Journal: Advanced Science

Article Title: Shear Stress Drives the Cleavage Activation of Protease‐Activated Receptor 2 by PRSS3/Mesotrypsin to Promote Invasion and Metastasis of Circulating Lung Cancer Cells

doi: 10.1002/advs.202301059

Figure Lengend Snippet: SS induced cell death and elevated cell colony formation, migration and invasion abilities compared with both 0 h and suspension conditions. A) Phase images of A549 cells at 0 h and after 10 h of suspension and SS treatment. Scale bar: 100 µm. B) The MTT assay was used to evaluate the viability of A549 cells under the indicated conditions. C,D) Representative images and quantification results of the colony formation assay for A549 cells under the indicated conditions. 1000 cells were seeded in each well of 6‐well plates and allowed to grow for 10 days. Scale bar: 2 mm. E,F) Representative images and quantification results of the Transwell migration assay for A549 cells under the indicated conditions. 10 000 cells were seeded and allowed to migrate for 14 h. Scale bar: 100 µm. G,H) Representative images and quantification results of the Transwell invasion assay for A549 cells under the indicated conditions. 20 000 cells were seeded and allowed to invade for 14 h. Scale bar: 100 µm. I) Venn diagrams of differentially expressed genes in the indicated comparisons. The thresholds were set as p < 0.05, a fold change of ≥2 for upregulated genes and a fold change of ≤0.5 for downregulated genes. J) Gene Set Enrichment Analysis (GSEA) of differentially expressed genes in the SS group compared with the suspension group (SUS). Gene sets with the top 10 highest normalized enrichment scores are shown. K) Expression heatmap of the 10 most upregulated genes in RNA‐seq that have been reported to promote cancer progression. L) qPCR results showing the relative mRNA levels of JUN, JUNB, JUND, FOS, FOSL1, FOSL2, PRSS3, PAR1, and PAR2 in A549 cells under suspension and SS conditions in comparison with those at 0 h. M) Schematics of generating the SS‐resistant cell line A‐SSP6 from the parental lung cancer cell line A549‐C3 through six rounds of circulation. N) qPCR results showing the relative mRNA levels of JUN, FOSL1, PRSS3, and PAR2 in A‐SSP6 cells compared to A549‐C3 cells. The quantification results are the means ± SD from three independent experiments. Significant differences were determined by one‐way ANOVA (B,D,F,H) and two‐way ANOVA (L,N). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. ns, not significant.

Article Snippet: The PRSS3 inhibitor diminazene (#18678‐1) was obtained from Cayman Chemical (USA).

Techniques: Migration, Suspension, MTT Assay, Colony Assay, Transwell Migration Assay, Transwell Invasion Assay, Expressing, RNA Sequencing, Comparison

Journal: Advanced Science

Article Title: Shear Stress Drives the Cleavage Activation of Protease‐Activated Receptor 2 by PRSS3/Mesotrypsin to Promote Invasion and Metastasis of Circulating Lung Cancer Cells

doi: 10.1002/advs.202301059

Figure Lengend Snippet: Upregulation of PRSS3, PAR2, FOSL1, and JUN induced by SS was further verified. A) Western blots showing the protein levels of PRSS3, PAR2, FRA1, p‐FRA1, cJUN, and p‐cJUN in A549 cells under the indicated conditions (left) and in A549‐C3 and A‐SSP6 cells (right). B) The cleavage of N‐mCherry‐PAR2 in A549 cells was observed under a confocal microscope at 0 h and after 1, 2, or 10 h of treatment (upper). Red fluorescence appeared on the cell membrane when PAR2 was not cleaved, while its significant reduction indicated cleavage. “PAR2 (R36A)” refers to A549 cells expressing N‐mCherry‐PAR2 with an R36A mutation to prevent cleavage under SS, while A549 cells expressing wild‐type PAR2 were used under other conditions. Cells were pretreated and cocirculated with or without 10 µ m PRSS3 inhibitor diminazene. Scale bar: 5 µm. The red fluorescence intensity of the membrane under each condition was measured (lower; n ≥ 50 cells). C) Representative immunofluorescence (IF) staining images showing the subcellular localization of FRA1, p‐FRA1, cJUN, and p‐cJUN before and after 4 h of suspension and SS treatment. Scale bar: 5 µm. D) Quantified nuclear fluorescence intensity for A549 cells in (C) ( n ≥ 100 cells). The quantification results are the means ± SD from three independent experiments. Significant differences were determined by one‐way ANOVA (B,D). ** p < 0.01 and **** p < 0.0001. ns, not significant.

