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Figure legends
Figure 1 Immunohistochemical analysis of ITLN1 in colorectal cancer (CRC) tissue
specimens. (a) ITLN1 immunostaining in the non-neoplastic colonic mucosa and CRC.
ITLN1 staining was observed in the cytoplasm of goblet cells in non-neoplastic colonic
mucosa, whereas ITLN1 staining was reduced in CRC cells (original magnification × 100).
(b) ITLN1 immunostaining in CRC (original magnification × 100). Some ITLN1 staining
was observed in the cytoplasm of CRC cells. (c) Kaplan-Meier plot of the survival of CRC
patients. (d) Prognostic value of ITLN1 in CRC patients and the survival curve as plotted
using the OncoLnc database.
Figure 2 Effects of the inhibition of ITLN1 on cell growth in colorectal cancer (CRC) cells.
(a) Western blotting of ITLN1 in the cell lysates from three CRC cell lines. (b) Western
blotting of ITLN1 in cell lysates from DLD-1 and WiDr transfected with ITLN1 siRNA or
negative control siRNA. β-Actin was included as a loading control. (c, d) Effect of ITLN1
knockdown on the cell growth of DLD-1 (c) and WiDr (d). Cell growth was assessed by an
MTT assay at 1, 2 and 4 days after seeding on 96-well plates. The mean (bars) and standard
deviation (SD; error bars) of three independent experiments are shown. OD, optical density.
*P < 0.05.
Figure 3 Effect of the downregulation of ITLN1 on the epidermal growth factor receptor
(EGFR) signaling pathway. (a, b) Western blotting of ITLN1, EGFR, phospho-EGFR
(pEGFR), Erk1/2, phospho-Erk1/2 (pErk1/2), Akt and phospho-Akt (pAkt) in cell lysates
from DLD-1 (c) and WiDr (d) transfected with ITLN1 siRNA or negative control siRNA.
Katsuya N et al. 20
β-actin was included as a loading control. We used three filters in each cell line. We
confirmed that total proteins are almost the same amounts in all lanes as monitored by βactin. In this figure, representative western blots for β-actin are shown.
Figure 4 Analysis of the correlation between the expression of ITLN1 and colorectal cancer
(CRC)-related molecules in the invasive front. (a) Expression levels of ITLN1 (b–g) CRCrelated molecules, including β-catenin, matrix metalloproteinase 7 (MMP7), CDX2, claudin18, MUC5AC and CD44 were examined. A serial section showed that β-catenin, MMP7,
CDX2, claudin-18, MUC5AC and CD44 at the invasive front were partially adjacent to the
area in which ITLN1 was reduced.
Figure 5 Analysis of the expression of ITLN1 in pre-cancerous lesions of each colorectal
carcinogenesis pathway. (a–d) Immunostaining of adenoma in colorectal polyps (CRPs). (a)
Conventional adenoma low grade, n=32; (b) conventional adenoma high grade (CAHG),
n=64; (c) traditional serrated adenoma (TSA), n=27 and (d) sessile serrated adenoma/polyp
(SSA/P), n=20. (e, f) ITLN1 immunostaining scores in CRP. The graph indicates the
percentage of sections with different scores (negative, weak, moderate and strong).
Katsuya N et al. 21
Supporting Information
Supporting Figure 1 Quantitative reverse transcriptase PCR analysis of intelectin-1
(ITLN1). (a) ITLN1 mRNA expression level in 13 normal tissues and 5 colorectal cancer
(CRC) cell lines. (b) T/N ratio of ITLN1 mRNA level between CRC tissue (T) and
corresponding nonneoplastic mucosa (N) in 20 CRC cases. Underexpression was defined as a
T/N ratio < 1.0. Underexpression of the ITLN1 gene was observed in 16 (80%) of the 20 GC
cases.
Supporting Table 1 Univariate and multivariate analysis of factors influencing survival in 148
patients with colorectal cancer
Katsuya N et al. 1
Table 1. Relationship between ITLN1 expression and clinicopathologic characteristics in the 148
CRC cases
ITLN1 expression
P-value
Reduced (%)
Preserved
≦ 65 (n = 70)
41 (59%)
29
≧ 66 (n = 78)
46 (59%)
32
Female (n = 89)
55 (62%)
34
Male (n = 59)
32 (54%)
27
T1/T2 (n = 25)
11 (44%)
14
T3/T4 (n = 123)
76 (62%)
47
N0 (n = 81)
46 (57%)
35
N1/2/3 (n =67)
41 (61%)
26
M0 (n = 129)
70 (54%)
59
M1 (n = 19)
17 (89%)
Stage I / II (n = 40)
16 (40%)
24
Stage III/IV (n = 108)
71 (66%)
37
Low(Grade1) (n = 98)
48 (49%)
50
High(Grade2/3) (n = 50)
39 (78%)
11
84 (58%)
60
3 (75%)
Age
NS
Sex
NS
T grade
NS
N grade
NS
M grade
0.0017
Stage
0.005
Budding Grade
0.001
Histlogic classification
Well/moderate (n = 144)
Poor/mucinous (n = 4)
P values were calculated with Fisher’s exact test.
NS, not significant.
