towards the further spread of tumor cells from liver metastases warrant further investigations into the roles of cytoskeletal proteins in metastatic disease. Further dissection of the mechanisms by which gelsolin and other cytoskeletal proteins regulate invasive pathways could contribute towards the understanding of how cancer progresses, and the development of effective strategies which counteract its spread. Materials and Methods Cell Lines and Reagents HCT116, HT29, WiDr, RCM-1, RKO, Caco-2, SW837, SW480, SW403, SW620, DLD-1, LS513, COLO201 and COLO205 are human colon cancer cell lines obtained from ATCC. The in vivo-derived metastatic E1 cell line was developed from HCT116, as previously described [31]. HCT116, E1, HT29 and WiDr were cultured in McCoy’s 5A modified medium; Caco- 2, SW480, SW837, SW403 in Dulbecco’s Modified Eagle’s Medium and DLD-1, RKO, LS513, RCM-1, COLO201, COLO205 in RPMI 1640 (All media from Sigma-Aldrich). All media were supplemented with 10% fetal bovine serum (FBS) (Hyclone). Stable HCT116 cell lines overexpressing gelsolin and empty vector control cell lines were grown using the McCoy’s 5A medium with addition of 500 mg/ml Geneticin (Gibco). Cells were maintained at 37uC in a humidified incubated with 5% CO2. Antibodies used include mouse antibodies against human gelsolin (Abcam), b-actin (Sigma-Aldrich), GAPDH (Santa Cruz Bio- technology), pan-cytokeratin (AE1/3) (Dako), uPA (America Diagnostica), uPAR, mouse IgG1 and goat IgG (R&D Systems). Secondary antibody goat anti-mouse IgG conjugated with HRP (Santa Cruz Biotechnology) was used. Metalloproteinase (MMP) inhibitors include GM6001, MMP-2 Inhibitor I, MMP-2/MMP-9 Inhibitor IV, MMP-3 Inhibitor II, MMP-8 Inhibitor I (Calbio- chem) as well as anti-MMP7 antibody (R&D Systems). Construction and Transfection of Gelsolin-overexpression Plasmids A coding sequence of human gelsolin was amplified from a PMW172 expression plasmid containing human cytoplasmic gelsolin cDNA, by polymerase chain reaction (PCR) using the forward primer 59CG GAA TTC ATG GTG GTG GAA CAC CCC GAG TTC 39 and reverse primer 59 CG CCG CGG TCA GGC AGC CAG CTC AGC CAT GGC 39, followed by cloning of gelsolin insert into pIRES2-EGFP vector (Becton Dickinson) at Eco RI and Sac II enzyme restriction sites. The resulting vectors were transformed into competent Escherichia coli XL1B MRF’ cells (Stratagene) and clones were selected based on kanamycin resistance. Sequencing of the cloned gelsolin cDNA verified that the cDNA sequence encodes the human cytoplasmic gelsolin protein (Swiss-Prot accession P06396). To generate stable cell lines, gelsolin- pIRES2-EGFP vector or empty vector were transfected into HCT116 using FuGENE 6 (Promega) and selected using 500 mg/ml G418. Immunohistochemistry 24 primary colorectal tumors and 26 colorectal liver metastases tissues as well as 15 adjacent normal colonic tissues were obtained for use from the Department of Pathology, National University Hospital, Singapore, with approval from the National University of Singapore Institutional Review Board. Briefly, the 4 mm-thick tissue specimens were deparaffinized, boiled in citric acid, and treated with hydrogen peroxide, before incubation with anti- gelsolin primary antibody overnight at 4uC, followed by in- cubation with polymer-horse radish peroxidase-conjugated sec- Gelsolin Induces Cancer Invasion via uPA Cascade ondary antibody at room temperature. Sections were developed with diaminobenzidine (DAB) and counterstained with hematox- ylin. Images were acquired using Olympus BX43F microscope equipped with a DP70 camera. Gelsolin staining in the muscularis propria and stroma [28,29] were used as internal positive controls. The intensity of staining was graded from 0 (undetectable) to 3 (intense staining), whilst the proportion of positive staining tumor cells within a tissue was scored from 0 to 3 where: 1 = ,30%, 2 = 30–60%, 3 = .60% of tumor cells identified. The staining score was expressed as the product of intensity of staining and proportion of tumor positivity. Both cytoplasmic and nuclear staining were scored and summed. The maximum possible score for a sample is therefore 18, the sum of the maximum cytoplasmic (9) and nuclear scores (9). siRNA Transfection 10 nM of the Stealth siRNA of the sequence 59 AAA CGU CCA AUC UUG UUG GAG CAG G 39 (Invitrogen), complexed with lipofectamine, was used to silence gelsolin expression in cells. Medium GC control siRNA which matched the GC content of the gelsolin siRNA used and no-siRNA treatment were included as controls. Cells were harvested at 48 or 72 hours, and treated as used in other assays as described. Matrigel Invasion Assay The transwell assay was carried out as previously described [31]. 56104 cells were seeded for HCT116 and E1 cell lines and 36104 cells for DLD-1, for 48 hours. 10% FBS was used as chemoattractant in the bottom transwell chamber. For function- blocking experiments, cells were seeded with 200 mg/mL anti-uPA or 80 mg/mL anti-uPAR (American Diagnostica). For MMP inhibition treatments, 0.156106 gelsolin-overexpressing HCT116 cells were seeded for 24 hours with 40 mM of MMP inhibitor (either GM6001 or MMP-2, MMP-2/-9, MMP-3 inhibitor), except for MMP-8 inhibitor which was used at 1 mM. Mouse IgG1and goat IgG were included as controls for anti-uPA/anti- uPAR and anti-MMP7 respectively whereas the vehicle control dimethyl sulfoxide (DMSO) was used alongside with the remaining MMP inhibitor treatments. The same protocol was used for migration studies but without the addition of matrigel coating on the transwell membrane. ELISA For measurement of secreted uPA under standard culture condition, cells were incubated in serum-free medium for 48 hours before harvest of culture supernatant for assay. For stimulated conditions, cells were serum-starved overnight prior to incubation with either 5 or 10 ng/mL of human recombinant TNF- a (PeproTech) in fresh serum-free media for 24 hours. As a control for TNF-a stimulation, cells were incubated with serum-free media in parallel. All harvested cell culture supernatants were kept at 220uC until analysis by ELISA. For measurement of secreted uPA under gelsolin-knockdown conditions, cells were treated with siRNA before the serum-starvation step on the following day. Levels of uPA present in the neat supernatants were determined by the human Duoset ELISA Development Kit (R&D Systems), according to the manufacturer’s protocol. Microarray Analysis Microarray analysis was performed on the wild-type HCT116 cell line as well as stably-transfected HCT116 cells including four gelsolin-overexpressing cell lines and two empty vector-transfected control cell lines using Sentrix HumanRef-8 Beadchips (Illumina), PLOS ONE | www.plosone.org 10 August 2012 | Volume 7 | Issue 8 | e43594