Hiromune Shimamura

In vivo evaluation of the early events associated with liver metastasis of circulating cancer cells

The mechanism of metastasis formation remains still largely unknown. Many studies underline the importance and complexity of the initial arrest of the circulating tumour cells in the target organ, a key stage in metastasis occurrence. In our study, we evaluated by visual means the metastasis formation using an in vivo microscopy system in a murine model. Moreover, we investigated the involvement of P-selectin in these processes using immunohistochemistry and P-selectin knockout mice. The present study offers direct evidence of distinct pathways for tumour metastasis formation by a lymphoma cell – EL-4 and a solid tumour cell – C26. Off-line analysis of the images and histological data confirmed that mechanical entrapment of the solid tumour cell, which had a bigger diameter than that of the liver sinusoids, promoted metastasis without any detectable involvement of adhesion molecules. On the other hand, we observed that lymphoma cells, in spite of their smaller diameter as compared to the sinusoids, promoted liver metastasis as well, but with the essential participation in their arrest of P-selectin, indicating an adhesion molecule-mediated pathway.

Do peritoneal macrophages play an essential role in the progression of acute pancreatitis in rats

Introduction Macrophages are considered to play an essential role in the events leading to systemic inflammatory response. Some are known to reside in the peritoneal cavity but there are no reports defining the participation of peritoneal macrophages (PMs) in the progression of acute pancreatitis. Aim To clarify the role of PMs in the progression of acute pancreatitis. Methodology Acute pancreatitis was induced in rats from which macrophages other than PMs were greatly depleted, and in rats greatly depleted of macrophages including PMs. Macrophages were depleted by the injection of liposome encapsulated dichloromethylene bisphosphonate. After the induction of acute pancreatitis, local pancreatic inflammation, intraperitoneal inflammation and lung injury were compared between the 2 groups. Results Local pancreatic inflammation did not differ between the 2 groups. However, intraperitoneal inflammation was clearly improved by the depletion of PMs. Serum cytokine level and lung injury were also improved by the depletion of PMs. Conclusion Peritoneal macrophages extend inflammation from the pancreas to the peritoneal cavity and subsequently induce lung injury in acute pancreatitis. Peritoneal macrophages play an essential role in the systemic inflammatory response and the progression of acute pancreatitis in the rat.

Ipsilateral Percutaneous Transhepatic Portal Vein Embolization with Gelatin Sponge Particles and Coils in Preparation for Extended Right Hepatectomy for Hilar Cholangiocarcinoma

PURPOSE To evaluate the effectiveness and safety of ipsilateral percutaneous transhepatic portal vein embolization (PTPVE) with gelatin sponge particles and coils to induce lobar hypertrophy in patients with hilar cholangiocarcinoma in preparation for extended right hepatectomy. MATERIALS AND METHODS Between 1999 and 2004, PTPVE was performed in 22 patients with hilar cholangiocarcinoma (mean age, 67 years; range, 57-77 y; 16 men and six women). Percutaneous puncture of the right portal vein was performed under ultrasound guidance. A reverse-curve catheter was used for right portal vein embolization. Coils were used to occlude second-order branches. The future liver remnant volume was assessed by comparing computed tomographic scans before and 14-24 days after PTPVE. RESULTS PTPVE was technically successful in all cases. The average increase in ratio of future liver remnant volume to total liver volume was 8.6%. Liver function tests after PTPVE but before surgery showed no significant changes. Nineteen patients underwent hepatic resection without liver failure. In three patients, tumors could not be removed because of detection of extrahepatic disease. One patient who underwent successful hepatic resection had an abscess in the removed right lobe. CONCLUSION Ipsilateral PTPVE with gelatin sponge and coils appears to be effective and safe for extended right hepatectomy for hilar cholangiocarcinoma.

The antiangiogenesis effect of interleukin 12 during early growth of human pancreatic cancer in SCID mice.

Interleukin 12 (IL-12) is a heterodimeric cytokine that exerts a potent antitumor effect through its pleiotropic actions. It was recently reported that IL-12 has also a potent antiangiogenic effect through the induction of IFN-&ggr;, which triggers the production of chemokines such as IP-10 that has been shown to have antiangiogenesis properties. In this study we transfected the IL-12 gene into a human pancreatic adenocarcinoma cell line (PK-1). PK-1 cells transfected with the green fluorescence protein (gfp) gene were used as positive controls. The in vitro growth curve and in vivo tumor growth of transfectants (IL-12/PK-1 and gfp/PK-1) were compared with those of parental cells. The SCID mice used in this study were administered antiasialo GM-1 Ab (100 &mgr;g, i.p., twice weekly) to deplete the remaining immunoeffector cells, NK cells. Using a skinfold chamber model, we observed and recorded tumor angiogenesis by intravital microscopy. In vitro growth of IL-12/PK-1 and gfp/PK-1 cells was not different from that of wild-type PK-1 cells (wt/PK-1). However, IL-12 transfected PK-1 cells did not develop into tumors as did the wt/PK-1 cells after subcutaneous inoculation in antiasialo GM-1 Ab administered SCID mice. The growth of IL-12/PK-1 tumors was restored in mice treated with anti-IL-12 antibody. We found that IL-12/PK-1, in contrast to gfp/PK-1 and wt/PK-1, failed to initiate an angiogenic response, as observed in the skinfold chamber model. These results indicate that the antiangiogenesis effect of IL-12 alone, without immune system involvement, is sufficient to block the growth of human pancreatic cancer.

