Cell lines and animal experiments
CT26 human colon adenocarcinoma cells were cultured in RPMI supplemented with 10% FCS, 2 mM glutamine, 100 IU/mL penicillin, and 50 mg/mL streptomycin. Liver metastases were induced in 6 to 8-week-old BALB/c mice by intrasplenic injection of tumor cells. Briefly, a small upper quadrant incision was used to expose the spleen, and 1 × 106 cells in 50 μl were injected into the lower splenic pole with a 30.5 gauge needle. The spleen was returned to the abdominal cavity, the peritoneum was closed by suture, and the skin with wound clips. Two weeks after tumor cell inoculation, the animals were sacrificed, and total liver weights were determined. Animal experiments were performed according to official guidelines, with permission (by regional board Karlsruhe) under File No. 35-9185.81/G-50/05.
Tissue preparation, laser microdissection (LMD) and microarray hybridization
Frozen tissue blocks were cut into 15 μm sections using a cryostat (Leica, Wetzlar, Germany) and stained using cresyl violet, according to the Ambion LCM staining kit protocol (Austin, TX, USA). Four distinct cell populations were separately microdissected with LCM equipment (Molecular Machines & Industries, Eching, Germany; or PALM, Bernried, Germany): (a) pure liver tissue, at least 5 cm away from the invasive front; (b) liver invasive front tissue, extending up to 10 cell layers into the liver; (c) tumor invasive front tissue, extending up to 10 cell layers into the tumor; and (d) pure tumor tissue, at least 100 cell layers away from the invasive front. These compartments were arbitrarily selected due to prior experience and results from immunostaining of up-regulated genes (unpublished data). Microdissection was performed to yield sufficient material for microarray and qPCR analysis.
Total RNA from microdissected samples was extracted (RNeasy mini kit; Qiagen, Hilden, Germany), and quality was evaluated using an Agilent 2100 bioanalyzer (Waldbronn, Germany). For microarray analysis, 30 ng of RNA corresponding to 2500-3500 cells from each microdissected group were amplified (RiboAmp HS RNA amplification kit; Arcturus, Sunnyvale, CA, USA), labeled, and the resulting biotinylated cRNA targets were used to probe the murine genome MOE430 set (A + B) (Affymetrix, Santa Clara, CA). Hybridization was performed in duplicates. Altogether, 8 chips were hybridized (2 compartments × 2 sub-chips [A + B] × 2 [duplicates] = 8).
For estimation of the percentage of tumor tissue as compared to pre-existent liver parenchyma, whole livers were embedded in paraffin and hematoxylin/eosin stained according to standard procedures.
Raw files (cel files) from the scanned images of the Affymetrix chips (run in duplicates) were normalized using Affymetrix GCOS software, and fold changes were calculated using Excel (Microsoft, Seattle, USA) software.
Relative quantitative real time-PCR
Microdissection and RNA isolation for relative qPCR were essentially performed as for hybridization experiments; however, independent samples were used. Thirty nanograms of total RNA, corresponding to 2500-3500 cells were used for quantification. Reverse transcription, qPCR, normalization (on 18S RNA), and efficiency correction (on 18S RNA) were performed. Oligonucleotides for 18S RNA and IL-8 qPCR were designed using the Primer3 software (Whitehead Institute, Cambridge, MA, USA). The sequences for 18S RNA were as follows: forward primer, 5'-AAA CGG CTA CCA CAT CCA AG-3'; reverse primer, 5'-CCT CCA ATG GAT CCT CGT TA-3'. Primers for TIMP-1 were purchased (Cat No. QT00996282; QuantiTect® primers, Qiagen GmbH, Hilden, Germany). All the experiments were done in triplicate and repeated twice.
ShRNA-mediated down-regulation of TIMP-1
Cells were transfected with pSM2C plasmids containing 2 different TIMP-1 shRNAmir constructs (TIMP-1 ShRNA-317 and TIMP-1 ShRNA-234) (Open Biosystems, AL, Huntsville, USA) or a non-silencing shRNAmir construct, according to the manufacturer's instructions. Briefly, 2 × 105 CT-26 cells were seeded into 6 well plates and subsequently transferred using Arrest-In™ transfection reagent (Open Biosystems) with the indicated shRNAmir (10 μg transfection solution containing 2 μg TIMP-1 shRNAmir or 2 μg control siRNA). After 6 h, the medium was replaced by standard culture medium. The cells were returned to the CO2 incubator at 37°C. Forty-eight hours later, the cells were grown in a complete medium supplemented with puromycin (2 μg/mL) for selection. Single clones were then selected from pooled populations by serial dilutions using standard protocols. Successful shRNA transfection was confirmed by qRT-PCR and ELISA. All the experiments were performed in triplicates and repeated twice.
Over-expression of TIMP-1
Lentiviral TIMP-1 viruses (LvhuTIMP-1) were generated by co-transfection of pLenti6/V5-DESThuTIMP-1 vector containing human TIMP-1 under the control of the CMV promoter with the ViraPower™ packaging plasmid mixture: pLP1, pLP2, and pLP/VSV-G (Invitrogen) into 293FT cells using Lipofectamine 2000 (Invitrogen). CT26 cells were infected with LvhuTIMP-1 viruses and transduced cells were selected by blasticidin (5 μg/mL).
Enzyme-linked immunosorbent assay (ELISA) was used to determine the concentration of TIMP-1 in TIMP-1 over-expressing and down-regulated cells compared to the wild type control cells and non-coding shRNA control cells using Quantikine human and mouse TIMP-1 assay kit according to the manufacturer's instructions (R&D systems, Wiesbaden, Germany). All the experiments were performed in triplicates and repeated twice.
Statistical analysis and software
Data are presented as the mean ± standard deviation. The statistical comparison between groups of animal experiments was accomplished with the non-parametric Wilcoxon test.