Supplementary Table 1: Clinico-pathological data of primary CRCs
Parameter
Category
n (%)
Age (years)
Median
Range
Male
Female
Alive
Dead (cancer related)
Dead (unrelated causes)
Unknown
Adenocarcinoma
Mucinous adenocarcinoma
Columnar adenocarcinoma
Signet ring mucinous
adenocarcinoma
Unknown
Well differentiated
Moderately differentiated
Poorly differentiated
Unknown
Colon
Rectal
Unknown
A
B
C1
C2
D
Unknown
0 (Tis)
1
2
3
4
Unknown
Negative
Positive
Unknown
72
57-89
257 (57%)
192 (43%)
167 (37%)
221 (49%)
60 (13%)
1
382 (85%)
49 (11%)
4 (1%)
6 (1%)
Sex
Status
Histological type
Histological grade
Tumour site
Duke’s stage
TNM stage
Extramural vascular
invasion
8 (2%)
28 (6%)
345 (77%)
67 (15%)
9 (2%)
230 (52%)
177 (39%)
42 (9%)
66 (15%)
175 (39%)
133 (30%)
20 (4%)
52 (11%)
3 (1%)
3 (1%)
67 (15%)
172 (38%)
149 (33%)
51 (11%)
7 (2%)
219 (49%)
121 (27%)
109 (24%)
The patients had a median age of 72 years (range 45–80 years) with a median follow-up of 37
months (range 0-116). Fifty percent of patients were male while 43% were female. Regarding
the histological type, the majority of tumours 345 (77%) were moderately differentiated
adenocarcinomas, 28 (6%) well differentiated tumours, and 67 (15%) were poorly
differentiated. Vascular invasion was present in 121 (27%) of cases, while 219 (49%) had no
evidence of vascular invasion, and this information was not available for the remaining 109
(24%) of cases. A total of 221 (49%) patients died from CRC, 60 (13%) died due to other
causes, while 167 (37%) of patients were alive at the end of follow-up.
1
Supplementary Table 2: Association of nuclear Cten expression with
tumour metastasis
Type of tumour deposit
Low nuclear Cten
expression
High nuclear Cten
expression
Primary
Metastasis
36
25
4
15
A series of 40 paired primary and metastatic tumours were evaluated for Cten expression by
immunohistochemistry. Positive nuclear expression of Cten was associated with liver
metastasis (Fisher’s exact test, p=0.002)
2
Materials and methods
Tissue microarray analysis (TMA) and Immunohistochemistry (IHC)
A tissue microarray analysis (TMA) of primary tumours was prepared from a
series of 462 cases of primary operable CRC from patients who underwent
elective surgery between 1st January 1993 and 31st December 2000 at the
Nottingham University Hospitals, Queen’s Medical Centre, Nottingham as has
been previously described(1, 2). This resource has been well characterised
and a prospectively maintained database was used to record relevant clinical
and pathological data. The length of follow-up was determined from the date
of primary tumour resection, with surviving cases censored for analysis on the
31st December 2003. Disease-specific survival (DSS) was used as the
primary end-point. A TMA was also prepared from a series of 40 patients
from whom tumour material from both primary tumour and corresponding
hepatic metastasis was available. Immunohistochemistry (IHC) for Cten was
performed with full local ethical approval. Slides were stained using the water
bath antigen retrieval method with EDTA Buffer (Sigma) as previously
described(3). The primary antibody Cten (Abcam,ab57940.1:75), was applied
for 45 minutes at room temperature and diaminobenzidine (DAB) (Sigma) was
used as the chromogen. Slides were then counterstained with haematoxylin
(Dako).
Evaluation of IHC
The IHC was scored by two pathologists (AA and WF) using the Hscoring system. Briefly, the slides were assessed for the intensity of
tumour cell staining ranging from zero to three, (zero = no staining, one
= weak, 2 = moderate, three = strong) and for the percentage of tumour
cells staining within each intensity category. The two numbers obtained
were multiplied and their product represents the H-score.
Cell culture and transfection
The CRC cell lines used in this study were kindly donated by Prof I Tomlinson.
All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM;
Invitrogen) supplemented with 10% foetal calf serum (Invitrogen) and
penicillin/streptomycin (respectively at concentrations of 100 units per ml
3
and 100g per ml, Invitrogen) in 5% CO2 in a humidified atmosphere. In
order to induce Cten expression, cells were transfected with a CMV promoter
driven expression construct containing Green Fluorescent Protein (GFP)
tagged Cten (GFP-Cten) using Lipofectamine 2000 (Invitrogen) as previously
described(3). Control cells were transfected with a GFP empty vector. In
order to knock down Cten and ILK, cells were transfected with small
interfering RNA (siRNA) duplexes using Lipofectamine 2000 (Invitrogen) as
previously described(4). The cells were transfected with each siRNA duplex
at a final concentration of 100nM and compared with cells transfected with
sequence scrambled control (ssc) duplexes (i.e. duplexes with the same base
composition arranged in a random order). Sequences for all the duplexes
used are available from the authors.
