Manabu Onimaru

Skills assessment using a virtual reality simulator, LapSim™, after training to develop fundamental skills for endoscopic surgery

Abstract Education and training to maintain medical safety are very important within clinical settings. We have established a training center for endoscopic surgery and we regularly hold a unique training course, which focuses on the development of fundamental skills. One hundred and ninety-four surgeons who participated in our training course were divided into four groups according to their experience in performing laparoscopic procedures. Group 1: 0–19 laparoscopic procedures (n=44). Group 2: 20–49 laparoscopic procedures (n=53). Group 3: 50–99 laparoscopic procedures (n=46). Group 4: more than 100 laparoscopic procedures (n=55). All subjects underwent evaluation for “Lifting & Grasping” using a virtual reality (VR) simulator, LapSim™, before and after the training course. The mean efficiency score, time to completion and tissue damage after training were significantly improved after the training as compared with before training. Before training, subjects with greater experience had better scores. However, the only significant difference in the score was between the low experience group and greatest experience group. After training, the score increased in all groups compared with that before training, and there was no significant difference between groups. This study demonstrates the feasibility of using a VR simulator to assess fundamental skills for endoscopic surgery after training. We found that the scores for the task were associated with the level of experience of the surgeons.

Radiation Enhances Adenoviral Gene Therapy in Pancreatic Cancer via Activation of Cytomegalovirus Promoter and Increased Adenovirus Uptake

Purpose: Adenovirus-mediated gene therapy combined with radiation is expected to be a new approach to treat pancreatic cancer. However, there are no reports of definitive effects of radiation on adenovirus-mediated gene therapies. In the present study, we investigated the effect of radiation on the transduction efficiency of an adenovirus-based gene therapy. Experimental Design: We used adenovirus expressing NK4 (Ad-NK4), an antagonist for hepatocyte growth factor, as a representative gene therapy. Pancreatic cancer cells preinfected with Ad-NK4 were irradiated, and NK4 levels in culture media of these cells were measured. We investigated cytomegalovirus (CMV) promoter activity and uptake of adenovirus in these cells. To examine the effect of radiation in vivo, Ad-NK4 was given to irradiated subcutaneous tumors in nude mice, and NK4 levels in tumors were measured. Results: NK4 levels in culture media of irradiated cells were 4.5-fold (P < 0.01) higher than those of nonirradiated cells. Radiation enhanced activation of the CMV promoter and adenovirus uptake (P < 0.01), leading to increased levels of NK4. We found that activation of p38 mitogen-activated protein kinase and up-regulation of dynamin 2 may be involved in the radiation-induced activation of the CMV promoter and adenovirus uptake, respectively. NK4 levels in irradiated tumors were 5.8-fold (P = 0.017) higher than those in nonirradiated tumors. Conclusions: The present findings suggest that radiation significantly improves the efficiency of adenovirus-mediated gene transfer in pancreatic cancer and probably contributes to decreasing the dose of adenovirus required for gene transfer and controlling side effects of adenovirus infection in nonirradiated normal tissue.

Prospectively Isolated Cancer-Associated CD10+ Fibroblasts Have Stronger Interactions with CD133+ Colon Cancer Cells than with CD133− Cancer Cells

Although CD133 has been reported to be a promising colon cancer stem cell marker, the biological functions of CD133+ colon cancer cells remain controversial. In the present study, we investigated the biological differences between CD133+ and CD133− colon cancer cells, with a particular focus on their interactions with cancer-associated fibroblasts, especially CD10+ fibroblasts. We used 19 primary colon cancer tissues, 30 primary cultures of fibroblasts derived from colon cancer tissues and 6 colon cancer cell lines. We isolated CD133+ and CD133− subpopulations from the colon cancer tissues and cultured cells. In vitro analyses revealed that the two populations showed similar biological behaviors in their proliferation and chemosensitivity. In vivo analyses revealed that CD133+ cells showed significantly greater tumor growth than CD133− cells (P = 0.007). Moreover, in cocultures with primary fibroblasts derived from colon cancer tissues, CD133+ cells exhibited significantly more invasive behaviors than CD133− cells (P<0.001), especially in cocultures with CD10+ fibroblasts (P<0.0001). Further in vivo analyses revealed that CD10+ fibroblasts enhanced the tumor growth of CD133+ cells significantly more than CD10− fibroblasts (P<0.05). These data demonstrate that the in vitro invasive properties and in vivo tumor growth of CD133+ colon cancer cells are enhanced in the presence of specific cancer-associated fibroblasts, CD10+ fibroblasts, suggesting that the interactions between these specific cell populations have important roles in cancer progression. Therefore, these specific interactions may be promising targets for new colon cancer therapies.

