Cancer Peritoneal Dissemination Research Articles

Enhanced cytotoxicity with a novel system combining the paclitaxel-2′-ethylcarbonate prodrug and an HSV amplicon with an attenuated replication-competent virus, HF10 as a helper virus

We previously demonstrated that HF10, which is a natural, non-engineered HSV-1, has potent oncolytic activity in the treatment of solid malignant tumors in vitro and in vivo [H. Takakuwa, F. Goshima, N. Nozawa, T. Yoshikawa, H. Kimata, A. Nakao, et al., Oncolytic viral therapy using a spontaneously generated herpes simplex virus type 1 variant for disseminated peritoneal tumor in immunocompetent mice, Arch. Virol. 148 (2003) 813–825; S. Kohno, C. Lou, F. Goshima, Y. Nishiyama, T. Sata, Y. Ono, Herpes simplex virus type 1 mutant HF10 oncolytic viral therapy for bladder cancer, Urology 66 (2005) 1116–1121; D. Watanabe, F. Goshima, I. Mori, Y. Tamada, Y. Matsumoto, Y. Nishiyama, Oncolytic virotherapy for malignant melanoma with herpes simplex virus type 1 mutant HF10, J. Dermatol. Sci. 50 (2008) 185–196; A. Nawa, C. Luo, L. Zhang, Y. Ushijima, D. Ishida, M. Kamakura, et al., Non-engineered, naturally oncolytic herpes simplex virus HSV1 HF10: applications for cancer gene therapy, Curr. Gene. Ther. 8 (2008) 208–221]. Previous reports have also shown that a combination of HF10 and paclitaxel (TAX) was more efficacious than either regimen alone for some types of malignant tumors [S. Shimoyama, F. Goshima, O. Teshigahara, H. Kasuya, Y. Kodera, A. Nakao, et al., Enhanced efficacy of herpes simplex virus mutant HF10 combined with paclitaxel in peritoneal cancer dissemination models, Hepatogastroenterology 54 (2007) 1038–1042]. In this study, we investigated the efficacy of gene-directed enzyme prodrug therapy (GDEPT) using a novel system that combines the paclitaxel-2′-ethylcarbonate prodrug (TAX-2′-Et) and an HSV amplicon expressing rabbit-carboxylesterase (CES) with HF10 as a helper virus. This GDEPT system aims to produce high level of CES at the tumor site, resulting in efficient local conversion of the TAX-2′-Et prodrug into the active drug TAX [A. Nawa, T. Tanino, C. Lou, M. Iwaki, H. Kajiyama, K. Shibata, et al., Gene directed enzyme prodrug therapy for ovarian cancer: could GDEPT become a promising treatment against ovarian cancer?, Anti-Cancer Agents Med Chem 8 (2008) 232–239]. We demonstrated that the green fluorescent protein (GFP) gene, as a trace maker, was more efficiently introduced by the HSV amplicon compared to the expression vector, pHGCX, and that the HSV amplicon system expressed an active CES enzyme that could convert TAX-2′-Et to TAX in Cos7 cells. Furthermore, although the cytotoxicity of this amplicon system was not enhanced in virus-sensitive tumor cells, it was significantly enhanced in low virus-sensitive tumor cells in the presence of the prodrug in a concentration-dependent manner, compared to the control virus alone ( p < 0.05). These results indicate that the addition of a prodrug converting enzyme may be a feasible approach to further enhance the efficacy of HF10 as a cancer therapeutics in low HF10-sensitive malignancies.

Changes in the expression of E-cadherin repressors, Snail, Slug, SIP1, and Twist, in the development and progression of ovarian carcinoma: the important role of Snail in ovarian tumorigenesis and progression

Changes in the expression of E-cadherin have been reported to be important in the tumorigenesis and progression of epithelial ovarian carcinoma. To further examine the mechanisms regulating E-cadherin expression in ovarian tumorigenesis, we investigated the immunohistochemical expression of transcriptional repressors for E-cadherin, such as Snail, Slug, SIP1, and Twist, in the ovarian surface epithelium (OSE) and 95 cases of epithelial ovarian tumors. OSE cells were negative for SIP1 and Slug, whereas weak expression of Snail and Twist was observed in 8 (73%) and 3 (27%) cases, respectively. Of 95 ovarian tumors, the expression of Snail, Slug, SIP1, and Twist increased stepwise in benign, borderline, and malignant tumors. Among them, the expression of Snail showed significantly inverse correlation with that of E-cadherin. Regarding the FIGO stage classification, the expressions of Snail and Twist were significantly increased in advanced cases. The prognosis of ovarian carcinoma patients positive for Snail expression was poorer than that of negative patients. Our results indicate that the expression of E-cadherin transcriptional repressors increased with malignancy in ovarian epithelial neoplasms and that the expression of E-cadherin and its negative regulators is altered during ovarian cancer development and peritoneal dissemination.

