All cases and samples were obtained from subjects residing in the Taihang mountain region of north central China. The study was approved by the Institutional Review Boards of the collaborating institutions: Shanxi Cancer Hospital and Institute, Taiyuan, Shanxi Province, China; and the National Cancer Institute, Bethesda, MD, USA.
Resection specimens from 24 ESCC patients (for clinical data refer to Table ) treated at the Shanxi Cancer Hospital in Taiyuan, Shanxi Province were blocked and stored at -70°C until assays could be performed. Serial 8-micron frozen sections were cut from each tissue block using a Leica Cryostat and representative foci of patient-matched normal mucosa (N = 24) and invasive squamous cell carcinoma (N = 24) were chosen based on histological review of hematoxylin-and-eosin-stained slides by two pathologists (J.R.C. and R.F.C.) using accepted criteria.
Gelatin zymography was performed as previously described with some modifications [14
]. 10 μl of tissue lysate containing 8 μg of protein, determined using the Micro BCA™ Protein Assay kit (Thermo Scientific/Pierce, Rockford, IL), was mixed with an equal volume of Novex®
Tris-glycine SDS native sample buffer (Invitrogen™ Carlsbad, CA, USA) and the mixture was loaded into wells of pre-cast 10% Novex®
zymogram gelatin gels (Invitrogen™). Pre-stained molecular weight standards were also run on each gel. The gels were electrophoresed at a constant voltage of 125 V for approximately 2 h.
Following electrophoresis, the gels were rinsed in distilled water and then gently shaken in a renaturing solution of 2.7% Triton X-100 (Novex® zymogram renaturing buffer, Invitrogen™) for 1 h at 37°C to reactivate MMPs. The gels were then incubated on a rotary shaker in a developing buffer (Novex® zymogram developing buffer, Invitrogen™) for 24 h at 37°C to allow denatured MMPs to digest the gelatin substrate. After the digestion phase, the gels were rinsed and stained by incubation with Coomassie Blue Rapid stain (Diversified Biotech, Boston, MA, USA) for 1 h. Gels were destained with a solution of acetic acid, methanol and water (10: 50: 40) to maximize contrast between proteolytic areas and non-digested areas. Proteolytic activity was visualized as areas of clear bands against a dark blue background. The identity of the proteases was determined by analysis of the distance that the bands migrated on the gels, compared with the distance for migration of molecular weight standards.
Laser Capture Microdissection
Serial frozen 8-μm sections were cut using a Leica Cryostat and placed onto uncharged glass slides. Every sixth slide was stained using hematoxylin-and-eosin and the histology confirmed by a pathologist (R.F.C. or J.R.C.). The remaining slides were stored at -80°C, not to exceed two weeks prior to dissection. The slides were placed on dry ice and then were stained as follows: 70% ethanol for 15 seconds, Mayer's hematoxylin (Sigma-Aldrich, St. Louis, MO) for 15 seconds, deionized water and bluing solution (Sigma-Aldrich) for 10 seconds each, and eosin (Sigma-Aldrich) for five seconds followed by dehydration using increased concentrations of ethanol (95%, 95%, 100% and 100%) for 10 seconds each. Tissue was then placed in xylenes for 20 seconds to complete the dehydration process.
LCM was performed using the PixCell IIe (Arcturus Engineering, Inc., Mountain View, CA) to isolate neoplastic epithelium and tumor stroma separately. Tumor-associated stromal fibroblasts and matrix were collected from locations proximate to epithelial tumor cells, being within 5 mm of an epithelial tumor nodule. Normal epithelial and stromal cells were similarly collected from histologically normal tissues. The time from slide removal from dry ice to completion of LCM did not exceed 30 minutes. On average, epithelial dissections required 3,000 shots (laser spot specifications: 30 μm spot size, 45-55 mW power, 3.0-4.0 ms duration); whereas stromal dissections required 4000 - 5000 shots.
Total RNA was isolated with the PicoPure RNA Isolation kit (Arcturus Engineering) as suggested by the manufacturer. RNA quantity was assessed using NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE). RNA quality, both 28S/18S ratio and RNA integrity number (RIN), was measured using the 2100 Bioanalyzer (Agilent Technologies, Inc., Palo Alto, CA) (Table-2).
Total RNA was used to generate complementary DNA (cDNA) using the Taqman High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Inc., Foster City, CA, USA Cat # 4374966) as suggested by the manufacturer to get the maximum expression of transcripts. Singleplex qPCR was performed after first strand cDNA synthesis using 2× Taqman Universal PCR Master Mix (Applied Biosystems, Inc., Cat#4364338) and Amplitaq Gold DNA polymerase, LD (Applied Biosystems, Inc., Cat#4338857) and specific primer/probe sets (Applied Biosystems, Inc.). Five cases were tested with commercially available optimized primer/probe sets for MMP-3 [TaqMan Gene Expression Assays, Inventoried Assay ID: Hs00233962 for MMP-3 (stromelysin-1, progelatinase), Applied BioSystems, Inc., Cat.# 4331182] and MMP-10 [TaqMan Gene Expression Assays, Inventoried Assay ID: Hs00233987 for MMP-10 (stromelysin-2), Applied BioSystems, Inc., Cat.# 4331182] gene expression levels. All primer and probe sets are cDNA specific. All qPCR assays were performed in triplicate after reverse transcription. Beta-actin (ACTB), a known housekeeping gene, was used for normalization. Taqman primer/probe sets and master mix reagents were procured from Applied Biosystems (Foster City, CA).
Each reaction was conducted in a 20 μl volume using Applied Biosystems 7500 Real-Time PCR system (Foster City, CA). Cycling conditions consisted of one cycle of 50°C for 2 min followed by 95°C for 10 min, and then 50 cycles of 95°C for 15 seconds followed by 60°C for 1 min. Controls consisting of total human esophageal RNA (100 ηg/μl; Ambion, Austin, TX, USA) were positive in all runs, and controls consisting of sterile molecular grade water were negative in all runs. Critical threshold (Ct) cycle numbers were obtained for amplification of MMP-3, MMP-10, and ACTB. ΔCt values were calculated by subtracting the average Ct value of ACTB from the average Ct value of MMP-3 and MMP-10 in each case. Relative quantitation analysis of gene expression data was conducted according to the 2-ΔΔCT