Journal: Oncogene

Article Title: Lymph node metastasis-derived gastric cancer cells educate bone marrow-derived mesenchymal stem cells via YAP signaling activation by exosomal Wnt5a

doi: 10.1038/s41388-021-01722-8

Figure Lengend Snippet: a The flow chart illustrates the process of treating BM-MSCs with GC-CM, and evaluation of the resulting BM-MSC phenotype and function. b , c The ability to educate BM-MSCs was compared between LNM-GCs (SGC-7901 and HGC-27) and primary GCs (AGS and MGC-803). d LNM-GC exosomes and exosome-free CM were prepared as indicated in the flow chart. e , f BM-MSCs were treated with CM, exosomes, or exosome-free CM from LNM-GCs. The educational effect of the three treatments on BM-MSCs was then compared. b , e Immunofluorescence detection of α-SMA in BM-MSCs was conducted to detect their tumor-promoting phenotype (magnification, ×200; scale bars, 50 μm). c , f GC migration and invasiveness analysis, and HLEC tubule formation assay, were performed to analyze the tumor-promoting function of BM-MSCs. The number of migrated and invaded GCs and tubule junctions were counted and represented in column charts. Data are presented as the mean ± SD of three independent experiments. Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Tukey’s test. *** P < 0.0001; ** P < 0.01; * P < 0.05; ns, non significant.

Article Snippet: GFP and Wnt5a in cells and their exosomes were individually detected by western blot. h Exosome internalization analysis in BM-MSCs after incubation with exosomes from the abovenamed two stably transfected AGS (magnification, ×400; scale bars, 25 μm). i Screening for the most efficient siRNA against Wnt5a (si-Wnt5a) in SGC-7901. j Western blotting analysis of Wnt5a expression in SGC-7901 cells, and analysis of their secreted exosomes after transfection of SGC-7901 with si-Wnt5a and NC. k – o si-Wnt5a- and NC-transfected SGC-7901 exosomes were separately used to treat BM-MSCs in order to compare their effects in education of BM-MSCs. p , q Western blotting analysis of Wnt5a in AGS and in their secreted exosomes after AGS were infected with the pLV-Wnt5a lentivirus. r – v Exosomes derived from pLV-Wnt5a- and pLV-vec-lentivirus-infected AGS were separately used to treat BM-MSCs to observe their BM-MSC-reprogramming abilities. k , r Detection of α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). l , s Numbers of migrated and invaded GCs and formed tubule junctions. m – o , t – v In vivo analysis of tumor-promoting properties in BM-MSCs. m , t The draining popliteal LNs harvested from the above groups. n , u Weight of popliteal LNs. o , v Pan-cytokeratin (AE1/AE3) expression in popliteal LNs detected using immunohistochemistry (magnification, ×100, scale bars, 100 μm; magnification, ×400, scale bars, 20 μm).

Techniques: Immunofluorescence, Migration, Tube Formation Assay

Journal: Oncogene

Article Title: Lymph node metastasis-derived gastric cancer cells educate bone marrow-derived mesenchymal stem cells via YAP signaling activation by exosomal Wnt5a

doi: 10.1038/s41388-021-01722-8

Figure Lengend Snippet: a , b Comparison of cellular and exosomal levels of the Wnt5a protein in AGS and SGC-7901 using western blotting. c Detection of Wnt5a protein in BM-MSCs after treatment with CHX (15 μg/ml) alone or with exosomes at the indicated times. d – g AGS stably transfected with pCMV-GFP and pCMV-Wnt5a-GFP were separately established. GFP and Wnt5a in cells and their exosomes were individually detected by western blot. h Exosome internalization analysis in BM-MSCs after incubation with exosomes from the abovenamed two stably transfected AGS (magnification, ×400; scale bars, 25 μm). i Screening for the most efficient siRNA against Wnt5a (si-Wnt5a) in SGC-7901. j Western blotting analysis of Wnt5a expression in SGC-7901 cells, and analysis of their secreted exosomes after transfection of SGC-7901 with si-Wnt5a and NC. k – o si-Wnt5a- and NC-transfected SGC-7901 exosomes were separately used to treat BM-MSCs in order to compare their effects in education of BM-MSCs. p , q Western blotting analysis of Wnt5a in AGS and in their secreted exosomes after AGS were infected with the pLV-Wnt5a lentivirus. r – v Exosomes derived from pLV-Wnt5a- and pLV-vec-lentivirus-infected AGS were separately used to treat BM-MSCs to observe their BM-MSC-reprogramming abilities. k , r Detection of α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). l , s Numbers of migrated and invaded GCs and formed tubule junctions. m – o , t – v In vivo analysis of tumor-promoting properties in BM-MSCs. m , t The draining popliteal LNs harvested from the above groups. n , u Weight of popliteal LNs. o , v Pan-cytokeratin (AE1/AE3) expression in popliteal LNs detected using immunohistochemistry (magnification, ×100, scale bars, 100 μm; magnification, ×400, scale bars, 20 μm). Data are presented as the mean ± SD of three independent experiments. Statistical significance was assessed using Student’s t test. *** P < 0.0001; ** P < 0.01.