激光诱导荧光技术在体探测裸鼠腹膜胃癌播散

激光诱导荧光技术在体探测裸鼠腹膜胃癌播散

Suppression of ovarian cancer by muscle‐mediated expression of soluble VEGFR‐1/Flt‐1 using adeno‐associated virus serotype 1‐derived vector

Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis in a variety of tumors. A soluble form of Flt-1 (sFlt-1), a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidences suggest the applicability of sFlt-1 in tumor suppression by means of anti-angiogenesis. We previously demonstrated the efficacy of sflt-1 gene expression in situ to suppress tumor growth and ascites in ovarian cancer. Here, we demonstrate the therapeutic applicability of muscle-mediated expression of sFlt-1 in tumor-bearing mice. Initially, tumor suppressive action was confirmed by inoculating sFlt-1-expressing ovarian cancer (SHIN-3) cells into mice, both subcutaneously and intraperitoneally. To validate the therapeutic efficacy in a more clinically relevant model, adeno-associated virus vectors encoding sflt-1 were introduced into mouse skeletal muscles and were subsequently inoculated with tumor cells. As a result, high serum sFlt-1 levels were constantly observed, and the growth of both subcutaneously- and intraperitoneally-inoculated tumors was significantly suppressed. No delay in wound healing or adverse events of neuromuscular damage were noted, body weight did not change, and laboratory data, such as those representing liver and renal functions, were not affected. These results indicate that sFlt-1 suppresses growth and peritoneal dissemination of ovarian cancer by the inhibition of angiogenesis, and thus suggest the usefulness of gene therapy for ovarian cancer.

Targeted chemotherapy against intraperitoneally disseminated colon carcinoma using a cationized gelatin–conjugated HVJ envelope vector

The hemagglutinating virus of Japan envelope (HVJ-E; Sendai virus) vector derived from inactivated HVJ particles can be used to deliver DNA, proteins, and drugs into cells both in vitro and in vivo. HVJ-E is capable of delivering bleomycin, an anticancer drug, to various cancer cell lines, thereby producing 300-fold greater cytotoxicity than administration of bleomycin alone. In a mouse model of peritoneally disseminated colon cancer, we injected HVJ-E containing the luciferase gene into the peritoneum. Unexpectedly, luciferase gene expression was not observed within the tumor deposits or any organs. However, when combined with cationized gelatin (CG), CG-HVJ-E produced a high level of luciferase gene expression primarily within the tumor deposits. Forty-eight hours after introducing colon cancer cells into the peritoneum of experimental mice, CG-HVJ-E with or without bleomycin was injected into the abdominal cavity. Following six injections of bleomycin-incorporated CG-HVJ-E, complete responses were observed in 40% of the mice examined. All of the mice that received either empty CG-HVJ-E or bleomycin alone died within 40 days of having cancer cells introduced into the peritoneum. When the mice with complete responses were rechallenged with colon cancer cells from the same cell line, no tumors developed. Thus, CG-HVJ-E may suppress peritoneal dissemination of cancer.

Enhanced efficacy of conditionally replicating herpes simplex virus (G207) combined with 5‐fluorouracil and surgical resection in peritoneal cancer dissemination models

The therapeutic efficacy of G207, a replication-competent herpes simplex virus, for malignancies is increased when combined with certain chemotherapies, but the mechanism is unclear and the interaction between G207 and surgical resection has not been extensively studied. The goals of the current study were to examine the performance of combination treatments for peritoneal disseminated cancers and to explore the mechanism of effective combinations. Hamsters and SCID and BALB/c mice harboring peritoneal dissemination of gallbladder, gastric or colon cancer cells were treated with G207, 5-fluorouracil (5FU), or surgical resection alone, or G207 combined with 5FU or surgery. Animal survival, antiviral immunity, intratumoral ribonucleotide reductase activity, and viral spread were compared between the groups. The combination of G207 and 5FU prolonged the survival of hamsters bearing peritoneal dissemination of gallbladder cancer compared with the controls, G207 alone and 5FU alone. 5FU did not suppress the production of neutralizing antibodies against G207, but increased ribonucleotide reductase activity and viral spread in subcutaneous gallbladder tumors. The enhanced efficacy of the combination treatment was also observed in immunodeficient mice with disseminated gastric cancer. Although surgical resection did not significantly prolong animal survival or increase the intratumoral activity of ribonucleotide reductase, long-term survivors emerged from groups of animals treated with surgical resection and G207 for gallbladder and colon disseminated cancers. These results indicate that the increased activity of ribonucleotide reductase in tumors mediated by 5FU and the decreased tumor burden resulting from surgical resection may enhance the therapeutic efficacy of oncolytic herpes virus for peritoneal disseminated cancer.