Article Snippet: The PRSS3 inhibitor diminazene (#18678‐1) was obtained from Cayman Chemical (USA).

Techniques: Western Blot, Microscopy, Fluorescence, Membrane, Expressing, Mutagenesis, Immunofluorescence, Staining, Suspension

Journal: Advanced Science

Article Title: Shear Stress Drives the Cleavage Activation of Protease‐Activated Receptor 2 by PRSS3/Mesotrypsin to Promote Invasion and Metastasis of Circulating Lung Cancer Cells

doi: 10.1002/advs.202301059

Figure Lengend Snippet: Knockdown of PRSS3, PAR2, and FOSL1 impaired the metastatic ability of A‐SSP6 cells. A,B) Representative images and quantification results of migrated A‐SSP6 cells after knocking down PRSS3, PAR2, and FOSL1 using shRNAs. 10 000 cells were seeded and allowed to migrate for 18 h. Scale bar: 100 µm. C,D) Representative images and quantification results of the invaded A‐SSP6 cells transfected with shRNAs. 20 000 cells were seeded in each insert and allowed to invade for 18 h. Scale bar: 100 µm. E) Representative fluorescent and H&E staining images and quantification results of colonies per left lung 28 days post injection. One million cells were injected into the tail vein of each NOD/SCID mouse ( n = 6 mice per group). Scale bar: 1 mm for GFP images and 400 µm for H&E staining images. F) Representative fluorescent images of primary tumors in the lungs and metastatic tumors in the liver, intestine, brain and skin ( n = 6–8 mice per group). Four million cancer cells were orthotopically injected into the right lungs of each NOD/SCID mouse. Scale bar: 2 mm (lungs, liver, and intestine) and 200 µm (brain and skin). White arrowheads indicate metastatic tumors. G) The tumor area in all five lungs of each mouse was measured by ImageJ. H) The quantified percentage of mice with metastatic tumors in each group 4 weeks after orthotopic injection was calculated. I) The body weights of each mouse at 0, 1, 2, 3, and 4 weeks after orthotopic implantation were measured. The quantification results are the means ± SD from three independent experiments or from more than five mice. Significant differences were determined by one‐way ANOVA (B,D,E,G) and two‐way ANOVA (I). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. ns, not significant.

Article Snippet: The PRSS3 inhibitor diminazene (#18678‐1) was obtained from Cayman Chemical (USA).

Techniques: Knockdown, Transfection, Staining, Injection

Journal: Advanced Science

Article Title: Shear Stress Drives the Cleavage Activation of Protease‐Activated Receptor 2 by PRSS3/Mesotrypsin to Promote Invasion and Metastasis of Circulating Lung Cancer Cells

doi: 10.1002/advs.202301059

Figure Lengend Snippet: Overexpression of PRSS3, PAR2, and FOSL1 promoted invasion and metastasis of A549‐C3 cells. A) Representative images and quantification results of invaded A549‐C3 cells overexpressing PRSS3, PAR2, or FOSL1. 20 000 cells were seeded in each insert and allowed to invade for 18 h. EV, empty vector. Scale bar: 100 µm. B) Representative fluorescent images and quantification results of colonies per left lung 28 days post injection. One million A549‐C3 cells overexpressing the empty vector, PRSS3, PAR2, FOSL1 or double overexpressing FOSL1 and JUN were injected into the tail vein of each NOD/SCID mouse ( n = 5 mice per group). Scale bar: 1 mm. C) Representative images of primary tumors in the lungs and metastatic tumors in the liver, intestine and brain ( n = 8 mice per group). Scale bar: 2 mm (lungs and liver) and 200 µm (intestine and brain). White arrowheads indicate metastatic tumors. D) The tumor area in all five lungs of each mouse was measured by ImageJ. E) Quantified percentage of mice with metastatic tumors in each group 4 weeks after orthotopic injection was calculated. F) The body weights of each mouse at 0, 1, 2, 3, and 4 weeks after orthotopic implantation were measured. The quantification results are the means ± SD from three independent experiments or from more than five mice. Significant differences were determined by one‐way ANOVA (A,B,D) and two‐way ANOVA (F). * p < 0.05, ** p < 0.01, and *** p < 0.001. ns, not significant.

Article Snippet: The PRSS3 inhibitor diminazene (#18678‐1) was obtained from Cayman Chemical (USA).

