The study material consisted of 136 squamous cell carcinomas, 108 adenocarcinomas, 21 small cell carcinomas, 11 large cell carcinomas and 13 carcinoid tumors. Material was collected from the files of the Oulu University Hospital, Oulu, Finland. The diagnosis of the cases was based on the WHO classification  of lung and pleural tumors. The presence of metastases was determined at the time of the operation.
Immunohistochemistry for claudins, snail and MMPs
Immunohistochemistry for claudins was performed as described earlier Merikallio et al. (2011) . For the immunohistochemical staining of MMPs, four-micrometer sections were cut on slides coated with poly-L-lysine (Sigma-Aldrich, St Louis, MO, USA) and incubated overnight at 37°C. The slides were deparaffinized and hydrated and treated with 0.5% pepsin (2000 FIP-U/g, Merck, Darmstadt, Germany) at 37°C. Endogenous peroxidase activity was blocked by incubating the slides in 3% hydrogen peroxide in absolute methanol and non-specific binding was blocked with 10% goat serum. Mouse monoclonal antibody (24 μg/ml), for MMP 2 (CA-4001, Diabor, Oulu, FI) and mouse monoclonal antibody to MMP 9 (10 μq/ml) (GE-231 Diabor, Oulu, FI) were used as primary antibodies. The slides were incubated with primary antibody at RT overnight after which the Histostain bulk kit was used (Zymed Laboratory Inc., San Fransisco, Ca, USA). The sections were counterstained with haematoxylin and mounted with Immuno-mount (Shanon Inc., Pittsburgh, PA, USA). For negative controls, the primary antibody was replaced by mouse non-immune IgG or PBS.
For snail the staining procedure was as follows. The sections were first deparaffinized and rehydrated in graded alchohol, then heated in microwave oven for 2 × 5 min in Tris-EDTA buffer (pH 9.0), and incubated in Tris-EDTA buffer for 20 min. After being washed two times for 5 min in phosphate buffered saline (PBS), endogenous peroxidase was blocked with 5% hydrogen peroxide for 5 min. Non-specific binding was blocked with 1.5% normal serum in PBS for 35 min at RT (room temperature). The sections were incubated overnight at 4°C with the primary antibody for SNAI1 (monoclonal anti-SNAI1 antibody was a kind gift from the Department of Anatomy, University of Helsinki, Helsinki, Finland) 1: 1000 dilution (Takkunen et al. 2006) and incubated with the biotinylated secondary antibody and avidin-biotinylated peroxidase complex (ABC Vectastain Elite Kit, Vector Laboratories, Burlingame, CA, USA). The colour was developed with diaminobenzidine (DAB) (Sigma-Aldrich). The slides were counterstained with haematoxylin, and mounted with Depex (BDH, Poole, UK). Negative control stainings were carried out by substituting non-immune mouse serum and PBS for the primary antibodies.
The evaluation of stainings was performed by two experienced pathologists for snail and claudins (RK, YS) and for MMPs pathologist (YS) and an experienced investigator (TTH).
For claudins, localisation of reaction product to cell membrane and the characteristics of the immunostaining were recorded and the staining was quantified as follows
0 = negative, 1 = less than 25% of positivity, 2 = 25–50% positivity, 3 = 50–75% positivity and 4 = over 75% positivity.
In the evaluation of the associations the results were divided into weak (0,1) and strong (2–4) positivity.
The immunostainings for MMPs were first assessed by dividing them into five groups (0 = negative, 1–25% = weak, 25–50% = moderate, 50–75% = strong, over 75% = very strong). For the final analysis, the cases with less than 25% of cells were considered as negative and the data was divided in two groups; negative (< 25%) and positive (≥ 25%). In cases of disagreement in the staining assessment, a consensus decision was reached by reviewing the case once more.
With respect to snail, nuclear expression for tumor cells was calculated in the array samples thus giving a value of positivity per array area. In each case, two separate array samples from each case were studied. We also recorded positivity or negativity based on the presence of snail positive nuclei in tumors.
