1

Calreticulin

2

1. 3

2. 3

3. 4

4. 5

5. 10

6. 12

7. 14

8. 14

9. 14

10. 17

3

cancer stem-like cells: CSLCs

CSLCs

CSLCs(Pancreatic CSLCs: P-CSLCs)

P-CSLCs 2

P-CSLCs

P-CSLCs

n=80 Cox

P-CSLCs Calreticulin (CRT)

CRT P-CSLCs P-CSLCs CD44v9

CRThigh/CD44v9low ATP binding cassette

CRTlow/CD44v9high CRT

CRT P-CSLCs CRT P-CSLCs

P-CSLCs

CRT P-CSLCs

4

2014

15-20% 1

4

cancer

stem-like cells (CSLCs) 2-4

CSLCs

pancreatic CSLCs P-CSLCs CD24 CD44 epithelial-specific antigen (ESA)

5 CD133 6 aldehyde dehydrogenase 1

(ALDH1) 7 c-Met 8 doublecortin-like kinase 1 (DCLK1) 9 P-CSLCs

CSLCs CD44 variant isoform CD44v9

CD44v9 reactive oxygen species

(ROS) 10

P-CSLCs 11

CD24 CD44 ESA CD44v9

P-CSLCs

CD44v9 P-CSLCs 10, 12

P-CSLCs

Calreticulin (CRT) 46–65-kDa

Ca2+ HLA class

mitoxantrone oxaliplatin CRT

13, 14

CRT 15

CRT

P-CSLCs Proteomics P-CSLCs

5

4-1

YPK2 YPK516 (Life Technologies,

Tokyo, Japan) 10% DMEM-F12 (Sigma-Aldrich Japan, Tokyo, Japan) 37 °C 5%

CO2

4-2 P-CSLCs

LIF (Merck Millipore, Darmstadt, Germany) NSF-1 (Lonza, Tokyo, Japan) N-acetyl-L-cysteine

(NAC; Sigma-Aldrich) 1 sphere

sphere B27 supplement (Life Technologies) EGF

(Sigma-Aldrich Japan) bFGF (Merck Millipore)

1 1-2

YPK2-Lm YPK5-Lm

Proteomics P-CSLCs

4-3 2

Dead Cell Removal MicroBeads (Miltenyi Biotec, Gladbach, Germany) MidiMACS™

Separator (Miltenyi Biotec) CD44v9 rat IgG (clone RV3, Cosmo bio, Tokyo, Japan)

anti-rat mouse IgG (eBioscience, San Diego, CA, USA) microbeads anti-biotin mouse

IgG (Miltenyi Biotec) MidiMACS™ Separator CD44v9

YPK YPK-Lm CD44v9 0.2% pharmalyte

5M Urea, 2M Thiourea, 2% CHAPS, 2% SB3-10, 1% DTT

0.34ml 18 cm Immobiline Drystrip(pH 3–10, GE Healthcare,

Tokyo, Japan) Immobiline Drystrip Multiphor II

6

Electrophoresis Unit CoolPhoreStar IPG-IEF Type-P (Anatech, Tokyo, Japan)

500 V 1 3,500 V 7.5 A 50 mM Tris-HCl

(pH 6.8), 6 M urea, 32% glycerol, 10% SDS, 0.25% DTT B 50 mM Tris-HCl

(pH 6.8), 6 M urea, 32% glycerol, 10% SDS, 4.5% , 0.125%

Immobiline Drystrip (9–18% acrylamide; Towa

Environment Science, Osaka, Japan) ANDERSON ISO-DALT Multiple Electrophoresis System

(Hoefer, Holliston, MA, USA) 80 V 16 SDS

SYPRO Ruby protein gel stain (S21900;

Thermo Fisher Scientific, Waltham, MA, USA) Molecular Imager FX

(Bio-Rad, Tokyo, Japan) ImageMaster 2D Platinum software (GE Healthcare)

