Journal: Science translational medicine

Article Title: Detection of early pancreatic ductal adenocarcinoma with thrombospondin-2 and CA19-9 blood markers.

doi: 10.1126/scitranslmed.aah5583

Figure Lengend Snippet: Fig. 1. Phase 1 validation studies and THBS2 expression in PDAC and other human tumors. (A) AUC analysis of blinded ELISA data for the proteins MMP2, MMP10, and THBS2 in plasma samples from 10 patients with PDAC at various stages of disease compared to 10 healthy controls. CI, confidence interval. (B) Box plots of THBS2 mRNA expression measured in various human tumors (sample sizes in parentheses) assessed by RNA-seq. Tumors are sorted in order of decreasing median expression of THBS2 mRNA. Of the pancreatic cancer samples from the TCGA database (n = 179), we analyzed only PDAC (n = 134). All expression values are log2(RSEM values = 1)–transformed.

Article Snippet: Two primary antibodies for THBS2 were used for our study: goat polyclonal THBS2 antibody (dilution 1:25; sc-7655, Santa Cruz Biotechnology) and rabbit polyclonal THBS2 antibody (dilution 1:100; TA590658, OriGene).

Techniques: Biomarker Discovery, Expressing, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, RNA Sequencing, Transformation Assay

Journal: Science translational medicine

Article Title: Detection of early pancreatic ductal adenocarcinoma with thrombospondin-2 and CA19-9 blood markers.

doi: 10.1126/scitranslmed.aah5583

Figure Lengend Snippet: Fig. 2. THBS2 and CA19-9 concentrations in plasma samples from patients with PDAC versus healthy controls. (A and B) Scatter plots of THBS2 concentra- tions in plasma samples from patients at all stages of PDAC versus controls for the phase 2a (A) and phase 2b (B) validation studies. (C and D) ROC curves for THBS2, CA19-9, and THBS2 + CA19-9 concentrations in plasma samples from patients with all stages of PDAC versus healthy controls for phase 2a (PDAC, n = 81; controls, n = 80) (C) and phase 2b (PDAC, n = 197; controls, n = 140) (D) studies. (E and F) Scatter plots showing THBS2 and CA19-9 concentrations in plasma samples from patients with all stages of PDAC versus healthy controls for phase 2a (E) and phase 2b (F) studies.

Article Snippet: Two primary antibodies for THBS2 were used for our study: goat polyclonal THBS2 antibody (dilution 1:25; sc-7655, Santa Cruz Biotechnology) and rabbit polyclonal THBS2 antibody (dilution 1:100; TA590658, OriGene).

Techniques: Clinical Proteomics, Biomarker Discovery

Journal: Science translational medicine

Article Title: Detection of early pancreatic ductal adenocarcinoma with thrombospondin-2 and CA19-9 blood markers.

doi: 10.1126/scitranslmed.aah5583

Figure Lengend Snippet: Fig. 3. THBS2 and CA19-9 concentrations in plasma samples from all-stage PDAC cases versus benign pan- creatic disease cases. (A to D) ROC curves for THBS2, CA19-9, and THBS2 + CA19-9 concentrations in plasma samples from patients with PDAC in the phase 2b study (n = 197) versus pancreatitis (n = 55) (A), PDAC versus intraductal papillary mucinous neoplasm (IPMN) (n = 115) (B), PDAC versus PNET (n = 30) (C), and PNET (n = 30) versus healthy controls (n = 140) (D) are shown.

Article Snippet: Two primary antibodies for THBS2 were used for our study: goat polyclonal THBS2 antibody (dilution 1:25; sc-7655, Santa Cruz Biotechnology) and rabbit polyclonal THBS2 antibody (dilution 1:100; TA590658, OriGene).

Techniques: Clinical Proteomics

Journal: Science translational medicine

Article Title: Detection of early pancreatic ductal adenocarcinoma with thrombospondin-2 and CA19-9 blood markers.

doi: 10.1126/scitranslmed.aah5583

Figure Lengend Snippet: Fig. 4. Expression of THBS2 in human PanIN tissue and PDAC tumor tissue. (A and B) Representative THBS2 immunohistochemistry analysis of incidental PanIN1/2 tissue derived from the head and neck of a pancreas from a patient with pancreatic periampullary cancer using two different antibodies (Ab). The arrows indicate that PanIN2 tissue stained positive for THBS2; dotted arrows indicate weak or negative staining of PanIN1 tissue. (C to K) THBS2 expression, designated by arrows, was also confirmed in stage II (C to E) and stage III (F to K) PDAC pancreatic cancer tissue arrays. Competitive assays were performed for antibody #2 by preincubating the antibody with a 10-fold excess of antigen peptide (E, H, and K) to confirm target specificity. Brown color indicates THBS2 staining, and blue color indicates hematoxylin nuclear staining. THBS2 was detected in the epithelial cells of noninvasive lesions (PanINs and intraductal papillary mucinous neoplasms) and poorly differentiated PDAC tissue as well as in fibroblasts in invasive PDAC tissue (see table S8 and fig. S6).

Article Snippet: Two primary antibodies for THBS2 were used for our study: goat polyclonal THBS2 antibody (dilution 1:25; sc-7655, Santa Cruz Biotechnology) and rabbit polyclonal THBS2 antibody (dilution 1:100; TA590658, OriGene).

Techniques: Expressing, Immunohistochemistry, Derivative Assay, Staining, Negative Staining

Journal: Cell reports

Article Title: THBS2-producing matrix CAFs promote colorectal cancer progression and link to poor prognosis via the CD47-MAPK axis.

doi: 10.1016/j.celrep.2025.115555

Figure Lengend Snippet: Figure 2. THBS2+ mCAFs exhibit strong interactions and close spatial proximity to tumor cells, correlating with poor prognosis in CRC (A) Dimensionality reduction plot showing the distribution of six CAF subtypes across all, primary CRC, and liver metastases samples. (B) Functional enrichment analysis of CAF subtypes. (C) Pseudotime analysis and CytoTRACE results of CAF subtypes. (D) Network plots illustrating cell-cell communication between CAFs and PTC-like (HS) and PTC-like (LS) tumor cells. (E) Heatmap visualizing cell-cell interactions between CAFs and other cell types across CRC scRNA-seq datasets. (F) Multiplex immunohistochemistry (mIHC) staining of primary CRC tissue, showing spatial proximity of THBS2+ mCAFs and tumor cells. (G) Spatial transcriptomic map showing the distribution of distinct cell types within the tissue. (H) Quantification of spatial distances between different cell types. (I) Heatmap depicting the spatial distribution of various cell populations within the TME, derived from robust cell type decomposition analysis of Visium HD spatial transcriptomics data.

Article Snippet: Thbs2-floxed mice, also on a C57BL/6J background, were generated by Cyagen using the following method: The THBS2 gene, which includes multiple exons, was targeted by two gRNAs designed to bind upstream of the promoter and flanking exon3.

Techniques: Functional Assay, Multiplex Assay, Immunohistochemistry, Staining, Derivative Assay

Journal: Cell reports

Article Title: THBS2-producing matrix CAFs promote colorectal cancer progression and link to poor prognosis via the CD47-MAPK axis.

doi: 10.1016/j.celrep.2025.115555

Figure Lengend Snippet: Figure 3. THBS2 promotes CRC progression through CAF-tumor interactions in vitro and in vivo (A) Heatmap showing THBS signaling probabilities between THBS2+ mCAFs (sender) and tumor cells (receiver) across scRNA-seq datasets. (B) Dot plot showing THBS-family gene expression (THBS1, THBS2, THBS3, THBS4, and COMP) in THBS2+ mCAFs across CRC datasets. (C) Schematic of recombinant protein generation and lentiviral transduction of CAFs with THBS1, THBS2, and COMP. (D) Proliferation assay measuring optical density (OD) values of tumor cells (SW480) treated with recombinant proteins or conditioned medium (CM) from CAF cell lines. (E–H) Representative images of EdU incorporation assays (E), Transwell migration assays (F), Transwell invasion assays (G), and adhesion assays (H) for tumor cells (SW480) treated with recombinant proteins or CAF CM (scale bars, 100 mm). (I) Quantification of EdU incorporation, migration, invasion, and adhesion assay results, comparing the effects of recombinant proteins and CAF CM on tumor cells (SW480). (J) Representative images of subcutaneous xenografts from SW480 and HCT116 CRC cells mixed with CAF-NC, CAF-COMP, CAF-THBS1, and CAF-THBS2. (K) Tumor growth curves and final tumor weights of subcutaneous xenografts in SW480 models. Tumors from the CAF-THBS2 group exhibit significantly larger volumes and weights compared to other groups. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Thbs2-floxed mice, also on a C57BL/6J background, were generated by Cyagen using the following method: The THBS2 gene, which includes multiple exons, was targeted by two gRNAs designed to bind upstream of the promoter and flanking exon3.

