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: TP-0903 treatment suppresses the population of CD206 + macrophages in human IBC xenograft and murine TNBC syngeneic models. A Tumors from a SUM149 xenograft mouse model treated with vehicle or TP-0903 for 7 days were dissociated to obtain a single-cell suspension and stained with antibodies. Flow cytometric analysis showed a decreased CD206 + macrophage population (CD45 + CD11b + Ly6C − Ly6G − F4/80 + CD206 + cells) after TP-0903 treatment. B IHC staining of CD206 on slides from the above tumor sections. TP-0903 decreased the population of CD206 + cells in SUM149 tumor tissues. Left panel: representative IHC staining images. Scale bar = 200 μm. Right panel: quantification of CD206 expression by ImageJ. C Tumor growth curves for the vehicle- and TP-0903–treated groups in murine TNBC syngeneic mouse models. TP-0903 suppressed the growth of 4T1.2 and E0771 mammary tumors in vivo. D TP-0903 treatment reduced CD206 + macrophages in murine mammary tumors from the 4T1.2 mouse model. E IHC staining for CD206 showed reduced CD206 + cells in TP-0903–treated tumor tissues from E0771 mice. Left panel: representative IHC staining images. Scale bar = 200 μm. Right panel: quantification of CD206 expression. F Flow cytometric analysis showed a decrease in Tregs (CD45 + CD3 + CD4 + CD25 + FOXP3 + cells) in TP-0903–treated 4T1.2 and E0771 mice. 4T1.2 syngeneic model: n = 10 mice; E0771 syngeneic model: n = 15 mice. Data were summarized as means ± SD in A , B , and D to F and means ± SEM in C . A 2-tailed Student t test was used to calculate P values. * P < 0.05; ** P < 0.01

Article Snippet: The human IBC cell line SUM149 was purchased from Asterand (Detroit, MI), and the human IBC cell line BCX010 was provided by Dr. Funda Meric-Bernstam (The University of Texas MD Anderson Cancer Center).

Techniques: Suspension, Staining, Immunohistochemistry, Expressing, In Vivo

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: TP-0903 treatment inhibits AXL signaling and reduces IBC cell migration and mammosphere formation in vitro and tumor growth in vivo. A TP-0903 reduced phospho-AXL and total AXL protein expression in SUM149 and BCX010 IBC cells as determined using Western blotting. B TP-0903 reduced SUM149 and BCX010 cell migration as tested using transwell migration assay. The mean number of migrated cells was determined for the 4 vehicle replicates in SUM149 and 3 vehicle replicates in BCX010. For each sample (12 each for SUM149 and 9 each for BCX010), the number of migrated cells, relative to the mean in the vehicle group, was plotted as a percentage (“% of vehicle”). C TP-0903 reduced primary and secondary mammosphere formation of SUM149 and BCX010 cells as determined using a surrogate mammosphere formation assay. D Tumor growth curves for vehicle- and TP-0903–treated groups in human IBC xenograft mouse models. TP-0903 decreased SUM149 and BCX010 tumor volumes in mice. SUM149 xenograft model: n = 12 mice; BCX010 xenograft model: n = 14 mice. Data were summarized as means ± SD in B and C and means ± SEM in D . One-way analysis of variance followed by Dunnett’s multiple comparison test ( B and C ) or 2-tailed Student t test ( D ) was used to calculate P values. * P < 0.05; ** P < 0.01

Article Snippet: The human IBC cell line SUM149 was purchased from Asterand (Detroit, MI), and the human IBC cell line BCX010 was provided by Dr. Funda Meric-Bernstam (The University of Texas MD Anderson Cancer Center).

Techniques: Migration, In Vitro, In Vivo, Expressing, Western Blot, Transwell Migration Assay, Tube Formation 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 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: The human IBC cell line SUM149 was purchased from Asterand (Detroit, MI), and the human IBC cell line BCX010 was provided by Dr. Funda Meric-Bernstam (The University of Texas MD Anderson Cancer Center).

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: The human IBC cell line SUM149 was purchased from Asterand (Detroit, MI), and the human IBC cell line BCX010 was provided by Dr. Funda Meric-Bernstam (The University of Texas MD Anderson Cancer Center).

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: The human IBC cell line SUM149 was purchased from Asterand (Detroit, MI), and the human IBC cell line BCX010 was provided by Dr. Funda Meric-Bernstam (The University of Texas MD Anderson Cancer Center).

