Journal: The American Journal of Pathology

Article Title: Bone Marrow Stromal Cell Antigen 2 Promotes Esophageal Squamous Cell Carcinoma Progression by Mediating the Cell-Cell Interaction between Cancer Cells and Cancer-Associated Fibroblasts

doi: 10.1016/j.ajpath.2026.01.012

Figure Lengend Snippet: Up-regulation of BST2 in both esophageal squamous cell carcinoma (ESCC) cells and cancer-associated fibroblast (CAF)–like cells. A: A schematic overview of the experimental design of direct co-culture and cDNA microarray analysis. ESCC cell lines (TE-9, TE-10, and TE-15) were co-cultured with mesenchymal stem cells (MSCs) in the same dish for 4 days, whereas monocultured ESCC cells and MSCs served as controls. Following co-culture, cells were separated using epithelial cell adhesion molecule (EpCAM) microbeads, with EpCAM-positive and EpCAM-negative fractions from the co-culture designated as TE co (TE-9 co, TE-10 co, and TE-15 co) and CAF-like cells (CAF9, CAF10, and CAF15), respectively. Similarly, monocultured ESCC cells and MSCs were applied to a cell separator and classified as TE mono (TE-9 mono, TE-10 mono, and TE-15 mono) or MSC mono, respectively. A cDNA microarray was performed between TE-9 mono and TE-9 co or between MSC mono and CAF9 in previous studies. B: Venn diagram of 188 genes with TE-9 co [signal intensity (global normalization) > 1000]/TE-9 mono ratio >2 and 59 genes with CAF9 [signal intensity (global normalization) > 1000]/MSC mono ratio >2 in the cDNA microarray analysis. A total of 32 overlapping genes were identified between the two groups. Among them, BST2, which facilitates direct cell-cell interactions, was selected for further investigation in this study. C: The mRNA expression levels of BST2 in MSC mono and CAF-like cells were analyzed and compared by quantitative real-time PCR. D: Western blot analysis was performed to compare BST2 protein expression levels in MSCs and CAF-like cells using SDS sample buffer containing 2-mercaptoethanol (2-ME), with β-actin serving as the loading control. E: Western blot analysis was performed to compare BST2 protein expression levels in MSCs and CAF-like cells using SDS sample buffer lacking 2-ME, with β-actin serving as the loading control. F: The mRNA expression levels of BST2 in TE monoculture and TE co-culture were analyzed and compared by quantitative real-time PCR. G: Western blot analysis was performed to compare BST2 protein expression levels in TE monoculture and TE co-culture using SDS sample buffer containing 2-ME, with β-actin serving as the loading control. H: Western blot analysis was performed to compare BST2 protein expression levels in MSCs and CAF-like cells using SDS sample buffer lacking 2-ME, with β-actin serving as the loading control. C – H: Experiments were performed in triplicate and repeated at least three times. C and F: Two-sided t -tests were performed for the statistical analysis of parametric data. Data are expressed as means ± SEMs ( C and F ). ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Human bone marrow–derived MSCs were obtained from ATCC (PCS-500-012; Manassas, VA) and cultured in low-glucose Dulbecco's modified Eagle's medium (DMEM; Fujifilm Wako Pure Chemical Corp.) supplemented with 10% FBS and a 1% penicillin‒streptomycin‒amphotericin B suspension under identical incubation conditions.

Techniques: Co-Culture Assay, Microarray, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: The American Journal of Pathology

Article Title: Bone Marrow Stromal Cell Antigen 2 Promotes Esophageal Squamous Cell Carcinoma Progression by Mediating the Cell-Cell Interaction between Cancer Cells and Cancer-Associated Fibroblasts