In order to functionally evaluate Cten, a number of different cellular conditions
were created using a series of co-transfections (see results). For cotransfection of GFP-Cten with an siRNA, the protocol for siRNA transfection
was used with appropriate amounts of plasmid mixed in with the siRNA prior
to adding to Lipofectamine 2000. For simultaneous knockdown of two
separate targets, each of the relevant siRNAs were admixed to maintain a
final concentration of 100nM for each duplex. Cells were analysed 72 hours
later by Western blot or functional assays.
Western blotting
Whole cell extracts were prepared using lysis buffer (20 mM Tris, pH 7.5, 150
mM NaCl, 1% TritonX-100, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1%
SDS, supplemented with protease and phosphatase inhibitors (Sigma)). 30g
protein was loaded on a 10% SDS–PAGE gel and transferred onto PVDF
membranes by semi-dry transfer. After blocking, membranes were incubated
overnight at room temperature with the indicated primary antibody. Antibodies
were used at the following dilution: anti-Cten (Sigma, WH0084951M1,1:1000),
anti-ILK antibody (Abcam, ab19025, 1:500) and mouse anti--actin (Sigma,
1:2000). After three washes in TBS/Tween-20 (0.05%), blots were incubated
for 1 hr at room temperature with the appropriate horseradish peroxidaselinked secondary antibody. After three further washes, detection was
4
performed using the Enhanced Chemiluminescence Kit (Pierce). Bands were
visualised using X-Ray films (Kodak).
Cell Migration/ Invasion Assays
Transwell cell migration was measured using a Boyden chamber containing a
polycarbonate filter with an 8m pore size (Costar). 5 X 104 cells were seeded
and cell migration was assessed after 48 hr by fixing the cells attached to the
lower surface in 10% methanol for 30 min, staining for 30 min with methylene
blue and manually counting the stained cells. Cell invasion was measured in
the same way as described for migration, except that prior to cell seeding the
upper chamber was prepared by coating the filter with 100l Matrigel (5 mg/ml;
BD Biosciences) and the cells attached to the lower surface were fixed,
stained and counted. All assays were performed at least twice in triplicate.
In vivo metastasis model
Male MF1 nude mice (Harlan-Olac) were used. Animal procedures were run
under the Home Office project PPL 40/2962 following local ethical approval.
For analysis of tumor growth and metastasis, the CRC cell line HCT116 was
stably transfected with either a GFP-Cten expression vector (HCT116GFP-Cten)
or empty vector control (HCT116evc) expressing GFP only. Cells were
injected into the spleens of the animals using standard techniques. Briefly, a
left abdominal flank incision was made and 1 x106 cells were injected into the
subcapsular region of the spleen of nude mice (n=10 in each group). On day
42, mice were sacrificed by approved S1 method and the spleen, liver and
lung were dissected and examined for orthotopic tumors and distant
metastases. Organs were formalin-fixed and paraffin-embedded and 4m
sections were stained with Haematoxylin and Eosin. All tissue sections were
scanned at x 20 using the NanoZoomer 2.0 (Hamamatsu Corporation, Japan)
and the tumour volume was obtained using the NanoZoomer Digital
Pathology Image (ndpi) software (Hamamatsu Corporation, Japan).
Statistical analysis
5
Data were analysed using SPSS 16.0 statistical software (SPSS Inc. USA).
For the IHC, determination of the optimal Cten cut-off was performed using Xtile bioinformatics software (version 3.6.1, 2003–2005, Yale University, USA)
(5). This program basically divides the total patient cohort randomly into two
separate equal training and validation sets ranked by patients’ follow-up time.
The optimal cut-points are determined by locating the brightest pixel on the Xtile plot diagram of the training set and the scoring are dichotomised into “low
Cten” and “high Cten”. Associations between Cten expression (i.e. low or
high) and clinicopathological parameters were evaluated using Pearson
2
test. Patients whose deaths were attributed to colorectal cancer, were
considered in the disease-specific survival calculations. Deaths as a result of
non-colorectal cancer related causes without evidence of recurrence were
censored at the time of death. Survival curves were analysed by the Kaplan–
Meier plot with a log rank test to assess significance. Multivariate Cox
proportional hazards model was used to test the statistical independence and
adjust for confounders. A two-tailed p-value of <0.05 was considered
significant. All other evaluations were done using the unpaired two-tailed
Student’s t–test and p< 0.05 was considered to indicate a significant
difference.
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Durrant LG. Overexpression of FLIPL is an independent marker of poor
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Ahmed MA, Al-Attar A, Kim J, Watson NF, Scholefield JH, Durrant LG,
et al. CD24 shows early upregulation and nuclear expression but is not a
prognostic marker in colorectal cancer. J Clin Pathol. 2009 Dec;62(12):111722.
3.
Albasri A, Seth R, Jackson D, Benhasouna A, Crook S, Nateri AS, et al.
C-terminal Tensin-like (CTEN) is an oncogene which alters cell motility
possibly through repression of E-cadherin in colorectal cancer. J Pathol. 2009
May;218(1):57-65.
4.
Elsaba TM, Martinez-Pomares L, Robins AR, Crook S, Seth R,
Jackson D, et al. The stem cell marker CD133 associates with enhanced
colony formation and cell motility in colorectal cancer. PLoS One.5(5):e10714.
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Camp RL, Dolled-Filhart M, Rimm DL. X-tile: A new bio-informatics tool
for biomarker assessment and outcome-based cut-point optimization. Clinical
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