Techniques: In Vitro, In Vivo, Gene Expression, Recombinant, Transduction, Proliferation Assay, Migration, Cell Adhesion Assay

Journal: Cell reports

Article Title: THBS2-producing matrix CAFs promote colorectal cancer progression and link to poor prognosis via the CD47-MAPK axis.

doi: 10.1016/j.celrep.2025.115555

Figure Lengend Snippet: Figure 4. Stroma-derived THBS2 is associated with mesenchymal activation and poor prognosis in CRC (A) Dot plot showing THBS2 expression across different cell types in multiple scRNA-seq datasets. (B and C) Representative images of mIHC and IHC staining for THBS2 in normal and tumor tissue sections (scale bars, 100 and 50 mm). (D) Boxplots showing THBS2 expression in epithelial and stromal compartments of normal and tumor tissues (GSE35602). (E and F) Boxplots displaying the THBS2 expression levels of normal and tumor tissues in the TCGA-CRC dataset (E) and a tissue microarray (TMA) (F). (G) ELISA quantification of THBS2 levels in the supernatant of CAF-THBS2 and normal CAF cultures, showing significantly higher secretion of THBS2 in CAF- THBS2. (H) Representative images of IHC, RNA-ISH for THBS2, Masson trichrome staining, and aSMA IHC on CRC samples, comparing low and high THBS2 expression groups (scale bars, 100 mm). (I) Correlation of THBS2 expression with stroma-related genes in TCGA-CRC, revealing a positive association between THBS2 and mesenchymal markers. (J and K) Violin plots demonstrating that high THBS2 expression is linked to increased aSMA+ cell abundance (J) and stromal content (K) in tumor samples, as assessed by TMA analysis.

Article Snippet: Thbs2-floxed mice, also on a C57BL/6J background, were generated by Cyagen using the following method: The THBS2 gene, which includes multiple exons, was targeted by two gRNAs designed to bind upstream of the promoter and flanking exon3.

Techniques: Derivative Assay, Activation Assay, Expressing, Immunohistochemistry, Microarray, Enzyme-linked Immunosorbent Assay, Staining

Journal: Cell reports

Article Title: THBS2-producing matrix CAFs promote colorectal cancer progression and link to poor prognosis via the CD47-MAPK axis.

doi: 10.1016/j.celrep.2025.115555

Figure Lengend Snippet: Figure 5. CREB3L1 promotes the transformation of normal fibroblasts into THBS2+ mCAFs (A) Heatmap showing transcription factor activity across CAF subtypes identified by SCENIC analysis. (B) Regulon specificity score (RSS) plot showing CREB3L1 as the top-ranked transcription factor in THBS2+ mCAFs. (C) Violin plot comparing the RSS of CREB3L1 across CAF subtypes, with THBS2+ mCAFs exhibiting a significantly higher RSS than other subtypes. (D) Dot plot showing CREB3L1 expression in various cell types across CRC datasets, with THBS2+ mCAFs showing the highest expression levels. (E) Importance score plot from SCENIC analysis across CRC scRNA-seq datasets, showing CREB3L1 as the top regulator in THBS2+ mCAFs. (F) Dimensionality reduction plot of normal fibroblasts (NFs) and THBS2+ mCAFs, showing distinct clustering and separation between these two populations. (G and H) Monocle pseudotime trajectory plots showing the progression from NFs to THBS2+ mCAFs. Cells are ordered along a differentiation trajectory, with NFs at the beginning and THBS2+ mCAFs at the end. (I and J) GeneSwitches analysis identifying key genes transitioning during the NF-to-THBS2+ mCAF transformation. The unit in (J) is log2(gene count). (K and L) Correlation analysis showing strong positive correlations between CREB3L1 regulon activity and pseudotime as well as THBS2 expression. (M) ChIP-qPCR analysis showing significant enrichment of CREB3L1 at the promoter region of THBS2. (N) Luciferase reporter assay showing that CREB3L1 enhances the transcriptional activity of the wild-type THBS2 promoter compared to a mutant form. Green and red represent the pmirGLO-NC and pmirGLO-THBS2 groups, respectively. (legend continued on next page)

Article Snippet: Thbs2-floxed mice, also on a C57BL/6J background, were generated by Cyagen using the following method: The THBS2 gene, which includes multiple exons, was targeted by two gRNAs designed to bind upstream of the promoter and flanking exon3.

Techniques: Transformation Assay, Activity Assay, Expressing, ChIP-qPCR, Luciferase, Reporter Assay, Mutagenesis

Journal: Cell reports

Article Title: THBS2-producing matrix CAFs promote colorectal cancer progression and link to poor prognosis via the CD47-MAPK axis.

doi: 10.1016/j.celrep.2025.115555

Figure Lengend Snippet: Figure 6. THBS2 promotes tumor progression by interacting with CD47 (A) mIHC images showing spatial proximity between THBS2+ mCAFs and CD47+ tumor cells alongside SDC1+/SDC4+ tumor cells (scale bars, 50 mm). (B) Schematic of the TimeCCI pipeline, showing the calculation of the Spearman correlation coefficient (SCC) for co-varying ligand-receptor pairs between THBS2+ mCAFs and tumor cells. THBS2-CD47 is the top ligand-receptor pair with the highest SCC among THBS interactions. (C) Spatial transcriptomics data showing widespread THBS2-CD47 interactions. (D) Molecular dynamics simulation of the THBS2-CD47 complex with structural visualization of key interacting residues. (E) Quantitative analysis of the THBS2-CD47 complex stability over a 100-ns simulation. (F) Co-immunoprecipitation (coIP) assays confirming the physical interaction between THBS2 and CD47 in SW480 and HCT116 cell lines. (G and H) CCK8 (SW480 and HCT116) and EdU (SW480) assays showing that si-CD47 transfection in CRC cell lines treated with rhTHBS2 significantly reduced tumor cell proliferation. Transwell and adhesion assays demonstrated that si-CD47 treatment also inhibited cell migration, invasion, and adhesion (scale bars, 100 mm). (I) Quantification of the reduction in tumor cell proliferation, migration, invasion, and adhesion after si-CD47 treatment in HCT116 cells following rhTHBS2 treatment.

Article Snippet: Thbs2-floxed mice, also on a C57BL/6J background, were generated by Cyagen using the following method: The THBS2 gene, which includes multiple exons, was targeted by two gRNAs designed to bind upstream of the promoter and flanking exon3.

Techniques: Immunoprecipitation, Transfection, Migration

Journal: Cell reports

Article Title: THBS2-producing matrix CAFs promote colorectal cancer progression and link to poor prognosis via the CD47-MAPK axis.

doi: 10.1016/j.celrep.2025.115555

Figure Lengend Snippet: Figure 7. THBS2 induces EMT and tumor progression through activation of the MAPK/ERK5 pathway (A) Volcano plot displaying differentially expressed genes (DEGs) between CAF-THBS2 and CAF-NC. (B) Gene ontology (GO) enrichment analysis of DEGs highlights significant enrichment in ECM remodeling pathways. (C) Ridge plot showing the enriched pathway results from proteomics analysis. (D) Heatmap of phosphoproteomic data involved in the MAPK pathway. (E) Correlation analysis showing a positive association between THBS2 expression and MAPK7 expression (top) and phosphorylation (bottom) in CRC samples from the CPTAC-COAD dataset. (F) Bar plot depicting correlations between THBS2 expression and genes associated with cell proliferation and EMT in the CPTAC-COAD and TCGA-CRC datasets.