Article Snippet: GFP and Wnt5a in cells and their exosomes were individually detected by western blot. h Exosome internalization analysis in BM-MSCs after incubation with exosomes from the abovenamed two stably transfected AGS (magnification, ×400; scale bars, 25 μm). i Screening for the most efficient siRNA against Wnt5a (si-Wnt5a) in SGC-7901. j Western blotting analysis of Wnt5a expression in SGC-7901 cells, and analysis of their secreted exosomes after transfection of SGC-7901 with si-Wnt5a and NC. k – o si-Wnt5a- and NC-transfected SGC-7901 exosomes were separately used to treat BM-MSCs in order to compare their effects in education of BM-MSCs. p , q Western blotting analysis of Wnt5a in AGS and in their secreted exosomes after AGS were infected with the pLV-Wnt5a lentivirus. r – v Exosomes derived from pLV-Wnt5a- and pLV-vec-lentivirus-infected AGS were separately used to treat BM-MSCs to observe their BM-MSC-reprogramming abilities. k , r Detection of α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). l , s Numbers of migrated and invaded GCs and formed tubule junctions. m – o , t – v In vivo analysis of tumor-promoting properties in BM-MSCs. m , t The draining popliteal LNs harvested from the above groups. n , u Weight of popliteal LNs. o , v Pan-cytokeratin (AE1/AE3) expression in popliteal LNs detected using immunohistochemistry (magnification, ×100, scale bars, 100 μm; magnification, ×400, scale bars, 20 μm).

Techniques: Comparison, Western Blot, Stable Transfection, Transfection, Incubation, Expressing, Infection, Derivative Assay, In Vivo, Immunohistochemistry

Journal: Oncogene

Article Title: Lymph node metastasis-derived gastric cancer cells educate bone marrow-derived mesenchymal stem cells via YAP signaling activation by exosomal Wnt5a

doi: 10.1038/s41388-021-01722-8

Figure Lengend Snippet: a – d Characterization of serum exosomes isolated from healthy participants (H), GC patients without LNM (NM), and GC patients with LNM (M). a Representative TEM images of exosomes (magnification, ×60,000; scale bars, 100 μm). b Size distribution of exosomes analyzed using NTA; c Detection of CD81 expression by flow cytometry. d Western blotting analysis of CD63 and CD9 expression. e , f Comparison of BM-MSC-educating capacity among exosomes from the three groups described above. e Detection α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). f Analysis of tumor-promoting properties of BM-MSCs. g , h Wnt5a expression in exosomes isolated from the pooled sera was detected by western blotting. g Representative image. h Fold changes in relative gray values of Wnt5a; i , j Serum concentration of exosomal Wnt5a was detected using ELISA. i Serum concentration of exosomal Wnt5a in clinical serum samples (20 individuals per group). j Wnt5a concentration in serum samples from the LNM model described in Figs. , . Data are presented using means ± SD of three independent experiments. Statistical significance was determined using one-way ANOVA followed by Tukey’s test. *** P < 0.0001; ** P < 0.01; * P < 0.05; ns, non significant.

Article Snippet: GFP and Wnt5a in cells and their exosomes were individually detected by western blot. h Exosome internalization analysis in BM-MSCs after incubation with exosomes from the abovenamed two stably transfected AGS (magnification, ×400; scale bars, 25 μm). i Screening for the most efficient siRNA against Wnt5a (si-Wnt5a) in SGC-7901. j Western blotting analysis of Wnt5a expression in SGC-7901 cells, and analysis of their secreted exosomes after transfection of SGC-7901 with si-Wnt5a and NC. k – o si-Wnt5a- and NC-transfected SGC-7901 exosomes were separately used to treat BM-MSCs in order to compare their effects in education of BM-MSCs. p , q Western blotting analysis of Wnt5a in AGS and in their secreted exosomes after AGS were infected with the pLV-Wnt5a lentivirus. r – v Exosomes derived from pLV-Wnt5a- and pLV-vec-lentivirus-infected AGS were separately used to treat BM-MSCs to observe their BM-MSC-reprogramming abilities. k , r Detection of α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). l , s Numbers of migrated and invaded GCs and formed tubule junctions. m – o , t – v In vivo analysis of tumor-promoting properties in BM-MSCs. m , t The draining popliteal LNs harvested from the above groups. n , u Weight of popliteal LNs. o , v Pan-cytokeratin (AE1/AE3) expression in popliteal LNs detected using immunohistochemistry (magnification, ×100, scale bars, 100 μm; magnification, ×400, scale bars, 20 μm).

