mesenchymal stem cells bm mscs  (ATCC)

Journal: International Journal of Molecular Sciences

Article Title: BGN Secreted by Cancer-Associated Fibroblasts Promotes Esophageal Squamous Cell Carcinoma Progression via Activation of TLR4-Mediated Erk and NF-κB Signaling Pathways

doi: 10.3390/ijms262412024

Figure Lengend Snippet: Biglycan (BGN), highly expressed in cancer-associated fibroblasts (CAFs), promotes the proliferation and migration of esophageal squamous cell carcinoma (ESCC) cells. ( A ) Euler diagram showing the overlap between genes meeting the signal intensity value criteria (CAF9 > 1000, TE-9 mono ≤ 100, and TE-9 co ≤ 100) and the expression ratio criteria (CAF9/MSC mono > 2 and TE-9 co/TE-9 mono < 2) in the cDNA microarray analysis ( GSE244020 and GSE274064 ). Eight overlapping genes were identified, with BGN showing the highest signal intensity value in CAF9. ( B ) Violin plot showing BGN expression across cell types based on single-cell RNA sequencing (scRNAseq) data from the GSE160269 dataset. ( C ) Uniform Manifold Approximation and Projection (UMAP) and BGN expression in fibroblasts based on scRNAseq data. Violin plot visualization shows significantly higher BGN expression in CAFs compared to normal fibroblasts. ( D – F ) The mRNA expression, protein expression, and secreted protein levels of BGN in mesenchymal stem cell (MSC) mono and CAFs were compared using quantitative reverse transcription polymerase chain reaction (qRT-PCR) ( D ) and Western blotting of cell lysates and supernatants ( E , F ), respectively. ( G ) The effects of recombinant human BGN (rhBGN) (100 ng/mL) on the proliferation of ESCC cells were evaluated using the MTS assay. ( H , I ) The effect of rhBGN (100 ng/mL) on ESCC cell migration ( H ) and the effect of BGN neutralizing antibody (220 ng/mL) on CAF-mediated ESCC cell migration ( I ) were assessed using the Transwell migration assay. In ( H ), ESCC cells were seeded in the upper chamber; in ( I ), ESCC cells were seeded in the upper chamber, while direct co-culture of ESCC cells and MSCs was performed in the lower chamber. The rhBGN ( H ) and BGN neutralizing antibody (220 ng/mL) or control immunoglobulin G (IgG; 220 ng/mL) ( I ) were added to the lower chamber. Migrated cells were counted in five representative microscopic fields after 48 h, and representative images are shown below the graphs. The data are presented as the mean ± standard error of the mean (SEM) from three independent experiments ( D , G – I ). * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars: 100 μm ( H , I ). FRC, fibroblastic reticular cells.

Article Snippet: Human bone marrow-derived MSCs (PCS-500-012, ATCC, Manassas, VA, USA) were cultured in low-glucose Dulbecco’s Modified Eagle Medium (DMEM; FUJIFILM Wako Pure Chemical Corporation) containing 10% FBS and 1% antibiotics.

Techniques: Migration, Expressing, Microarray, RNA Sequencing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Recombinant, MTS Assay, Transwell Migration Assay, Co-Culture Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: BGN Secreted by Cancer-Associated Fibroblasts Promotes Esophageal Squamous Cell Carcinoma Progression via Activation of TLR4-Mediated Erk and NF-κB Signaling Pathways

