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