Yeh et al. BMC Cancer 2011, 11:139 http://www.biomedcentral.com/1471-2407/11/139 (ECL) detection system (Perkin Elmer Life Sciences, USA). siRNA transfection Specific siRNA sense sequences were as follows: c-Met siRNA: 5`-AAGTGCAGTATCCTCTGACAG-3`, Axl siRNA: 5`-CGTGGAGAACAGCGAGATTTA-3`, and PDGFRa siRNA: 5`-CGAGACGATTGATGCAG- GATA-3`. The cells (5 × 105) were seeded into a 6-cm cell culture dish and incubated in DMEM medium with- out antibiotics. Lipofectamine 2000 reagent (10 μl) was diluted in 500 μl of DMEM serum-free media and incu- bated for 5 min at RT. The siRNA was diluted in 500 μl of DMEM serum-free medium to the assigned concen- trations. Mock transfection was conducted in parallel using distilled water as the negative control. Then cells were incubated at 37°C in the 5% CO2 incubator for 4 h. The media were replaced with normal media and cells were incubated for additional 48 h before protein extraction. Trans-well migration assay The effect of RTK cross-talk on cell migration was ana- lyzed in TSGH8301 bladder cancer cells using a 24-well Transwell™ system (Corning inc., Lowell, MA). Briefly, cells were cultured in a 6-cm plate and transfected with c-Met, Axl, or PDGFR-a siRNA for 24 h, respectively. Then, cells were resuspended with serum-free medium and added into the upper chamber of the trans-well insert (2 × 105 cells/well). The 10% FBS-containing DMEM was added in the lower chamber. Cells were incubated h at 37°C for 36 h. Migrated cells were fixed with 4% formaldehyde in PBS and stained with 2% crys- tal violet in 2% ethanol. The non-migrated cells in the upper chamber were removed by wiping with a cotton swab. The cells on the lower surface of the filter, repre- senting migration of the cells through the membrane, were counted under a light microscope. Clinicopathological characteristics of study cases Since c-Met is important in the progression of bladder cancer, both locally advanced and metastatic bladder tumors were recruited to evaluate the significance of co- expression patterns of c-Met and other RTKs. Archival material of 65 patients (44 men and 21 women; age range, 40 to 84 yr old; mean ± SD, 61.5 ± 9.4 yrs) with locally advanced or metastatic urothelial bladder cancer (21pT2, 27pT3, 17pT4) was analyzed for RTK expres- sion. These patients were diagnosed and treated in the National Cheng Kung University Hospital, Tainan, Tai- wan, between 1990 and 1999 years. The numbers with low or high grade urothelial carcinoma were 20 and 45, respectively, according to definitions described pre- viously [34]. Seven patients received partial cystectomy Page 3 of 12 and remaining fifty-eight patients received radical cystectomy and bilateral pelvic lymph node dissection. Among them, 23 (35.4%) patients had pelvic lymph node involvement. An adjuvant systemic chemotherapy, including methotrexate, vinblastine, epirubicin, and cis- platin (M-VEC regimen), was given to 20 patients (30.8%) after radical cystectomy. The survival status was determined by outpatient clinic records and/or con- firmed by interview with patients’ families. Clinical fol- low-up ranged from 26 to 140 months (mean ± SD: 50.02 ± 6.46 months). The time of the first tumor recur- rence and for disease specific survivals were counted. The time is calculated until the death of the patient due to bladder cancer. Patients who died of other causes or were still alive at the last follow-up were censored. Immunohistochemical staining Immunostaining procedures were described in detail previously [6]. Briefly, tissue sections were incubated at RT for 2 h with monoclonal anti-cMet (1:100 dilution; Santa Cruz), anti-AXL (1:10 dilution; Santa Cruz) and anti-PDGFR-a (1:200 dilution; Santa Cruz) antibodies raised against the membrane protein. The optimal dilu- tion was determined by using human kidney as a posi- tive control [5]. Then StrAviGen Super Sensitive MultiLink kit (BioGenex Laboratories, Inc., San Ramon, California) was used to detect the resulting immune complex. Peroxidase activity was visualized using an aminoethyl carbazole substrate kit (Zymed Laboratories, Inc., San Francisco, Califonia). Because no apparent difference of staining intensity was detected, only the proportion of tumor cells stained for c-Met, Axl or PDGFR-a was considered in classifica- tion [6]. “High level of expression” indicates > 50% of the tumor cells were immunostained, “low level of expression” indicates 10%-50% reactivity; and “negative” indicates < 10% staining for RTK protein. Statistical analysis The association between tumor staging or gross charac- teristics with expression status of c-Met, Axl, and PDGFR-a was analyzed by Chi-square test as appropri- ate. The correlation between co-expression patterns of RTKs and disease-specific survival of cancer patients was constructed according to Kaplan-Meier method by Log rank test. Results Establishment of stable cell lines harboring inducible c- Met gene Two stable cell lines, designated as NIH-Met5 (mouse fibroblast) and T24-Met3 (human bladder cancer cell), were established to harbor the inducible c-Met gene, which was expressed only in the absence of tetracycline