doi: 10.1016/j.ajpath.2026.01.012

Figure Lengend Snippet: Direct co-culture with esophageal squamous cell carcinoma (ESCC) cells promotes the survival, growth, migration, and adhesion of cancer-associated fibroblast (CAF)–like cells via BST2. A: The mRNA expression levels of BST2 in mesenchymal stem cells (MSCs) and CAFs transfected with either negative control siRNA (siNC) or siRNA targeting BST2 (siBST2) during direct co-culture were analyzed by quantitative real-time PCR and compared. B: The protein expression levels of BST2 in MSCs and CAFs transfected with either siNC or siBST2 during direct co-culture were analyzed and compared by Western blot analysis. β-Actin was used as a loading control. C: The protein expression levels of extracellular signal-regulated kinase (ERK) and phosphorylated ERK (pERK) in MSCs and CAFs transfected with either siNC or siBST2 during direct co-culture were analyzed and compared by Western blot analysis. β-Actin was used as a loading control. D and E: The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was used to evaluate the survival ( D ) or growth ( E ) of MSCs and CAFs transfected with either siNC or siBST2 during direct co-culture. F: Transwell migration assays were used to evaluate the migration of MSCs and CAFs transfected with either siNC or siBST2 during direct co-culture. MSCs or CAFs were seeded in the upper chamber, and after 48 hours, the migrated cells were counted in five representative fields of view under a microscope. Representative images for each condition are shown in . G: An adhesion assay was used to evaluate the number of attached MSCs and CAFs transfected with either siNC or siBST2 during direct co-culture. A – G: Experiments were performed in triplicate and repeated at least three times. A and C – G: Two-sided t -tests were performed for the statistical analysis of parametric data. Data are expressed as means ± SEMs ( A and D – G ). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. NS, not significant; tERK, total ERK.

Article Snippet: Human bone marrow–derived MSCs were obtained from ATCC (PCS-500-012; Manassas, VA) and cultured in low-glucose Dulbecco's modified Eagle's medium (DMEM; Fujifilm Wako Pure Chemical Corp.) supplemented with 10% FBS and a 1% penicillin‒streptomycin‒amphotericin B suspension under identical incubation conditions.

Techniques: Co-Culture Assay, Migration, Expressing, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Western Blot, Control, MTS Assay, Microscopy, Cell Adhesion Assay

Journal: The American Journal of Pathology

Article Title: Bone Marrow Stromal Cell Antigen 2 Promotes Esophageal Squamous Cell Carcinoma Progression by Mediating the Cell-Cell Interaction between Cancer Cells and Cancer-Associated Fibroblasts

doi: 10.1016/j.ajpath.2026.01.012

Figure Lengend Snippet: Overexpression of BST2 promotes cellular survival, growth, migration, and adhesion in mesenchymal stem cells (MSCs) through activation of the extracellular signal-regulated kinase (ERK) signaling pathway. A: The mRNA expression levels of BST2 in MSCs transfected with either negative control vector (oeNC) or a BST2 overexpression vector (oeBST2) were analyzed by quantitative real-time PCR and compared. B: The protein expression levels of BST2 in MSCs transfected with either oeNC or oeBST2 were analyzed by Western blot analysis and compared. The protein expression levels of ERK and phosphorylated ERK (pERK) in MSCs transfected with either oeNC or oeBST2 were analyzed by Western blot analysis and compared. β-Actin was used as a loading control. C and D: The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was used to evaluate the survival ( C ) or growth ( D ) of MSCs transfected with either oeNC or oeBST2. E: Transwell migration assays were used to evaluate the migration of MSCs transfected with either oeNC or oeBST2. MSCs were seeded in the upper chamber, and after 48 hours, the migrated cells were counted in five representative fields of view under a microscope. Representative images for each condition are shown below the graphs. F: An adhesion assay was used to evaluate the number of attached MSCs transfected with either oeNC or oeBST2. A – F: Experiments were performed in triplicate and repeated at least three times. A and C – F: Two-sided t -tests were performed for the statistical analysis of parametric data. Data are expressed as means ± SEMs ( A and C – F ). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. Scale bars = 100 μm ( E ). tERK, total ERK.

Article Snippet: Human bone marrow–derived MSCs were obtained from ATCC (PCS-500-012; Manassas, VA) and cultured in low-glucose Dulbecco's modified Eagle's medium (DMEM; Fujifilm Wako Pure Chemical Corp.) supplemented with 10% FBS and a 1% penicillin‒streptomycin‒amphotericin B suspension under identical incubation conditions.