Human non-malignant bronchial BEAS-2B cells which are SV40 transformed cells and human lung carcinoma SK-MES-1 and SK-LU-1 cell lines were obtained from American Type Culture Collection (Rockville, MD). Cells were cultured according to the instructions of the American type culture collection.
Snail knockdown cell lines
Lentiviruses were produced in Phoenix-GP packaging cells (PhGP-cells). PhGP-cells were transfected with lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Cells were given low glucose medium just before transfection. For transfection, a mix of Optimem (Invitrogen, Carlsbad, CA, USA) was prepared containing lipofectamine 2000, the desired retroviral vector Slug 865 (Sigma-Aldrich, St.Louis, MO, USA) and core protein plasmids (REV, pVSV-G and Gag/Pol). The mix was added to the cells and the 24 hours post transfection medium was removed and changed to fresh medium for virus-collection. Viruses were collected three times. BEAS-2B, SK-LU-1 and SK-MES-1 cells were infected with fresh virus-stocks. Polybrene (Sigma-Aldrich, St.Louis, MO, USA) was added in each infection to enhance the efficiency of the infection. After the infections, the infected cells were separated from the normal cells by antibiotic selection. Puromycin (Sigma-Aldrich, St.Louis, MO, USA) was added to the normal mediums of the cell lines to exterminate cells that were not infected.
RT-PCR and result analysis were carried out as described before .
Cells were grown in serum free media and it was collected from the cells, centrifuged and the supernatants were and concentrated with 10 000 cut-off concentration tubes (Millipore) to final volume of 150 μl. Proteins were measured with DC protein assay-kit (BIO-RAD) and 3 μg of each sample were applied to 1,5 mm 10% SDS polyacrylamide slab gels casted in the presence of 1 mg/ml fluorescently (2-methoxy-2,4-diphenyl-3-[2H]furanone) labeled gelatin (Fluka Ronkonkoma, NY) according to the method described by O’Grady et al. 1984. After electrophoresis, SDS was removed by 2.5% Triton X-100 to reactivate the gelatinases. Gels were incubated in 50 mM Tris–HCl buffer (pH 7.8, 150 mM NaCl, 5 mM CaCl2, 1 M ZnCl2) overnight at 37°C. A set of corresponding gels were incubated overnight at 37°C with 10 mM EDTA in 50 mM Tris–HCl to inhibit the metalloproteinase activities. The gelatin degradation was visualized under long-wave ultraviolet illumination followed by 0.5% Coomassie Blue R-250 staining of the gels. The gels were photographed and cleavage rates of gelatin were estimated by determining the rates of disappearance of gelatin by densitometric scanning of the photographed gels.
Invasion of the cells were studied by two different assays, the gel matrix assay and myoma organotypic model. Both original and blocked cell lines were studied in these models. As the gel matrix assay, the Culturex BME cell invasion assay from Trevigen (Gaithersburg, MD) was used and carried out according to the instructions of the manufacturer.
In the myoma organotypic assay 400 000 cells were cultured on the myoma tissue for ten days and fixed with formalin for the histochemistry and immunohistochemistry (Nurmenniemi et al. 2009). The 4 μm thick slides were stained with haematoxylin and eosin and with anti-human cytokeratin (DakoCytomation, Glostrup, Denmark) to evaluate the distance for the maximal invasion depth (the distance from the lower surface of the non-invasive cell layer to the deepest invaded cell) by light microscopy.
Comparison between groups, was made by Mann–Whitney U-test, Kruskall-Wallis test and t-test The significance of the associations was determined by Fisher’s exact probability test and correlation analysis. Survival data were studied by Kaplan-Meier method and Cox regression model. Probability values ≤ 0.05 were considered significant.
The study was approved by the ethical committee of Northern Ostrobothnia Hospital District. The survival data was obtained from the Finnish Cancer Register after receiving permission from the Ministry of Health and Social Welfare and the National Supervisory Authority for Welfare and Health.