Article Snippet: Thbs2-floxed mice, also on a C57BL/6J background, were generated by Cyagen using the following method: The THBS2 gene, which includes multiple exons, was targeted by two gRNAs designed to bind upstream of the promoter and flanking exon3.

Techniques: Activation Assay, Expressing, Phospho-proteomics

Journal: Clinical Proteomics

Article Title: Proteomic profiling identifies markers for inflammation-related tumor–fibroblast interaction

doi: 10.1186/s12014-017-9168-7

Figure Lengend Snippet: Proteom analysis of CRC. a Protein abundances in tumor versus normal tissue samples. Volcano plot representing differences in LFQ values (log2FC, fold-changes in logarithmic scale to the base of two) of proteins including corresponding p values (logarithmic scale). Red labeled proteins are higher abundant in the tumor tissue whereas green labeled proteins are higher abundant in the normal tissue samples. b Stromal contribution. Proteomics derived data of significantly regulated proteins were plotted against stromal contribution of their respective mRNA . Red indicates upregulated proteins of stromal origin. Blue indicates the 2 stromal proteins THBS2 and SPARC plus the parenchymal SerpinB5 that were chosen for IHC staining

Article Snippet: If not otherwise stated, primary antibodies were diluted in PBST containing 1% goat serum (Dako) as follows: THBS2 (TA590658, Origene) 1:150, αSMA (1A4, Dako) 1:100, SerpinB5 (sc-271,694, Santa Cruz) 1:100, SPARC (D10F10, Cell Signaling) 1:2400 in SignalStain Antibody Diluent (Cell Signaling).

Techniques: Labeling, Derivative Assay, Immunohistochemistry

Journal: Clinical Proteomics

Article Title: Proteomic profiling identifies markers for inflammation-related tumor–fibroblast interaction

doi: 10.1186/s12014-017-9168-7

Figure Lengend Snippet: Proteins of mainly stromal origin that are increased in abundance

Article Snippet: If not otherwise stated, primary antibodies were diluted in PBST containing 1% goat serum (Dako) as follows: THBS2 (TA590658, Origene) 1:150, αSMA (1A4, Dako) 1:100, SerpinB5 (sc-271,694, Santa Cruz) 1:100, SPARC (D10F10, Cell Signaling) 1:2400 in SignalStain Antibody Diluent (Cell Signaling).

Techniques:

Journal: Clinical Proteomics

Article Title: Proteomic profiling identifies markers for inflammation-related tumor–fibroblast interaction

doi: 10.1186/s12014-017-9168-7

Figure Lengend Snippet: Tissue localization of selected proteins. Serial sections of normal mucosa and tumor tissue were stained using antibodies directed against SerpinB5 ( a , b ), THBS2 ( e , f ), SPARC ( i , j ), and αSMA ( m , n ). a , e , i , m representative area of normal intestinal mucosa. b , f , j , n representative area of tumor tissue. Scale bars correspond to 500 µm. A selected area was magnified ×20 and is shown in the inserts. Staining intensity for all images was quantified using Definiens software. The diagrams depict the pooled quantification obtained from 26 tumor samples and corresponding normal tissue with regard to overall tumor and normal tissue ( c ) or tumor and normal epithelium for SerpinB5 ( d ). For THBS2, quantification results are depicted for total tumor and normal areas ( g ) or tumor and normal stromal compartments ( h ). For SPARC, quantification is presented for tumor and normal stromal compartments ( k ) and specifically for low/high staining intensities ( l ). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: not significant, according to Mann–Whitney U test

Article Snippet: If not otherwise stated, primary antibodies were diluted in PBST containing 1% goat serum (Dako) as follows: THBS2 (TA590658, Origene) 1:150, αSMA (1A4, Dako) 1:100, SerpinB5 (sc-271,694, Santa Cruz) 1:100, SPARC (D10F10, Cell Signaling) 1:2400 in SignalStain Antibody Diluent (Cell Signaling).

Techniques: Staining, Software, MANN-WHITNEY

Journal: Cell reports

Article Title: THBS2-producing matrix CAFs promote colorectal cancer progression and link to poor prognosis via the CD47-MAPK axis.

doi: 10.1016/j.celrep.2025.115555

Figure Lengend Snippet: Figure 2. THBS2+ mCAFs exhibit strong interactions and close spatial proximity to tumor cells, correlating with poor prognosis in CRC (A) Dimensionality reduction plot showing the distribution of six CAF subtypes across all, primary CRC, and liver metastases samples. (B) Functional enrichment analysis of CAF subtypes. (C) Pseudotime analysis and CytoTRACE results of CAF subtypes. (D) Network plots illustrating cell-cell communication between CAFs and PTC-like (HS) and PTC-like (LS) tumor cells. (E) Heatmap visualizing cell-cell interactions between CAFs and other cell types across CRC scRNA-seq datasets. (F) Multiplex immunohistochemistry (mIHC) staining of primary CRC tissue, showing spatial proximity of THBS2+ mCAFs and tumor cells. (G) Spatial transcriptomic map showing the distribution of distinct cell types within the tissue. (H) Quantification of spatial distances between different cell types. (I) Heatmap depicting the spatial distribution of various cell populations within the TME, derived from robust cell type decomposition analysis of Visium HD spatial transcriptomics data.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Anti -Ki67 mouse mAb servicebio Cat # GB121141; RRID: AB_3083641 Anti-mouse CD47/IAP antibody MCE Cat # MIAP301; RRID: AB_3668803 Mouse IgG antibody Beyotime Cat # A7028; RRID: AB_2909433 Beta actin monoclonal antibody Proteintech Cat #66009-1-Ig; RRID: AB_2687938 HRP-conjugated Goat Anti-Mouse IgG(H + L) Proteintech Cat #SA00001-1; RRID: AB_2722565 HRP-conjugated Goat Anti-Rabbit IgG(H + L) Proteintech Cat #SA00001-2; RRID: AB_2722564 HRP conjugated Goat Anti-Rabbit IgG (H + L) Servicebio Cat # GB23303; RRID: AB_2811189 HRP conjugated Goat Anti-Mouse IgG (H + L) Servicebio Cat # GB23301; RRID: AB_2904020 Cy3 conjugated Goat Anti-Rabbit IgG (H + L) Servicebio Cat # GB21303; RRID: AB_2861435 Cy3 conjugated Goat Anti-mouseIgG(H + L) Servicebio Cat # GB21301; RRID: AB_2923552 Chemicals, peptides, and recombinant proteins Human Fibronectin MCE Cat #HY-P70593 Tamoxifen Merck Cat #10540-29-1 Corn oil Aladdin Cat #8001-30-7 Lipofectamine 3000 Thermo Scientific Cat #L3000015 MIAP301 MCE Cat #HY-P990131 Critical commercial assays THBS2 ELISA kit CUSABIO Cat #CSB-EL023488HU Dual Luciferase Reporter Gene Assay Kit Beyotime Cat #RG027 CCK-8 Cell Proliferation and Cytotoxicity Assay Kit Solarbio Cat #CA1210 PierceTM Crosslink Magnetic IP/Co-IP Kit Thermo Fisher Scientific Cat # 88805 Cell-Light EdU Apollo567 In Vitro Kit RiboBio Cat #C10310-1 Crystal Violet Ammonium Oxalate Solution, 1% Solarbio Cat #G1062 DAB chromogenic reagent kit Servicebio Cat #G1212 Hematoxylin-eosin (H&E) HD constant dye kit Servicebio Cat #G1076 Masson dye solution set Servicebio Cat #G1006 AlphaTSA fluorescent staining kit Alpha X Bio Cat#AXT37100041 Deposited data Transcriptomic data Present Study https://doi.org/10.7303/syn62787929 Proteomic data Present Study https://doi.org/10.7303/syn62787929 Phosphorylation data Present Study https://doi.org/10.7303/syn62787929 Experimental models: Cell lines SW480 Pricella Cat#CL-0223 HCT116 Pricella Cat#CL-0096 MC38 CTCC Cat#CTCC-001-0975 HEK-293T Pricella Cat# CL-0005 CCD-18Co CTCC Cat# CTCC-001-0080 (Continued on next page) 18 Cell Reports 44, 115555, April 22, 2025

Techniques: Functional Assay, Multiplex Assay, Immunohistochemistry, Staining, Derivative Assay