Techniques: Over Expression, Migration, Activation Assay, Expressing, Transfection, Negative Control, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Control, MTS Assay, Microscopy, Cell Adhesion Assay

Journal: The American Journal of Pathology

Article Title: Bone Marrow Stromal Cell Antigen 2 Promotes Esophageal Squamous Cell Carcinoma Progression by Mediating the Cell-Cell Interaction between Cancer Cells and Cancer-Associated Fibroblasts

doi: 10.1016/j.ajpath.2026.01.012

Figure Lengend Snippet: Suppression of BST2 mediates cell adhesion between esophageal squamous cell carcinoma (ESCC) cells and mesenchymal stem cells (MSCs) in direct co-culture. A , C , E , G , I , and K: Immunofluorescence staining of TE-9 ( A and G ), TE-10 ( C and I ), and TE-15 ( E and K ) cells was conducted by direct co-culture of ESCC cells and MSCs to detect epithelial cell adhesion molecule (EpCAM; red), fibroblast activation protein (FAP; green), and BST2 (blue). The nuclei of the cells were counterstained with DAPI (white). B , D , F , H , J , and L: The numbers of TE-9 ( B and H ), TE-10 ( D and J ), and TE-15 ( F and L ) cells attached to MSCs or cancer-associated fibroblasts (CAFs) were counted in accordance with EpCAM expression. A – F: Experiments were performed in triplicate and repeated at least three times. B , D , F, H, J , and L: Two-sided t -tests were performed for the statistical analysis of parametric data. Data are expressed as means ± SEMs ( B , D , F, H, J , and L ). ∗∗∗ P < 0.001. Scale bars = 50 μm ( A , C , E , G , I , and K ). siBST2, siRNA targeting BST2; siNC, negative control siRNA.

Article Snippet: Human bone marrow–derived MSCs were obtained from ATCC (PCS-500-012; Manassas, VA) and cultured in low-glucose Dulbecco's modified Eagle's medium (DMEM; Fujifilm Wako Pure Chemical Corp.) supplemented with 10% FBS and a 1% penicillin‒streptomycin‒amphotericin B suspension under identical incubation conditions.

Techniques: Co-Culture Assay, Immunofluorescence, Staining, Activation Assay, Expressing, Negative Control

Journal: The American Journal of Pathology

Article Title: Bone Marrow Stromal Cell Antigen 2 Promotes Esophageal Squamous Cell Carcinoma Progression by Mediating the Cell-Cell Interaction between Cancer Cells and Cancer-Associated Fibroblasts

doi: 10.1016/j.ajpath.2026.01.012

Figure Lengend Snippet: Overexpression of BST2 mediates cell adhesion between esophageal squamous cell carcinoma (ESCC) cells and mesenchymal stem cells (MSCs) in direct co-culture. A , C , E , G , I , and K: Immunofluorescence staining of TE-9 ( A and G ), TE-10 ( C and I ), and TE-15 ( E and K ) cells was conducted to detect epithelial cell adhesion molecule (EpCAM; red), fibroblast activation protein (FAP; green), and BST2 (blue). The nuclei of the cells were counterstained with DAPI (white). B , D , F, H, J , and L: The numbers of TE-9 ( B and H ), TE-10 ( D and J ), and TE-15 ( F and L ) cells attached to MSCs were counted in accordance with EpCAM expression. A – F: Experiments were performed in triplicate and repeated at least three times. B , D , F, H, J , and L: Two-sided t -tests were performed for the statistical analysis of parametric data. Data are expressed as means ± SEMs ( B , D , F, H, J , and L ). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. Scale bars = 50 μm ( A , C , E , G , I , and K ). oeBST2, BST2 overexpression vector; oeNC, negative control vector.

Article Snippet: Human bone marrow–derived MSCs were obtained from ATCC (PCS-500-012; Manassas, VA) and cultured in low-glucose Dulbecco's modified Eagle's medium (DMEM; Fujifilm Wako Pure Chemical Corp.) supplemented with 10% FBS and a 1% penicillin‒streptomycin‒amphotericin B suspension under identical incubation conditions.

