Supplementary methods
RNA extraction from tissues and construction of cDNA
RNA was isolated from tissue using 1 ml of TRIzol reagent (Invitrogen, Carlsbad, CA, USA).
The tissue was homogenized in a 5 ml glass tube and the homogenate was transferred to a 1.5
ml tube and mixed with 200 μl of chloroform. After incubation for 5 minute at 4°C, the
homogenate was centrifuged for 13 minutes at 13,000 × g at 4°C. The upper aqueous phase
was transferred to a clean tube and then 500 μl of isopropanol was added. The mixture was
incubated for 60 minutes at 4°C, and the tube was centrifuged for 8 minutes at 13,000 × g at
4°C. The upper aqueous phase was discarded and mixed with 500 μl of 75% ethanol, and
centrifuged for 5 minutes at 13,000 × g at 4°C. After discarding the upper aqueous layer, the
pellet was dried at room temperature, dissolved in diethylpyrocarbonate (DEPC)-treated
water, and then stored at -80°C. The quality and integrity of the RNA were confirmed by
agarose gel electrophoresis and visual inspection upon ethidium bromide staining under
ultraviolet light. cDNA was prepared from 1 μg of total RNA using the First Strand cDNA
Synthesis kit (Amersham Biosciences Europe GmbH, Freiburg, Germany), according to the
manufacturer’s instructions.
nucleic acid extraction from urine samples
S100A8 and S100A9 urinary nucleic acids were extracted using the QIAquickR○ Gel Extraction
Kit (Qiagen GmbH, Hilden, Germany). Each frozen urine sample (1 ml) was melted down at
room temperature and treated with 500 µl of QG buffer. After incubation for 10 minute at
50°C, 500 µl of isopropanol was added and the sample was mixed. The sample was
transferred onto a QIAquick column, which binds nucleic acids. The column was placed into
a 2 ml centrifuge tube and centrifuged for 1 minute at 13,000 rpm. The aqueous flow-through
was discarded and the QIAquick column was placed back into the same collection tube.
Then, 500 µl of QG buffer was added to the QIAquick column and centrifuged for 1 minute
at 13,000 rpm. The nucleic acids bound to the column were washed by centrifuging the
column with 750 µl of PE buffer for 1 minute. The aqueous flow-through was discarded and
the QIAquick column was centrifuged for an additional 1 minute at 13,000 rpm. The
QIAquick column was placed into a clean 1.5 ml microcentrifuge tube. To elute the nucleic
acids, 50 µl of EB buffer was added to the center of the QIAquick membrane and the column
was centrifuged for 1 minute at 13,000 rpm. The nucleic acids dissolved in EB buffer were
stored at -20°C until use.
real-time PCR
To quantify the expression of S100A8 and S100A9, real-time PCR was performed using a
Rotor Gene 6000 instrument (Corbett Research, Mortlake, Australia). Real-time PCR assays
were carried out in micro-reaction tubes (Corbett Research, Mortlake, Australia) using SYBR
Premix EX Taq (TAKARA BIO INC., Otsu, Japan). The primers used for amplification of
S100A8 (185 base pairs), S100A9 (151 base pairs) and GAPDH (156 base pairs) in the
prostate tissues were as follows: sense, 5'-CATCGACGTCTACCACAAGT-3' and antisense,
5'-GAATGAGGAACTCCTGGAAG-3'; sense, 5'-CACCCAGACACCCTGAACCA-3' and
antisense, 5'-CCTCGAAGCTCAGCTGCTTG-3'; and sense, 5'CATGTTCGTCATGGGTGTGA-3' and antisense, 5'-ATGGCATGGACTGTGGTCAT-3',
respectively. The primers used for amplification of S100A8 (85 base pairs), S100A9 (90 base
pairs) and GAPDH (83 base pairs) in urine were as follows: sense, 5'CATCGACGTCTACCACAAGT-3' and antisense, 5'-GGTCTCTAGCAATTTCTTCAG-3';
sense, 5'-CACGAGAAGATGCACGAGG-3' and antisense, 5'TTGGCCACTGTGGTCTTAGG-3'; and sense, 5'-ACCCAGAAGACTGTGGATGG-3' and
antisense, 5'-GTAGAGGCAGGGATGATGTTC-3', respectively. The PCR products then
were cloned into pGEM-T Easy vector (Promega, Wisconsin, USA) to make standard
plasmid after confirming correct product by sanger sequencing. The standard plasmid was
included in PCR run with S100A8/A9 in order to produce standard curve using copy number
(with copy number of 105, 104, 103, 102, and 10) and threshold cycle (Ct) value. The Ct value
of S100A8/A9 from the PCR run was plotted on the standard curve to calculate copy number.
GAPDH was analyzed in parallel as an endogenous RNA reference gene and the expression
of each gene of interest was normalized to that of GAPDH. All samples were run in
triplicates.
immunohistochemical staining
Paraffin blocks from 19 prostate cancer cases and 20 BPH controls were used for
immunohistochemical analysis. Tissue sections were cut, placed on Superfrost® Plus
microscope slides (Fisher Scientific, Pittsburgh, PA) and stained using the Benchmark XT
automated immunohistochemistry stainer (Ventana Medical Systems, Inc., Tucson, AZ).
Detection was conducted using the Ventana Ultraview DAB Kit (Ventana Medical Systems).
