Title Page
Abstract
Contents
Introduction 11
Materials and methods 13
1. Case selection 13
2. Gene expression array using NanoString 13
3. Tissue microarray (TMA) for IHC validation 14
4. Immunohistochemistry 14
5. Tissue immunolabeling and 3D imaging 15
6. Cell culture 15
7. siRNA transfection 16
8. Cancer cell invasion assay 16
9. Western blot analysis 17
10. Statistical analysis 17
Results 19
1. Clinicopathologic characteristics 19
2. Candidate genes for venous invasion through NanoString gene expression assay 19
3. Protein expression of TIMP1 and CXCR4 genes 19
4. Association between TIMP1 and CXCR4 in cell migration and invasion 20
5. The effect on TIMP1 in venous invasion ability of pancreatic cancer cell line 21
6. The association of TIMP1 expression of pancreatic cancer cell with TIMP1/PI3K/Akt signaling pathway in venous invasion 21
Discussion 39
References 42
국문요약 48
Table 1. Clinicopathologic characteristics of cases used in this study 22
Table 2. Clinicopathologic factors according to TIMP1 expression status 23
Table 3. Clinicopathologic factors according to CXCR4 expression status 24
Figure 1. Representative image of uncovered hematoxylin and eosin-stained slide for manual microdissection. Portal/ superior mesenteric vein with cancer invasion area... 25
Figure 2. Scatter plot showing gene expression fold change of vascular invasion group (VI) compared to cancer (CA) and normal vessel (NV) group. 26
Figure 3. Representative images of portal/ superior mesenteric vein with cancer invasion (A) showing diffuse strong immunoreactivity for TIMP1 (B) and CXCR4 (C) (X2.3, X20 in inlet) 27
Figure 4. TIMP1 immunohistochemical (IHC) sections with different intensity (A) TIMP1 IHC intensity 3 (200x); (B) TIMP1 IHC intensity 2 (200x); (C) TIMP1 IHC intensity 1 (200x);... 28
Figure 5. Number of strong TIMP1-expression cases in VI and CA set. The proportion of strong TIMP1-expression cases in the VI set is 86.7% (13/15), which is higher than the CA... 29
Figure 6. Overall survival according to TIMP1 status (5-year overall survival rate: TIMP1 strong, 8.6%; TIMP1 weak, 15.4%; P=0.027). 30
Figure 7. CXCR4 immunohistochemical (IHC) sections with different scores in tumor, inflammatory, and stromal cells. (A) CXCR4 IHC score 2, in tumor, inflammatory and,... 31
Figure 8. Number of cases for each CXCR4 immune-score in VI and CA set. The proportion of VI set in the immune-score 6 is 55% (11/20), which is higher than the CA... 32
Figure 9. Box-plot of CXCR4 Immune-score in CA and VI set. The immune-score of the VI set (4.73±2.22) is higher than that of CA set (1.14±1.48, p value<0.001) 33
Figure 10. Overall survival according to CXCR4 expression status (5-year overall survival rate: CXCR4 strong, 11.8%; CXCR4 weak, 10.6%; P = 0.8). 34
Figure 11. Representative image of multicolor immunofluorescent labeling of pancreatic ductal adenocarcinoma tissue with vascular invasion. TIMP1 expression (Red) is well... 35
Figure 12. Scatter plot showing association between CXCR4 (tumor, stroma, and inflammatory cells) and TIMP1 using immune-score (IS). There is a correlation between IS... 36
Figure 13. Invasion ability assay by boyden chamber system. (A) Inhibition of TIMP1 by siRNA reduced Panc1 cell invasion ability in the presence of CAF. (B) The effect of the... 37
Figure 14. Western blot analysis in co-culture condition. Expression of TIMP1 increased along with PI3Kp110, phospho-AKT, and phospho-p42 MAPK as co-incubation time... 38