breast adenocarcinoma mcf 7 are tumor cell lines were obtained  (ATCC)

Journal: Experimental & Molecular Medicine

Article Title: Hirudin suppresses hematogenous metastasis by targeting desmosome junction transition in circulating tumor cell clusters via HIF-1α–DSG2 signaling

doi: 10.1038/s12276-025-01598-8

Figure Lengend Snippet: a The flow chart of animal experiments. b , c Tumor size ( b ) and tumor weight ( c ) in an orthotopic tumor model mice established with 4T1-GFP-luc cells after hirudin or heparin administration. n = 7. d Representative lung tissue images and metastatic nodule counts after killing. n = 5. e – g H&E staining ( e ), quantification ( f ) and micro-CT ( g ) analysis of lung metastases in a 4T1-GFP-luc tumor model after hirudin or heparin treatment. h A volcano plot of differentially expressed genes in the primary tumor tissues from the model and the 5 mg/kg hirudin groups. i A heat map of metastasis-related genes in primary tumor tissues from model and 5 mg/kg hirudin groups. j KEGG enrichment analysis of differentially expressed genes in primary tumor tissues from model and 5 mg/kg hirudin groups. n = 4. k The migration of MCF-7 and 4T1 cells treated with hirudin for 24 h was determined by the transwell assay. n = 4. l A volcano plot of differentially expressed genes between para-cancer and tumor tissue in model mice. n = 4. m KEGG enrichment analysis showing CTC cluster formation-related pathways based on differentially expressed genes between para-cancer and tumor tissue. n The number of CTC clusters in 1 ml of peripheral blood was determined from orthotopic tumor models established using 4T1-GFP-luc cells. n = 3. o KEGG enrichment analysis conducted on differentially expressed genes in primary tumor tissues from the model group and 5 mg/kg hirudin group identified pathways related to the formation of CTC clusters. n = 4. Data are mean ± s.d., * P < 0.05, ** P < 0.01 versus model group. i.p., intraperitoneal; ns, not significant.

Article Snippet: Breast tumor cell lines MCF-7, MDA-MB-231 and 4T1 were from the American Type Culture Collection and cultured in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C and 5% CO2.

Techniques: Staining, Micro-CT, Migration, Transwell Assay

Journal: Experimental & Molecular Medicine

Article Title: Hirudin suppresses hematogenous metastasis by targeting desmosome junction transition in circulating tumor cell clusters via HIF-1α–DSG2 signaling

doi: 10.1038/s12276-025-01598-8

Figure Lengend Snippet: a MCF-7-GFP cells were used to create in vitro CTC clusters. After treating with 50 μg/ml or 100 μg/ml hirudin for 24 h, the formation of CTC clusters was observed. n = 3. b The protein expression of vimentin and CEACAM6 after 24-h treatment of hirudin or PBS. n = 3. c Quantitative analysis of vimentin and CEACAM6 protein expression. d Quantitative analysis of the relative cluster area of MCF-7 cell clusters. n = 4. e CTC clusters were blown apart and the proliferation of tumor cells within the clusters was examined by Edu staining. n = 3. f Transmission electron micrographs displaying the morphological characteristics of tumor cells in different states. The arrow indicates cells in apoptosis or premature apoptosis. g A schematic illustrating the establishment of the lung metastasis model via tail vein injection of 4T1-luc single cells or cell clusters pre-treated with hirudin or PBS for 24 h. h Representative bioluminescence images (IVIS spectral system) monitoring lung metastases on days 1, 2, 3, 7 and 14 after injection. n = 7. i – k Ki67 ( i, j ) and pan-CK ( k ) immunohistochemical images of lung tissue in retention model, and quantitative analysis of Ki67 ( j ). n = 3. l , m Ki67 immunohistochemical images ( l ) and quantitative analysis ( m ) of lung tissue in the colonization model. n = 3. n , o Representative HE staining ( n ) and quantitative analysis ( o ) of lung metastases in colonization model mice. n = 3. Data are mean ± s.d., * P < 0.05, ** P < 0.01, *** P < 0.001 versus clusters.

Article Snippet: Breast tumor cell lines MCF-7, MDA-MB-231 and 4T1 were from the American Type Culture Collection and cultured in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C and 5% CO2.

