Animals
Male BALB/c nu/nu mice with 4-6 weeks old were obtained from the Shanghai Institute of Materia Medica, Chinese Academy of Science (CAS), China, and housed in laminar-flow cabinets under specific pathogen-free conditions. The mice were kept for 5-7 days as an adaptation period before being subjected to experimental use. The study protocol on mice was approved by the Shanghai Medical Experimental Animal Care Committee. All animals received humane care according to the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23 revised 1985).
Cell culture
HCCLM3 was established by Dr. Li et al. in our institute [11]. HCCLM3-R with high metastatic potential, used as parental cell line in the study, was maintained in high-glucose Dulbecco’s modified eagle medium (D-MEM; GibcoBRL, Grand Island, NY) supplemented with 10 % fetal bovine serum (GibcoBRL, Grand Island, NY) in a humidified atmosphere of 5 % CO2 at 37 °C.
Human ethics
Human ethics approval was not required for any aspect of this study.
Establishment of HCCLM3-RFP xenograft models
Male athymic BALB/c nude mice with 4 weeks old (Institute of Materia, CAS, Shanghai, China) were injected each with 1 × 107 HCCLM3-R cells in 200 μl 0.9 % sodium chloride solution on the right upper flank region to establish subcutaneous xenograft model. When it reached 1 cm in diameter 3 or 4 weeks later, the subcutaneous tumor was removed, cut into pieces in a size of 2 × 2 × 2 mm3 and implanted into the livers of another 8 nude mice, as described previously [11].
Colonization of single-cell derived HCC population
By virtue of RFP signaling, metastatic tissues were biopsied directly from the lung and lymph node of orthotropic HCCLM3-R xenograft models. The first descendant cells from lung and lymph node metastatic foci, namely LM-1 and LnM-1 respectively, were trypsinized, washed with PBS, and resuspended at 1000 cells/ml. The suspensions were serially diluted in 10-fold volume of fresh media. One hundreds μl of such diluted suspension was added into the well of 96-well plate. After 24 h, these wells harboring a single cell were identified and labeled under microscope. Two weeks later, only colonies derived from one single cell were trypsinized and sub-cultured gradually using a 12-well plate, 6-well plate, and then a 15 cm tissue flask.
Metastatic tropism of monoclonal HCC cells
Each monoclonal HCC cell line was used to establish xenograft models in 6 nude mice. On the 6th week after orthotopic transplantation, all animals were sacrificed for autopsy. Spontaneous metastasis to liver, lung and celiac lymph nodes were imaged under stereomicroscope (Leica, Heerbrugg, Switzerland). The integrated optical density (IOD) of the tumor fluorescence was quantitated by Image Pro Plus software 6.0 (Media Cybernetics, Bethesda, MD). And the metastatic tropism of each monoclonal HCC cell line was plotted using IOD.
Pathological examination
Orthotopic tumor and its metastatic foci were resected for histopathological studies. After fixed with 4 % formaldehyde, lung metastasis tissue were embedded in paraffin and serially cut for hemotoxylin and eosin staining. Tumor metastatic potential in each monoclonal HCC xenograft was analyzed by the following semi-quantitative scoring system as described by Dr. Gao [16], which is advantage to evaluate tumor metastatic potential, even when the metastatic foci in a given organ are overlapped into a cluster. In detail, Grade I: the diameter of metastatic foci in lung were less than 0.5 mm; Grade II: the diameter of foci was between 0.5 and 1 mm; Grade III: the diameter of foci was between 1 and 2 mm; Grade IV: the diameter of foci was more than 2 mm. For a large metastatic tumor, the number of foci was multiple of grade IV. Then, the total metastatic foci number of each monoclonal HCC cell line = grade I × 1+ grade II × 2+ grade III × 3+ grade IV × 4.
Lung colonization assays
A tail vein injection model was employed for lung colonization assays [16]. In brief, nude mice were injected with 1 × 105 of viable parent and monoclonal HCC cells via a lateral tail vein. Three days later, the lungs were removed for making continuous frozen section. The colonized foci of HCCLM3-R and its derived monoclonal cells in lung were quantified by fluorescence microscope.
Cell proliferation assays
Cell proliferation was determined by the Cell Counting Kit-8 assay (Dojin Laboratories, Kumamoto, Japan). In brief, HCCLM3-R and its derived monoclonal cells in exponential growth phase were trypsinized to give single-cell suspension. Then the cell concentration was adjusted into 2 × 104 /ml, and added 100 μl of cell suspension into each well of a 96-well plate. After a cell culture of 0-, 24-, 48-, 72- and 96-h at 37 °C with 5 % of CO2, 10 μl of the kit reagent was added to each well. Two hours later, all plates were scanned by a microplate reader (Thermo Fisher Scientific, Waltham, MA) at 450 nm. Cell proliferation was calculated on the basis of absorbency.