Sections were deparaffinized using the EZ Prep solution. CC1 standard (pH 8.4, buffer
containing Tris/Borate/EDTA) was used for antigen retrieval. DAB inhibitor (3% H2O2,
endogenous peroxidase) was blocked for 4 minute at 37°C. Sections were incubated with
anti-S100A8 and -S100A9 primary antibodies (Abcam Inc., San Diego, CA) for 40 minutes
at 37°C, and with a secondary antibody conjugated to Universal HRP Multimer for 8 minute
at 37°C. Slides and then DAB with H2O2 substrate for 8 minutes followed by hematoxylin
and bluing reagent counterstain at 37°C. Reaction buffer (pH 7.6 Tris buffer) was used as
washing solution. The staining intensity and proportion of positively stained epithelial cells
were evaluated. S100A8 and S100A9 were localized primarily to the cytoplasm. Staining
intensity was classified as follows: none, weak, moderate or strong. Each specimen was
examined and scored separately by three investigators, and discrepancies were discussed until
agreement was reached.
Supplementary Table 1. Clinicopathological characteristics and tissue S100A8/9 expression in
CaP and BPH
Characteristics
Controls
Cases
No.
90
132
Age (years; range)
69.3 (46‒85)
69.6 (48‒87)
0.809
PSA ± SD (ng/ml)
4.61 ± 5.21
101.47 ± 254.32
< 0.001a
Prostate size ± SD (gram)
38.42 ± 22.56
41.31 ± 23.32
60 (100)
63 (47.7)
Operation (%)
TURP
Radical prostatectomy
69 (52.3)
No. Gleason score (%)
6 or less
6 (4.5)
7
49 (37.1)
8
38 (28.8)
9
33 (25.0)
P-value
0.411
10
6 (4.5)
No. stage (%)
Nonmetastatic
75 (56.8)
Metastatic
57 (43.2)
S100A8 expression, median
(IQR; × 103 copies/μg)a
S100A9 expression, median
(IQR; × 103 copies/μg)a
240.6
(82.3‒1402.0)
4332.3
(147.2‒2058.9)
31.0
< 0.001b
(8.8‒213.5)
110.1
< 0.001b
(25.5‒523.3)
Abbreviations: CaP, prostate cancer; BPH, benign prostatic hyperplasia; PSA, prostatespecific antigen; SD, standard deviation; TURP, transurethral resection of prostate; IQR,
interquartile range.
a
Copy number of corresponding transcirpt per μg of total RNA used for cDNA synthesis
b
Mann-Whitney U test was used to compare the expression levels to clinical variables.
Supplementary Table 2. Clinicopathological characteristics and S100A8/A9 urinary nucleic
acid levels in CaP and BPH
Characteristics
Controls
Cases
No.
363
283
Age (years; range)
69.5 (49‒85)
68.9 (52‒86)
P-value
0.243
PSA ± SD (ng/ml)
5.31 ± 5.75
Prostate size ± SD (gram)
42.89 ± 22.20
56.01 ± 193.14
37.07 ± 18.85
< 0.001a
0.119
Operation (%)
TURP
363 (100)
Radical prostatectomy
66 (23.3)
217 (76.7)
No. Gleason score (%)
6 or less
10 (3.5)
7
163 (57.6)
8
55 (19.4)
9
50 (17.7)
10
5 (1.8)
No. stage (%)
Nonmetastatic
214 (75.6)
Metastatic
44 (15.5)
Unknown
25 (8.8)
Urinary S100A8, median (IQR;
11.3 (7.4‒18.5)
9.1 (6.6‒15.8)
0.001b
14.6 (6.0‒76.5)
10.2 (5.1‒28.0)
< 0.001b
× 103 copies/μg)a
Urinary S100A9, median (IQR;
× 103 copies/μg)a
Abbreviations: CaP, prostate cancer; BPH, benign prostatic hyperplasia; PSA, prostatespecific antigen; SD, standard deviation; TURP, transurethral resection of prostate; IQR,
interquartile range.
a
Copy number of corresponding transcirpt per μg of total RNA used for cDNA synthesis
b
Mann-Whitney U test was used to compare the expression levels to clinical variables.
Supplementary Table 3. Comparison of clinicopathological outcomes and S100A8/9 urinary
nucleic acid levels in prostate cancer
S100A8 mRNA
S100A9 mRNA
No. of
expression level,
P-
expression level,
P-
patients
median (IQR; × 103
valueb
median (IQR; × 103
valueb
Variables
copies/μg)a
Age
copies/μg)a
0.021
< 70
155
≥ 70
128
10.0 (6.9‒16.9)
11.3 (5.3‒37.5)
8.5 (6.1‒13.7)
PSA
0.025
8.0 (5.0‒19.7)
0.294
0.020
< 20
191
9.5 (6.7‒16.3)
11.2 (5.5‒31.5)
≥ 20
92
8.9 (6.5‒13.8)
7.8 (4.4‒18.9)
Gleason score
0.675
≤7
173
≥8
110
9.5 (6.7‒16.2)
10.1 (5.1‒27.8)
8.8 (6.6‒14.5)
Stage
0.906
10.8 (5.1‒30.4)
0.662
0.310
Nonmeta
214
9.0 (6.7‒16.1)
10.6 (5.3‒31.3)
static
Metastati
44
9.7 (6.9‒16.3)
8.7 (4.7‒32.5)
c
Abbreviations: PSA, prostate-specific antigen; IQR, interquartile range
a
Copy number of corresponding transcirpt per μg of total RNA used for cDNA synthesis
b
Mann-Whitney U test was used to compare the expression levels to clinical variables.