Overexpression of the ITGAV Gene Is Associated with Progression and Spread of Colorectal Cancer

Patients and Methods

Patients and tumor samples. We studied 114 patients with CRC who underwent primary tumor resection at the Barretos Cancer Hospital (São Paulo, Brazil) between August 2006 and July 2009. All patients underwent the analysis of the expression of genes of interest in real-time PCR (RT-PCR) and immunohistochemistry (IHC) using the tissue microarray technique (TMA).

We included patients of both genders, aged ≥18 years old. Patients who had received neoadjuvant treatment (chemotherapy or radiotherapy) were excluded, as well as those without primary CRC site resection, those with previous or current diagnosis of another primary malignancy in any location of the body other than non-melanoma skin cancer or in situ carcinoma of the cervix and the patients with a known history of familial CRC.

Sixty-three (55.3%) patients were male and 51 (44.7%) female. Their median age was 60 years old (24-83). Both the right and left colons were affected in 41 cases (36.0%) each and the rectum was the primary tumor site in 32 cases (28.0%). Twenty-five (21.9%) patients were in tumor-node-metastasis (TNM) stage I, 39 (34.2%) were classified as TNM stage II, 34 (29.8%) as TNM stage III and 16 (14.0%) as TNM stage IV.

Table I shows the distribution of patients according to the categorization of the clinic and pathologic co-variables.

Covariates' analysis. Patients were classified according to the following clinicopathological characteristics: age group (<60 and ≥60 years old), histologic classification (adenocarcinoma not otherwise specified vs. mucinous adenocarcinoma), subtype tubular or villous, tumor grade [low (grades I and II) vs. high (grades III and IV)] and peritumoral lymphocyte infiltration (presence vs. absence).

Histological characteristics commonly associated with tumor dissemination and progression have been categorized as follows: venous invasion (presence vs. absence), lymphatic vessel invasion (presence vs. absence), perineural invasion (presence vs. absence), tumor stage (T1-2 vs. T3-4), lymph node metastasis (presence vs. absence), distant metastases (presence vs. absence) and TNM staging (I-II vs. III-IV) (14).

RNA extraction and cDNA synthesis by RT-PCR. Cryopreserved samples were embedded in a special medium for frozen tissue specimen conservation (Tissue-Tek OCT; Sakura Finetek, Torrance, CA, USA). After discarding inappropriate areas for RNA extraction, the tissue was mechanically macerated with liquid nitrogen and transferred to 1.5-ml microtubes free of RNAse and DNAse containing 1.000 μl TRIzol (Invitrogen, Carlsbad, CA, USA). RNA was extracted according to the manufacturer's instructions and its quantification was performed using a spectrophotometer (Thermo Scientific NanoDrop 2000, Waltham, MA USA). The quality and integrity of the RNA were verified by the presence of 28S and 18S bands in agarose gel stained with 1% ethidium bromide to guarantee the absence of RNA samples degradation. RNA was purified with the RNeasy mini kit (Qiagen, Valencia, CA, USA) following the manufacturer's recommendations, eluted with 30 ml of water RNAse- and DNAse-free (Qiagen, Valencia, CA, USA), quantified in the spectrophotometer at a wavelength of 260 nm (NanoVue; GE Healthcare, Chicago, IL, USA) and stored at −80°C until use. RT-PCR was performed using the Super-Script™ III first-strand synthesis SuperMix (Invitrogen, Carlsbad, CA, USA), as recommended by the manufacturer. The reaction was carried out in a 20-μl final volume containing 2 μg of total RNA with oligo (dT) (20) as a primer. The transcription phase was carried out in a thermal cycler (Mastercycler® ep Gradient S; Eppendorf, Hamburg, Germany) and the cDNA was stored at −20°C for future reactions.

Analysis of the genes of interest. For each sample, an ECM and adhesion molecule PCR array (PAHS-013; SABiosciences, Qiagen, Valencia, CA, USA) plate was used. A mixture containing 1,275 μl of buffer with SYBR Green (2× Master Mix RT2 qPCR; SABiosciences, Qiagen, Valencia, CA, USA), 1,173 μl RNAse-free H₂O and 102 μl of cDNA sample was prepared. Afterwards, 25-μl aliquots were added to each well of a 96-well plate. Reactions were performed in a thermal cycler (ABI 7500; Applied Biosystems, Foster City, CA, USA), according to the following protocol: 95°C for 10 min, 40 cycles at 95°C for 15s and 60°C for 1 min. Data analysis was performed according to http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php. Expression of each gene was classified as ‘high’ or ‘low’, considering the level of expression obtained after grouping patients by the covariates of interest after categorizing patients into the control or interest groups according to the covariates studied.

Tissue microarray (TMA) block construction. Original paraffin blocks were sectioned at 4-μm thickness and hematoxylin and eosin (HE) stained. All sections were reviewed to confirm the diagnosis of CRC and the histopathology findings were re-evaluated.