Techniques: Over Expression, Co-Culture Assay, Immunofluorescence, Staining, Activation Assay, Expressing, Plasmid Preparation, Negative Control

Journal: The American Journal of Pathology

Article Title: Bone Marrow Stromal Cell Antigen 2 Promotes Esophageal Squamous Cell Carcinoma Progression by Mediating the Cell-Cell Interaction between Cancer Cells and Cancer-Associated Fibroblasts

doi: 10.1016/j.ajpath.2026.01.012

Figure Lengend Snippet: Schematic illustration depicting the role of BST2 in esophageal squamous cell carcinoma (ESCC) cell–cancer-associated fibroblast (CAF) interactions. BST2 expression in ESCC cells and CAFs is up-regulated through direct contact between ESCC cells and mesenchymal stem cells (MSCs), facilitating cell survival, growth, migration, and adhesion using the extracellular signal-regulated kinase (ERK) signaling pathway.

Article Snippet: Human bone marrow–derived MSCs were obtained from ATCC (PCS-500-012; Manassas, VA) and cultured in low-glucose Dulbecco's modified Eagle's medium (DMEM; Fujifilm Wako Pure Chemical Corp.) supplemented with 10% FBS and a 1% penicillin‒streptomycin‒amphotericin B suspension under identical incubation conditions.

Techniques: Expressing, Migration

Journal: BioMedical Engineering OnLine

Article Title: Synergistic effects of dexamethasone and vitamins D and K on mesenchymal stem cell differentiation

doi: 10.1186/s12938-026-01556-z

Figure Lengend Snippet: Morphological changes in BM-MSCs during osteogenic differentiation over 28 days in media with different supplement compositions. Cells were differentiated in DM1–DM4 media. Cell morphology was assessed by bright-field microscopy on days 0, 7, 14, and 28. Representative images show progressive changes in cell shape and density indicative of osteogenic differentiation. Scale bar = 200 μm

Article Snippet: Normal human bone marrow-derived mesenchymal stem cells (BM-MSCs) (ATCC PCS-500-012 TM ) (Sigma Aldrich, St. Louis, MO, USA) were used for experiments.

Techniques: Microscopy

Journal: BioMedical Engineering OnLine

Article Title: Synergistic effects of dexamethasone and vitamins D and K on mesenchymal stem cell differentiation

doi: 10.1186/s12938-026-01556-z

Figure Lengend Snippet: Von Kossa staining of BM-MSCs cultured for 28 days in different osteogenic media reveals varying degrees of mineralization. Cells were differentiated in DM1–DM4 media. Black precipitates indicate calcium phosphate deposits, demonstrating the extent of ECM mineralization. Scale bar = 500 μm

Article Snippet: Normal human bone marrow-derived mesenchymal stem cells (BM-MSCs) (ATCC PCS-500-012 TM ) (Sigma Aldrich, St. Louis, MO, USA) were used for experiments.

Techniques: Staining, Cell Culture

Journal: BioMedical Engineering OnLine

Article Title: Synergistic effects of dexamethasone and vitamins D and K on mesenchymal stem cell differentiation

doi: 10.1186/s12938-026-01556-z

Figure Lengend Snippet: Alizarin Red staining of BM-MSCs cultured in different osteogenic media (DM1–DM4) at various time points reveals differences in calcium deposition. Cells were cultured for 7, 14, and 28 days in DM1–DM4 media. Red staining indicates the presence of calcium-rich deposits. The most extensive mineralization was observed in DM4, particularly on day 14 and 28, whereas DM2 (lacking DEX after day 7) showed the weakest staining. Scale bar = 200 μm

Article Snippet: Normal human bone marrow-derived mesenchymal stem cells (BM-MSCs) (ATCC PCS-500-012 TM ) (Sigma Aldrich, St. Louis, MO, USA) were used for experiments.