Journal: Cell reports

Article Title: THBS2-producing matrix CAFs promote colorectal cancer progression and link to poor prognosis via the CD47-MAPK axis.

doi: 10.1016/j.celrep.2025.115555

Figure Lengend Snippet: Figure 3. THBS2 promotes CRC progression through CAF-tumor interactions in vitro and in vivo (A) Heatmap showing THBS signaling probabilities between THBS2+ mCAFs (sender) and tumor cells (receiver) across scRNA-seq datasets. (B) Dot plot showing THBS-family gene expression (THBS1, THBS2, THBS3, THBS4, and COMP) in THBS2+ mCAFs across CRC datasets. (C) Schematic of recombinant protein generation and lentiviral transduction of CAFs with THBS1, THBS2, and COMP. (D) Proliferation assay measuring optical density (OD) values of tumor cells (SW480) treated with recombinant proteins or conditioned medium (CM) from CAF cell lines. (E–H) Representative images of EdU incorporation assays (E), Transwell migration assays (F), Transwell invasion assays (G), and adhesion assays (H) for tumor cells (SW480) treated with recombinant proteins or CAF CM (scale bars, 100 mm). (I) Quantification of EdU incorporation, migration, invasion, and adhesion assay results, comparing the effects of recombinant proteins and CAF CM on tumor cells (SW480). (J) Representative images of subcutaneous xenografts from SW480 and HCT116 CRC cells mixed with CAF-NC, CAF-COMP, CAF-THBS1, and CAF-THBS2. (K) Tumor growth curves and final tumor weights of subcutaneous xenografts in SW480 models. Tumors from the CAF-THBS2 group exhibit significantly larger volumes and weights compared to other groups. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Anti -Ki67 mouse mAb servicebio Cat # GB121141; RRID: AB_3083641 Anti-mouse CD47/IAP antibody MCE Cat # MIAP301; RRID: AB_3668803 Mouse IgG antibody Beyotime Cat # A7028; RRID: AB_2909433 Beta actin monoclonal antibody Proteintech Cat #66009-1-Ig; RRID: AB_2687938 HRP-conjugated Goat Anti-Mouse IgG(H + L) Proteintech Cat #SA00001-1; RRID: AB_2722565 HRP-conjugated Goat Anti-Rabbit IgG(H + L) Proteintech Cat #SA00001-2; RRID: AB_2722564 HRP conjugated Goat Anti-Rabbit IgG (H + L) Servicebio Cat # GB23303; RRID: AB_2811189 HRP conjugated Goat Anti-Mouse IgG (H + L) Servicebio Cat # GB23301; RRID: AB_2904020 Cy3 conjugated Goat Anti-Rabbit IgG (H + L) Servicebio Cat # GB21303; RRID: AB_2861435 Cy3 conjugated Goat Anti-mouseIgG(H + L) Servicebio Cat # GB21301; RRID: AB_2923552 Chemicals, peptides, and recombinant proteins Human Fibronectin MCE Cat #HY-P70593 Tamoxifen Merck Cat #10540-29-1 Corn oil Aladdin Cat #8001-30-7 Lipofectamine 3000 Thermo Scientific Cat #L3000015 MIAP301 MCE Cat #HY-P990131 Critical commercial assays THBS2 ELISA kit CUSABIO Cat #CSB-EL023488HU Dual Luciferase Reporter Gene Assay Kit Beyotime Cat #RG027 CCK-8 Cell Proliferation and Cytotoxicity Assay Kit Solarbio Cat #CA1210 PierceTM Crosslink Magnetic IP/Co-IP Kit Thermo Fisher Scientific Cat # 88805 Cell-Light EdU Apollo567 In Vitro Kit RiboBio Cat #C10310-1 Crystal Violet Ammonium Oxalate Solution, 1% Solarbio Cat #G1062 DAB chromogenic reagent kit Servicebio Cat #G1212 Hematoxylin-eosin (H&E) HD constant dye kit Servicebio Cat #G1076 Masson dye solution set Servicebio Cat #G1006 AlphaTSA fluorescent staining kit Alpha X Bio Cat#AXT37100041 Deposited data Transcriptomic data Present Study https://doi.org/10.7303/syn62787929 Proteomic data Present Study https://doi.org/10.7303/syn62787929 Phosphorylation data Present Study https://doi.org/10.7303/syn62787929 Experimental models: Cell lines SW480 Pricella Cat#CL-0223 HCT116 Pricella Cat#CL-0096 MC38 CTCC Cat#CTCC-001-0975 HEK-293T Pricella Cat# CL-0005 CCD-18Co CTCC Cat# CTCC-001-0080 (Continued on next page) 18 Cell Reports 44, 115555, April 22, 2025

Techniques: In Vitro, In Vivo, Gene Expression, Recombinant, Transduction, Proliferation Assay, Migration, Cell Adhesion Assay

Journal: Cell reports

Article Title: THBS2-producing matrix CAFs promote colorectal cancer progression and link to poor prognosis via the CD47-MAPK axis.

doi: 10.1016/j.celrep.2025.115555

Figure Lengend Snippet: Figure 4. Stroma-derived THBS2 is associated with mesenchymal activation and poor prognosis in CRC (A) Dot plot showing THBS2 expression across different cell types in multiple scRNA-seq datasets. (B and C) Representative images of mIHC and IHC staining for THBS2 in normal and tumor tissue sections (scale bars, 100 and 50 mm). (D) Boxplots showing THBS2 expression in epithelial and stromal compartments of normal and tumor tissues (GSE35602). (E and F) Boxplots displaying the THBS2 expression levels of normal and tumor tissues in the TCGA-CRC dataset (E) and a tissue microarray (TMA) (F). (G) ELISA quantification of THBS2 levels in the supernatant of CAF-THBS2 and normal CAF cultures, showing significantly higher secretion of THBS2 in CAF- THBS2. (H) Representative images of IHC, RNA-ISH for THBS2, Masson trichrome staining, and aSMA IHC on CRC samples, comparing low and high THBS2 expression groups (scale bars, 100 mm). (I) Correlation of THBS2 expression with stroma-related genes in TCGA-CRC, revealing a positive association between THBS2 and mesenchymal markers. (J and K) Violin plots demonstrating that high THBS2 expression is linked to increased aSMA+ cell abundance (J) and stromal content (K) in tumor samples, as assessed by TMA analysis.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Anti -Ki67 mouse mAb servicebio Cat # GB121141; RRID: AB_3083641 Anti-mouse CD47/IAP antibody MCE Cat # MIAP301; RRID: AB_3668803 Mouse IgG antibody Beyotime Cat # A7028; RRID: AB_2909433 Beta actin monoclonal antibody Proteintech Cat #66009-1-Ig; RRID: AB_2687938 HRP-conjugated Goat Anti-Mouse IgG(H + L) Proteintech Cat #SA00001-1; RRID: AB_2722565 HRP-conjugated Goat Anti-Rabbit IgG(H + L) Proteintech Cat #SA00001-2; RRID: AB_2722564 HRP conjugated Goat Anti-Rabbit IgG (H + L) Servicebio Cat # GB23303; RRID: AB_2811189 HRP conjugated Goat Anti-Mouse IgG (H + L) Servicebio Cat # GB23301; RRID: AB_2904020 Cy3 conjugated Goat Anti-Rabbit IgG (H + L) Servicebio Cat # GB21303; RRID: AB_2861435 Cy3 conjugated Goat Anti-mouseIgG(H + L) Servicebio Cat # GB21301; RRID: AB_2923552 Chemicals, peptides, and recombinant proteins Human Fibronectin MCE Cat #HY-P70593 Tamoxifen Merck Cat #10540-29-1 Corn oil Aladdin Cat #8001-30-7 Lipofectamine 3000 Thermo Scientific Cat #L3000015 MIAP301 MCE Cat #HY-P990131 Critical commercial assays THBS2 ELISA kit CUSABIO Cat #CSB-EL023488HU Dual Luciferase Reporter Gene Assay Kit Beyotime Cat #RG027 CCK-8 Cell Proliferation and Cytotoxicity Assay Kit Solarbio Cat #CA1210 PierceTM Crosslink Magnetic IP/Co-IP Kit Thermo Fisher Scientific Cat # 88805 Cell-Light EdU Apollo567 In Vitro Kit RiboBio Cat #C10310-1 Crystal Violet Ammonium Oxalate Solution, 1% Solarbio Cat #G1062 DAB chromogenic reagent kit Servicebio Cat #G1212 Hematoxylin-eosin (H&E) HD constant dye kit Servicebio Cat #G1076 Masson dye solution set Servicebio Cat #G1006 AlphaTSA fluorescent staining kit Alpha X Bio Cat#AXT37100041 Deposited data Transcriptomic data Present Study https://doi.org/10.7303/syn62787929 Proteomic data Present Study https://doi.org/10.7303/syn62787929 Phosphorylation data Present Study https://doi.org/10.7303/syn62787929 Experimental models: Cell lines SW480 Pricella Cat#CL-0223 HCT116 Pricella Cat#CL-0096 MC38 CTCC Cat#CTCC-001-0975 HEK-293T Pricella Cat# CL-0005 CCD-18Co CTCC Cat# CTCC-001-0080 (Continued on next page) 18 Cell Reports 44, 115555, April 22, 2025