With the aid of Beecher™ equipment (Beecher Instruments, Silver Spring, MD, USA), TMA blocks were prepared according to the manufacturer's specifications. The samples were cut to 4-μm thickness and a small roll was used to press the section on the tape. The tape with the attached histological section was then placed on a resin-coated slide (part of the adhesive system kit) and pressed with the same roll for better adherence. Afterwards, the slides were placed under UV light for 20 min and then exposed to a solvent solution (TPC) for 20 additional min. The slides were dried and the tapes removed. Subsequently, the slides were paraffin embedded and sent for storage in ideal cooling conditions.

IHC technique. Endogenous peroxidase activity was blocked by incubating the sections in a methanol bath containing 3% hydrogen peroxide for 20 min, followed by a washing in distilled water. The sections were initially submitted to heat-induced epitope retrieval using citrate buffer (pH 9.0) in an uncovered pressure cooker (Eterna®; Nigro, Araraquara, SP, Brazil).

Blocking of endogenous peroxidase was obtained with 3% H₂O₂ (10 vol.), with 3 washes of 10-min each. The slides were again washed in distilled running water and then in phosphate-buffered saline (10 mM; pH 7.4) for 5 min. Subsequently, the primary antibody was applied and the slides were incubated overnight at 8°C.

Primary antibodies. Three primary antibodies to ECM components were imported from Abcam (Cambridge, MA, USA): anti-SPARC antibody, rabbit IgG isotype, polyclonal, 1: 400 (code: ab14174); ITGAV, mouse IgG1 isotype, clone 272-17E6, 1:400 (code: ab16821); THBS1, mouse isotype IgG1, clone A6, 1:400 (code: ab1823); and VCAM-1. One of them was imported by Santa Cruz Biotechnology (Dallas, TX, USA): mouse IgG1 isotype, clone B-N8, 1:400 (code: BMS 141).

Additionally, the following non-ECM primary antibodies were used in this study: anti-p53 antibody, IgG2b class, clone DO-7, 1:300 (reference: M7001); anti-Bcl-2 antibody, mouse IgG1 isotype, clone 124, 1:600 (reference: M0887); anti-VEGF antibody, mouse IgG1 isotype, clone VG 1, 1:100 (reference: M7273); anti Ki67 antibody, mouse IgG1 isotype, clone MIB-1, 1:500 (reference: M7240), all of them from DAKOCytomation (Glostrup, Denmark); and anti-EGFR antibody, mouse IgG1 isotype, clone EGFR-25, 1:100 (reference: NCLEGFR-384) from Novocastra (Newcastle, UK).

The IHC analysis positive controls were the following: osteosarcoma for SPARC; human tonsils for ITGAV, THBS1, VCAM-1, VEGF, Ki67, p53, and Bcl-2, and placenta for EGFR.

Immunostaining analysis. Tissue expression of SPARC, ITGAV, THBS1, VCAM-1, EGFR, VEGF, p53, Bcl-2 and Ki67 markers were categorized dichotomously as ‘overexpression’ or ‘low expression’ according to the ‘quick score’ method (15, 16). This score system uses a combination of the percentage of stained cells (P) and intensity of staining (I); the ‘quick score’ was calculated by multiplying both values.

The scores used for the percentage of stained tumor cells were as follows: 0 points (absence of stained cells), 1 point (stained cells ≥25%), 2 points (stained cells from 26 up to 50%) and 3 points (stained cells >50%). Scores used for the intensity of the cancer cell staining were as follows: 1 point (mild intensity), 2 points (moderate intensity) and 3 points (intense staining). As a result, expression of a gene product in tumor cells was considered to be high (overexpressed) when the final score was >4 (P × I >4) and the markers that presented a final score ≤4 were considered to have low expression. Stromal and tumor cells were not treated separately during IHC analysis and only the level of expression of markers on tumor cells was considered for scoring.

Statistical analysis. Statistical associations between gene and protein expression levels of SPARC, ITGAV, THBS1 and VCAM-1, as well as the clinicopathological factors were determined using a non-parametric Mann-Whitney U-test for quantitative variables and a Chi-square (χ²) test for qualitative variables or frequencies and proportions. When the two assumptions were not met, the Fisher's exact test was used.

To measure the association between ECM markers (SPARC, ITGAV, THBS1 and VCAM-1) and non-ECM markers (EGFR, VEGF, p53, Bcl-2 and Ki67) as ordinal variables, the Spearman correlation coefficient was used. The Shapiro-Wilk test was used to verify the data's normal distribution.

The statistical significance level was set at 5% (p<0.05) and the data were analysed using the SPSS software™ (Statistical Package for Social Sciences; SPSS, Chicago, IL, USA), version 15.0.