**5.3 Delayed NK4 therapy for attenuation of end-stage pancreas carcinoma**

Anti-tumor effect of NK4 is also observed in a mouse model of advanced pancreas carcinoma (Tomioka *et al*., 2001). When NK4 treatment was initiated on day 10, a time when cancer cells were already invading surrounding tissues, NK4 potently inhibited the tumor growth, peritoneal dissemination, and ascites accumulation at 4 weeks after the inoculation. Such an anti-tumor effects of NK4 correlated with decreased vessel density in pancreatic tumors. In an end-stage of pancreas cancer, NK4 inhibited the malignant phenotypes, such as peritoneal dissemination, invasion of cancer cells into the peritoneal walls and ascites accumulation (Tomioka *et al*., 2001). As a result, NK4 prolonged the survival time of mice at an end-stage of cancer (**Fig. 4**). Because effective systemic therapy for pancreatic cancer is currently not available, and diagnosing pancreatic cancer in its early stages is difficult, the highly invasive and metastatic behaviors of pancreatic cancer lead to difficulty in attaining a

Endocrine Delivery System of NK4, an HGF-Antagonist and

for anti-tumor immunotherapy.

(Cell lines and treatment)

A. Digestive system**:**

B. Respiratory system**:**

Anti-Angiogenic Regulator, for Inhibitions of Tumor Growth, Invasion and Metastasis 129

Recently, it was demonstrated that NK4-mediated tumor regression depends on the infiltration of cytotoxic T lymphocytes (Kubota *et al*., 2009b). Importantly, depletion of CD8+ cells markedly abrogated the anti-tumor activity of NK4 in a mouse model of colon cancer. NK4 enhances immune responses in dendritic cells *in vitro*. Thus, NK4 may also have utility

There is now ample evidence that NK4 is useful for the inhibition of growth, invasion and metastasis in various types of tumors, such as gastric carcinoma (Hirao *et al*., 2002), pancreas cancer (Tomioka *et al*., 2001), prostate cancer (Davies *et al*., 2003), multiple myeloma (Du *et al*., 2007) and melanoma (Kishi *et al*., 2009) (**Table-1**). These results support our hypothesis

that HGF is a key determinant of tumor malignancy (Matsumoto *et al.*, 1996b).

Tumor diseases NK4 therapy Outcome Literature

Gastric carcinoma Adeno-NK4, ip Inhibitions of growth Ueda K *et al*., (TMK1 cells, and metastasis, Eur J Cancer ip, Mouse) Anti-angiogenesis, 40: 2135-2142

Hepatic carcinoma Adeno-NK4, iv Inhibitions of growth, Son G *et al*., (HUH7 cells, Anti-angiogenesis, J Hepatol 45: portal vein, Mouse) Prolonged survival 688-695 (2006)

Gallbladder cancer NK4, sc Inhibitions of growth Date K *et al*., (GB-d1 cells, and invasion Oncogene 17: sc, Mouse) 3045-354 (1998)

Pancreatic carcinoma r-NK4, ip Inhibitions of growth, Tomioka D*et al*., (SUIT-2 cells, invasion and metastasis, Cancer Res 61: intra-pancreas, Anti-angiogenesis, 7518-7524 Mouse) Reduced ascites, (2001)

Colon carcinoma NK4 cDNA, Inhibitions of growth, Wen J *et al*., (MC-38 cells, bolus iv invasion and metastasis, Cancer Gen Ther intra-spleen, (hydrodynamics) Anti-angiogenesis, 11: 419-430 Mouse) Prolonged survival (2004)

Lung carcinoma r-NK4, sc Inhibitions of growth Kuba K *et al*., (Lewis carcinoma, and metastasis, Cancer Res 60: sc, Mouse) Anti-angiogenesis, 6737-6743

Lung carcinoma Adeno-NK4, Inhibition of growth, Maemondo M (A549 cells, intra-tumor Anti-angiogenesis *et al*., Mol Ther 5: sc, Mouse) or ip 177-185 (2002)

 Mesothelioma Adeno-NK4, Inhibition of growth, Suzuki Y *et al*., (EHMES-10 cells, intra-tumor Enhanced apoptosis, Int J Cancer 127: sc, Mouse) Anti-angiogenesis 1948-1957

Reduced ascites (2004)

Prolonged survival

Enhanced apoptosis (2000)

(2010)

Fig. 4. Anti-tumor effects of NK4 on advanced pancreas cancer in mice. (A) Schedules for NK4 treatment of mice with pancreatic cancer. NK4 was injected into mice between 3 and 28 days after the inoculation of human pancreatic cancer cells (SUIT-2). (B) Inhibition of primary tumor growth by NK4. Photographs show appearance of the primary pancreatic cancer. (C) Histological analysis of the effect of NK4-treatment on tumor angiogenesis (left) and apoptosis (right). NK4-treatment reduced the number of vessel numbers, while apoptotic death of cancers was enhanced by NK4. (D) Inhibitory effects of NK4 on peritoneal metastasis. Left: Typical macroscopic findings. Middle: Changes in the number of metstatic nodules. Right: Changes in the ascite volumes. (E) Prolonged survival of tumorbearing mice treated with NK4.

long-term survival and a recurrence-free status. Targeting tumor angiogenesis and blockade of HGF-mediated invasion of cancer cells may prove to be potential therapy for patients with pancreatic cancer.
