Prognostic relevance of LGALS3BP in human colorectal carcinoma

Cell lines and culture

The HCT-116 human colon cancer cell line was obtained from ATCC (Rockville, MD, USA).
Cells were maintained in culture for fewer than 6 months after thawing. Cells were
maintained in RPMI-1640 medium (Invitrogen, Carlsbad, CA. USA) with 10% heat-inactivated
fetal bovine serum (FBS; Invitrogen), L-glutamine and antibiotics (Sigma Aldrich Corporation, St. Louis, MO, USA). The cells
were maintained in a humidified chamber with 95% air and 5% CO
₂
at 37°C.

LGALS3BP gene knockdown

A 21-nucleotide sequence corresponding to nucleotide 2216–2236 of human LGALS3BP mRNA
(NCBI Accession NM-005567.3) or a 21-nucleotide sequence with no significant homology
to any mammalian gene sequence serving as a non-silencing control (OligoEngine, Hercules,
CA, USA) were inserted into the pSUPER.retro.puro (OligoEngine). After transformation
of DH5? competent cells (Invitrogen), the recombinant plasmids were confirmed by PCR
amplification, restriction enzymes digestion and DNA sequencing.

The generation of HCT-116 knock-down cells was performed according to the methods
described in our previous report 17].

Enzyme-linked immunosorbent assay (ELISA)

A sandwich-type ELISA (Diesse, Siena, Italy) was used to determine the concentration
of LGALS3BP in the conditioned medium of control- and LGALS3BP-knock-down HCT-116
cells. Culture medium was used a blank control.

Generation of recombinant LGALS3BP

Human recombinant LGALS3BP was immunoaffinity-purified 18] from serum-free supernatant of human embryonic kidney EBNA-293 cells (Invitrogen)
transfected with LGALS3BP cDNA 19]. In brief, the supernatant of the cells (2 L) added with Pefabloc (Boehringer Mannheim,
Germany) and EDTA (1 and 0.4 mM, respectively), was concentrated with a Vivaflow 200
system (Sartorius Biotech Goettingen, Germany) to 50 mL and passed over an affinity
column made of 20 mg of the anti-LGALS3BP antibody (SP-2) covalently coupled to 12 mL
of cyanogen bromide activated Sepharose CL-4B (Sigma Aldrich Corporation). After washing
the column with PBS, bound proteins (95% LGALS3BP) were eluted with 20 mL of 0.1 M
glycine buffer, pH 2.8. Pooled LGALS3BP-containing fractions were dialysed against
PBS and stored in small aliquots at ?80°C. SDS-PAGE showed a major band (90%) migrating
at ~97 kDa. The endotoxin level of the final preparation was 5 EU/?g, as evaluated
by the Lymulus Amebocyte Lysate (LAL) test (Clongen Labs, Germantown, MD, USA).

Confocal microscopy

HCT-116shctrl and HCT-116shLGALS3BP were seeded on glass coverslips and allowed to
grow for 24 h at 37°C in 5% CO
₂
. Cells were incubated with LGALS3BP (10 ?g/mL) for the indicated times, fixed with
4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.25% Triton
X-100 for 5 min and blocked with 0.1% bovine serum albumin for 1 h at room temperature.
Coverslips were then incubated for 2 h at room temperature with a mouse anti ?-catenin
antibody (clone 14/?-catenin, Becton–Dickinson, Franklin Lakes, NJ, USA) followed
by Alexa-Fluor 488 goat anti-mouse secondary antibody (Molecular Probes, Life Technologies,
Paisley, UK). DRAQ5 (Vinci Biochem, Firenze, Italy) was used to visualize nuclei.
Images were acquired with a Zeiss LSM 510 meta-confocal microscope (Zeiss, Oberkochen,
Germany) using 488 and 633 nm lasers. Detector gain voltages and pinhole were set
at the beginning of the experiment and maintained constant during the acquisition
of all samples.

Western blotting

Cells were lysed with RIPA buffer containing protease and phosphatase inhibitors (Sigma
Aldrich Corporation). Lysates were clarified by centrifugation at 14,000× rpm for
15 min at 4°C, subjected to 10% SDS-PAGE and Western blotting using a mouse anti-?-catenin
antibody (Becton–Dickinson), a mouse anti-actin antibody (Sigma Aldrich Corporation)
or a mouse monoclonal antibody against LGALS3BP (3C12.2). Incubation was performed
overnight at 4°C. After washing with PBS containing 0.1% Tween-20, blots were incubated
with a goat anti-mouse HRP-conjugated IgG as a secondary antibody (Biorad, Berkeley,
CA, USA) at room temperature for 2 h and developed with a chemiluminescence detection
system (Perkin-Elmer, Waltham, MA, USA).