Combination with low-dose gemcitabine and hTERT-promoter-dependent conditionally replicative adenovirus enhances cytotoxicity through their crosstalk mechanisms in pancreatic cancer

To overcome the limited clinical efficacy of conditionally replicative adenoviruses (CRAds), we investigated the effects of combination therapy with gemcitabine (GEM) and the hTERT-promoter-dependent CRAd (hTERT-CRAd), Ad5/3hTERTE1. This combination therapy exhibited enhanced cytotoxic effects on pancreatic cancer both in vitro and in vivo. Furthermore, we revealed that this enhancement effect was due to the multiple bidirectional interactions between hTERT-CRAd and GEM. The GEM-sensitizing effect of E1 expression derived from hTERT-CRAd, and the enhancement effect by GEM on hTERT promoter activity which led to the increase of adenovirus E1 and viral infectivity. This combination therapy may be a promising therapeutic approach for pancreatic cancer.

Predicting the chemosensitivity of pancreatic cancer cells by quantifying the expression levels of genes associated with the metabolism of gemcitabine and 5-fluorouracil

Gemcitabine (GEM) is the standard treatment for advanced/metastatic pancreatic cancer. However, there is a substantial subset of patients in whom the efficacy of GEM, when used as a single agent, is inadequate. Recently, the 5-fluorouracil (5-FU) prodrugs capecitabine and S-1 have been used as an alternative, either alone or in combination with GEM. The aim of the present study was to investigate the expression pattern of genes that render pancreatic cancer cells sensitive to GEM and 5-FU, and to identify markers for individualized chemotherapy, even in patients who have developed resistance. We investigated the correlation between the expression of genes associated with the metabolism of GEM and 5-FU, and sensitivity to these drugs in 15 human pancreatic cancer cell lines. We also established GEM- and 5-FU-resistant pancreatic cancer cell lines to investigate changes in the expression levels of these genes and the effects of one drug on cells resistant to the other. We found no correlation between pancreatic cancer cell sensitivity to either GEM- or 5-FU. GEM-resistant cells did not become resistant to 5-FU and vice versa. High expression of RRM1 (P=0.048) and TS x DPD (P=0.035) correlated significantly with sensitivity to GEM and 5-FU, respectively. 5-FU-resistant cells expressed significantly higher levels of TP than parental cells (P<0.05). In conclusion, pancreatic cancer cells showed no cross-resistance to GEM and 5-FU. Quantitative analyses of RRM1, TP, DPD and TS mRNA levels in pancreatic cancer cells may be useful for predicting their sensitivity to GEM and 5-FU.

High EGFR mRNA expression is a prognostic factor for reduced survival in pancreatic cancer after gemcitabine-based adjuvant chemotherapy

Pancreatic ductal adenocarcinoma (PDAC) still presents a major therapeutic challenge and a phase III clinical trial has revealed that the combination of gemcitabine and a human epidermal growth factor receptor type I (HER1/EGFR) targeting agent presented a significant benefit compared to treatment with gemcitabine alone. The aim of this study was to investigate EGFR mRNA expression in resected PDAC tissues and its correlation with patient prognosis. We obtained formalin-fixed paraffin-embedded (FFPE) tissue samples from 88 patients with PDAC who underwent pancreatectomy, and measured EGFR mRNA levels by quantitative real-time reverse transcription-polymerase chain reaction. The high-level EGFR group had significantly shorter disease-free-survival (p=0.029) and overall-survival (p=0.014) as shown by univariate analyses, although these did not reach statistical significance, as shown by multivariate analyses. However, we found that high EGFR expression was an independent prognostic factor in patients receiving gemcitabine-based adjuvant chemotherapy (p=0.023). Furthermore, we measured EGFR mRNA levels in 20 endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) cytological specimens. Altered EGFR levels were distinguishable in microdissected neoplastic cells from EUS-FNA cytological specimens compared to those in whole cell pellets. In conclusion, quantitative analysis of EGFR mRNA expression using FFPE tissue samples and microdissected neoplastic cells from EUS-FNA cytological specimens could be useful in predicting prognosis and sensitivity to gemcitabine in PDAC patients.

hTERT‐promoter‐dependent oncolytic adenovirus enhances the transduction and therapeutic efficacy of replication‐defective adenovirus vectors in pancreatic cancer cells

(Cancer Sci 2010; 101: 735–742)

