1) Nestin is an intermediate filament protein expressed in pancreatic cancer cells and progenitor cells. It forms heterodimers that assemble into intermediate filaments. 2) Nestin expression was found in 30% of pancreatic cancer cases. Knockdown of nestin in pancreatic cancer cell lines decreased cancer cell migration, invasion, and metastasis in mice. 3) Nestin is also strongly expressed in the blood vessels of pancreatic tumors and promotes tumor angiogenesis. Inhibition of nestin using siRNA decreased blood vessel proliferation and tumor growth, suggesting it may be a novel therapeutic target for pancreatic cancer.

1. Expression and Roles of Nestin in Pancreatic Cancer
Departments of Pathology and Integrative Oncological
Pathology, Nippon Medical School, Tokyo, Japan

2. Nestin: Neuroepithelial Stem Cell Marker (Cell, 1990)
Class Intermediate filament protein
I Acidic keratin
II Neutral keratin, Basic keratin
III Vimentin, Desmin, GFAP, Peripherin
IV Neurofilaments (L, M, H chains)
V Lamin
VI Nestin, Tanabin, Transitin, Paranemin, Synemin
Nestin is a member of the class VI intermediate filament protein.

3. Structure of Intermediate Filament Proteins
α-helical rod domain
N-terminal C-terminal
Monomer
Nestin forms heterodimers
with vimentin or internexin.
Heterodimer
Tetramer
Intermediate filament 10 nm
Nestin forms heterodimers with other types of intermediate filament proteins, and these
heterodimers combine to form intermediate filaments.

4. Expression of Nestin in Stem/Progenitor Cells
Pancreas Endocrine
Islets
progenitors
Foregut Pancreatic Pdx1(+)
endoderm Pdx1 progenitors
p48 Exocrine
Acini
progenitors
p48(+)
Nestin(+)
p48(+) Ducts
Nestin is also expressed in progenitor cells of brain, heart, muscle, testis and
blood vessels.

5. Expression of Nestin in Tumors
Brain tumor Gastrointestinal tract tumor
Glioblastoma Pancreatic ca, GIST
Gynecological tumor Skin tumor
Ovarian ca, Uterine ca Melanoma, SCC
Nestin
Lung tumor
Non small cell ca, Endocrine or metabolic tumor
Small cell ca Breast ca, Thyroid ca
Urinary tumor Otorhinolaryngological tumor
Prostate ca Salivary gland ca
Orthopedic tumor
Osteosarcoma
Overexpression of nestin has been reported in various types of malignant tumors, and our
group has found a higher expression of nestin in glioblastoma, cervical cancer, melanoma and
pancreatic cancer.

6. Expression of Nestin in Pancreatic Cancer
Immunohistochemistry of Nestin in
Pancreatic Cancer
Nestin was strongly expressed in 30% of
pancreatic cancer cases.
Kawamoto et al. Human Pathol 2009 6

7. Expression Levels of Nestin
PANC-1 PK- 45H
1.0
250
Nestin/18S rRNA
1.0
150
0.5 *
100 0.5
*
75 *
*
0 0
Sc-1 Sc-4 Sh-1 Sh-10 Sc-13 Sc-16 Sh-7 Sh-8
Nestin
GAPDH
GAPDH
Nestin shRNA–transfected clones (Sh) showed lower expression levels of nestin than
sham cells (Sc). Matsuda et al., Cancer Biol Ther. 2011

8. Phase Contrast Images
PANC-1 PK- 45H
Sc Sc
Sh Sh
Sham cells showed round or short spindle-like appearance with loose cell adhesion, while Nestin
shRNA-transfected cells showed a sheet-like appearance with tight cell adhesion.

9. Expression of F-actin
PANC-1 PK- 45H
Sc Sc
Sh Sh
The Nestin shRNA-transfected cells showed a markedly higher expression of F-actin
(arrows) than sham cells.

10. 1 2 3 4 5 6 7
Expression Levels of E-cadherin
PANC-1 PK- 45H
E-cadherin/18S rRNA
* 250 *
4 *
* 150
3
100 0.5
2
75
1
0 0
β-ac
E-cadherin
GAPDH GA
Sc Sh Sc Sh
Expression levels of E-cadherin were higher in nestin shRNA-transfected cells than in
sham cells. Matsuda et al., Cancer Biol Ther. 2011

11. Scratch Assay (Wound Healing Assay)
Sc PANC-1 Sh Sc PK- 45H Sh
0h 0h
8h 12 h
% of wounded area filled
20 30
* *
* *
20
10
10
0 Sc-1 Sc-4 Sh-1 Sh-10 0 Sc-13 Sc-16 Sh-7 Sh-8
The percentage of the wounded area filled was statistically lower in nestin shRNA-
transfected PANC-1 and PK-45H cells than in sham cells.

12. Boyden Chamber Assay
PANC-1 PK- 45H
300
Migrated cells
500 200
* *
*
*
100
0 Sc-1 Sc-4 Sh-1 Sh-10 0 Sc-13 Sc-16 Sh-7 Sh-8
100
Invaded cells
200
*
50 100 *
** **
0
Sc-1 Sc-4 Sh-1 Sh-10 0 Sc-13 Sc-16 Sh-7 Sh-8
Migrated and invaded cells were fewer in nestin shRNA-transfected cells than sham
cells.

13. In vivo Metastasis Assay
Sc Sh
There was less metastasis to the liver by nestin shRNA-transfected cells (Sh) compared
with sham cells (Sc) in NOG mice.
Matsuda et al., Cancer Biol Ther. 2011

14. Immunohistochemical Analysis of Nestin in
Pancreatic Cancer Tissue
Ca
Nestin is strongly expressed in vascular endothelial cells adjacent to pancreatic cancer
cells.

15. Analysis of Nestin or CD34-positive Blood Vessels
using Software (WinROOF ver. 6.1)
Nestin
B
CD34

16. Proliferation Assay of Blood Vessels using
PCNA-positive Endothelial Cells
Nestin
**
PCNA
Yamahatsu et al. Ｉnt J Oncol,

17. Inhibition of Nestin in Mouse Endothelial Cells (TKD2)
TKD2 siNegative siNestin
*
Cell Proliferation Assay in vitro
TKD2
×105
6 *
Cells/well
5
4
3
2
1
0

18. Injection of siRNA targeting Mouse Nestin mRNA in Subcutaneous Human
PDAC Tumors in Nude Mice
siNegative siNestin
siNestin
(Day)
siNegative siNestin Yamahatsu et al.Ｉnt J Oncol, 2012

19. Conclusion
Nestin positive PDAC cells
(Metastasis)
siRNA targeting nestin
Nestin positive tumor vessels
（tumor angiogenesis）
Liver and Lung metastases
Inhibition of nestin using siRNA may be a novel therapeutic option for PDAC patients.

20. Thank you for your attention