human fascin 1  (Addgene inc)

Journal: Science Advances

Article Title: Mesothelial cells promote peritoneal invasion and metastasis of ascites-derived ovarian cancer cells through spheroid formation

doi: 10.1126/sciadv.adu5944

Figure Lengend Snippet: ( A ) Schematic showing the comparison between RNA expression in OV90 and mesothelial cells. ( B and C ) PCA plot of (B) OV90 and (C) HPMCs. ( D ) Volcano plot and clustering of RNA expression changes in OV90. The red line indicates an adjusted P value <0.05. ( E ) Volcano plot and clustering of RNA expression changes in HPMCs. The red line represents an adjusted P value <0.05. The right side of the volcano plot represents a fold change. ( F ) Significant up-regulated pathway changes in mesothelial cells after interaction with OV90 in KEGG. ( G ) Significant up-regulated pathway changes in mesothelial cells after interaction with OV90 in the GO term. ( H ) Significant down-regulated pathway changes in mesothelial cells after interaction with OV90 in KEGG. ( I ) Significant down-regulated pathway changes in mesothelial cells after interaction with OV90 in the GO term. ( J and K ) PROGENy pathway activity analysis of the ascites samples of the Zheng et al. EOC scRNA-seq dataset revealed high TGF-β pathway activity in both EOC and mesothelial cells. ( L ) Bar plot showing the concentration of TGF-β1 in the supernatant from HPMCs, TGF-β1–stimulated HPMCs, and OV90 cells. ( M ) Scheme of an invadopodium in a mesothelial cell. ( N ) Immunofluorescence images of a single cell invading the collagen layer using invadopodium formation. Green, cortactin; red, phalloidin. Scale bars, 10 μm. ( O ) The number of invadopodia was significantly higher in TGF-β1–stimulated mesothelial cells. ( P ) Strategy to detect candidates with a high invasion ability in mesothelial cells. ( Q ) Western blot analysis of fascin-1 and several proteins related to invadopodium formation. ( R ) Immunofluorescence images of fascin-1 or myosin X (green) in TGF-β1–stimulated mesothelial cells. Scale bars, 5 μm. FACS, fluorescence-activated cell sorting; FC, fold change. *** P < 0.001.

Article Snippet: The following primary antibodies were used: fascin-1 (Merck Millipore, MAB3582), myosin X (Novus Biologicals, 22430002), integrin β1 (BD Biosciences, 610467), cortactin (BD Biosciences, 610049), Tks5 (Santa Cruz Biotechnology, sc-30122), and HIF1A (R&D Systems, 241809).

Techniques: Comparison, RNA Expression, Activity Assay, Concentration Assay, Immunofluorescence, Single Cell, Western Blot, Fluorescence, FACS

Journal: Science Advances

Article Title: Mesothelial cells promote peritoneal invasion and metastasis of ascites-derived ovarian cancer cells through spheroid formation

doi: 10.1126/sciadv.adu5944

Figure Lengend Snippet: ( A and B ) Violin plots showing the expression of invadopodium-related genes across cell components in ascites on the basis of two different scRNA-seq datasets from Izar et al. and Zheng et al. . In the dataset of (A), mesothelial cells are classified as fibroblasts. ( C ) Collagen degradation assay. The thickness represents the cell invasion ability. Scale bars, 400 μm. ( D ) Bar graph showing the thickness of remnant collagen 48 hours after incubation. ( E ) Bar graph showing the number of invadopodia. sh-Fascin-1 or sh-myosin X inhibited invadopodium maturation. ( F and G ) 3D images and bar graph showing that spheroids invade collagen with shRNA-induced mesothelial cells (green) and OV90 (red). The invasion ability of mesothelial cells was significantly inhibited by sh- FSCN1 or sh- MYO10 . Scale bars, 200 μm. ( H ) Scheme of the malignant ascites in vivo model using shRNA-treated HPMCs. ( I and J ) Images and bar graph showing the differences in the metastasis area on the omentum from mice 1 week after the injection of OV90 with or without sh-induced mesothelial cells. Scale bars, 1 mm. ( K ) Representative IHC image of mouse tissue with fascin-1. Invasive stromal cells strongly expressed fascin-1. Scale bar, 100 μm. ( L ) IHC of metastasis samples in clinical samples. Fascin-1–positive stromal cells were present in the tumor-invasive regions. Scale bar, 100 μm. ( M ) Kaplan-Meier plot showing the patient’s progression-free survival depending on fascin-1 expression in stromal cells or cancer cells. Fascin-1 expression in stromal cells in metastasis samples was significantly related to a worse prognosis ( P = 0.030). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The following primary antibodies were used: fascin-1 (Merck Millipore, MAB3582), myosin X (Novus Biologicals, 22430002), integrin β1 (BD Biosciences, 610467), cortactin (BD Biosciences, 610049), Tks5 (Santa Cruz Biotechnology, sc-30122), and HIF1A (R&D Systems, 241809).

Techniques: Expressing, Degradation Assay, Incubation, shRNA, In Vivo, Injection

Journal: Science Advances

Article Title: Mesothelial cells promote peritoneal invasion and metastasis of ascites-derived ovarian cancer cells through spheroid formation

doi: 10.1126/sciadv.adu5944

Figure Lengend Snippet: Almost all the EOC cells identified in the ascites were in a spheroids formation and 65% were accompanied by mesothelial cells, referred to as ACMSs. The formation of ACMSs enabled EOC cells to alter the RNA expression profiles of mesothelial cells via TGF-β related pathway. These alternations increased the expression of fascin-1 in this pathway, which caused invadopodia formations in mesothelial cells to mature, and this degraded collagen with MMP14. Mesothelial cells interacted with EOC cells, which aggressively invaded the collagen and mesothelial layer. These results show that EOC cells can induce peritoneal metastasis without direct dynamic RNA expression changes. EOC cells then followed the route created by the mesothelial cells. This model explains that EOC cells control the unique tumor microenvironment in ascites to rapidly induce abdominal dissemination.

Article Snippet: The following primary antibodies were used: fascin-1 (Merck Millipore, MAB3582), myosin X (Novus Biologicals, 22430002), integrin β1 (BD Biosciences, 610467), cortactin (BD Biosciences, 610049), Tks5 (Santa Cruz Biotechnology, sc-30122), and HIF1A (R&D Systems, 241809).

Techniques: RNA Expression, Expressing, Control