Gene therapy for intraperitoneally disseminated pancreatic cancers by Escherichia coli uracil phosphoribosiltransferase (UPRT) gene mediated by restricted replication-competent adenoviral vectors

Although patients with unresectable pancreatic tumors have been treated with 5‐fluorouracil (5FU)‐based combination chemotherapy, the drug resistance of cancer cells presents a crucial therapeutic problem. It was reported that UPRT overcomes 5FU resistance. UPRT catalyzes the synthesis of 5‐fluorouridine monophosphate (FUMP) from Uracil and phosphoribosylpyrophosphate (PRPP). The antitumor effect of 5FU is enhanced by augmenting 5‐fluorodeoxyuridine monophosphate (FdUMP) converted from FUMP, which inhibits thymidylate synthetase (TS). We first demonstrated that injecting an E1‐deficient adenoviral vector (Adv) expressing UPRT (AxCAUPRT) followed by 5‐FU treatment resulted in a volume reduction of xenotransplanted human tumors. In examining the therapeutic effect of AxCAUPRT/5‐FU against peritoneal dissemination, we found that non‐selective gene transduction of AxCAUPRT caused severe adverse effects arising from the increase of F‐dUMP in normal intestine. Because the therapeutic gene delivered by a restricted replication‐competent Adv lacking 55 kDa E1B protein (AxE1AdB) is speculated to be expressed selectively in tumors, mice with established tumors were injected with AxE1AdB and E1‐deleted Adv expressing the lacZ reporter gene (AxCAlacZ). The expression of the reporter gene (lacZ) was selectively enhanced in disseminated tumors. The therapeutic advantage of restricted replication competent Adv that expresses UPRT (AxE1AdB‐UPRT) was evaluated in an intraperitoneal disseminated tumor model. To study the anti‐tumor effect of AxE1AdB‐UPRT/5FU, mice with disseminated AsPC‐1 tumors were administered the Adv, followed by the 5FU treatment. It was shown that the treatment with AxE1AdB‐UPRT/5FU caused a dramatic reduction of the disseminated tumor burden without toxicity in normal tissues. Our results showed that the AxE1AdB‐UPRT/5FU system is a promising tool for intraperitoneal disseminated pancreatic cancer.

Adjuvant immunotherapy using fibroblasts genetically engineered to secrete interleukin 12 prevents recurrence after surgical resection of established tumors in a murine adenocarcinoma model

BACKGROUND To explore effective therapeutic strategy against cancer of the gastrointestinal tract, tumor vaccination using fibroblasts secreting interleukin-12 (IL-12) was developed as an adjuvant therapy against murine tumor after surgical resection. METHODS Initially, IL-12 was genetically engineered into fibroblasts (IL-12/3T3 cells), and then we evaluated in vivo and in vitro antitumor effects. In the vaccination model, irradiated C-26 tumor mass was reinoculated intradermally with IL-12/3T3 cells in mice as a tumor vaccine to examine how much it suppresses tumor recurrence. RESULTS IL-12/3T3 cells producing 7.2 ng/10(6) cells/24 h murine IL-12 in vitro exerted dose-dependent potent tumor suppression when coinoculated with C-26 cells in vivo. Specific immunity was also acquired in 63% of mice in vivo. In the vaccination model, protective immunity was developed in 70% of mice that were inoculated with irradiated tumor mass and IL-12/3T3 cells. In addition, local recurrence was not observed in vaccinated mice, although 44% of control mice had recurrence. CONCLUSIONS Coinoculation of genetically engineered fibroblasts secreting IL-12 with irradiated tumor mass was proved to be an effective tumor vaccine. This system of vaccination is easily applicable to clinical situations, particularly to human gastrointestinal tract cancers.

