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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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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