Tumor xenografts

All animal studies were approved by the Institutional Animal Ethics Committee. Female
athymic (nu+/nu+) mice (6-week old) (Charles River Laboratories, Milan, Italy) were
acclimatized for 2 weeks before the start of the experiments and housed under specific
pathogen-free conditions. Mice were given ad libitum access to food and water. HCT-116shLGALS3BP
or HCT-116shctrl cells (5 × 10
⁶
) were implanted s.c. into the right flank of the mice (15 mice for HCT-116shLGALS3BP
cells; 9 mice for HCT-116shctrl cells). Tumor volume was monitored twice a week for
a total of 6 weeks by a caliper and calculated using the following formula: tumor
volume (mm
³
) = (length × width
²
)/2. In another set of experiments, animals harboring HCT-116shLGALS3BP xenografts
(approximately 200 mm
³
) were randomly divided into two groups of 10 animals each; one group was injected
intra-tumorally with 50 ?L LGALS3BP (100 ?g), while the other group was injected with
the same volume of PBS. Injections were made twice a week. Animals received a total
of nine injections.

Patient information and tissue specimens

A total of 196 assessable CRCs were collected from patients who received surgical
treatment at the University “G. D’Annunzio”, Chieti, Italy between 1996 and 2010.
Inclusion criteria were: (a) CRC primary cancer; (b) CRC with pathological diagnosis;
(c) informed consent or waiver of consent; (d) age ?18 years; (e) receipt of at least
one follow-up within 5 years. To avoid possible interactions between response to treatment
and LGALS3BP status, only patients not receiving any adjuvant systemic therapy were
included in the study. The clinico-pathological classification and the stage were
determined according to the American Joint Committee on Cancer (AJCC) TNM staging
system. Each lesion was graded histologically according to the WHO classification
criteria. Patients and tumor characteristics are summarized in Table 1. The median follow-up was of 45 months (range 1–176 months). During follow-up, 63
out of 196 (32%) patients developed relapses and deaths were observed in 50 out of
196 (26%) patients. The study was reviewed and approved by Institutional Research
Ethics Committee and written informed consent was obtained from all patients.

Table 1. Clinico-pathological data of 196 patients with CRC

Immunohistochemistry

For the evaluation of ?-catenin expression in mouse xenografts, formalin-fixed and
paraffin-embedded tumor xenografts of HCT-116shctrl (n = 9) and HCT-116shLGALS3BP
(n = 15), were sectioned at 5 ?m and stained using anti-human ?-catenin mouse monoclonal
antibody (BD Transduction Laboratories) at 1:3,000 dilution for 60 min. Antigen retrieval
was performed by microwave treatment at 750 W for 10 min in 10 mmol/L sodium citrate
buffer (pH 6.0). EnVision kit (K4001, Dako, Glostrup, Denmark) was used for signal
amplification. In control sections the specific primary antibody was replaced with
isotype-matched immunoglobulins (Dako).

Tissue microarrays (TMA) were constructed by extracting 2-mm diameter cores of histologically
confirmed neoplastic areas from 196 invasive primary human CRC, as previously detailed
20] TMA sections were stained using the monoclonal mouse anti-human LGALS3BP as previously
reported 8]. Staining of LGALS3BP was quantified as percentage of stained tumor cells. To dichotomize
LGALS3BP expression, a cut-off value of 69% was chosen, which corresponded to the
75th percentile. Therefore, tumors whose percentage of stained cells was ?69% were
considered as low LGALS3BP, all the others as high LGALS3BP. Immunohistochemical analysis
was done by two pathologists (MP, RL) who were blinded to the clinical data of the
patients.

Statistical methods

Two-tailed unpaired T-test was used to compare the statistical significance of the
differences in data from two groups, where appropriate. Disease-free survival (DFS)
was defined as the time from surgery to the first one of the following events: recurrence
at local or distant sites, or intercurrent death without recurrence. Overall survival
(OS) was defined as the interval between the date of surgery and date of death or
the last known follow up. Survival curves were plotted by the Kaplan–Meier method
and compared using the log-rank test. The association of LGALS3BP expression with
outcome, adjusted for other prognostic factors, was tested by Cox’s proportional hazards
model. The following covariates were included in the multivariate models: gender,
tumor location, grade and LGALS3BP status. All statistical analyses were performed
using by the SPSS 15.0 statistical software package (SPSS Inc., Chicago, IL, USA);
p  0.05 was considered as statistically significant.