doi: 10.3390/ijms262412024

Figure Lengend Snippet: Biglycan (BGN) promotes proliferation, migration, and activation of macrophages and fibroblasts. ( A ) Cell–cell communication network analysis of BGN -expressing cancer-associated fibroblasts (CAFs) in esophageal squamous cell carcinoma (ESCC) tissues. Fibroblasts in single-cell RNA sequencing (scRNAseq) datasets from ESCC tissues were stratified into CAF_ BGN _High (scaled expression > 3) and CAF_ BGN _Low (≤3) groups based on BGN expression levels. Cell–cell communication inferred by CellChat revealed that CAF_ BGN _High cells exhibited strong outgoing signaling toward epithelial and myeloid cells, as well as prominent autocrine signaling within the CAF_ BGN _High population. ( B , C ) MTS assay ( B ) and Transwell migration assay ( C ) showing increased proliferation and migration of mesenchymal stem cells (MSCs) treated with recombinant human BGN (rhBGN; 100 ng/mL), respectively. ( D ) Western Blot analysis showing that fibroblast activation protein (FAP) and α-smooth muscle actin (αSMA) (CAF markers) were upregulated in MSCs treated with rhBGN for 24 h, whereas interleukin-6 (IL6) expression remained unchanged. TLR4 expression was detected; however, phosphorylation of NF-κB and Erk was not increased by rhBGN. ( E , F ) MTS assay ( E ) and Transwell migration assay ( F ) showing that rhBGN-induced proliferation and migration of MSCs were not suppressed by a TLR4-neutralizing antibody (1 μg/mL). ( G , H ) MTS assay ( G ) and Transwell migration assay ( H ) showing increased proliferation and migration of macrophages treated with rhBGN (100 ng/mL), respectively. ( I ) Western Blot analysis showing that rhBGN treatment upregulated the expression of CD163 and CD206 (M2 macrophage markers) and increased NF-κB phosphorylation, while TLR4 expression was also detected, but phosphorylated Erk was not changed. ( J , K ) MTS assay ( J ) and Transwell migration assay ( K ) showing that rhBGN-induced proliferation and migration of macrophages were attenuated by treatment with a TLR4-neutralizing antibody (1 μg/mL), respectively. ( L , M ) MTS assay ( L ) and Transwell migration assay ( M ) showing that treatment with the NF-κB pathway inhibitor Bay 11-7082 (1 μM) attenuated rhBGN-induced proliferation and migration to a similar extent, respectively. Data are presented as the mean ± standard error of the mean (SEM) from three independent experiments ( B , C , E – H , J – M ). * p < 0.05, ** p < 0.01, *** p < 0.001. N.S., not significant. Scale bars: 100 μm ( C , F , H , K , M ).

Article Snippet: Human bone marrow-derived MSCs (PCS-500-012, ATCC, Manassas, VA, USA) were cultured in low-glucose Dulbecco’s Modified Eagle Medium (DMEM; FUJIFILM Wako Pure Chemical Corporation) containing 10% FBS and 1% antibiotics.

Techniques: Migration, Activation Assay, Expressing, RNA Sequencing, MTS Assay, Transwell Migration Assay, Recombinant, Western Blot, Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: BGN Secreted by Cancer-Associated Fibroblasts Promotes Esophageal Squamous Cell Carcinoma Progression via Activation of TLR4-Mediated Erk and NF-κB Signaling Pathways

doi: 10.3390/ijms262412024

Figure Lengend Snippet: A schematic illustration of the role of BGN in the tumor–stromal interactions among ESCC cells, CAFs, and macrophages. BGN secreted from MSCs that have undergone CAF transition upon direct contact with ESCC cells promotes ESCC cell proliferation and migration through the TLR4–Erk/NF-κB signaling pathways. In addition, BGN enhances the proliferation and migration of MSCs and induces their differentiation into CAFs. Furthermore, BGN promotes the proliferation and migration of macrophages and drives their polarization toward the M2 phenotype through the TLR4–NF-κB signaling pathways.

Article Snippet: Human bone marrow-derived MSCs (PCS-500-012, ATCC, Manassas, VA, USA) were cultured in low-glucose Dulbecco’s Modified Eagle Medium (DMEM; FUJIFILM Wako Pure Chemical Corporation) containing 10% FBS and 1% antibiotics.

Techniques: Migration, Protein-Protein interactions