HIF-1α expression in liver metastasis but not primary colorectal cancer is associated with prognosis of patients with colorectal liver metastasis

The HCT116 colon cancer cell line was obtained from the Riken Cell Bank, and the hepatic stellate cell line LX2 was obtained from Cellular Engineering Technologies Inc. HCT116 cells were cultured in McCoy’s 5A Modified Medium (Life Technologies Ltd., Tokyo, Japan) with 10% fetal bovine serum (FBS) (Life Technologies Ltd.). LX2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies Ltd.) with 10% FBS. Both cell lines were cultured under 37 °C in 5% CO_2.

HCT116 cells (3.0 × 10⁶ cells) were cultured in McCoy medium in a 10-cm dish until cell numbers reached 3.0 × 10⁵ cells. The cell culture media was then changed to CM from cancer cells for 24 h of culture. The medium of HCT116 (3.0 × 10⁶ cells) changed to activated HSCs conditioned medium (HSC-CM) or DMEM (control) for 24 h culture. After that, the FBS-free medium culture was followed. After 24 h, HCT116 cells were collected for experimental analyses.

Cells were plated in 6-cm dishes at 3.0 × 10⁶ cells/dish. The medium was replaced with activated HSC-CM or DMEM (control) for 24 h. After the cells had reached confluency, a plastic pipette tip was drawn across the center of the plate to produce a scratch that was 1 mm in width. After 24 h of culture in medium with 1% FBS, a phase contrast microscope was used to examine cell movement into the wound area.

RNA was extracted from samples using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cDNA was synthesized using a reverse transcription kit (Applied Biosystems, Foster City, CA, USA). The HIF-1α TaqMan gene expression assay (Hs00153153_m1, Applied Biosystems) was used, and GAPDH (4326317E, Applied Biosystems) was selected as an internal control. The StepOnePlus Real-Time PCR System (Applied Biosystems) was used to perform qRT-PCR.

RIPA buffer (Thermo Fisher Scientific Inc.) containing both protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA) and the PhosSTOP phosphatase inhibitor cocktail (Roche, Tokyo, Japan) was used to lyse cells. Protein concentrations were measured with the BCA kit (Thermo Fisher Scientific Inc.), and equal amounts of extracted proteins were separated on 10% SDS-PAGE gels and transferred onto PVDF membranes (Bio-Rad Inc., Hercules, CA, USA). The membranes were incubated with the indicated primary antibody, followed by the appropriate HRP-conjugated secondary antibody. The bands were detected by chemiluminescence (Thermo Fisher Scientific Inc.). Primary antibody against HIF-1α (diluted 1:1000; HPA001275) was purchased from Sigma-Aldrich, and primary antibody against β-actin was obtained from Sigma Chemical (St Louis, MO, USA).

Seventy-five CRLM patients who underwent an initial hepatectomy at our institute from 1994 to 2015 with available surgical specimens of primary CRCs and the matched liver metastases were enrolled in this study. This study was authorized in advance by the Institutional Review Board of the University of Tokushima Graduate School (approval ID number: 2392), and all patients provided written informed consent. The participants in this study included 47 males and 28 females with a mean age of 66.5 years, ranging from 33 to 90 years in age. The numbers of patients with synchronous and metachronous liver metastases were 32 (43%) and 43 (57%), respectively. Staging and curability was defined according to the Classification of Primary Colorectal Cancer by the Colorectal Cancer Study Group of Japan [30]. T-factor was determined by tumor number, size, and vascular infiltration. Tumor stage was determined by T-, N-, and M-factors. We defined H class as the following classification: H0 class, No metastasis to liver; H1 class, ≦ 4 lesions and ≦ 5 cm; H2 class, other than H1 and H3; H3 class, > 5 lesions and > 5cm [31]. We divided surgical procedure into minor and major hepatectomy, and major hepatectomy was defined as resection of four or more liver segments [32]. All patients had not received neoadjuvant chemotherapy and follow-up period had started after hepatectomy. The mean follow-up period was 41.3 months (range 4.4–191.3 months). We examined clinicopathological features, prognosis, molecular biological malignancy, 5-year overall survival (OS), and disease-free survival (DFS).

Paraffin sections (4 μm) were cut from archival formalin-fixed paraffin-embedded tissue blocks. The samples were deparaffinized and dehydrated using a graded series of ethanol solutions. Endogenous peroxidase activity was stopped through the administration of 0.3% hydrogen peroxidase and methanol for 20 min. After rinsing in phosphate-buffered saline (PBS; Fisher Scientific, Pittsburgh, PA, USA), the tissue sections were processed in a 0.01 M citrate buffer (pH 6.0) inside a heat-resistance plastic container. The sections were irradiated in a microwave oven for 25 min and then allowed to cool at room temperature. The sections were incubated with primary mouse monoclonal antibody against HIF-1α (1:500; HPA001275, Sigma-Aldrich, MO, USA) overnight at 4 °C in a humidified chamber. The sections were incubated using Daco REAL^TM Envision^TM/HRP, Rabbit/Mouse (ENV), for 45 min followed by three washes in PBS. Peroxidase labeling was developed by incubating the section in 3,3′-diaminobenzidine tetrahydrochloride (DAB) for 5 min. Nuclear counterstaining was completed using Mayer’s hematoxylin solution. Cell counts were performed using a Nikon Digital Camera DXM 1200F photomicroscope at a magnification of × 200 (× 20 objective and × 10 eyepiece). The area counted in each section was randomly selected from the representative tumor field. For each section, eight areas were assessed. The staining score for HIF-1α was determined based on staining intensity (0 negative, 1 low, 2 medium, 3 high) and staining area (0, 0%; 1, 0–25%; 2, 26–50%; 3, ≥ 51%). Scores over 4 points were defined as positive expression (Fig. 1).