Techniques: Derivative Assay, Activation Assay, Expressing, Immunohistochemistry, Microarray, Enzyme-linked Immunosorbent Assay, Staining

Journal: Cell reports

Article Title: THBS2-producing matrix CAFs promote colorectal cancer progression and link to poor prognosis via the CD47-MAPK axis.

doi: 10.1016/j.celrep.2025.115555

Figure Lengend Snippet: Figure 5. CREB3L1 promotes the transformation of normal fibroblasts into THBS2+ mCAFs (A) Heatmap showing transcription factor activity across CAF subtypes identified by SCENIC analysis. (B) Regulon specificity score (RSS) plot showing CREB3L1 as the top-ranked transcription factor in THBS2+ mCAFs. (C) Violin plot comparing the RSS of CREB3L1 across CAF subtypes, with THBS2+ mCAFs exhibiting a significantly higher RSS than other subtypes. (D) Dot plot showing CREB3L1 expression in various cell types across CRC datasets, with THBS2+ mCAFs showing the highest expression levels. (E) Importance score plot from SCENIC analysis across CRC scRNA-seq datasets, showing CREB3L1 as the top regulator in THBS2+ mCAFs. (F) Dimensionality reduction plot of normal fibroblasts (NFs) and THBS2+ mCAFs, showing distinct clustering and separation between these two populations. (G and H) Monocle pseudotime trajectory plots showing the progression from NFs to THBS2+ mCAFs. Cells are ordered along a differentiation trajectory, with NFs at the beginning and THBS2+ mCAFs at the end. (I and J) GeneSwitches analysis identifying key genes transitioning during the NF-to-THBS2+ mCAF transformation. The unit in (J) is log2(gene count). (K and L) Correlation analysis showing strong positive correlations between CREB3L1 regulon activity and pseudotime as well as THBS2 expression. (M) ChIP-qPCR analysis showing significant enrichment of CREB3L1 at the promoter region of THBS2. (N) Luciferase reporter assay showing that CREB3L1 enhances the transcriptional activity of the wild-type THBS2 promoter compared to a mutant form. Green and red represent the pmirGLO-NC and pmirGLO-THBS2 groups, respectively. (legend continued on next page)

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Anti -Ki67 mouse mAb servicebio Cat # GB121141; RRID: AB_3083641 Anti-mouse CD47/IAP antibody MCE Cat # MIAP301; RRID: AB_3668803 Mouse IgG antibody Beyotime Cat # A7028; RRID: AB_2909433 Beta actin monoclonal antibody Proteintech Cat #66009-1-Ig; RRID: AB_2687938 HRP-conjugated Goat Anti-Mouse IgG(H + L) Proteintech Cat #SA00001-1; RRID: AB_2722565 HRP-conjugated Goat Anti-Rabbit IgG(H + L) Proteintech Cat #SA00001-2; RRID: AB_2722564 HRP conjugated Goat Anti-Rabbit IgG (H + L) Servicebio Cat # GB23303; RRID: AB_2811189 HRP conjugated Goat Anti-Mouse IgG (H + L) Servicebio Cat # GB23301; RRID: AB_2904020 Cy3 conjugated Goat Anti-Rabbit IgG (H + L) Servicebio Cat # GB21303; RRID: AB_2861435 Cy3 conjugated Goat Anti-mouseIgG(H + L) Servicebio Cat # GB21301; RRID: AB_2923552 Chemicals, peptides, and recombinant proteins Human Fibronectin MCE Cat #HY-P70593 Tamoxifen Merck Cat #10540-29-1 Corn oil Aladdin Cat #8001-30-7 Lipofectamine 3000 Thermo Scientific Cat #L3000015 MIAP301 MCE Cat #HY-P990131 Critical commercial assays THBS2 ELISA kit CUSABIO Cat #CSB-EL023488HU Dual Luciferase Reporter Gene Assay Kit Beyotime Cat #RG027 CCK-8 Cell Proliferation and Cytotoxicity Assay Kit Solarbio Cat #CA1210 PierceTM Crosslink Magnetic IP/Co-IP Kit Thermo Fisher Scientific Cat # 88805 Cell-Light EdU Apollo567 In Vitro Kit RiboBio Cat #C10310-1 Crystal Violet Ammonium Oxalate Solution, 1% Solarbio Cat #G1062 DAB chromogenic reagent kit Servicebio Cat #G1212 Hematoxylin-eosin (H&E) HD constant dye kit Servicebio Cat #G1076 Masson dye solution set Servicebio Cat #G1006 AlphaTSA fluorescent staining kit Alpha X Bio Cat#AXT37100041 Deposited data Transcriptomic data Present Study https://doi.org/10.7303/syn62787929 Proteomic data Present Study https://doi.org/10.7303/syn62787929 Phosphorylation data Present Study https://doi.org/10.7303/syn62787929 Experimental models: Cell lines SW480 Pricella Cat#CL-0223 HCT116 Pricella Cat#CL-0096 MC38 CTCC Cat#CTCC-001-0975 HEK-293T Pricella Cat# CL-0005 CCD-18Co CTCC Cat# CTCC-001-0080 (Continued on next page) 18 Cell Reports 44, 115555, April 22, 2025

Techniques: Transformation Assay, Activity Assay, Expressing, ChIP-qPCR, Luciferase, Reporter Assay, Mutagenesis

Journal: Cell reports

Article Title: THBS2-producing matrix CAFs promote colorectal cancer progression and link to poor prognosis via the CD47-MAPK axis.