Techniques: Over Expression, Plasmid Preparation, Injection

Journal: Advanced Science

Article Title: Shear Stress Drives the Cleavage Activation of Protease‐Activated Receptor 2 by PRSS3/Mesotrypsin to Promote Invasion and Metastasis of Circulating Lung Cancer Cells

doi: 10.1002/advs.202301059

Figure Lengend Snippet: Clinical significance of PRSS3, PAR2, and FOSL1 in NSCLC. A,B) Kaplan–Meier plots of OS and PPS curves in NSCLC patients. C) Representative images and quantified IHC scores of PRSS3, PAR2, and FRA1 in adjacent tissues and lung tumors of NSCLC patients ( n = 30). Scale bar: 100 µm (adjacent tissue and lung tumor) and 25 µm (enlarged images of lung tumors). Significant differences were determined by Student's t ‐test (C). **** p < 0.0001.

Article Snippet: The PRSS3 inhibitor diminazene (#18678‐1) was obtained from Cayman Chemical (USA).

Techniques:

Journal: Advanced Science

Article Title: Shear Stress Drives the Cleavage Activation of Protease‐Activated Receptor 2 by PRSS3/Mesotrypsin to Promote Invasion and Metastasis of Circulating Lung Cancer Cells

doi: 10.1002/advs.202301059

Figure Lengend Snippet: Signaling pathways induced by SS and related to PRSS3, PAR2, and FOSL1. A) Western blots showing increases in p‐Src, p‐ERK, p‐p38, p‐JNK, snail, slug, N‐cadherin (CDH2), and MMP1 in A549 cells under SS. B) Western blots showing the effects of knocking down PRSS3 and PAR2 in A‐SSP6 cells and overexpressing PRSS3 and PAR2 in A549‐C3 cells on potential downstream molecules. The quantification results are the means from three independent experiments.

Article Snippet: The PRSS3 inhibitor diminazene (#18678‐1) was obtained from Cayman Chemical (USA).

Techniques: Protein-Protein interactions, Western Blot

Journal: Advanced Science

Article Title: Shear Stress Drives the Cleavage Activation of Protease‐Activated Receptor 2 by PRSS3/Mesotrypsin to Promote Invasion and Metastasis of Circulating Lung Cancer Cells

doi: 10.1002/advs.202301059

Figure Lengend Snippet: Validating the regulation of FOSL1 through the G α i‐Src‐ERK/p38/JNK axis and downstream molecules of FOSL1. A,B) Representative phase images and quantification results showing the percentage of viable A‐SSP6‐shPRSS3 cells after 10 h of circulation treated with 50 µ m PAR2 activating peptide (PAR2‐AP) and 0.1 µg mL −1 G α i inhibitor PTX. Cells were starved in DMEM with or without adding PTX for 24 h, treated with the control peptide or PAR2‐AP for 1 h, and then cocirculated for 10 h with the indicated reagents. Scale bar: 100 µm. C,D) Representative images and quantification results of the invaded A‐SSP6‐shPRSS3 cells treated with 50 µ m PAR2‐AP and 0.1 µg mL −1 of the G α i inhibitor PTX. Cells were starved in DMEM with or without adding PTX for 24 h and treated with the control peptide or PAR2‐AP for 1 h. 20 000 cells were seeded in each insert and allowed to invade for 18 h along with the treatment of PAR2‐AP and PTX as described. Scale bar: 100 µm. E) Western blots showing the changes in downstream molecules after treating A‐SSP6‐shPRSS3 cells with PTX for 24 h and PAR2‐AP for 1 h (left) and treating A‐SSP6 cells with the Src inhibitor dasatinib for 24 h (right). F–H) Western blots showing the reduction in FRA1, p‐FRA1, cJUN, p‐cJUN, and PRSS3 after treating A‐SSP6 cells with the MEK inhibitor trametinib, p38 inhibitor SB202190 and JNK inhibitor SP600125 for 24 h. I) Western blots showing the impacts of knocking down FOSL1 in A‐SSP6 cells and overexpressing FOSL1 in A549‐C3 cells on related proteins. The quantification results are the means ± SD from three independent experiments. Significant differences were determined by one‐way ANOVA (B,D). ** p < 0.01, *** p < 0.001.

Article Snippet: The PRSS3 inhibitor diminazene (#18678‐1) was obtained from Cayman Chemical (USA).

