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Calreticulinは膵癌幹細胞様細胞に高発現している

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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

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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

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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,

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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

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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

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

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

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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

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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 % )

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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

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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)

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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 =

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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

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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

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36 CRT CRT P-CSLCs CRT P-CSLCs CD24, CD44, ESA UV P-CSLCs CRT

[1] Ryan DP, Hong TS, Bardeesy N. Pancreatic adenocarcinoma. N Engl J Med. 2014; 371: 1039-49. [2] Clevers H. The cancer stem cell: premises, promises and challenges. Nat Med. 2011; 17: 313-9.

[3] Li X, Lewis MT, Huang J, et al. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst. 2008; 100: 672-9.

[4] Zhou BB, Zhang H, Damelin M, Geles KG, Grindley JC, Dirks PB. Tumour-initiating cells: challenges and opportunities for anticancer drug discovery. Nat Rev Drug Discov. 2009; 8: 806-23.

[5] Li C, Heidt DG, Dalerba P, et al. Identification of pancreatic cancer stem cells. Cancer research. 2007; 67: 1030-7.

[6] Hermann PC, Herrler T, Aicher A, et al. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell. 2007; 1: 313-23.

[7] Rasheed ZA, Yang J, Wang Q, et al. Prognostic significance of tumorigenic cells with mesenchymal features in pancreatic adenocarcinoma. J Natl Cancer Inst. 2010; 102: 340-51.

[8] Li C, Hynes M, Dosch J, et al. c-Met is a marker of pancreatic cancer stem cells and therapeutic target. Gastroenterology. 2011; 141: 2218-27.

[9] Sureban SM, May R, Qu D, et al. DCLK1 regulates pluripotency and angiogenic factors via microRNA-dependent mechanisms in pancreatic cancer. PLoS ONE. 2013; 8: e73940.

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[10] Ishimoto T, Nagano O, Yae T, et al. CD44 variant regulates redox status in cancer cells by stabilizing the xCT subunit of system xc(-) and thereby promotes tumor growth. Cancer cell. 2011; 19: 387-400.

[11] Watanabe Y, Yoshimura K, Yoshikawa K, et al. A stem cell medium containing neural stimulating factor induces a pancreatic cancer stem-like cell-enriched population. Int J Oncol. 2014; 45: 1857-66.

[12] Yoshida GJ, Saya H. Therapeutic strategies targeting cancer stem cells. Cancer Sci. 2016; 107: 5-11. [13] Obeid M, Tesniere A, Ghiringhelli F, et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med. 2007; 13: 54-61.

[14] Yamamura Y, Tsuchikawa T, Miyauchi K, et al. The key role of calreticulin in immunomodulation induced by chemotherapeutic agents. Int J Clin Oncol. 2015; 20: 386-94.

[15] Sheng W, Chen C, Dong M, et al. Overexpression of calreticulin contributes to the development and progression of pancreatic cancer. J Cell Physiol. 2014; 229: 887-97.

[16] Yamamoto K, Yahara N, Gondo T, Ishihara T, Oka M. Establishment and characterization of a new human pancreatic cancer cell line, YPK-1. Bull Yamaguchi Med Sch. 2002; 49: 33-42.

[17] Soga F, Katoh N, Inoue T, Kishimoto S. Serotonin activates human monocytes and prevents apoptosis. J Invest Dermatol 2007;127:1947-55.

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[19] Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med. 1996; 183: 1797 - 806.

[20] Japan Pancreas Society. General Rules for the Study of Pancreatic Cancer, The 6th Edition, Revised Version edn. Tokyo, Japan: Kanehara, 2013

[21] Lee HJ, Xu X, Choe G, et al. Protein overexpression and gene amplification of epidermal growth factor receptor in nonsmall cell lung carcinomas: Comparison of four commercially available antibodies by immunohistochemistry and fluorescence in situ hybridization study. Lung Cancer. 2010; 68: 375-82.

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Fig. 1. Calreticulin

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

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Fig. 2.

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

CD44v9

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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

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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

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(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,

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A B C Fig.6: Calreticulin (CRT) CD44v9 islet acinus duct

Normal tissue Absent Weak

Strong Moderate HE CRT CD44v9 Merged Merged-DAPI islet acinus duct

Normal tissue Absent Weak

Strong Moderate

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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)

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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.

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Performed 32 35 CD44v9 expression, No.

Low 25 15 0.004

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Fig. 1.  Calreticulin
Fig. 4. YPK-Lm sort Side population (SP)
Table 2. CRT

参照

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