doi: 10.1016/j.celrep.2025.115555

Figure Lengend Snippet: Figure 6. THBS2 promotes tumor progression by interacting with CD47 (A) mIHC images showing spatial proximity between THBS2+ mCAFs and CD47+ tumor cells alongside SDC1+/SDC4+ tumor cells (scale bars, 50 mm). (B) Schematic of the TimeCCI pipeline, showing the calculation of the Spearman correlation coefficient (SCC) for co-varying ligand-receptor pairs between THBS2+ mCAFs and tumor cells. THBS2-CD47 is the top ligand-receptor pair with the highest SCC among THBS interactions. (C) Spatial transcriptomics data showing widespread THBS2-CD47 interactions. (D) Molecular dynamics simulation of the THBS2-CD47 complex with structural visualization of key interacting residues. (E) Quantitative analysis of the THBS2-CD47 complex stability over a 100-ns simulation. (F) Co-immunoprecipitation (coIP) assays confirming the physical interaction between THBS2 and CD47 in SW480 and HCT116 cell lines. (G and H) CCK8 (SW480 and HCT116) and EdU (SW480) assays showing that si-CD47 transfection in CRC cell lines treated with rhTHBS2 significantly reduced tumor cell proliferation. Transwell and adhesion assays demonstrated that si-CD47 treatment also inhibited cell migration, invasion, and adhesion (scale bars, 100 mm). (I) Quantification of the reduction in tumor cell proliferation, migration, invasion, and adhesion after si-CD47 treatment in HCT116 cells following rhTHBS2 treatment.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Anti -Ki67 mouse mAb servicebio Cat # GB121141; RRID: AB_3083641 Anti-mouse CD47/IAP antibody MCE Cat # MIAP301; RRID: AB_3668803 Mouse IgG antibody Beyotime Cat # A7028; RRID: AB_2909433 Beta actin monoclonal antibody Proteintech Cat #66009-1-Ig; RRID: AB_2687938 HRP-conjugated Goat Anti-Mouse IgG(H + L) Proteintech Cat #SA00001-1; RRID: AB_2722565 HRP-conjugated Goat Anti-Rabbit IgG(H + L) Proteintech Cat #SA00001-2; RRID: AB_2722564 HRP conjugated Goat Anti-Rabbit IgG (H + L) Servicebio Cat # GB23303; RRID: AB_2811189 HRP conjugated Goat Anti-Mouse IgG (H + L) Servicebio Cat # GB23301; RRID: AB_2904020 Cy3 conjugated Goat Anti-Rabbit IgG (H + L) Servicebio Cat # GB21303; RRID: AB_2861435 Cy3 conjugated Goat Anti-mouseIgG(H + L) Servicebio Cat # GB21301; RRID: AB_2923552 Chemicals, peptides, and recombinant proteins Human Fibronectin MCE Cat #HY-P70593 Tamoxifen Merck Cat #10540-29-1 Corn oil Aladdin Cat #8001-30-7 Lipofectamine 3000 Thermo Scientific Cat #L3000015 MIAP301 MCE Cat #HY-P990131 Critical commercial assays THBS2 ELISA kit CUSABIO Cat #CSB-EL023488HU Dual Luciferase Reporter Gene Assay Kit Beyotime Cat #RG027 CCK-8 Cell Proliferation and Cytotoxicity Assay Kit Solarbio Cat #CA1210 PierceTM Crosslink Magnetic IP/Co-IP Kit Thermo Fisher Scientific Cat # 88805 Cell-Light EdU Apollo567 In Vitro Kit RiboBio Cat #C10310-1 Crystal Violet Ammonium Oxalate Solution, 1% Solarbio Cat #G1062 DAB chromogenic reagent kit Servicebio Cat #G1212 Hematoxylin-eosin (H&E) HD constant dye kit Servicebio Cat #G1076 Masson dye solution set Servicebio Cat #G1006 AlphaTSA fluorescent staining kit Alpha X Bio Cat#AXT37100041 Deposited data Transcriptomic data Present Study https://doi.org/10.7303/syn62787929 Proteomic data Present Study https://doi.org/10.7303/syn62787929 Phosphorylation data Present Study https://doi.org/10.7303/syn62787929 Experimental models: Cell lines SW480 Pricella Cat#CL-0223 HCT116 Pricella Cat#CL-0096 MC38 CTCC Cat#CTCC-001-0975 HEK-293T Pricella Cat# CL-0005 CCD-18Co CTCC Cat# CTCC-001-0080 (Continued on next page) 18 Cell Reports 44, 115555, April 22, 2025

Techniques: Immunoprecipitation, Transfection, Migration

Journal: Cell reports

Article Title: THBS2-producing matrix CAFs promote colorectal cancer progression and link to poor prognosis via the CD47-MAPK axis.

doi: 10.1016/j.celrep.2025.115555

Figure Lengend Snippet: Figure 7. THBS2 induces EMT and tumor progression through activation of the MAPK/ERK5 pathway (A) Volcano plot displaying differentially expressed genes (DEGs) between CAF-THBS2 and CAF-NC. (B) Gene ontology (GO) enrichment analysis of DEGs highlights significant enrichment in ECM remodeling pathways. (C) Ridge plot showing the enriched pathway results from proteomics analysis. (D) Heatmap of phosphoproteomic data involved in the MAPK pathway. (E) Correlation analysis showing a positive association between THBS2 expression and MAPK7 expression (top) and phosphorylation (bottom) in CRC samples from the CPTAC-COAD dataset. (F) Bar plot depicting correlations between THBS2 expression and genes associated with cell proliferation and EMT in the CPTAC-COAD and TCGA-CRC datasets.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Anti -Ki67 mouse mAb servicebio Cat # GB121141; RRID: AB_3083641 Anti-mouse CD47/IAP antibody MCE Cat # MIAP301; RRID: AB_3668803 Mouse IgG antibody Beyotime Cat # A7028; RRID: AB_2909433 Beta actin monoclonal antibody Proteintech Cat #66009-1-Ig; RRID: AB_2687938 HRP-conjugated Goat Anti-Mouse IgG(H + L) Proteintech Cat #SA00001-1; RRID: AB_2722565 HRP-conjugated Goat Anti-Rabbit IgG(H + L) Proteintech Cat #SA00001-2; RRID: AB_2722564 HRP conjugated Goat Anti-Rabbit IgG (H + L) Servicebio Cat # GB23303; RRID: AB_2811189 HRP conjugated Goat Anti-Mouse IgG (H + L) Servicebio Cat # GB23301; RRID: AB_2904020 Cy3 conjugated Goat Anti-Rabbit IgG (H + L) Servicebio Cat # GB21303; RRID: AB_2861435 Cy3 conjugated Goat Anti-mouseIgG(H + L) Servicebio Cat # GB21301; RRID: AB_2923552 Chemicals, peptides, and recombinant proteins Human Fibronectin MCE Cat #HY-P70593 Tamoxifen Merck Cat #10540-29-1 Corn oil Aladdin Cat #8001-30-7 Lipofectamine 3000 Thermo Scientific Cat #L3000015 MIAP301 MCE Cat #HY-P990131 Critical commercial assays THBS2 ELISA kit CUSABIO Cat #CSB-EL023488HU Dual Luciferase Reporter Gene Assay Kit Beyotime Cat #RG027 CCK-8 Cell Proliferation and Cytotoxicity Assay Kit Solarbio Cat #CA1210 PierceTM Crosslink Magnetic IP/Co-IP Kit Thermo Fisher Scientific Cat # 88805 Cell-Light EdU Apollo567 In Vitro Kit RiboBio Cat #C10310-1 Crystal Violet Ammonium Oxalate Solution, 1% Solarbio Cat #G1062 DAB chromogenic reagent kit Servicebio Cat #G1212 Hematoxylin-eosin (H&E) HD constant dye kit Servicebio Cat #G1076 Masson dye solution set Servicebio Cat #G1006 AlphaTSA fluorescent staining kit Alpha X Bio Cat#AXT37100041 Deposited data Transcriptomic data Present Study https://doi.org/10.7303/syn62787929 Proteomic data Present Study https://doi.org/10.7303/syn62787929 Phosphorylation data Present Study https://doi.org/10.7303/syn62787929 Experimental models: Cell lines SW480 Pricella Cat#CL-0223 HCT116 Pricella Cat#CL-0096 MC38 CTCC Cat#CTCC-001-0975 HEK-293T Pricella Cat# CL-0005 CCD-18Co CTCC Cat# CTCC-001-0080 (Continued on next page) 18 Cell Reports 44, 115555, April 22, 2025

Techniques: Activation Assay, Expressing, Phospho-proteomics

Journal: American Journal of Cancer Research

Article Title: THBS2 promotes gastric cancer progression and stemness via the Notch signaling pathway

doi: 10.62347/UXWK4038

Figure Lengend Snippet: THBS2 expression is upregulated in GC and predicts poor survival in GC patients. A. A comparison between three datasets with 1213, 1738, and 1305 DEGs, revealed a total of 137 common DEGs between GC and normal tissues. B. Network of the top 10 genes, with a higher ranking indicated by a more intense red. C. Genetic mutation analysis of 10 hub genes in GC. D. Expression of THBS2 mRNA in different tumor types including gastric cancer (GC) and normal adjacent tissue, according to TCGA database analysis. E. Relationship between THBS2 and T stage of tumor. F. Expression of THBS2 mRNA in GC according to GEO databases analysis. G, H. Expression of THBS2 mRNA and protein in gastric cancer cell line and GES-1. I. The relative expression level of THBS2 mRNA in 30 pairs of GC and normal adjacent tissues. J, K. THBS2 protein expression and IHC analysis in GC and adjacent normal tissues. L. The prognostic survival of THBS2 was analyzed based on K-M plotter (OS, n=875; FPS, n=640; PPS, n=498). Data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ns = no significance.

Article Snippet: After PBS washes, sections were blocked with 3% BSA and incubated overnight at 4°C with primary antibody THBS2 (#BA3616-2, BOSTER, Wuhan, China).