Migration and invasion assays
Cell migration and invasion was analyzed by a Transwell™ Permeable Supports system (Corning, Corning, NY) according to the manufacturer’s instruction. HCCLM3-R and its derived monoclonal cells were seeded into uncoated upper insert at 5 × 104 cells for migration assay and seeded into a Matrigel (BD Biosciences, San Jose, CA) coated upper insert at 1 × 105 cells for invasion assay. Then, the medium containing 10 % fetal bovine serum as a chemoattractant was added into the lower well. Following a culture of 24 or 48 h, non-invading cells were removed from the upper surface by wiping with a cotton swab. The membrane was fixed with 4 % formaldehyde for 15 min at room temperature. The invading cells were stained with Giemsa (Sigma, St Louis, MO) for 25 min, and its numbers in ten fields of each triplicate filter were analyzed by inverted microscope.
RNA extraction and real-time PCR
Total RNA was purified using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA) following the manufacturer’s instruction. The quality of RNA was examined by A260 absorption. And 500 ng of total RNA was reversely transcribed into first-strand cDNA using PrimeScript RT reagent kit (Takara, Tokyo, Japan). Real-time PCR in triplicate was performed on a DNA Engine Opticon system (MJ Research, Reno, NV) using SYBR Premix Ex Taq (TaKaRa, Tokyo, Japan). And the reaction was evaluated with the Opticon Monitor software (Version 1.02). The primer sequences for interested mRNAs were synthesized by Sangon Biotech (Sangon Biotech, Shanghai, China; Additional file 1: Table S1). The threshold cycle (Ct) values were analyzed using the comparative Ct (ΔΔCt) method as previously reported [17]. The level of target mRNA was obtained by normalizing to the endogenous reference and relative to a control.
Western blot analysis
Total cell protein was extracted from HCCLM3-R and its derived monoclonal cells with ProteoJETTM Mammalian Cell Lysis Reagent (Fermentas, Waltham, MA), supplemented with phenylmethanesulfonyl fluoride (PMSF; Roche, Indianapolis, IN). Equal protein amount was loaded onto 10 % SDS-PAGE gels. The protein samples were separated and transferred onto PVDF membranes (Millipore, Billerica, MA) for 2.5 h at 110 V. After blocked with 5 % milk solution in TBS for 1 h at room temperature, the membrane was incubated with rabbit antibodies against VCAM1 (1:1000, Epitomics, Burlingame, CA), SPARC (1:1000, Epitomics, Burlingame, CA), ANGPTL4 (1:1000, Abgent, San Diego, CA) and ITGA1 (1:1000, Abgent, San Diego, CA) overnight at 4 °C respectively. Membranes were washed extensively and then incubated for 1 h with anti-rabbit IgG conjugated to HRP (1:5000). The protein bands were visualized using enhanced chemiluminescence reagents (Thermo Fisher Scientific, Waltham, MA), acquired by Molecular Imager ChemiDox XRS + Imaging System (Bio-Rad Laboratories, Hercules, CA) and quantified with Gel-Pro Analyzer (United Bio, Sanborn, NY).
Immunohistochemistry assays
Immunohistochemistry for target molecules was performed on single serial section made from primary tumor samples. The slides probed with a primary antibody against VCAM1 (1:200, Epitomics, Burlingame, CA), SPARC (1:200, Epitomics, Burlingame, CA), ANGPTL4 (1:100, Abgent, San Diego, CA) and ITGA1 (1:100, Abgent, San Diego, CA), and then incubated with HRP (horseradish peroxidase) -conjugated IgG (1:500, Invitrogen) and the proteins in situ were visualized with 3, 3’-diaminobenzidine. Density of target proteins was determined as previous report [18].
Statistical analysis
All values are expressed as mean ± standard deviation. Data were analyzed by the computer program Graphpad Prism 5 software [16]. The number of lung metastasis in histopathological examinations, were shown by median. Quantitative variables were analyzed by One-way ANOVA, Kruskal-Wallis test or Fisher exact test to compare the qualitative variables. Other comparisons were performed by using unpaired 2-sided Student’s t test without equal variance assumption or nonparametric Mann-Whitney test. Results were considered statistically significant at p < 0.05.