Techniques: Staining, Cell Culture

Journal: BioMedical Engineering OnLine

Article Title: Synergistic effects of dexamethasone and vitamins D and K on mesenchymal stem cell differentiation

doi: 10.1186/s12938-026-01556-z

Figure Lengend Snippet: A Quantification of calcium deposition in BM-MSCs cultured in osteogenic media DM1–DM4 using Alizarin Red extraction. Absorbance was measured at 562 nm and expressed in arbitrary units. B ALP activity in BM-MSCs cultured in different osteogenic media (DM1–DM4) at various time points. ALP activity was normalized to total protein content and expressed as enzyme activity per milligram of protein per milliliter of lysate. All data represent means ± standard deviations from three independent biological replicates. Statistical significance was assessed using a two-tailed unpaired t -test and is indicated by asterisks (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Normal human bone marrow-derived mesenchymal stem cells (BM-MSCs) (ATCC PCS-500-012 TM ) (Sigma Aldrich, St. Louis, MO, USA) were used for experiments.

Techniques: Cell Culture, Extraction, Activity Assay, Two Tailed Test

Journal: BioMedical Engineering OnLine

Article Title: Synergistic effects of dexamethasone and vitamins D and K on mesenchymal stem cell differentiation

doi: 10.1186/s12938-026-01556-z

Figure Lengend Snippet: Visualization of actin cytoskeleton and mitochondrial network in BM-MSCs undergoing osteogenic differentiation in various media (DM1–DM4). Cells were stained with Phalloidin–TRITC (red) to visualize F-actin filaments and MitoTracker™ Red CMXRos (red) to detect mitochondria. Cell nuclei were counterstained with NucBlue™ (blue). Images were processed using ImageJ software (W. S. Rasband, U.S. National Institutes of Health, Bethesda, MD, USA). Scale bar = 100 μm

Article Snippet: Normal human bone marrow-derived mesenchymal stem cells (BM-MSCs) (ATCC PCS-500-012 TM ) (Sigma Aldrich, St. Louis, MO, USA) were used for experiments.

Techniques: Staining, Software

Journal: BioMedical Engineering OnLine

Article Title: Synergistic effects of dexamethasone and vitamins D and K on mesenchymal stem cell differentiation

doi: 10.1186/s12938-026-01556-z

Figure Lengend Snippet: Relative expression levels of osteogenic marker genes during BM-MSCs osteodifferentiation in different supplementation conditions. Transcript levels of A RUNX2 , B ALP , C OCN , and D BMP2 were measured by qRT-PCR at days 7, 14, and 28 in BM-MSCs cultured in osteogenic media DM1–DM4. Gene expression was normalized to the housekeeping gene GAPDH and expressed relative to undifferentiated control levels. All data are presented as means ± standard deviation from three biological replicates. Asterisks indicate statistically significant differences (* P < 0.05, ** P < 0.01; unpaired two-tailed t -test).

Article Snippet: Normal human bone marrow-derived mesenchymal stem cells (BM-MSCs) (ATCC PCS-500-012 TM ) (Sigma Aldrich, St. Louis, MO, USA) were used for experiments.

Techniques: Expressing, Marker, Quantitative RT-PCR, Cell Culture, Gene Expression, Control, Standard Deviation, Two Tailed Test

Journal: BioMedical Engineering OnLine

Article Title: Synergistic effects of dexamethasone and vitamins D and K on mesenchymal stem cell differentiation

doi: 10.1186/s12938-026-01556-z

Figure Lengend Snippet: Overview of experimental workflow. Timeline of BM-MSCs culture, induction of osteogenic differentiation in four different media (DM1–DM4), and time points of sample collection. The diagram illustrates key methodological steps, including morphological assessment, von Kossa and Alizarin Red staining, ALP activity and total protein quantification, immunofluorescence staining (F-actin and mitochondria), and gene expression analysis by qRT-PCR. Sampling was performed at days 0, 7, 14, and 28, as indicated. Created in Biorender.com

Article Snippet: Normal human bone marrow-derived mesenchymal stem cells (BM-MSCs) (ATCC PCS-500-012 TM ) (Sigma Aldrich, St. Louis, MO, USA) were used for experiments.

Techniques: Staining, Activity Assay, Immunofluorescence, Gene Expression, Quantitative RT-PCR, Sampling