Techniques: Expressing, Comparison, Mutagenesis

Journal: American Journal of Cancer Research

Article Title: THBS2 promotes gastric cancer progression and stemness via the Notch signaling pathway

doi: 10.62347/UXWK4038

Figure Lengend Snippet: THBS2 regulates the progression and stemness of GC cells in vitro. A, B. WB and qRT-PCR validate the efficiency of constructed THBS2 knockdown and overexpression gastric cancer cell lines. C, D. The migration and invasion ability of sh-THBS2 and OE-THSB2 groups were detected by Transwell assay. E, F. Wound healing assay was used to detect the migration distance of sh-THBS2 and OE-THSB2 groups. G, H. The proliferation ability of sh-THBS2 and OE-THSB2 groups was detected by CCK8 assay. I, J. The proliferative potential of sh-THBS2 and OE-THSB2 groups was verified by colony formation experiment. K, L. Apoptosis assay showed the proportions of apoptotic HGC-27 and AGS cells in sh-THBS2 and OE-THSB2 groups. M, N. The cell cycle of sh-THBS2 and OE-THSB2 groups was detected by flow cytometry. O. The Sphere-forming abilities was detected in HGC-27 and AGS cells after THBS2 intervention. P. The expression of CD44 in HGC-27 and AGS cells after THBS2 intervention was detected by flow cytometry. Q. Expression of proliferation, apoptosis, invasion and EMT-related proteins in HGC-27 and AGS cells after THBS2 intervention. Data represent the mean ± SD. **P < 0.01, ***P < 0.001.

Article Snippet: After PBS washes, sections were blocked with 3% BSA and incubated overnight at 4°C with primary antibody THBS2 (#BA3616-2, BOSTER, Wuhan, China).

Techniques: In Vitro, Quantitative RT-PCR, Construct, Knockdown, Over Expression, Migration, Transwell Assay, Wound Healing Assay, CCK-8 Assay, Apoptosis Assay, Flow Cytometry, Expressing

Journal: American Journal of Cancer Research

Article Title: THBS2 promotes gastric cancer progression and stemness via the Notch signaling pathway

doi: 10.62347/UXWK4038

Figure Lengend Snippet: MiR-29b-3p directly targeted the 3’-UTR of THBS2 mRNA and down-regulated THBS2 in GC. A. Bioinformatics predicts THBS2-targeting miRNAs. B. THBS2 mRNA expression of 4 candidate miRNAs transfected into gastric cancer cells. C. The heatmap was drawn to exhibit the decrease extent of THBS2 mRNA. D. The protein expression of THBS2 GC cells after transfection with different kinds of miRNA mimics. E, F. Relative expression of miR-29b-3p in GC and normal tissues in TCGA. G. Expression of THBS2 protein after miR-29b-3p transfected into GC cell lines and GES-1. H. The relative expression level of miR-29b-3p in 30 pairs of GC and normal adjacent tissues. I, J. Correlation between miR-29b-3p andTHBS2 mRNA expression in GC tissues in TCGA and 30 pairs clinic tissues. K, L. The mRNA Expression of THBS2 in HGC-27 and AGS after transfection with miR-29b-3p inhibitor and mimics. Data represent the mean ± SD. **P < 0.01. M. The potential miR-29b-3p binding sites in 3’-UTR of THBS2 mRNA predicted by TargetScan and the sequences of the WT-THBS2 and MUT-THBS2 3’-UTR reporter plasmids. N. Dual luciferase reporter assay validated of miR-29b-3p targeting THBS2. O, P. Colony formation experiment for AGS and HGC-27 cells with miR-29b-3p mimic or inhibitor. Q, R. CCK8 assay for AGS and HGC-27 cells with miR-29b-3p mimic or inhibitor. S, T. Wound healing assay for AGS and HGC-27 cells with miR-29b-3p mimic or inhibitor. U, V. Transwell assay for AGS and HGC-27 cells with miR-29b-3p mimic or inhibitor. Data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: After PBS washes, sections were blocked with 3% BSA and incubated overnight at 4°C with primary antibody THBS2 (#BA3616-2, BOSTER, Wuhan, China).

Techniques: Expressing, Transfection, Binding Assay, Luciferase, Reporter Assay, CCK-8 Assay, Wound Healing Assay, Transwell Assay

Journal: American Journal of Cancer Research

Article Title: THBS2 promotes gastric cancer progression and stemness via the Notch signaling pathway

doi: 10.62347/UXWK4038

Figure Lengend Snippet: THBS2 reversed Mir-29b-3p-mediated inhibition of GC. A, B. The protein expression of THBS2 in HGC-27 and AGS that co-transfected miR-29b-3p mimic or inhibitor and THBS2 knockdown or overexpression plasmid. C, D. The proliferation of HGC-27 and AGS cells that co-transfected miR-29b-3p mimic or inhibitor and THBS2 knockdown or overexpression plasmid was determined by CCK-8 assay. E, F. The proliferation of HGC-27 and AGS cells that co-transfected miR-29b-3p mimic or inhibitor and THBS2 knockdown or overexpression plasmid was determined by colony formation experiment. G, H. The migration and invasion ability of HGC-27 and AGS cells that co-transfected miR-29b-3p mimic or inhibitor and THBS2 knockdown or overexpression plasmid was determined by transwell assay. I, J. The migration distance of HGC-27 and AGS cells that co-transfected miR-29b-3p mimic or inhibitor and THBS2 knockdown or overexpression plasmid was determined by wound healing assay. Data represent the mean ± SD. **P < 0.01, ***P < 0.001.

Article Snippet: After PBS washes, sections were blocked with 3% BSA and incubated overnight at 4°C with primary antibody THBS2 (#BA3616-2, BOSTER, Wuhan, China).

Techniques: Inhibition, Expressing, Transfection, Knockdown, Over Expression, Plasmid Preparation, CCK-8 Assay, Migration, Transwell Assay, Wound Healing Assay

Journal: American Journal of Cancer Research

Article Title: THBS2 promotes gastric cancer progression and stemness via the Notch signaling pathway

doi: 10.62347/UXWK4038

Figure Lengend Snippet: THBS2 affects the progression and stemness of GC through Notch signaling pathway. A. GSEA showed the relationship between the expression of THBS2 and Notch signaling pathway. B. Correlations among THBS2 and Notch1, Notch2, Notch3, and Notch4 analyzed on the basis of TCGA database. C. The protein level of Notch3, Notch target genes or EMT markers (such as NICD, Hey1, Hes1, E-cadherin, N-cadherin, and Vimentin) was assessed in sh-THBS2 and OE-THBS2 groups vs. control by Western Blotting. D. The Sphere-forming abilities was detected in Vector, THBS2 or THBS2 + DAPT groups. Data represent the mean ± SD. ***P < 0.001. E. The Sphere-forming abilities was detected in knockdown THBS2 HGC-27 cells with or without overexpressing Notch3. Data represent the mean ± SD. **P < 0.01. F. The expression levels of Notch3, Hey1 and Hes1 proteins in AGS cell with Vector, OE-THBS2 or OE-THBS2 + DAPT groups were detected by WB. G. The Notch3, Nanog, Sox2, and OCT4 protein expression levels in knockdown THBS2 HGC-27 cells with or without overexpressing Notch3 were analyzed by WB.

Article Snippet: After PBS washes, sections were blocked with 3% BSA and incubated overnight at 4°C with primary antibody THBS2 (#BA3616-2, BOSTER, Wuhan, China).

Techniques: Expressing, Control, Western Blot, Plasmid Preparation, Knockdown

Journal: American Journal of Cancer Research

Article Title: THBS2 promotes gastric cancer progression and stemness via the Notch signaling pathway

doi: 10.62347/UXWK4038

Figure Lengend Snippet: Down-regulation of THBS2 inhibits GC tumorigenesis in vivo. A. Images of tumors from nude mice in the sh-Ctrl and sh-THBS2 groups. B, C. Tumor weights and volumes in the two groups. Data represent the mean ± SD. **P < 0.01, ***P < 0.001. D. Images of liver metastatic tumors after injection of HGC-27 cells (sh-Ctrl and sh-THBS2 groups) into the spleen. E. H&E staining about liver metastasis of spleen injected mice, 2× and 20×. F. The mRNA level of THBS2, EMT markers and Notch3 in established xenograft model assessed by qRT-PCR. Data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. G. The protein level of THBS2, EMT markers and Notch3 in established xenograft model assessed by WB. H. Overall schematic presentation of miR-29b-3p/THBS2/NOTCH3 cascade in gastric carcinogenesis.

