Expression of intestinal stem cell and cancer stem cell markers in submucosal invasion and its prognostic significance in gastric cancers

(1)

Expression of intestinal stem cell and cancer stem

cell markers in submucosal invasion and its

prognostic significance in gastric cancers

Supplementary Information

Hye Sung Kim

1

, Hyun Joo Song

2

, In Ho Jeong

3

, Bo Gun Jang

1

1_{Department of Pathology, Jeju National University School of Medicine and Jeju National University Hospital, 63241, Korea}

2_{Department of Internal Medicine, Jeju National University School of Medicine and Jeju National University Hospital, 63241, Jeju, Korea}

(2)

Supplementary Table S1Clinicopathological characteristics of 91 early gastric cancers

Age 40 - 85 years (median: 67; s.d.: 11.0)

Tumor size 0.4 - 10cm (median: 2.5; s.d.: 1.96)

Sex

Male 62 (68%)

Female 29 (32%)

Depth

Lamina propria 14 (15%)

Muscularis mucosa 10 (11%)

Submucosa 67 (74%)

Histology

WD 23 (25%)

MD 53 (58%)

PD 8 (9%)

PCC 7 (8%)

Lauren

Intestinal 72 (79%)

Diffuse 11 (12%)

Mixed 8 (9%)

LN invasion

Absent 77 (85%)

Present 14 (15%)

LN metastasis*

Absent 59 (80%)

Present 15 (20%)

* Lymph node dissection was performed in 74 cases.

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Supplementary Table S2 Association between clinicopathologic parameters and lymph node metastasis in 74 early gastric cancers

Characteristics Total No.(%) Lymph node metastasis p-value

Negative (%) Positive (%)

74 (100) 59 (80) 15 (20)

Age

<65 34 (46) 24 (71) 10 (29)

0.071†

≥65 40 (54) 35 (88) 5 (12)

Sex

Female 24 (32) 16 (67) 8 (33)

0.053†

Male 50 (68) 43 (86) 7 (14)

Size

<2.5 28 (38) 26 (93) 2 (7)

0.028†

≥2.5 46 (62) 33 (56) 13 (28)

Depth

Mucosa 20 (27) 19 (95) 1 (5)

0.047†

Submucosa 54 (73) 40 (74) 14 (26)

Histology

WD 17 (23) 15 (88) 2 (12)

0.752†

MD 42 (57) 33 (79) 9 (21)

PD 8 (11) 6 (75) 2 (25)

PCC 7 (9) 5 (71) 2 (29)

Lauren

Intestinal 55 (74) 45 (82) 10 (18)

0.743†

Diffuse 11 (15) 8 (73) 3 (27)

Mixed 8 (11) 6 (75) 2 (25)

Lymphatic invasion

Absent 60 (81) 54 (90) 6 (10)

< 0.001†

Present 14 (19) 5 (36) 9 (64)

†

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Supplementary Figure S1 Immunohistochemistry for intestinal stem cell markers (EPHB2 and OLFM4) and candidate cancer

stem cell markers (CD44, CD133, and ALDHA1). Scale bars: 50mm.

EPHB2

CD44

CD133

OLFM4

ALDH1A

Str

o

n

g

M

o

d

er

at

e

W

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EPHB2

H&E OLFM4 CD133

CD44 ALDH1A

LGR5

12

0 0

1 1

1 2

2

2 2 6

5

9

LGR5 EPHB2

CD133 OLFM4

Supplementary Figure S2 Gastric cancers with basal expression of multiple intestinal stem cell and cancer stem cell markers. (a) Representative case of gastric cancer with basal EPHB2, LGR5, OLFM4, CD133, ALDH1A, and CD44. (b) A diagram showing the numbers of gastric cancer cases with basal expression of EPHB2, LGR5, OLFM4, and CD133 (n = 43).

Higher magnification views of the white boxed areas are shown in the lower pictures. Scale bars: 50mm.

a

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Supplementary Figure S3 Symmetrical distribution of multiple intestinal stem cell markers in the gastric cancer with submucosal invasion.Sequential immunostaining of EPHB2 and OLFM4, and RNA in situ hybridization of LGR5. The basally located cancer cells right above muscularis mucosa (indicated by red dotted line) and cancer cells invading into submucosa are positive for EPHB2, LGR5, and OLFM4. White boxed areas are shown at higher magnification in lower

panel. Scale bars: 50mm.

EPHB2

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Mu Submu 0 20 40 60 80 100 ns P ro p o rt io n (% ) Mu Submu 0 20 40 60 80 100 ns P ro p o rt io n (% ) Mu Submu 0 20 40 60 80 100 ns P ro p o rt io n (% ) Mu Submu 0 20 40 60 80 100 ns P ro p o rt io n (% ) Mucosa Submu 0 20 40 60 80 100 ns P ro p o rt io n (% ) Mu Submu 0 20 40 60 80 100 ns P ro p o rt io n (% ) Mucosa Submu 0 20 40 60 80 100 ns P ro p o rt io n (% ) Mu Submu 0 20 40 60 80 100 ns P ropor tion (% ) Mucosa Submu 0 20 40 60 80 100

**

P ro p o rt io n (% ) Mucosa Submu 0 20 40 60 80 100 ns P ro p o rt io n (% )

