Elsevier

European Journal of Cancer

Volume 47, Issue 14, September 2011, Pages 2230-2239
European Journal of Cancer

The inhibition of pancreatic cancer invasion-metastasis cascade in both cellular signal and blood coagulation cascade of tissue factor by its neutralisation antibody

https://doi.org/10.1016/j.ejca.2011.04.028Get rights and content

Abstract

Tissue factor (TF), the initiating cell surface receptor for the blood coagulation cascade, plays an important role in malignant transformation of the pancreas, although the precise mechanism remains unresolved. Here, we report that the TF – factor VIIa complex in human pancreatic cancer cells produced a significant amount of MMP-9 and promoted invasion ability in vitro and invasion and metastasis in vivo. For treatment, we successfully developed an anti-human TF monoclonal antibody that inhibits both cellular signalling and blood coagulation cascade via TF. Invasive capability and MMP-9 expression were significantly reduced by the antibody. The antibody inhibited not only tumour invasion in the orthotopic model, but also haematogenous metastasis in the portal-injection liver metastasis model. In conclusion, the TF-VIIa complex plays an important role in invasion-metastasis by enhancing tumour cell infiltration ability and forming microthrombi. The newly established anti-human TF neutralisation antibody may be useful for the treatment of pancreatic and other invasive cancers.

Introduction

In cancer invasion and metastasis, the cancer cells degrade the basement membrane and intravasate into lymphatic or blood microvessels. The cells are then transported to a new location and become clogged within the microvessels, proceeding to grow following extravasation.1 These steps include cancer cell invasion, degradation of the basement membrane and stromal extracellular matrix (ECM), and formation of microthrombi. The matrix metalloproteinase (MMP) family represents important enzymes that degrade ECM and facilitate tumour invasion.2 Amongst them, MMP-9 is well-known as one of the most important factors in facilitating invasion and metastasis in pancreatic cancer.3

Tissue factor (TF), the initiating cell surface receptor for the coagulation cascade, activates factor VIIa. The TF-VIIa complex activates factor X, and consequently this protease cascade forms fibrin clots.4, 5 The relationship between cancer and blood coagulation was initially described by the French surgeon Trousseau.6 Cancer patients, especially those with pancreatic, stomach, and glioma cancer, often suffer from a state of hypercoagulation and venous thrombosis, leading to patient morbidity and mortality.7, 8, 9 In another study using a fibrinogen-deficient transgenic mouse model, fibrinogen appeared to be an important element of the metastatic potential of circulating tumour cells.10 Meanwhile, TF plays an important role in not only blood coagulation but also cell signalling in which the TF-VIIa complex phosphorylates extracellular-regulated kinase 1/2 (ERK1/2) via protease-activated receptor-2 (PAR-2).11 Moreover, its complex promotes the expression of interleukin-8 (IL-8) and invasion in breast cancer cell lines.12 However, the concrete involvement of TF in tumour invasion-metastasis has not yet been fully evaluated.

Human pancreatic cancer has one of the worst prognoses amongst cancers.13 Invasion and metastasis advancing beyond the pancreas are typical. Direct invasion to nearby organs, such as the stomach, duodenum, colon, spleen and kidney frequently occurs. Distant metastasis to the liver and peritoneal dissemination are also commonly seen.14, 15 In terms of the relationship between TF and pancreatic cancer, TF expression is an important early event in malignant transformation of the pancreas.16 TF expression may contribute to the aggressiveness of pancreatic cancer that would stimulate tumour invasiveness, and evaluation of the primary tumour for TF expression may identify patients with a poor prognosis.17, 18

Therefore, elucidation of the relationship between TF and pancreatic cancer invasion-metastasis may lead to the development of new therapeutic strategies as well as a better understanding of pancreatic cancer biology.

Section snippets

Cell lines

Human pancreatic cancer cell lines BxPC3, Panc1, Capan1, and MIA PaCa-2 were purchased from the American Type Culture Collection (Rockville, MD, USA). The cell lines were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% faetal bovine serum (FBS) (Cell Culture Technologies, Gaggenau-Hoerden, Germany), 100 units/mL streptomycin, and 2 mmol/L L-glutamine (Sigma, St. Louis, MO, USA) in an atmosphere of 5% CO2 at 37 °C.

Immunocytochemistry

Cells (1 × 105) were seeded on a 4-well culture slide (BD

TF-positive human pancreatic cancer cells, BxPC3, enhance invasion potential in vitro and in vivo

We used the BxPC3 cell line in a series of our experiments because real-time PCR analysis and immunostaining of TF showed that BxPC3 strongly expressed TF amongst the four pancreatic cancer cell lines (Fig. 1A). Then, we examined the status of TF expression for BxPC3 in vitro and in vivo. Immunostaining revealed TF expression specifically in cancer cells contacting with stromal tissues, namely the invasive front in vivo, although TF expression was uniformly observed in all cells in vitro (Fig. 1

Discussion

Pancreatic cancer is the most refractory neoplasm and possesses several clinicopathological characteristics. First, pancreatic cancer exerts extensive invasion and metastasis to other organs.14, 15 Second, thrombosis occurs most frequently in patients with pancreatic cancer.7 Third, expression of TF may contribute to the aggressiveness of pancreatic cancer that stimulates tumour invasiveness, and evaluation of the primary tumour for TF expression may identify patients with a poor prognosis.17,

Grant support

This work was supported partly by a Grant-in-Aid from the Third Term Comprehensive Control Research for Cancer, the Ministry of Health, Labor and Welfare (Matsumura, H19-025), Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology (Matsumura, 17016087), and the Japanese Foundation for Multidisciplinary Treatment of Cancer (Matsumura), Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program) and the Princess

Conflict of interest statement

None declared.

Acknowledgements

We thank Dr. Miyawaki (RIKEN) for Venus. We thank N. Mie and M. Ohtsu for their technical assistance, and K. Shiina for her secretarial assistance.

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