Techniques: In Vitro, Expressing, Staining, Transmission Assay, Injection, Immunohistochemical staining

Journal: Experimental & Molecular Medicine

Article Title: Hirudin suppresses hematogenous metastasis by targeting desmosome junction transition in circulating tumor cell clusters via HIF-1α–DSG2 signaling

doi: 10.1038/s12276-025-01598-8

Figure Lengend Snippet: a Schematic diagram of the cell-cell adhesion assay. Upper layer: MCF-7-GFP; lower layer: MCF-7. b A cell–cell adhesion assay was performed to evaluate the intercellular adhesion in MCF-7 clusters following 24 h of hirudin treatment. Representative fluorescence images were taken, and adherent MCF-7-GFP cells were quantified. n = 3. c A cell–fibronectin adhesion assay was used to assess the extracellular matrix attachment capacity of cells within MCF-7 clusters following 24 h hirudin treatment. n = 3. d WB analysis of PG expression in 24 h hirudin-treated MCF-7 cell clusters with grayscale values normalized to GAPDH. n = 3. e , f Immunofluorescence staining of PG in 24 h hirudin-treated MCF-7 cell clusters ( e ) with mean fluorescence intensity quantification ( f ). Red, PG; blue, nucleus. n = 3. g WB analysis of ZO-1 and integrin-β5 expression in hirudin-treated MCF-7 cell clusters. n = 3. h – m Immunofluorescence staining ( h , j and l ) and mean fluorescence intensity quantification ( i , k and m ) of ZO-1 ( h and i ), integrin-β5 ( j and k ) and CEACAM6 ( l and m ) in 24 h hirudin-treated MCF-7 cell clusters. Red, ZO-1/integrin-β5/CEACAM6; blue, nucleus. n = 3. Data are mean ± s.d., * P < 0.05, ** P < 0.01, *** P < 0.001 versus control.

Article Snippet: Breast tumor cell lines MCF-7, MDA-MB-231 and 4T1 were from the American Type Culture Collection and cultured in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C and 5% CO2.

Techniques: Cell Adhesion Assay, Fluorescence, Expressing, Immunofluorescence, Staining, Control

Journal: Experimental & Molecular Medicine

Article Title: Hirudin suppresses hematogenous metastasis by targeting desmosome junction transition in circulating tumor cell clusters via HIF-1α–DSG2 signaling

doi: 10.1038/s12276-025-01598-8

Figure Lengend Snippet: a A schematic diagram of desmosomes and adherens junctions. b , c The expression of DSG2 and PG proteins in primary tumors ( b ) and blood samples ( c ) from patients with breast cancer. d WB analysis of DSG2 and PG expression in MCF-7 adherent cells and cell clusters. n = 3. e Immunofluorescence analysis of DSG2 and PG expression in MCF-7 adherent cells and cell clusters. n = 3. f WB analysis of the expression differences of E-cadherin and β-catenin in MCF-7 adherent cells and cell clusters, with grayscale quantification. n = 3. g , h Immunofluorescence analysis of E-cadherin ( g ) and β-catenin ( h ) expression in MCF-7 adherent cells and cell clusters, with quantification of mean fluorescence intensity. n = 3. i – k Immunofluorescence analysis showing the expression and colocalization of DSG2 and PG ( i ), E-cadherin and β-catenin ( j ) and E-cadherin and PG ( k ) in MCF-7 adherent cells and cell clusters. n = 3. l Transmission electron microscopy images of desmosomes (yellow arrows) and adherent junctions (AJ) in MCF-7 adherent cells and cell clusters. n = 3. m WB analysis of DSG2 expression. n = 3. n , o The formation of CTC clusters following DSG2 knockdown or recombinant DSG2 protein treatment. Representative bright‑field images of CTC clusters ( n ), and quantitative analysis of cluster area ( o ). n = 3. p Transmission electron microscopy showing the desmosomes (yellow arrows) and AJ in MCF-7 cell clusters with or without DSG2 knockdown. n = 3. q WB analysis of PG, E-cadherin and β-catenin expression after DSG2 knockdown or recombinant DSG2 treatment, with grayscale quantification. n = 3. Data are mean ± s.d., ** P < 0.01, *** P < 0.001 versus Si-DSG2.

Article Snippet: Breast tumor cell lines MCF-7, MDA-MB-231 and 4T1 were from the American Type Culture Collection and cultured in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C and 5% CO2.

Techniques: Expressing, Immunofluorescence, Fluorescence, Transmission Assay, Electron Microscopy, Knockdown, Recombinant

Journal: Experimental & Molecular Medicine

Article Title: Hirudin suppresses hematogenous metastasis by targeting desmosome junction transition in circulating tumor cell clusters via HIF-1α–DSG2 signaling