EPHB2 OLFM4 LGR5 CD133 CD44 ALDH1A

a

EPHB2 OLFM4 LGR5 CD133 CD44 ALDH1A

b

Supplementary Figure S4 Proportion of stem cell (SC) marker-positive cancer cells in the submucosal invading cells. Proportion of SC marker-expressing cells among submucosal cancer cells in the gastric cancers (GCs) with focal distribution pattern (a) and in GCs with diffuse pattern (b) for each marker. The proportion of CD44-positive gastric cancer cells significantly decreased in the submucosa than in the mucosa of gastric cancers with both focal and diffuse

patterns for CD44. ns, not significant. *p< 0.05, **p< 0.005

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Supplementary Figure S5 Association of EPHB2 expression with stem cell markers in gastric cancers (n = 37). Real-time PCR was performed to measure the expression of intestinal and cancer stem cell markers, and association

of EPHB2 with OLFM4(a), LGR5(b), CD133(c), CD44(d), and ALDH1A1(e)was evaluated.

0.00 0.05 0.10 0.15 0.20 0.00

0.01 0.02 0.03

0.04 _r2_{= 0.20,} _p_{= 0.007}

EPHB2 / GAPDH

L G R 5 / G A P D H

0.0 0.1 0.2 0.3 0.4

0 2 4 6 8

10 _r2_{= 0.55,} _p_{< 0.0001}

EPHB2 / GAPDH

O L F M 4 / G A P D H

0.0 0.1 0.2 0.3 0.4

0.00 0.05 0.10 0.15 0.20 0.25

r2 = 0.01, p = 0.47

EPHB2 / GAPDH

C D 1 3 3 / G A P D H

0.0 0.1 0.2 0.3 0.4

0.0 0.5 1.0 1.5

r2 = 0.04, p = 0.20

EPHB2 / GAPDH

C D 4 4 / G A P D H

0.0 0.1 0.2 0.3 0.4

0.00 0.02 0.04 0.06 0.08

r2 = 0.06, p = 0.15

EPHB2 / GAPDH

A L D H 1 A 1 / G A P D H

a

b

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SN U16 SN U62 0 MK N45 MK N74 SN U1 SN U66 8 MK N28 Kat o3 AG S SN U63 8 SN U21 6 NC I-N87 SN U71 9 MK N1 SN U60 1 0.00 0.02 0.04 0.06 0.08 0.10 CDX2 EPHB2 R e la ti v e m R N A e x p re s s io n

a

b

0.00 0.01 0.02 0.03 0.04 0.00 0.02 0.04 0.06 0.08 0.10

r2 = 0.42,p= 0.01

CDX2 / GAPDH

E P H B 2 / G A P D H CDX2 Ctl CDX2 0.0 0.5 1.0 1.5 2.0 2.5

**

C D X 2 / G A P D H Ctl CDX2 0

5.00× 10-8 1.00× 10-7 1.50× 10-7

L G R 5 / G A P D H Ctl CDX2 0.00000 0.00005 0.00010 0.00015

***

E P H B 2 / G A P D H Ctl CDX2 0.00 1.00e-006 2.00e-006 3.00e-006 ns C D 1 3 3 / G A P D H M K N -28 M KN -74 Ctl CDX2 0.0 0.2 0.4 0.6 0.8 1.0

***

C D X 2 / G A P D H Ctl CDX2 0

1.00× 10-7 2.00× 10-7 3.00× 10-7 4.00× 10-7 5.00× 10-7 ns

L G R 5 / G A P D H Ctl CDX2 0.00000 0.00002 0.00004 0.00006

***

E P H B 2 / G A P D H Ctl CDX2 0.00 2.00e-006 4.00e-006 6.00e-006 ns C D 1 3 3 / G A P D H LGR5 EPHB2 CD133

c

d

CDX2 EPHB2 HSP90 MKN-28 MKN-74 Ctl CDX2 Ctl CDX2

Supplementary Figure S6Effect of CDX2 on the expression of EPHB2 in gastric cancer (GC) cells. (a) CDX2 and EPHB2 expression in 15 GC cell lines. (b) Correlation of CDX2 with EPHB2 expression in GC cell lines. (c) Influence of CDX2 overexpression in MKN-28 and MKN-74 cells on the expression of EPHB2, LGR5, and CD133. (d) Western blot for CDX2

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Supplementary Table S3

The results of multivariate analysis for survival rate for all cases

Variables

HR

95% CI

p

value

a

Age

1.428

1.089 - 1.872

0.010

Histology

1.026

0.920 - 1.145

0.642

Lymphatic invasion

1.496

1.017 - 2.200

0.041

Venous invasion

1.374

1.032 - 1.830

0.029

TNM_7th

3.605

2.954- 4.399

< 0.001

CDX2

0.711

0.458 - 1.102

0.127

CD133

1.111

0.815 - 1.614

0.507

OLFM4

0.832

0.624 - 1.110

0.211

EPHB2

0.861

0.640 - 1.158

0.321

HR, Hazard ratio; CI, confidence interval

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Basal

Focal

Diffuse

EPHB2

LGR5

OLFM4

CD133

CD44

ALDH1A

OLFM4

Mucosa

Submucosa

Stem cell marker-negative cancer cell

Stem cell marker-positive cancer cell

Intestinal stem cell markers:

EPHB2, LGR5, OLFM4

Candidate cancer stem cell markers:

CD133, CD44, ALDH1A