Article Snippet: After PBS washes, sections were blocked with 3% BSA and incubated overnight at 4°C with primary antibody THBS2 (#BA3616-2, BOSTER, Wuhan, China).

Techniques: In Vivo, Injection, Staining, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: miR-29a-3p/THBS2 Axis Regulates PAH-Induced Cardiac Fibrosis

doi: 10.3390/ijms221910574

Figure Lengend Snippet: miR-29a-3p regulates THBS2 expression in vitro and in vivo. ( A , B ) MCT-induced expression of miR-29a-3p and exosomal THBS2 mRNA, determined by real-time PCR. ( C ) THBS2 expression in MCT-treated HCM-secreted exosomes determined by western blotting. HSP70 was used as the internal control for exosomal proteins. ( D ) Alignment of the miR-29a-3p with the THBS2 3′-untranslated region, showing the putative binding sites. ( E ) Real-time PCR analysis of miR-29a-3p expression in HCMs after transfection with an miR-29a-3p mimic or inhibitor. Data are representative of three independent experiments ( n = 3), and values are expressed as median and interquartile range. Mann–Whitney U-test was used to compare two independent groups (* p < 0.05). ( F , G ) After transfection, THBS2 expression was analyzed by real-time PCR and immunocytochemical staining. Data are representative of three independent experiments ( n = 3), and values are expressed as median and interquartile range. Kruskal–Wallis test was used to compare two independent groups (* p < 0.05). ( H , I ) MCT-induced expression of miR-29a-3p and THBS2 mRNA, determined by real-time PCR. Mann–Whitney U-test was used to compare two independent groups (** p < 0.01). ( J ) In situ hybridization (ISH) of miR-29a-3p expression (pink color) in myocardial tissues of MCT-induced PAH mice. U6, miRNA internal control. ( K , L ) THBS2 expression in myocardial tissues and circulating serum of MCT-induced PAH mice were examined by immunohistochemistry and western blotting, respectively. Transferrin was used as the internal control. Data are representative of three independent experiments ( n = 3), and values are expressed as median and interquartile range. Mann–Whitney U-test was used to compare two independent groups (* p < 0.05 and ** p < 0.01).

Article Snippet: Plasma THBS2 levels were determined using human THBS2 ELISA Kit PicoKineTM (Catalog# EK0642; Boster Biological Technology, Pleasanton CA, USA) according to the manufacturer’s instructions.

Techniques: Expressing, In Vitro, In Vivo, Real-time Polymerase Chain Reaction, Western Blot, Control, Binding Assay, Transfection, MANN-WHITNEY, Staining, In Situ Hybridization, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: miR-29a-3p/THBS2 Axis Regulates PAH-Induced Cardiac Fibrosis

doi: 10.3390/ijms221910574

Figure Lengend Snippet: Cardiomyocyte-derived exosomes regulate cardiac fibroblast to myofibroblast transformation. ( A ) Schematic diagram for studying the effects of human cardiomyocyte (HCM)-derived exosome uptake by human cardiac fibroblasts (HCF) using early endosome antigen 1(EEA1) staining. ( B ) Immunocytochemistry and confocal microscopy identified the time-dependent intake and regulation of HCM-derived exosomes by EEA1 in HCFs. Nuclear DNA, exosomes, and EEA1 were labeled with 4′,6-diamidino-2-phenylindole (DAPI, blue), Dil dye (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine, red), and GFP fluorescence dye (green), respectively. Data are representative of three independent experiments ( n = 3). ( C ) HCFs were treated with or without HCM-derived exosomes, HCM+MCT-derived exosomes, or rTHBS2 (THBS2 recombinant protein, 1 μg/mL) for 48 h to assess cardiomyocyte-derived exosomal THBS2-mediated fibroblast to myofibroblast transformation. Expression of the myofibroblast marker proteins, α-SMA, β-catenin, fibronectin, and vimentin was examined by immunocytochemistry and confocal microscopy with Imaris software for confocal image 3D reconstruction. ( D , E ) Myofibroblast marker protein expression in myocardial tissues of MCT-induced PAH mice was examined by western blotting and immunohistochemistry. Data are representative of three independent experiments ( n = 3), and values are expressed as median and interquartile range. Mann–Whitney U-test was used to compare two independent groups (* p < 0.05).

Article Snippet: Plasma THBS2 levels were determined using human THBS2 ELISA Kit PicoKineTM (Catalog# EK0642; Boster Biological Technology, Pleasanton CA, USA) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Transformation Assay, Staining, Immunocytochemistry, Confocal Microscopy, Labeling, Fluorescence, Recombinant, Expressing, Marker, Software, Western Blot, Immunohistochemistry, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: miR-29a-3p/THBS2 Axis Regulates PAH-Induced Cardiac Fibrosis

doi: 10.3390/ijms221910574

Figure Lengend Snippet: MiR-29a-3p/THBS2 axis regulates the progression of pulmonary artery hypertension (PAH). ( A ) Exosomal miR-29a-3p level in the serum of healthy controls (NR) and patients with PAH was determined by real-time PCR. ( B ) THBS2 expression in the serum was determined by ELISA. Data are expressed as median and interquartile range. Mann–Whitney U-test was used to compare two independent groups (** p < 0.01). ( C ) Correlation coefficient between the miR-29a-3p and THBS2 expression levels. R, Pearson’s correlation coefficient. ( D – F ) The correlation of miR-29a-3p with the mean pulmonary artery pressure (mPAP), tricuspid annular plane systolic excursion (TAPSE), and pulmonary vascular resistance (PVR) in patients with PAH. ( G – I ) The correlation of the serum THBS2 level with mPAP, TAPSE, and PVR in patients with PAH. ( J ) Suggested mechanism underlying PAH-induced cardiac fibrosis mediated by exosomal miR-29a-3p and THBS2. Serum miR-29a-3p and THBS2 (secreted by cardiomyocytes) might increase cardiomyocyte hypertrophy and promote cardiac fibroblast to myofibroblast transformation, enhance collagen production/deposition, and augment cardiac fibrosis.

Article Snippet: Plasma THBS2 levels were determined using human THBS2 ELISA Kit PicoKineTM (Catalog# EK0642; Boster Biological Technology, Pleasanton CA, USA) according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Transformation Assay

Journal: ecancermedicalscience

Article Title: High-risk gastrointestinal stromal tumour (GIST) and synovial sarcoma display similar angiogenic profiles: a nude mice xenograft study

doi: 10.3332/ecancer.2017.726

Figure Lengend Snippet: Supplementary. Genes and assays included in the low-density array for the study of the expression of angiogenic factors by quantitative RT-PCR.

Article Snippet: THBS2-Hs00170248_m1 , 0.24877942 , 0.00247538 , 0.5332035 , 0.46211198 , 7.79E-04 , , 0.99361634.

Techniques: Expressing, Amplification

Journal: ecancermedicalscience

Article Title: High-risk gastrointestinal stromal tumour (GIST) and synovial sarcoma display similar angiogenic profiles: a nude mice xenograft study

doi: 10.3332/ecancer.2017.726

Figure Lengend Snippet: Supplementary. 2- DDCt values corresponding to the Nu335 series.

Article Snippet: THBS2-Hs00170248_m1 , 0.24877942 , 0.00247538 , 0.5332035 , 0.46211198 , 7.79E-04 , , 0.99361634.

Techniques:

Journal: ecancermedicalscience

Article Title: High-risk gastrointestinal stromal tumour (GIST) and synovial sarcoma display similar angiogenic profiles: a nude mice xenograft study

doi: 10.3332/ecancer.2017.726

Figure Lengend Snippet: Supplementary. 2- DDCt values corresponding to the Nu407 series.

Article Snippet: THBS2-Hs00170248_m1 , 0.24877942 , 0.00247538 , 0.5332035 , 0.46211198 , 7.79E-04 , , 0.99361634.

Techniques: