1 and control cells, and the expression of M2 macrophage markers or cytokines/chemokines was examined using qRT-PCR. AXL KO in M2 macrophages derived from THP-1 cells reduced the mRNA expression of CD209 , IL13RA , and IL2RG A . AXL KO in M2 macrophages derived from THP-1 cells reduced the expression of the immunosuppressive cytokines/chemokines CCL20 , CCL26 , EREG , and IL1B B . AXL KO in M2 macrophages derived from THP-1 cells increased the expression of cytokines/chemokines involved in the interferon γ–mediated signaling pathway, such as IFNG , CXCL10 , and GBP2 , at the gene level C . D and E CM from and lysates of M2 macrophages polarized from AXL-KO THP-1 cells had decreased expression of CCL20, CCL26, and EREG protein D but increased expression of CXCL9 and CXCL10 protein E as determined using ELISA. F The migration of human IBC cells was assessed using a transwell migration assay, with CM collected from control and AXL-KO M2 macrophages with or without the addition of recombinant CCL20, CCL26, and EREG protein, serving as attractants. CCL20, CCL26, and EREG mitigated the inhibitory effect of AXL-KO M2 macrophages on SUM149 and BCX010 cell migration. G qRT-PCR was conducted to measure the mRNA expression level of CCL20 , CCL26 , and EREG in control, AXL-KO, and AXL-KO + STAT6-overexpressing M2 macrophages. Overexpression of STAT6 mitigated the suppressive effect of AXL KO in M2 macrophages on the expression of CCL20 , CCL26 , and EREG genes. All experiments were repeated at least three times. Data were summarized as means ± SD. Two-tailed Student t test ( A – E ) and 1-way analysis of variance followed by Dunnett’s multiple comparison test ( F and G ) were used to calculate P values. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Protein levels of human CCL20, CCL26, EREG, CXCL9, and CXCL10 in lysates or CM from AXL-KO or control M2 macrophages were quantified using a DuoSet ELISA kit, following the manufacturer’s instructions (R&D Systems).

Techniques: Expressing, Control, Quantitative RT-PCR, Derivative Assay, Enzyme-linked Immunosorbent Assay, Migration, Transwell Migration Assay, Recombinant, Over Expression, Two Tailed Test, Comparison

Journal: Breast Cancer Research : BCR

Article Title: AXL promotes inflammatory breast cancer progression by regulating immunosuppressive macrophage polarization

doi: 10.1186/s13058-025-02015-8

Figure Lengend Snippet: AXL expression correlates with an immunosuppressive TME of IBC. A – D Violin plots showed the absolute percentages of different immune cell subsets as defined by CIBERSORT according to high versus low AXL expression in IBC samples from the Inflammatory Breast Cancer International Consortium: M2 macrophages A , resting memory CD4 + T cells B , activated myeloid dendritic cells C , and follicular helper T cells D . A 2-tailed Student t test was used to calculate P values. E Proposed mechanistic model of AXL in the IBC TME. AXL regulated M2 macrophage polarization and the expression of immunosuppressive molecules and STAT6-modulated cytokines, which induced IBC’s aggressiveness

Article Snippet: Protein levels of human CCL20, CCL26, EREG, CXCL9, and CXCL10 in lysates or CM from AXL-KO or control M2 macrophages were quantified using a DuoSet ELISA kit, following the manufacturer’s instructions (R&D Systems).

Techniques: Expressing

Journal: Medical science monitor : international medical journal of experimental and clinical research

Article Title: The Function of the Novel Mechanical Activated Ion Channel Piezo1 in the Human Osteosarcoma Cells.