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

Journal: bioRxiv

Article Title: Small extracellular vesicles promote stiffness-mediated metastasis

doi: 10.1101/2023.07.01.545937

Figure Lengend Snippet: A and B , Gene expression fold change in ACTA2 , CCN2 , COL1A1 , IL6 , KGF , MMP1 , VIM , VEGFA , and S100 proteins assessed by qRT-PCR in IMR90 human lung fibroblasts exposed to PBS only, 25 kPa EVs, and 0.5 kPa EVs. Two biological repeats. One-way ANOVA. C , Schematic showing (left) the arrival of stiff EVs in the lung, a mechanically soft environment, and encountering resident normal lung fibroblasts; (middle) cancer cells are then recruited to the lung; and (right) the cells, now experiencing a soft environment, release soft EVs that transform the resident fibroblasts to a cancer-associated fibroblast (CAF) phenotype.

Article Snippet: Human breast cancer cell lines MDA-MB-231, SUM149, and MDA-MB-468, human pancreatic cancer cell line BxPC3 (all from ATCC), and IMR90 human lung fibroblasts (gift from Daniele Gilkes, Johns Hopkins University) were cultured in 013-CV DMEM (Corning) containing 10% fetal bovine serum (Corning) and 1% penicillin-streptomycin (Gibco).

Techniques: Gene Expression, Quantitative RT-PCR

Journal: Cancer discovery

Article Title: Combining a PI3K inhibitor with a PARP inhibitor provides an effective therapy for BRCA1 -related breast cancer

doi: 10.1158/2159-8290.CD-11-0336

Figure Lengend Snippet: PI3K pathway activation in BRCA1-related breast cancer in MMTV-CreBRCA1f/fp53+/−. Tumor-bearing females were euthanized, tissues harvested and processed for immunohistochemistry. Displayed are representative images of immunohistochemistry for phospho-AKT (S473), pospho-(Thr202/Tyr204)-ERK, and the tumor-suppressor phosphatases INPP4B and PTEN. Adjacent normal mammary gland tissue is on the left, tumor tissue on the right. 400 x magnification.

Article Snippet: BRCA1 -mutant human breast cancer cell line HCC1937 was from American Type Culture Collection; # CRL-2336, and maintained in DMEM/10% FBS and SUM149 a gift from Dr. Christina Gewinner, Division of Signal Transduction, BIDMC, maintained in Ham's F-12 with 5% fetal bovine serum (FBS), 5 µg/ml insulin, 2 µg/ml hydrocortisone, 5 µg/ml gentamicin and 2.5 µg/ml fungizone.

Techniques: Activation Assay, Immunohistochemistry

Journal: Cancer discovery

Article Title: Combining a PI3K inhibitor with a PARP inhibitor provides an effective therapy for BRCA1 -related breast cancer

doi: 10.1158/2159-8290.CD-11-0336

Figure Lengend Snippet: PI3K-inhibition increases poly-ADP-ribosylation and H2AX phosphorylation. A. Compensatory pathway activation induced by treatments with NVP-BKM120. HCC1937 or SUM149 cells were treated with NVP-BKM120, Olaparib or its combination as indicated for 72 hours, lysed and subjected to immunoblotting with antibodies against total AKT, EGFR, ERK and their phospho-specific epitopes. B. In vivo increase of γH2AX-positive cells after treatment with NVP-BKM120 and proliferative activity at the “pushing margin”. Tumor-bearing mice were subjected to a pre-treatment biopsy and then treated with NVP-BKM120 at 50 mg/kg/day. IHCs of pre-treatment biopsies and post-treatment tumor tissues were performed with antibodies as indicated. C. Effects of combined PI3K- and PARP-inhibition on BRCA1-mutant cells. Cells were treated with NVP-BKM120 at 1 µM and Olaparib 10 µM or their combination for 24 hours, lysed and subjected to immunoblotting with antibodies against PAR, pAKT, total AKT and γH2AX and Actin as indicated. D. BRCA1 mutant human HCC1937 or SUM149 cells were treated with vehicle control or NVP-BKM120 at the indicated concentrations for 24 hours, lysed and subjected to immunoblotting with antibodies against PAR, p-AKT (S473), γH2AX, Cleaved Caspase 3(CC3) as an apoptosis marker and Actin as a loading control.

Article Snippet: BRCA1 -mutant human breast cancer cell line HCC1937 was from American Type Culture Collection; # CRL-2336, and maintained in DMEM/10% FBS and SUM149 a gift from Dr. Christina Gewinner, Division of Signal Transduction, BIDMC, maintained in Ham's F-12 with 5% fetal bovine serum (FBS), 5 µg/ml insulin, 2 µg/ml hydrocortisone, 5 µg/ml gentamicin and 2.5 µg/ml fungizone.