NS
Katsuya N et al. 2
Table 2. Relationship between ITLN1 expression and cancer related molecules in 126 patients
with colorectal cancer
ITLN1 expression
Reduced (%)
Reserved
Positive (n = 87)
55 (63%)
32
Negative (n = 39)
15 (38%)
24
Positive (n = 40)
28 (70%)
12
Negative (n = 86)
42 (49%)
44
Positive (n = 69)
39 (57%)
29
Negative (n = 58)
31 (53%)
27
Positive (n = 73)
26 (36%)
47
Negative (n = 53)
44 (83%)
Positive (n = 79)
41 (52%)
38
Negative (n = 47)
29 (61%)
18
Positive (n = 67)
44 (65%)
23
Negative (n = 59)
26 (38%)
33
Positive (n = 15)
12 (80%)
Negative (n = 111)
58 (53%)
53
Positive (n = 79)
50 (63%)
29
Negative (n = 47)
20 (42%)
27
Positive (n = 70)
39 (56%)
31
Negative (n = 56)
31 (55%)
25
Positive (n = 78)
45 (57%)
33
Negative (n = 48)
25 (52%)
23
P-value
β-catenin (nuclear localization)
0.012
MMP7
0.026
P53 expression
NS
CDX2
<0.001
MUC2
NS
MUC5AC
0.02
Claudin-18
0.042
CD44
0.027
MLH1
NS
MSH2
P values were calculated with Fisher’s exact test.
NS, Not significant.
NS
Katsuya N et al. 1
Supporting Table 1. Univariate and multivariate analysis of factors influencing survival in 148 patients with
colorectal cancer
Univariate analysis
HR (95% CI)
Multivariate analysis
P-value
HR (95% CI)
P-value
Age
≦ 65
1 (Reference)
≧ 66
1.49 (0.79-2.92)
NS
Sex
Female
1 (Reference)
Male
0.87 (0.45-1.65)
NS
Classification
T grade
5.18 (0.97-5.03)
<0.0001
2.47 (0.47-2.12)
NS
N grade
4.89 (2.45-10.59)
<0.0001
2.05 (1.45-8.39)
NS
M grade
5.52 (2.78-10.51)
<0.0001
2.81 (1.39-5.67)
0.003
Stage I/II
1 (Reference)
<0.0001
1 (Reference)
0.0041
Stage III/IV
5.08 (0.10-12.04)
Tumor stage
3.34 (1.44-8.36)
Budding Grade
Low (Grade 1)
1 (Reference)
High (Grade 2/3)
3.31 (1.47-7.31)
0.0006
1 (Reference)
NS
1.56 (077-4.47)
ITLN1 expression
Reduced
1 (Reference)
Preserved
2.93 (1.09-4.81)
P values were calculated with Fisher's exact test.
NS, not significant
0.0126
1 (Reference)
2.57 (1.16-5.34)
0.0376
Figure 1
100
ITLN1 preserved n = 61
ITLN1 preserved n = 225
Overall survival
Overall survival (%)
80
60
40
ITLN1 reduced n = 87
Log rank P = 0.0155
20
Hiroshima cohort
Log rank P = 0.01149
ITLN1 reduced n = 223
TCGA cohort
20
40
60
Survival period (months)
80
Survival period (days)
ITLN1 -
- 31.3kDa
β-actin -
- 41kDa
siRNA-2
siRNA-1
Negative
control
siRNA-1
- 31.3kDa
β-actin -
- 41kDa
WiDr
0.120
control
0.100
siRNA1
siRNA2
0.030
0.020
0.080
O.D. 595
O.D. 595
0.040
WiDr
ITLN1 -
DLD-1
0.060
0.050
DLD-1
Negative
control
WiDr
DLD-1
COLO201
siRNA-2
Figure 2
control
siRNA1
siRNA2
* *
0.060
0.040
0.020
0.010
*P<0.05
0.000
DAY0
DAY1
DAY2
DAY4
*P<0.05
0.000
DAY0
DAY1
DAY2
DAY4
Figure 3
DLD-1
WiDr
ITLN1 siRNA
Negative
control
Negative
control
ITLN1 siRNA
ITLN1 -
- 31.3kDa
ITLN1 -
- 31.3kDa
AKT -
- 60kDa
AKT -
- 60kDa
pAKT -
- 60kDa
pAKT -
- 60kDa
- 44.4kDa
ERK-
ERK-
pERK -
- 44.4kDa
EGFR-
- 170kDa
pEGFR -
- 170kDa
β-actin -
- 41kDa
- 44.4kDa
pERK -
- 44.4kDa
EGFR-
- 170kDa
pEGFR -
- 170kDa
β-actin -
- 41kDa
Figure 4
ITLN1
β-catenin
MMP7
CDX2
Claudin-18
MUC5AC
CD44
Figure 5
CALG
SSA/P
CAHG
TSA
Immunostaining intensity(%)
100%
90%
strong
80%
moderate
70%
weak
60%
negative
50%
40%
30%
20%
10%
*P<0.05
0%
Normal
CALG
CAHG
Immunostaining intensity(%)
NS
100%
90%
strong
80%
moderate
70%
weak
60%
negative
50%
40%
30%
20%
10%
*P<0.05
0%
Normal
TSA
SSA/P
Fold difference (T/N)
2.5
1.5
0.5
LoVo
WiDr
COLO-320
COLO-201
DLD-1
Muscle
Spleen
Proatate
Kidney
Pancreas
Liver
Colon
Small intestine
Stomach
Lung
Heart
Spinal cord
ITLN1 mRNA expression level
Supporting Figure 1
Colon cancer cell lines
9 10 11 12 13 14 15 16 17 18 19 20
...