Techniques: Control, Western Blot

Journal: Advanced Science

Article Title: Shear Stress Drives the Cleavage Activation of Protease‐Activated Receptor 2 by PRSS3/Mesotrypsin to Promote Invasion and Metastasis of Circulating Lung Cancer Cells

doi: 10.1002/advs.202301059

Figure Lengend Snippet: SS promotes EMT and facilitates the invasion and metastasis of cancer cells through the regulation of PRSS3, PAR2, and AP‐1. A) Proposed signaling pathways. SS first induces the cleavage activation of PAR2 by PRSS3, and then activated PAR2 is coupled with G α i protein to upregulate the Src‐ERK/p38/JNK‐FRA1/cJUN pathway. Under the regulation of AP‐1, EMT‐promoting molecules including snail, slug, CDH2, and MMP1 are upregulated to promote invasion and metastasis. The level of PRSS3 also increases upon the activation of AP‐1, which is then secreted out of the cells to cleave PAR2 and further enhance the signaling axis. B) Western blots showing the levels of involved molecules in A‐SSP6‐shCtrl and A‐SSP6‐shPRSS3 cells at 0 h and after 10 h of suspension and SS treatment. The quantification results are the means from three independent experiments.

Article Snippet: The PRSS3 inhibitor diminazene (#18678‐1) was obtained from Cayman Chemical (USA).

Techniques: Protein-Protein interactions, Activation Assay, Western Blot, Suspension

Journal: BMC Genomics

Article Title: Identification of novel vascular markers through gene expression profiling of tumor-derived endothelium

doi: 10.1186/1471-2164-9-201

Figure Lengend Snippet: Transcriptional differences: tumor vs normal tissue derived EC

Article Snippet: In situ hybridization experiments were carried out with a mixture of specific biotin-labeled oligonucleotide antisense (or sense) probes for ADAM23, GPNMB and PRSS3 (Metabion International AG, Martinsried, Germany) listed in the supplementary Table 2 [see Additional file ].

Techniques: Derivative Assay, Membrane, Immunopeptidomics, Activation Assay, Binding Assay, Variant Assay, Dominant Negative Mutation, Wilms Tumor Assay

Journal: BMC Genomics

Article Title: Identification of novel vascular markers through gene expression profiling of tumor-derived endothelium

doi: 10.1186/1471-2164-9-201

Figure Lengend Snippet: Gene expression in different cell lines . Levels of expression of ADAM23, FAP, GPNMB and PRSS3 were evaluated in fibroblasts (HuFb and Malme3) and smooth muscle cells (USMAC) as well as in tumor cell lines (1A9, SKOV3, HT29 and MDA-MB-231). Gene expression of each target gene was normalized to 18s rRNA for each cell type (ΔCt = Ct target - Ct 18s ). Average of ΔCt of the target gene from tumor derived ECs (Figure 1) was assumed as reference (ΔCt ref ). Fold differences for each cell type were calculated according to the comparative ΔΔCt method (Fold difference = 2 -(ΔCt each celltype-ΔCt ref) ) and expressed as percentage relative to tumor-derived EC (100%).

Article Snippet: In situ hybridization experiments were carried out with a mixture of specific biotin-labeled oligonucleotide antisense (or sense) probes for ADAM23, GPNMB and PRSS3 (Metabion International AG, Martinsried, Germany) listed in the supplementary Table 2 [see Additional file ].

Techniques: Gene Expression, Expressing, Derivative Assay

Journal: BMC Genomics

Article Title: Identification of novel vascular markers through gene expression profiling of tumor-derived endothelium

doi: 10.1186/1471-2164-9-201

Figure Lengend Snippet: In situ hybridization of human tissues . Shown are representative samples of medulloblastoma (A), brain metastasis of an adenocarcinoma (B) and Ewing sarcoma (C), hybridized with antisense probes for ADAM23, GPNMB and PRSS3, respectively. Positively stained blood vessels are indicated by arrows. Related hybridization with control sense probes for ADAM23, GPNMB and PRSS3 is shown in panel D-E-F. Normal brain samples, from different donors, hybridized respectively with antisense probes for ADAM23 (G) and GPNMB (H) are shown. Alzheimer's-diseased brain hybridized with antisense probe for PRSS3 is shown (I).

Article Snippet: In situ hybridization experiments were carried out with a mixture of specific biotin-labeled oligonucleotide antisense (or sense) probes for ADAM23, GPNMB and PRSS3 (Metabion International AG, Martinsried, Germany) listed in the supplementary Table 2 [see Additional file ].

Techniques: In Situ Hybridization, Staining, Hybridization, Control