doi: 10.1038/s12276-025-01598-8

Figure Lengend Snippet: a WB analysis of HIF-1α and P300 expression in MCF-7 adherent cells and cell clusters with grayscale quantification. n = 3. b , c Immunofluorescence analysis of HIF-1α expression in MCF-7 adherent cells and cell clusters ( b ) with mean fluorescence intensity quantification ( c ). n = 3. d , e HIF-1α distribution in lung tissues at 0 h post-injection of 4T1 single cells/clusters ( d ) with positive area quantification ( e ). n = 3. f , g Validation of HIF-1α knockdown efficiency by WB ( f ) with quantification ( g ). n = 3. h Representative images of CTC clusters formed by MCF-7 cells with or without HIF-1α knockdown. i Representative images of cell–cell adhesion within these CTC clusters. j , k Quantification of cluster area ( j ) and adherent MCF-7-GFP cell number ( k ). n = 3. l Transmission electron microscopy showing desmosome (yellow arrows) and adherent junctions (AJ) in MCF-7 cell clusters with or without HIF-1α knockdown. n = 3. m WB analysis of PG, DSG2, E-cadherin and β-catenin expression in MCF-7 cell clusters with or without HIF-1α knockdown. n = 3. n CTC cluster formation across experimental groups: wild type (Si-Cont), HIF-1α-knockdown (Si-HIF-1α) and rescue (Si-HIF-1α + recombinant DSG2) using MCF-7 cells. n = 3. o Corresponding intercellular adhesion within CTC clusters. n = 3. p WB analysis of PG, DSG2, E-cadherin and β-catenin expression in CTC clusters across experimental groups. n = 3. Data are mean ± s.d., ** P < 0.01, *** P < 0.001.

Article Snippet: Breast tumor cell lines MCF-7, MDA-MB-231 and 4T1 were from the American Type Culture Collection and cultured in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C and 5% CO2.

Techniques: Expressing, Immunofluorescence, Fluorescence, Injection, Biomarker Discovery, Knockdown, Transmission Assay, Electron Microscopy, Recombinant

Journal: Experimental & Molecular Medicine

Article Title: Hirudin suppresses hematogenous metastasis by targeting desmosome junction transition in circulating tumor cell clusters via HIF-1α–DSG2 signaling

doi: 10.1038/s12276-025-01598-8

Figure Lengend Snippet: a Representative images and quantitative analysis of intercellular adhesion in MCF-7 cell clusters following 24-h hirudin treatment under three conditions: wild type (Si-Cont), DSG2-knockdown (Si-DSG2) and DSG2-reconstituted (+ recombinant DSG2) groups. Upper layer: MCF-7-GFP; lower layer: MCF-7. n = 3. Data are mean ± s.d., ** P < 0.01. b Transmission electron microscopy showing desmosomes (yellow arrows) and adherent junctions (AJ) in MCF-7 cell clusters after 24-h hirudin treatment. n = 3. c – e WB analysis and grayscale quantification of DSG2 and E-cadherin expression in MCF-7 cell clusters following 24-h hirudin treatment. n = 3. f Immunofluorescence analysis of DSG2 expression in MCF-7 cell clusters after 24-h hirudin treatment, with mean fluorescence intensity quantification. n = 3. g Immunofluorescence costaining of E-cadherin and DSG2 in MCF-7 cell clusters treated with hirudin for 24 h. n = 3. h Morphology of CTC clusters formed by DSG2 knockdown or recombinant DSG2-treated MCF-7 cells after 24-h hirudin treatment, with quantification of clusters area percentage. n = 3. i Transmission electron microscopy images of desmosomes (yellow arrows) and AJ in MCF-7 cell clusters with or without DSG2 knockdown following 24 h of hirudin treatment. n = 3. Data are mean ± s.d., * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Breast tumor cell lines MCF-7, MDA-MB-231 and 4T1 were from the American Type Culture Collection and cultured in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C and 5% CO2.

Techniques: Knockdown, Recombinant, Transmission Assay, Electron Microscopy, Expressing, Immunofluorescence, Fluorescence

Journal: Experimental & Molecular Medicine

Article Title: Hirudin suppresses hematogenous metastasis by targeting desmosome junction transition in circulating tumor cell clusters via HIF-1α–DSG2 signaling

doi: 10.1038/s12276-025-01598-8

Figure Lengend Snippet: a WB analysis of HIF-1α and P300 expression in MCF-7 cell clusters after 24-h hirudin treatment, with grayscale quantification. n = 3. b , c Immunofluorescence staining of HIF-1α in MCF-7 cell clusters following 24-h hirudin treatment ( b ) with mean fluorescence intensity quantification ( c ). n = 3. d , e Immunofluorescence images of lung tissue at 0 h and 24 h after tail vein injection of 4T1 cell clusters treated with hirudin or PBS, showing HIF-1α expression ( d ) and quantification of HIF-1α-positive areas ( e ). n = 3. f , g Morphological analysis of CTC clusters formed by control (Si-Cont) and HIF-1α-knockdown MCF-7 cells with or without 24-h hirudin treatment ( f ) with quantification of area percentage ( g ). n = 3. h , i Representative images of intercellular adhesion within CTC clusters ( h ) with MCF-7-GFP cell counts ( i ). Upper layer: MCF-7-GFP; lower layer: MCF-7. n = 3. j Transmission electron microscopy images of desmosomes (yellow arrows) and adherent junctions (AJ) in MCF-7 cell clusters with or without HIF-1α knockdown following 24 h of hirudin treatment. n = 3. Data are mean ± s.d., * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Breast tumor cell lines MCF-7, MDA-MB-231 and 4T1 were from the American Type Culture Collection and cultured in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C and 5% CO2.

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Injection, Control, Knockdown, Transmission Assay, Electron Microscopy