Techniques: Inhibition, Activation Assay, Western Blot, In Vivo, Activity Assay, Mutagenesis, Marker

Journal: Cancer discovery

Article Title: Combining a PI3K inhibitor with a PARP inhibitor provides an effective therapy for BRCA1 -related breast cancer

doi: 10.1158/2159-8290.CD-11-0336

Figure Lengend Snippet: Effects of NVP-BKM120, KU-55933 and their combination on the DNA damage response. A. HCC1937 were treated for 18 hours with NVP-BKM120 at 2.5 µM, KU55933 at 10 µM or their combination, subjected to ionizing irradiation with 10 Gy or mock, lysed 6 hours later and subjected to immunoblotting with antibodies as indicated. B-E Loss of Rad51 focus formation in response to ionizing radiation in the presence of NVP-BKM120. Breast cancer cells were isolated from primary tumors from MMTV-Cre MMTV-CreBRCA1f/fp53+/− mice and either treated with vehicle control (B, C) or NVP-BKM120 (D, E) for 18 hours, followed by irradiation with 10 Gy. 6 hours later cells were fixed and processed for immunofluorescence with antibodies against Rad51 and counterstained with DAPI. F. Induction of DNA-PK and H2AX phosphorylation and loss of RAD51 occur in response to PI3Kα, not PI3Kβ-inhibition. SUM149 cells were transfected with siRNA pools depleting PI3Kα (left panel) or PI3Kβ (right panel). Cells were lysed after 48 hrs and subjected to immunoblotting with antibodies as indicated.

Article Snippet: BRCA1 -mutant human breast cancer cell line HCC1937 was from American Type Culture Collection; # CRL-2336, and maintained in DMEM/10% FBS and SUM149 a gift from Dr. Christina Gewinner, Division of Signal Transduction, BIDMC, maintained in Ham's F-12 with 5% fetal bovine serum (FBS), 5 µg/ml insulin, 2 µg/ml hydrocortisone, 5 µg/ml gentamicin and 2.5 µg/ml fungizone.

Techniques: Irradiation, Western Blot, Isolation, Immunofluorescence, Inhibition, Transfection

Journal: Cancer discovery

Article Title: Combining a PI3K inhibitor with a PARP inhibitor provides an effective therapy for BRCA1 -related breast cancer

doi: 10.1158/2159-8290.CD-11-0336

Figure Lengend Snippet: Anti-tumor efficacy of PI3K inhibitor NVP-BKM120 alone and in combination with Olaparib. A–D Tumor-bearing MMTV-CreBRCA1 p53+/− were treated with either vehicle control (A), NVP-BKM120 (B, 50 mg/kg/day (n=11) or 30 mg/kg/day (n=10)), Olaparib (C, 50 mg/kg/day (n=8)) or the combination of NVP-BKM120 and Olaparib (D, NVP-BKM 50 mg/kg/day+Olaparib 50 mg/kg/day (n=8) or NVP-BKM 30 mg/kg/day+Olaparib 50 mg/kg/day (n=7)) and tumor volumes were measured every 2–3 days using calipers. Trendlines for vehicle control (red curve) and NVP-BKM120 treatments (green curve) were calculated using all data points to determine best fit. The functions of the best-fit curves were used to determine tumor doubling times for all three treatment modalities and controls. E, Stable body mass with PI3K-inhibitor and PARP-inhibitor treatments Mice were weighed before and after treatments. F, G. Target inhibition and pharmacokinetics in vivo. Tumor tissues harvested from animals treated with NVP-BKM120 (30 mg/kg/day) alone or in combination with Olaparib (50 mg/kg/day) as indicated were harvested 3 hours after the last treatment and subjected to immunoblotting with antibodies against Actin, p-AKT and γH2AX (F) or lysed and subjected to Mass Spectrometry (G). For standards used see Materials and Methods and Fig. S5. H, I. Responses of human BRCA1 -related breast cancers implanted as xenotransplants into nude mice to NVP-BKM120, Olaparib or their combination. Breast cancer tissues from two patients, one with a 185delAG germline mutation (H) and the other one with a 2080delA germline mutation (I) were propagated as subcutaneous implants in nude mice. Tumors were allowed to grow to a size of 5 mm when mice were randomized to treatments with either vehicle control (black, —), NVP-BKM120 (red, ), Olaparib (green, )or their combination (blue, ) (n=6 for each cohort, same dosing as in F). Tumor assessment with electronic calipers was done as described in Materials and Methods.

Article Snippet: BRCA1 -mutant human breast cancer cell line HCC1937 was from American Type Culture Collection; # CRL-2336, and maintained in DMEM/10% FBS and SUM149 a gift from Dr. Christina Gewinner, Division of Signal Transduction, BIDMC, maintained in Ham's F-12 with 5% fetal bovine serum (FBS), 5 µg/ml insulin, 2 µg/ml hydrocortisone, 5 µg/ml gentamicin and 2.5 µg/ml fungizone.

Techniques: Inhibition, In Vivo, Western Blot, Mass Spectrometry, Mutagenesis