Establishment of an experimental liver metastasis model by intraportal injection of a newly derived human pancreatic cancer cell line (KLM-1)

SummaryConclusionIt is suggested that this liver metastasis model formed by a highly metastatic variant (KLM-1) is valuable for the study of the liver metastatic processes of human pancreatic cancer.BackgroundLiver metastasis in the early postoperative period is one of the causes for the poor prognosis of patients with resected pancreatic cancer. Therefore, it is necessary to establish an experimental model to study the mechanisms of liver metastasis in pancreatic cancer.MethodsHuman pancreatic cancer cell lines (PK-1, PK-9, and KLM-1) were injected into the portal vein of nude mice with or without pretreatment with antiasialo GM1, and colonies of liver metastases were counted for comparison of metastatic ability of these cell lines. Biological and histopathological characteristics of the highly liver metastatic cell line (KLM-1) were compared with its parent cell line (PK-1).ResultsPK-1 cells and PK-9 cells rarely formed liver metastasis foci, but pretreatment with antiasialo GM1 promoted liver metastasis. KLM-1 cells formed liver metastases at the rate of 70% even without antiasialo GM1 pretreatment. KLM-1 cells had such biological characteristics as short doubling time, short lag phase, and resistance to NK cytotoxicity. After intraportal injection of125I-labeled KLM-1 cells, radioactivitiy as well as micrometastases were detected in the liver at 72 h.

Irradiated Pancreatic Cancer Cells Undergo both Apoptosis and Necrosis, and Could Be Phagocytized by Dendritic Cells

The interaction of immature dendritic cells (DC) with irradiated pancreatic cancer cells was examined. Flow cytometric analysis using annexin V and propidium iodide revealed that ionizing radiation (25–35 Gy X-ray) induced both apoptosis and necrosis in pancreatic cancer cell lines. After irradiation, PK-1 and Panc-1 cells were likely to undergo necrosis, whereas MIAPaCa-2 cells underwent apoptosis. When DiO-stained immature DCs were co-incubated with DiI-stained irradiated MIAPaCa-2, it was observed under fluorescent microscopy that DCs phagocytized dead tumor cells as early as 4 h after co-incubation. The DCs’ phagocytosis of irradiated tumor cells was also confirmed by flow cytometry. These results suggest that irradiated pancreatic cancer cells, which undergo both apoptosis and necrosis, could be a good source of tumor-associated antigens for cross-presentation by DCs.

Mucin-producing tumor of the pancreas — surgical treatment

To evaluate appropriate surgical treatment and to predict prognosis for mucin-producing tumors (MPTs) of the pancreas, we conducted a retrospective clinicopathological study in 25 patients with MPT. Eight patients who had mild dysplasia of the pancreas were diagnosed with benign disease and 17 patients with ductal lesions of the pancreas with severe dysplasia/carcinoma in situ or with invasive foci were diagnosed as malignant. Clinical symptoms and histories, tumor markers, cytological examination, and radiological and gross pathological features were compared in the benign and malignant groups. Clinical symptoms, history, and tumor markers were of no value for predicting prognosis. Cytological examination results were ambiguous because of too many falsenegatives. Radiological features, including the diameters of the cystic space and of the main pancreatic duct (MPD) were shown to be valuable. The significant maximal diameters, above which malignancy was seen, were 30mm for the diameter of the cystic space and 10mm for the MPD. In regard to gross features, the uniformly dilated main duct-type and focally dilated main duct-type were significant indicators of malignancy. Long term follow-up was available in 22 patients. Seven patients died of recurrence of the tumor or other malignancies. We could have predicted prognosis by the radiological features and gross appearance. Surgical treatment should be considered the primary choice for MPT. Total pancreatectomy would be ideal for patients with the uniformly dilated main duct-type. Because of the probability of multicentricity and the transforming potential of mild dysplasia, very careful follow-up is necessary for patients with MPT.

Cytotoxic Effect of Bone Marrow-Derived Dendritic Cells

Some reports suggest that cancer patients have a better prognosis when dendritic cells (DCs) infiltrate their tumors. Although the role of these DCs is not fully understood, we focused on a potential effector function of DCs and examined whether DCs could directly mediate tumor cell cytotoxicity. Murine DCs were propagated from bone marrow stem cells using granulocyte/monocyte colony-stimulating factor and interleukin-4 for 7 days and were sorted by a magnetic-activated cell sorter system. Purified CD11c+ DCs were shown to be cytotoxic against murine tumor cells such as MCA205 fibrosarcoma. The annexin V staining assay showed that MCA205 underwent apoptosis when incubated with DCs for 4h. This cytotoxic effect was not blocked by an anti-Fas ligand antibody but was partially abrogated by the Ca2+ chelator EGTA/MgCl2. These data suggest that DCs are directly cytotoxic to some kinds of tumor cell, and this effect is mediated via both calcium-dependent and calcium-independent pathways.