YPK2-Lm YPK5-Lm

4-4

100 mM 20

50 mM 5 mM 0.01 g/ l

37 16 5% TFA 5%TFA 50%

20 3 10 l

ZipTip C18 pipette tips (ZTC18S960, Merck Millipore)

50% 0.1%TFA 1 l

(0.3 g/l alpha-cyano-4-hydroxycinnamic acid, 33% acetone, 66% ethanol target plate

(MTP Anchorchip 600/384, Bruker Daltonics, Bremen, Germany) (Ultraflex

TOF/TOF; Bruker Daltonics) MS/MS NCBInr

Mascot database search engine (Matrix Science, London, UK)

4-5

YPK2-Lm YPK5-Lm CRT CD44v9 CD47

7

50ml 2% PBS

2 × 105 cells/100 l

1

1 isotype 20 4

1

rat anti-CD44v9 (clone RV3, Cosmo bio, Tokyo, Japan)

mouse Alexa Fluor 488 anti-CRT (clone #326203, R&D systems, Minneapolis, MN, USA)

Vioblue anti-CD47 (#130-101-359, Miltenyi Biotec, Gladbach, Germany)

CD44v9 2 allophycocyanin donkey anti-rat IgG (eBioscience)

2 CRT

CD44v9 Fix/Permeabilization buffer (eBioscience) 20

PBS permeabilization buffer (eBioscience) 10

Blocking buffer (2% normal rat serum in permeabilization buffer)

Alexa Fluor 488 anti-CRT (clone 326203, R&D systems)

isotype control 20 Permeabilization buffer 2% FBS

PBS MACSQuant analyzer (Miltenyi Biotec)

Relative fluorescence intensity (RFI)

= [mean fluorescence intensity (MFI) - MFI of corresponding isotype control]/MFI of corresponding

isotype control17

4-6

YPK-Lm CRT, CD44v9 BD FACSAriaII (BD Biosciences, San Jose, CA,

USA) CRThigh/CD44v9low CRTlow/CD44v9high CRThigh/CD44v9high 3

ATP

8

4-7 ATP ABC

ABC 18

side population (SP) ABC Hoechst 33342

19 ABC YPK2-Lm YPL5-Lm

5% FBS DMEM 5 g/ml Hoechst 33342 (Sigma-Aldrich Japan) 30 min

BD LSRFortessa X-20 cell analyzer (BD Biosciences)

Hoechst 33342 375-nm trigon violet laser 450/20 (Hoechst 33342-Blue) 670 LP

(Hoechst 33342-Red)

4-8

(1)

2001 6 2013 6 2007

3 2012 10 D2 20

IPMN

(

H27-007)

(2)

10 mM Target Retrieval Solution, pH

6.0 (Dako, Tokyo, Japan)

CRT 3% H2O2 10 CD44v9 0.3% H2O2

5 Protein block serum-free (Dako)

1 2

1 mouse anti-CRT monoclonal antibody (clone FMC75, Abcam,

Cambridge, MA, USA): 1:6000 1

2 House raddish peroxidase (HRP) anti-mouse IgG Dako

9

30

1 rat anti- CD44v9 monoclonal antibody (clone RV3, Cosmo bio)

1:200,4 °C, overnight

2 HRP anti-rat IgG (Nichirei Biosciences, Tokyo, Japan).

PBS 3,3 -diaminobenzidine tetrahydrochloride (DAB; Dako) 3

21

a) absent / weak :1 b) moderate: 2 c) strong: 3

IHC × % (=1×absent / weak % 2×moderate %

3×strong % )

(3)

citrate buffer (10 mM, pH 6.0) 95 °C 10 5% 0.3% Triton X-

100 PBS 60 CRT CD44v9

Anti-CRT antibody (FMC75, Abcam) 1:200

1 Alexa Fluor 488 anti-mouse IgG (#4408, Cell Signaling Technology, Denver, MA, USA)

1:1,000 1.5 anti-CD44v9 rat antibody (RV3, Cosmo bio) 1:100 1

Alexa Fluor 555 anti-rat IgG (#4417, Cell Signaling Technology) 1:1,000 1.5

DAPI ProLong Gold Antifade Reagent (#8961; Cell Signaling Technology)

