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: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: Isolation of Placenta-Derived MSCs. (A) Illustration of the MSC isolation process. (B) Representative morphology of MSCs during isolation. (C) Differentiation of MSCs into adipocytes (FABP-4 + ) and osteocytes (osteocalcin + ). (D) Flow cytometry analysis of MSC surface markers. Negative markers include CD34, CD45, CD11b, CD79A, and HLA-DR. (E) Size distribution of MSCs cultured in T-25 flasks at passages 4 (P4), 6 (P6), and 8 (P8).

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Isolation, Derivative Assay, Flow Cytometry, Cell Culture

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: 3D Spheroid Culture Reduces MSC Size: Identifying the Optimal Spheroid Diameter. (A) Representative images of MSC (P3) spheroids cultured for 3 days with varying initial cell numbers per spheroid. (B) Quantification of spheroid diameter over the 3-day culture period. (C) Representative images of individual MSCs following 2D and 3D spheroid culture. (D,E) Cell size distribution of MSCs after 2D and 3D spheroid culture. (F) Proportions of small (≤15 µm) and large (>15 µm) MSCs after 2D and 3D spheroid culture. Spheroid culture duration in (C–F) was 72 h.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Cell Culture

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: 3D Spheroid Culture Reduces MSC Size: Determining the Optimal Culture Duration. (A) Representative images showing morphological changes in MSC (P4) spheroids over a 7-day culture period. (B) Images of individual MSCs cultured in 2D or in 25 K-cell spheroids for varying durations. (C) Changes in the diameter of 25 K-cell MSC 3D spheroids over 7 days. (D) Comparison of mean MSC diameter in 2D versus 25 K spheroid cultures over 7 days (E,F) Size distribution of MSCs within 25 K spheroids across the 7-day culture period.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Cell Culture, Comparison

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: Effect of Chemically Defined Medium on MSC Viability and Size in Spheroid Culture. (A) Phase-contrast and dead cell staining (red) images of MSC (P6) spheroids cultured in various media. Three representative spheroids are shown in each condition. (B) MSC size distribution in 3D cultures across different media conditions. (C,D) Anti-inflammatory capability of MSCs cultured under varying conditions. RAW-Dual™ reporter cells were stimulated with 100 ng/mL LPS and 10 ng/mL IFNγ, then treated with MSCs. Dexamethasone (Dex, 1 µg/mL) was used as a positive control. Luciferase activity (C) and mouse IL-6 (D) were measured. Spheroid culture time was 48 h.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Staining, Cell Culture, Positive Control, Luciferase, Activity Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: Effects of Alternating 2D/3D Culture on MSC Size and Immunomodulatory Function. (A) MSCs were cultured in flasks for four passages, with an additional 2-day spheroid culture following each passage. Shown are the mean cell diameters immediately after 2D culture and after subsequent spheroid culture. (B) MSC diameters from P5 to P8 using conventional 2D culture versus the alternating 2D/3D method. P4 cells served as the starting population for both conditions. Diameters were measured after harvesting from 2D flasks. (C) Comparison of MSC sizes at P5–P8 between the two culture methods. (D) Comparison of doubling times at P5–P8 between the two methods. (E,F) Macrophages were activated with LPS and IFNγ, then treated with either early-passage (P4) or late-passage (P15) MSCs derived from conventional 2D culture or the alternating 2D/3D protocol. Levels of mouse IL-6 and IL-10 were measured to assess immunomodulatory effects.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Cell Culture, Comparison, Derivative Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: RGD-Modified Alginate Hydrogel Tubes (AlgTubes) for MSC Culture. (A) Schematic of alginate modification with RGD peptides. (B,C) Chemistry underlying hydrogel tube formation. (D,E) Process AlgTubes using a micro-extruder: a cell suspension and alginate solution are pumped into the central and side channels, respectively, creating coaxial core-shell flows. These are extruded through a nozzle into a CaCl 2 buffer, where Ca 2+ ions crosslink the outer alginate shell, forming hydrogel tubes instantly. (F) Illustration of growing MSCs within an AlgTube. (G,H) SEM images showing the porous structure of the AlgTubes.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Modification, Suspension

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: Dynamic Cell Adhesion in RGD-Modified AlgTubes. (A) MSCs were processed into RGD-modified AlgTubes. (B–D) Cells adhered to the inner surface and proliferated from day 0 to day 6. (E) On day 6, free RGD peptides were added to the culture medium, leading to cell contraction within 24 h. (F) By 48 h, cells had fully detached and formed spheroids (red arrows). (G,H) Removal of free RGD peptides from the medium resulted in MSC reattachment to the AlgTube surface and resumed cell growth (blue arrows). (I–L) As a comparison, in the absence of free RGD peptides, day 6 MSCs continued to grow and eventually covered the inner surface of the hydrogel tube.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Modification, 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 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