Chemotherapeutic agents potentiate adenoviral gene therapy for pancreatic cancer

Adenovirus‐mediated gene therapy combined with chemotherapeutic agents is expected to represent a new approach for treating pancreatic cancer. However, there have been no reports of definitive effects of chemotherapeutic agents on adenovirus‐mediated gene therapies. In the present study, we investigated the effects of chemotherapeutic agents on the transduction efficiency of an adenovirus‐based gene therapy. Adenovirus (Ad‐NK4) expressing NK4, which acts as a hepatocyte growth factor antagonist, was used as a representative gene therapy. Pancreatic cancer cells infected with Ad‐NK4 were treated with chemotherapeutic agents (5‐fluorouracil [5FU], cisplatin or etoposide), and the NK4 levels in their culture media were measured. To examine the effects of chemotherapeutic agents in vivo, Ad‐NK4 was administered to subcutaneous tumors in mice after treatment with the agents, and the tumor NK4 levels were measured. The NK4 levels in culture media from cells treated with 5FU, cisplatin and etoposide were 5.2‐fold (P = 0.026), 6‐fold (P < 0.001) and 4.3‐fold (P < 0.001) higher than those of untreated cells, respectively. The chemotherapeutic agents also increased Ad‐NK4 uptake. The NK4 levels in tumors treated with 5FU, cisplatin and etoposide were 5.4‐fold (P = 0.006), 11.8‐fold (P < 0.001) and 4.9‐fold (P = 0.017) higher than those in untreated tumors, respectively. The present findings suggest that chemotherapeutic agents significantly improve the efficiency of adenovirus‐mediated gene transfer in pancreatic cancer. Furthermore, they will contribute to decreases in the adenovirus doses required for gene transfer, thereby controlling the side‐effects of adenovirus infection in normal tissues. (Cancer Sci 2009; 100: 722–729)

Quantitative analysis of hTERT mRNA levels in cells microdissected from cytological specimens

Clinicians frequently require cytopathological assessment of tumor samples for preoperative diagnosis, but in some specimens, diagnosis remains inconclusive after cytological examination. To date, several molecular markers, including human telomerase reverse transcriptase (hTERT), have been assessed for the ability to detect malignancy. However, analyses using whole cytological specimens are generally affected by contamination of untargeted cells. The present study investigated the feasibility of more sensitive examination by quantitative mRNA analysis of target cells microdissected from cytological specimens. Laser capture microdissection (LCM) was used to obtain target cells from cytological specimens. hTERT mRNA levels were then measured in target cells by quantitative real‐time RT‐PCR (qRT‐PCR). The effect of RNA fragmentation on qRT‐PCR was also assessed. Total RNA from cytological specimens was sometimes fragmented to a large degree. To avoid the effect of RNA fragmentation, gene specific priming and PCR primers generating short PCR products were used and no difference in delta Ct values between fragmented and non‐fragmented RNA were found. hTERT mRNA levels were measured in cells microdissected from 33 cytological specimens. The levels of hTERT mRNA were significantly higher in malignant cases compared to those in non‐malignant cases (P = 0.0003). The sensitivity was 96.2%, even when the specificities were 100%. High levels of hTERT mRNA were also found in three cases that were not diagnosed as malignant by cytological examination. Quantitative assessment of hTERT mRNA levels in cells microdissected from cytological specimens is a potential diagnostic tool to potentiate cytological examination in diagnosing malignancy. (Cancer Sci 2008; 99: 2244–2251)

Gemcitabine synergistically enhances the effect of adenovirus gene therapy through activation of the CMV promoter in pancreatic cancer cells

Adenovirus-mediated gene therapy shows remarkable promise as a new strategy for advanced pancreatic cancer, but satisfactory clinical results have not yet been obtained. To improve this gene therapy, we investigated the effects of gemcitabine (GEM) on transgene expression by adenoviral vectors and their biological effects. We used Ad-lacZ and adenoviral vector-expressing NK4 (Ad-NK4) as representative adenoviral vectors. These vectors express β-galactosidase (β-gal) and NK4 (which inhibits the invasion of cancer cells), respectively, under the control of the CMV promoter. Cells were infected with the individual adenoviruses and then treated with GEM. GEM increased β-gal mRNA expression and β-gal activity, and increased NK4 expression in both culture media and within infected cells, in dose-dependent manners. The increased expression of NK4 delivered by Ad-NK4 had biological effects by inhibiting the invasion of cancer cells. GEM also enhanced NK4 expression in SUIT-2 cells transfected with an NK4-expressing plasmid, suggesting that GEM enhanced CMV promoter activity. In in vivo experiments, NK4 expression within subcutaneously implanted tumors was increased in GEM-treated mice compared with control mice. These results suggest that adenovirus-mediated gene therapy with GEM may be a promising approach for treating pancreatic cancer, and that this combination therapy may decrease the risks of side effects.