4-9

± Student’s t-test Chi-squared

Kaplan-Meier Wilcoxon test

Cox Statflex ver. 6 (Artec, Osaka,

10

Japan) p 0.05

5-1. Calreticulin

YPK2-Lm 4.43 YPK5-Lm 5.80

(Fig. 1A–D, ) Calreticulin (CRT) (NCBI accession No. gi|4757900) (Fig. 1E)

5-2. YPK-Lm CRT CD44v9

YPK2-Lm YPK5-Lm

CRT CD44v9 (Fig. 2A, B) YPK-Lm CRThigh/CD44v9low

CRTlow/CD44v9high 2 (Fig. 2C, D) CRT YPK-Lm

(Fig. 2E, F)

5-3.YPK-Lm CD47

CD47 CRT ”eat me signal” “anti-phagocytic signal” 22

CD47 YPK-Lm

Fig. 3A 22 CRT CD47

Fig. 3B

5-4. YPK-Lm ABC

YPK2 side population ( SP) 0.338% YPK2- Lm

34.0% (Fig. 2G) YPK5-Lm SP 12.9%

SP(1.72%) (Fig. 2H) YPK- Lm CRThigh/CD44v9low SP

(Fig. 4B, F) CRTlow/CD44v9high (Fig. 4C, G) CRThigh/CD44v9high (Fig. 4D, H)

SP P-CSLCs CRThigh/CD44v9low

11

subpopulation

5-5 CRT CD44v9

n=77 n=64

61 80

Fig. 5 1/3 (n = 26)

(n = 67) CRT Fig. 6A

CRT

CD44v9 Fig. 6B CD44v9

CRT CD44v9

IHC ( : 0.356 [0.148–0.534], p = 0.0012)

Cox

23-25 TNM T CRT

CD44v9 IHC CRT

IHC (p < 0.01) (p < 0.01) (p < 0.05)

(Table 1)

1 CRT IHC 150 CRT

(IHC score 150; n = 43) CRT (IHC score < 150; n = 37)

CD44v9 (IHC score 165; n = 40) CD44v9 (IHC score < 165; n = 40)

CRT Table 2 CRT

T3 Stage II CD44v9

CRT (p =

0.0127) (p = 0.0221) (Fig. 7)

12

CRT 26

CRT IHC CRT

4-7 CRT

5-5 CRT CD44v9

CRT CD44v9 CRT CD44v9

(Fig. 6C)

CRT P-CSLCs CRT

CRT ER Ca2+

folding

CRT

ER 27 CRT

ER fold

CRT ”unfolded protein response”

26

CRT CD44v9 (Fig. 6C)

CRT CD44v9 population

2 1 P-CSLCs

P-CSLCs CRT

CRT

SP CSLCs 28 SP

13

29 CRThigh/CD44v9low

CRTlow/CD44v9high SP CRT CD44v9 P-CSLCs

CRT CSLCs

22 CSLCs CRT

CRT

Chao 22 CRT

CRT

CRT “eat-me” signal 13

CRT 30 31 15

2 22 1 CRT anti-phagocytic

signal CD47 CRT CD47

CD47 YPK-Lm

P-CSLCs CD47 2 CRT

CRT neuropilin-1, matrix

metalloproteinase (MMP)2, MMP9, focal adhesion kinase (FAK) upregulate

32 phosphoinositide 3-kinase (PI3K)/Akt pathway

anoikis

33 CRT

CRT LDL-receptor related protein 1

(LRP1)/CD91 34

tumor-associated macrophages (TAMs) M2

35 CRT

CRT

14

36

CRT

CRT P-CSLCs CRT P-CSLCs

CD24, CD44, ESA

UV P-

CSLCs

CRT

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17

Fig. 1. Calreticulin

A-D: YPK2 A, C YPK2-Lm B, D 2

E: MALDI TOF/TOF MS calreticulin

18

Fig. 2.