doi: 10.12659/msm.906959

Figure Lengend Snippet: Figure 7. (A) Images of nuclei shrinking and evident nuclear condensation of osteosarcoma (OS) cells (shown by arrowhead) after mechanical stretch forces of 0, 2, 12, 24, and 48 hours. Notes: (a) 0-hour group 200×. (b) 0-hour plus shPiezo1RNA group 200x. (c) 2-hour group 200×. (d) 2-hour plus shPiezo1RNA group 200×. (e) 12-hour group 200×. (f) 12-hour plus shPiezo1RNA group 200×. (g) 24-hour group 200×. (h) 24-hour plus shPiezo1RNA group 200×. (i) 48-hour group 200×. (j) 48-hour plus shPiezo1RNA group 200×. (B) Results of flow cytometry detection of 0S cell apoptosis in 0, 2, 12, 24, and 48 hours and shPiezo1RNA groups. Q1: (AnnexinV-FITC)-/PI+, necrotic cells. Q2: (AnnexinV-FITC)+/PI+, late stage apoptotic cells. Q3: (AnnexinV-FITC)+/PI–, early stage apoptotic cells. Q4: (AnnexinV-FITC)-/PI-, live cells. Notes: (a) 0-hour group. (b) 0-hour group plus shPiezo1RNA. (c) 2-hour group. (d) 2-hour group plus shPiezo1RNA. (e) 12-hour group. (f) 12-hour group plus shPiezo1RNA. (g) 24-hour group. (h) 24-hour group plus shPiezo1RNA. (i) 48-hour group. (j) 48-hour group plus shPiezo1RNA. (C) Early stage apoptosis of human OS cells in 0, 2, 12, 24, 48 hors and shPiezo1RNA groups. After a 2 hour mechanical stretch, the rate of early stage apoptosis significantly increased. After a 12 hour mechanical stretch, the late stage apoptotic rate increased and peaked in the 24-hour group. The late stage apoptotic rate was lower in the 48-hour group compared to the 24-hour group. The results are presented as the means ±SD, n=3; ns means p>0.05 versus the control group; * p<0.05 versus the control group; ** p<0.01 versus the control group; one-way ANOVA.

Article Snippet: Cells were stained with FITC-conjugated Annexin V and propidium iodide (PI) according to the manufacturer’s instructions for the Apoptosis Detection Kit (R&D Systems, MN, USA).

Techniques: Flow Cytometry, Control

Journal: International Journal of Molecular Sciences

Article Title: Reduced Dendritic Cells Expressing CD200R1 in Children with Inflammatory Bowel Disease: Correlation with Th17 and Regulatory T Cells

doi: 10.3390/ijms161226143

Figure Lengend Snippet: Representative flow cytometry data to illustrate the gating strategy for FACS analysis of DC subsets. ( A ) Dendritic cell HLA-DR + Lin − cells were gated ( R1 ), and myeloid CD11c + mDCs ( R2 ), plasmacytoid CD123 + pDCs ( R3 ) were determined as a percentage of the total PBMC ( B ). Dot plots to illustrate the expression of CD200 and CD200R1 on the myeloid dendritic cell (mDC) subset of healthy controls and IBD patients ( C , D ) and on and plasmacytoid DCs (pDCs) of healthy controls and IBD patients ( E , F ).

Article Snippet: For regulatory T cell staining, 1 × 10 6 cells were resuspended in 100 μL flow cytometry staining buffer (R&D Systems, Minneapolis, MN, USA).

Techniques: Flow Cytometry, Expressing

Journal: Neuro-Oncology

Article Title: Agonist OX40 immunotherapy improves survival in glioma-bearing mice and is complementary with vaccination with irradiated GM-CSF–expressing tumor cells

doi: 10.1093/neuonc/nox125

Figure Lengend Snippet: Th1 vs Th2 BILs. (A) Flow cytometry demonstrating percent expression of T-bet, IFN-γ, and IL-12R β2 on CD4+ lymphocytes 18 days after GL261 tumor implantation by treatment group. Combination immunotherapy is associated with significant increases in all 3 Th1 markers. (***P < 0.0005, **P < 0.005, *P < 0.05; absence of an asterisk denotes not significant comparison.) (B) Flow cytometry demonstrating percent expression of GATA3, the IL-4 receptor, and IL-5 on CD4+ lymphocytes 18 days after GL261 tumor implantation by treatment group. Combination immunotherapy is associated with significant decreases in all 3 Th2 markers. (**P < 0.005, *P < 0.05, absence of an asterisk denotes not significant comparison.)

Article Snippet: For the analysis of T helper cell subsets, we used Multi-Color Flow Cytometry Kits (R&D Systems) for mouse Th1 cells (conjugated antibodies to T-bet-PerCP, IFN-gamma-fluorescein, IL-12 R beta 2-APC, and CD4-PE) and Th2 cells (conjugated antibodies to CD4-PerCP, IL-4 R-fluorescein, STAT6-APC, and IL-5-PE).

Techniques: Flow Cytometry, Expressing, Tumor Implantation, Comparison

Journal: Neuro-Oncology

Article Title: Agonist OX40 immunotherapy improves survival in glioma-bearing mice and is complementary with vaccination with irradiated GM-CSF–expressing tumor cells

doi: 10.1093/neuonc/nox125

Figure Lengend Snippet: Day 18 flow cytometry of BILs shows that vaccination improves the intratumoral CD8+/FoxP3+ lymphocyte ratio, which is little affected by treatment with anti-OX40 immunotherapy. (**P < 0.005, *P < 0.05; absence of an asterisk denotes not significant comparison.)