Techniques: Isolation, Expressing, Flow Cytometry, Western Blot, Comparison, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Lymph node metastasis-derived gastric cancer cells educate bone marrow-derived mesenchymal stem cells via YAP signaling activation by exosomal Wnt5a

doi: 10.1038/s41388-021-01722-8

Figure Lengend Snippet: a – d Characterization of AGS- and SGC-7901-exosomes. a Representative transmission electron microscopy (TEM) images (magnification, ×60,000; scale bars, 100 μm). b Size distribution shown by nanoparticle tracking analysis (NTA). c Flow cytometry analysis of CD81 expression. d Western blotting analysis of CD63 and CD9 expression. e – i The ability of AGS- and SGC-7901-exosomes to educate BM-MSCs was evaluated. e α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). f Quantification of migration, invasion, and tubule formation. g – i In vivo analysis of tumor-promoting properties of BM-MSCs. g The draining popliteal lymph nodes (LNs) from each group were harvested and imaged. h Weight of popliteal LNs. i Immunohistochemistry analysis of pan-cytokeratin (AE1/AE3) expression in popliteal LNs. Representative images from each group are shown (magnification, ×100, scale bars, 100 μm; magnification, ×400, scale bars, 20 μm). Data are expressed as the mean ± SD of three independent experiments. Statistical significance was calculated using one-way ANOVA followed by Tukey’s test. *** P < 0.0001; ** P < 0.01; ns, non significant.

Article Snippet: GFP and Wnt5a in cells and their exosomes were individually detected by western blot. h Exosome internalization analysis in BM-MSCs after incubation with exosomes from the abovenamed two stably transfected AGS (magnification, ×400; scale bars, 25 μm). i Screening for the most efficient siRNA against Wnt5a (si-Wnt5a) in SGC-7901. j Western blotting analysis of Wnt5a expression in SGC-7901 cells, and analysis of their secreted exosomes after transfection of SGC-7901 with si-Wnt5a and NC. k – o si-Wnt5a- and NC-transfected SGC-7901 exosomes were separately used to treat BM-MSCs in order to compare their effects in education of BM-MSCs. p , q Western blotting analysis of Wnt5a in AGS and in their secreted exosomes after AGS were infected with the pLV-Wnt5a lentivirus. r – v Exosomes derived from pLV-Wnt5a- and pLV-vec-lentivirus-infected AGS were separately used to treat BM-MSCs to observe their BM-MSC-reprogramming abilities. k , r Detection of α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). l , s Numbers of migrated and invaded GCs and formed tubule junctions. m – o , t – v In vivo analysis of tumor-promoting properties in BM-MSCs. m , t The draining popliteal LNs harvested from the above groups. n , u Weight of popliteal LNs. o , v Pan-cytokeratin (AE1/AE3) expression in popliteal LNs detected using immunohistochemistry (magnification, ×100, scale bars, 100 μm; magnification, ×400, scale bars, 20 μm).

Techniques: Transmission Assay, Electron Microscopy, Flow Cytometry, Expressing, Western Blot, Migration, In Vivo, Immunohistochemistry

Journal: Oncogene

Article Title: Lymph node metastasis-derived gastric cancer cells educate bone marrow-derived mesenchymal stem cells via YAP signaling activation by exosomal Wnt5a

doi: 10.1038/s41388-021-01722-8

Figure Lengend Snippet: a The flow chart shows removal of exosomal proteins by proteinase K alone or combined with Triton X-100, and purification of these treated exosomes by ultrafiltration centrifugal tubes. b SDS-PAGE and Coomassie brilliant blue stain were used to confirm that proteins were indeed removed from SGC-7901 exosomes. c , d The influence of protein removal on education of BM-MSCs by SGC-7901 exosomes. c Immunofluorescence detection of α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). d Quantification of migrated and invaded GCs, and formed tubule junctions. Data are presented as the mean ± SD of three independent experiments. Statistical significance was assessed using one-way ANOVA followed by Tukey’s test. *** P < 0.0001; ** P < 0.01; ns, non significant.