A, B: (A)YPK2 YPK2-Lm (B) YPK5 YPK5-Lm CRT

CD44v9

C, D: (C)YPK2 YPK2-Lm (D) YPK5 YPK5-Lm

19

CRT CD4v9

E, F: (E)YPK2 YPK2-Lm F (E)YPK5 YPK5-Lm

CRT

G,H: (G)YPK2 YPK2-Lm H (E)YPK5 YPK5-Lm

Hoechst33342

Fig.3. CD47

A: YPK YPK-Lm CD47

B: CRT CD47 -0.1585 (95% confidence interval: -0.6356 – 0.4063) p > 0.05

20

Fig. 4. YPK-Lm sort Side population (SP)

(A) YPK2-Lm calreticulin (CRT)high/CD44 variant isoform 9 (CD44v9)low ( ) CRTlow/CD44v9high

( ) CRThigh/CD44v9high ( ) 3 sort ATP-binding cassette transporter

(B) CRThigh/CD44v9low SP 88.5% (C) CRTlow/CD44v9high SP 2.42%

21

(D) CRThigh/CD44v9high SP 3.32% (E) YPK5-Lm CRThigh/CD44v9low

( ) CRTlow/CD44v9high ( ) CRThigh/CD44v9high ( ) sort ATP-binding cassette transporter

(F) CRThigh/CD44v9low SP 43.1% (G) CRTlow/CD44v9high SP

0.78% (H) CRThigh/CD44v9high SP 12.4%

Fig.5.

IHC; immunohistochemistry,

IPMN; intraductal papillary mucinous neoplasm,

22

A

B

C

Fig.6: Calreticulin (CRT) CD44v9

islet

acinusduct

Normal tissue Absent Weak

StrongModerate

HECRT CD44v9

Merged Merged-DAPI

islet

acinus

ductNormal tissue Absent Weak

StrongModerate

23

A: CRT

CRT

CRT

50 m

B: CD44v9

CD44v9

CD44v9

50 m

C: CRT CD44v9 50 m

Table 1. Cox

Variable SE p-value Hazard ratio (95% CI)

Age 0.051 0.017 0.002 1.053 (1.019-1.088)

CRT IHC score 0.007 0.002 0.004 1.007 (1.002-1.011)

Post-operative therapy -0.815 0.365 0.026 0.443 (0.216-0.905)

CRT, calreticulin; IHC, immunohistochemistry; SE, standard error; 95% CI, 95% confidence interval.

24

Table 2. CRT

Variable CRT expression P-value

Low (n = 37) High (n = 43)

Age, years.

mean ± SD 68.1 ± 7.6 65.6 ± 9.5 0.199

Gender, No.

Male 17 18 0.713

Female 20 25

Tumor location, No.

Head 24 31 0.487

Body – tail 13 12

Tumor size, mm

mean ± SD 25.7 ± 9.9 29.8 ± 17.8 0.356

Differentiation, No.

Well 1 5 0.139

Moderate – poor 36 38

Invasion depth, No.

T1 5 2 0.013

T2 5 0

T3 27 41

Lymph node metastasis, No.

Negative 21 20 0.361

Positive 16 23

TNM stage, No.

I 7 2 0.048

II 30 41

Perineural invasion

Negative 9 3 0.030

Positive 28 40

Portal invasion

Negative 24 26 0.685

Positive 13 17

Pre-operative therapy, No.

None 20 34 0.017

Performed 17 9

Post-operative therapy, No.

None 5 8 0.538

25

Performed 32 35

CD44v9 expression, No.

Low 25 15 0.004

High 12 28

26

Fig.7. CRT CRT