Article Snippet: For the analysis of T helper cell subsets, we used Multi-Color Flow Cytometry Kits (R&D Systems) for mouse Th1 cells (conjugated antibodies to T-bet-PerCP, IFN-gamma-fluorescein, IL-12 R beta 2-APC, and CD4-PE) and Th2 cells (conjugated antibodies to CD4-PerCP, IL-4 R-fluorescein, STAT6-APC, and IL-5-PE).

Techniques: Flow Cytometry, Comparison

Journal: Science advances

Article Title: Profiling of mature-stage human breast milk cells identifies six unique lactocyte subpopulations.

doi: 10.1126/sciadv.abm6865

Figure Lengend Snippet: Fig. 1. Maternal cells in breast milk were identified by protein, gene, and transcriptome analyses. Mature-stage breast milk from healthy donors was assessed for expression of cell marker proteins (e.g., EpCAM and CD45) in freshly isolated samples by flow cytometry. Then, RNA was isolated and used to conduct complementary gene expression analysis by RT-qPCR. Single-cell transcriptomics added depth to the analysis and defined the lactocyte population in breast milk.

Article Snippet: Fixable Yellow samples were subsequently fixed using Flow Cytometry Fixation Buffer (R&D Systems), centrifuged, and resuspended in 1% FBS in PBS.

Techniques: Expressing, Marker, Isolation, Flow Cytometry, Gene Expression, Quantitative RT-PCR, Single-cell Transcriptomics

Journal: Science advances

Article Title: Profiling of mature-stage human breast milk cells identifies six unique lactocyte subpopulations.

doi: 10.1126/sciadv.abm6865

Figure Lengend Snippet: Fig. 2. Mature-stage breast milk from donors contained epithelial lactocytes and a smaller population of immune cells. (A) Flow cytometry quantified expression of cell-specific markers: EpCAM+ (epithelial cells), CD45+ (immune cells), CK18+ (lactocytes), and VIM+ (mesenchymal cells). These data suggest that the CK18 antibody bounds poorly to lactocytes and likely underestimates true lactocyte percentages. FITC, fluorescein isothiocyanate. (B) The relative cellular composition in breast milk was affected by the health status of the mother and infant. Representative density plots are shown of EpCAM versus CD45 cell populations in milk from healthy donors (n = 10), donors with sick infants (n = 1), or donors with recent mastitis (n = 3). (C) Flow cytometry analysis of protein markers in all health statuses (n = 14) and the breakdown of (D) CD45+ immune cells and (E) VIM+ mesenchymal cells in different health statuses. (F) mRNA expression of gene markers corresponding to proteins in (C) indicates the presence of epithelial lactocytes (n = 6 to 7). Data are shown as means ± SEM. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant.

Article Snippet: Fixable Yellow samples were subsequently fixed using Flow Cytometry Fixation Buffer (R&D Systems), centrifuged, and resuspended in 1% FBS in PBS.

Techniques: Flow Cytometry, Expressing, Comparison

Journal: Science advances

Article Title: Profiling of mature-stage human breast milk cells identifies six unique lactocyte subpopulations.

doi: 10.1126/sciadv.abm6865

Figure Lengend Snippet: Fig. 3. Stem-like transcription factors were expressed in 5% of breast milk cells. Four stem-like transcription factors were assessed in breast milk epithelial cells. (A) From flow cytometry analysis, representative histograms of SOX2+, TRA-1-60+, NANOG+, and SSEA4+ stained and unstained samples are shown. (B) Flow cytometry analysis of EpCAM+ cells (n = 15) indicated high expression of SOX2 and TRA-1-60 with moderate to low expression of NANOG and SSEA4, respectively. (C) mRNA expression relative to a housekeeping gene was quantified for the genes corresponding to the proteins in (B) (n = 7). (D) Flow cytometry analysis of EpCAM+ cells and their increasing stemness based on positive expression of multiple stem-like markers (n = 15). Data are means ± SEM. One-way ANOVA with Tukey’s multiple comparison test; *P < 0.05; ****P < 0.0001.

Article Snippet: Fixable Yellow samples were subsequently fixed using Flow Cytometry Fixation Buffer (R&D Systems), centrifuged, and resuspended in 1% FBS in PBS.

Techniques: Flow Cytometry, Staining, Expressing, Comparison