Tissue inhibitor of metalloproteinase 1 as a biomarker of venous invasion in pancreatic ductal adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with a poor prognosis. While venous invasion is believed to contribute to liver metastasis and an unfavorable prognosis, the precise mechanisms involved remain unclear. Here, we conducted gene expression profiling on eight PDAC tissue samples exhibiting portal venous invasion (VI group) compared to PDAC samples without portal venous invasion (CA group) and normal portal vein tissues (NV group). A subset of genes, including tissue inhibitor of metalloproteinase 1 (TIMP1), C-X-C motif chemokine receptor 4 (CXCR4), olfactomedin-like 2B (OLFML2B), and cytochrome P450 family 1 subfamily B member 1 (CYP1B1), was found to be specifically expressed in the PDAC group with venous invasion. Immunohistochemical staining of 15 cases revealed significantly higher levels of TIMP1 (P=.026) and CXCR4 (P<.001) in the VI set compared to the CA set. In addition, the PDAC group with strong TIMP1 expression had a higher frequency of lymphovascular invasion (P<.001) and lower 5-year survival rates than the PDAC group with no/weak TIMP1 expression (P=.027). Specific TIMP1 expression in the venous invasion foci was highlighted on 3D reconstruction imaging. Invasion assays and/or Western blot analyses were performed on pancreatic cancer cells (Panc1), cancer-associated fibroblasts (CAFs), and human endothelial cells (EA.hy926). TIMP1 inhibition suppressed cancer cell invasion in the presence of CAFs. TIMP1 expression increased with PI3Kp110, phospho-AKT, and phospho-ERK1/2 in Panc1 cells co-cultured with CAFs and EA.hy926 endothelial cells. Our data demonstrate that TIMP1 in pancreatic cancer cells promotes venous invasion of PDACs by activating the PI3K/AKT and ERK1/2 pathways in collaboration with CAFs and endothelial cells. Therefore, TIMP1 may serve as a biomarker for venous invasion in PDACs.

Keywords: Pancreas; TIMP1; biomarker; cancer; invasion; vein.

Figure 1

A. Representative H&E staining images of areas before manual microdissection: 1) VI set, portal vein/superior mesenteric vein with cancer cell invasion; 2) CA set, PDAC tissue without portal vein/superior mesenteric vein invasion; and 3) NV set, portal vein/superior mesenteric vein located in close proximity to non-neoplastic pancreatic tissue. Dotted lines indicate the area of microdissection; PV, portal vein; PDAC, pancreatic ductal adenocarcinoma. B. Venn diagrams illustrating candidate genes that are either overexpressed or underexpressed in the VI or CA groups compared to the NV group, with P<0.05 and a difference in expression of more than two-fold. C. Scatter plot showing gene expression fold change in the VI group compared to the CA and NV groups. TIMP1, CXCR4, OLFMI2B, and CYP1B1 were overexpressed in the VI group relative to the CA and NV groups.

Figure 2

Representative histologic and immunohistochemical staining images of foci of venous invasion. (A) H&E, (B) diffuse strong cytoplasmic TIMP1 immunolabeling in the cytoplasm of cancer cells and CAFs, and (C) diffuse nuclear CXCR4 immunolabeling in the cancer cells and CAFs (all, magnification, 20×; inset, 2.3×).

Figure 3

Representative immunohistochemical staining images of TIMP1 in PDAC tissue exhibiting venous invasion. (A) Strong staining (intensity 3). (B) Intermediate staining (intensity 2). (C) Weak staining (intensity 1). (D) No staining (intensity 0; all 20× magnification). TIMP1 intensities of 2 and 3 were classified as strong, while intensities of 1 and 0 were categorized as weak or absent TIMP1 immunolabeling (A-D, 20× magnification). (E) Results of TIMP1 immunolabeling in the VI and CA sets. The proportion of cases exhibiting strong TIMP1 expression in the VI set was 86.7% (13/15), which was significantly higher than in the CA set (53.3%, 90/169; P=.026). (F) The PDAC group with strong TIMP1 expression demonstrated a poorer 5-year overall survival rate of 8.6% compared to 15.4% in the weak/no TIMP1 expression group (P=.027).

Figure 4

Multi-colour immunofluorescence labelling of 3D reconstruction of PDAC tissue at the foci of venous invasion. Green labelling for cytokeratin 19 highlights cancer cells, red indicates TIMP1 and blue labelling for desmin highlights the muscular layer of the muscular vein. A. Merged cytokeratin 19, desmin, and TIMP1 images. Cytoplasmic TIMP1 expression was clearly visible in tubule-forming cancer cells (arrows) inside the muscular venous wall, and CAFs (asterisks) surrounding scattered cancer cells (arrowhead) destroying the smooth muscle of the venous wall. B. Desmin image. C. Merged cytokeratin 19 and desmin image. D. Cytokeratin image. E. Merged TIMP1 and desmin image. F. TIMP1 image. V, vein.

Figure 5

TIMP1-induced invasion ability and related pathways in cell lines under conditions mimicking venous invasion of PDACs. (A-D) Western blot analysis for the expression of TIMP1 and its related proteins (PI3Kp110, phospho-AKT, total-AKT, phospho-ERK1/2, and total-ERK1/2) in each cell line through indirect co-culture of (A) Panc1, (B) CAFs, and (C) EA.hy926 cells at the indicated time points. (D) TIMP1 suppression in Panc1 cells co-cultured indirectly with CAFs and EA.hy926 cells at the indicated times. GAPDH was utilized as the loading control. (E-G) Assessment of invasion ability using endothelial cells coated in the Boyden chamber system. (E) Panc1 or (G) CAFs transfected with TIMP1 or scramble siRNA were allowed to penetrate the matrix and endothelial layer with or without CAFs for 24 hours. (F) The effects of TIMP1 pathway inhibitors (PI3K inhibitor LY294002 at 10 µM or ERK1/2 inhibitor PD98059 at 50 µM) with or without CAFs for 24 hours on Panc1 invasion capability. *P<.05, **P<.01, ***P<.001.

Figure 6

Illustration of TIMP1-induced venous invasion in PDAC. TIMP1 secreted by CAFs stimulates adjacent pancreatic cancer cells. In stimulated cancer cells, activation of EMT-related genes through the TIMP/PI3K/AKT enhances venous invasion, and TIMP1 signaling pathways promote CAFs activation. CAFs and endothelial cells facilitate venous invasion by promoting CAF activation and increasing vascular permeability via the PI3K/Akt and ERK1/2 signaling pathway.