Article Snippet: GFP and Wnt5a in cells and their exosomes were individually detected by western blot. h Exosome internalization analysis in BM-MSCs after incubation with exosomes from the abovenamed two stably transfected AGS (magnification, ×400; scale bars, 25 μm). i Screening for the most efficient siRNA against Wnt5a (si-Wnt5a) in SGC-7901. j Western blotting analysis of Wnt5a expression in SGC-7901 cells, and analysis of their secreted exosomes after transfection of SGC-7901 with si-Wnt5a and NC. k – o si-Wnt5a- and NC-transfected SGC-7901 exosomes were separately used to treat BM-MSCs in order to compare their effects in education of BM-MSCs. p , q Western blotting analysis of Wnt5a in AGS and in their secreted exosomes after AGS were infected with the pLV-Wnt5a lentivirus. r – v Exosomes derived from pLV-Wnt5a- and pLV-vec-lentivirus-infected AGS were separately used to treat BM-MSCs to observe their BM-MSC-reprogramming abilities. k , r Detection of α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). l , s Numbers of migrated and invaded GCs and formed tubule junctions. m – o , t – v In vivo analysis of tumor-promoting properties in BM-MSCs. m , t The draining popliteal LNs harvested from the above groups. n , u Weight of popliteal LNs. o , v Pan-cytokeratin (AE1/AE3) expression in popliteal LNs detected using immunohistochemistry (magnification, ×100, scale bars, 100 μm; magnification, ×400, scale bars, 20 μm).

Techniques: Purification, SDS Page, Staining, Immunofluorescence, Expressing

Journal: Oncogene

Article Title: Lymph node metastasis-derived gastric cancer cells educate bone marrow-derived mesenchymal stem cells via YAP signaling activation by exosomal Wnt5a

doi: 10.1038/s41388-021-01722-8

Figure Lengend Snippet: a – d Western blotting analysis of p-YAP s127 and YAP levels in BM-MSCs treated with GC exosomes ( a ), serum exosomes isolated from healthy individuals (H), GC patients without (NM), and those with regional LNM (M) ( b ), exosomes from lentivirus-infected AGS cells ( c ), and exosomes from oligonucleotide-transfected SGC-7901 cells ( d ). e – j BM-MSCs were pretreated with verteporfin before incubation with SGC-7901 exosomes ( e , g , h ) and serum exosomes isolated from GC patients with LNM ( f , i , j ). e , f Western blot analysis of CTGF and CYR61 in BM-MSCs. g , i Detection of α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). h , j Numbers of migrated and invaded GCs and formed tubule junctions. Data are presented as the mean ± SD of three independent experiments. Statistical significance was assessed using Student’s t test. *** P < 0.0001; ** P < 0.01.

Article Snippet: GFP and Wnt5a in cells and their exosomes were individually detected by western blot. h Exosome internalization analysis in BM-MSCs after incubation with exosomes from the abovenamed two stably transfected AGS (magnification, ×400; scale bars, 25 μm). i Screening for the most efficient siRNA against Wnt5a (si-Wnt5a) in SGC-7901. j Western blotting analysis of Wnt5a expression in SGC-7901 cells, and analysis of their secreted exosomes after transfection of SGC-7901 with si-Wnt5a and NC. k – o si-Wnt5a- and NC-transfected SGC-7901 exosomes were separately used to treat BM-MSCs in order to compare their effects in education of BM-MSCs. p , q Western blotting analysis of Wnt5a in AGS and in their secreted exosomes after AGS were infected with the pLV-Wnt5a lentivirus. r – v Exosomes derived from pLV-Wnt5a- and pLV-vec-lentivirus-infected AGS were separately used to treat BM-MSCs to observe their BM-MSC-reprogramming abilities. k , r Detection of α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). l , s Numbers of migrated and invaded GCs and formed tubule junctions. m – o , t – v In vivo analysis of tumor-promoting properties in BM-MSCs. m , t The draining popliteal LNs harvested from the above groups. n , u Weight of popliteal LNs. o , v Pan-cytokeratin (AE1/AE3) expression in popliteal LNs detected using immunohistochemistry (magnification, ×100, scale bars, 100 μm; magnification, ×400, scale bars, 20 μm).

Techniques: Western Blot, Isolation, Infection, Transfection, Incubation, Expressing