Prognostic Significance of Tumor Markers in Peritoneal Lavage in Advanced Gastric Cancer

Objective: Predicting peritoneal dissemination of cancer is very difficult whatever method of examination is used. Recently, a cytological examination of peritoneal lavage has been shown to be a feasible measure to predict an early state of peritoneal seeding. The predictive value of the levels of tumor markers in peritoneal lavage for peritoneal metastasis from gastric carcinoma was thus studied. Methods: In 229 patients gastric cancer tumor markers, CEA, CA 125, and CA 19-9, in peritoneal lavage were intraoperatively evaluated using a chemiluminescent enzyme immunoassay. Results: CEA in peritoneal lavage at a cutoff level of 0.5 ng/ml showed overall a higher sensitivity of 75.8% at a specificity of 90.8% for a diagnosis of peritoneal dissemination including cytologically positive peritoneal lavage [CY(+)] than CA 125 or CA 19-9 in peritoneal lavage. The CEA level in peritoneal lavage as well as both serosal invasion and the CA 125 level in peritoneal lavage were significant factors for the prediction of peritoneal dissemination including CY(+) with a relative risk of 6.6, 14.1 and 9.4. In patients undergoing curative operations, the recurrence rate for peritoneal dissemination and liver metastasis in cases with CEA levels in peritoneal lavage of ≧0.5 ng/ml was significantly higher than that in cases with CEA levels of <0.5 ng/ml (p < 0.0001, p < 0.002). Conclusions: These finding suggest that the CEA level in peritoneal lavage is thus considered to be a predictor of peritoneal dissemination including CY(+).

Differential gene expression profiles of gastric cancer cells established from primary tumour and malignant ascites.

Advanced gastric cancer is often accompanied by metastasis to the peritoneum, resulting in a high mortality rate. Mechanisms involved in gastric cancer metastasis have not been fully clarified because metastasis involves multiple steps and requires a combination of altered expressions of many different genes. Thus, independent analysis of any single gene would be insufficient to understand all of the aspects of gastric cancer peritoneal dissemination. In this study, we performed a global analysis of the differential gene expression of a gastric cancer cell line established from a primary main tumour (SNU-1) and of other cell lines established from the metastasis to the peritoneal cavity (SNU-5, SNU-16, SNU-620, KATO-III and GT3TKB). The application of a high-density cDNA microarray method made it possible to analyse the expression of approximately 21 168 genes. Our examinations of SNU-5, SNU-16, SNU-620, KATO-III and GT3TKB showed that 24 genes were up-regulated and 17 genes down-regulated besides expression sequence tags. The analysis revealed the following altered expression such as: (a) up-regulation of CD44 (cell adhesion), keratins 7, 8, and 14 (epitherial marker), aldehyde dehydrogenase (drug metabolism), CD9 and IP3 receptor type3 (signal transduction); (b) down-regulation of IL2 receptor γ, IL4-Stat (immune response), p27 (cell cycle) and integrin β4 (adhesion) in gastric cancer cells from malignant ascites. We then analysed eight gastric cancer cell lines with Northern blot and observed preferential up-regulation and down-regulation of these selected genes in cells prone to peritoneal dissemination. Reverse transcriptase–polymerase chain reaction confirmed that several genes selected by DNA microarray were also overexpressed in clinical samples of malignant ascites. It is therefore considered that these genes may be related to the peritoneal dissemination of gastric cancers. The results of this global gene expression analysis of gastric cancer cells with peritoneal dissemination, promise to provide a new insight into the study of human gastric cancer peritoneal dissemination.British Journal of Cancer (2002) 87, 1153–1161. doi:10.1038/sj.bjc.6600580 www.bjcancer.com© 2002 Cancer Research UK

Expression of vascular endothelial growth factor and E-cadherin in human ovarian cancer: association with ascites fluid accumulation and peritoneal dissemination in mouse ascites model.

Ascites formation and peritoneal dissemination are critical problems in patients with advanced ovarian cancer. Vascular endothelial growth factor (VEGF), also known as angiogenic growth factor, is a potent mediator of peritoneal fluid accumulation and angiogenesis of tumors. E‐Cadherin is an adhesion molecule that is important for cell‐to‐cell interaction. To elucidate the molecular mechanism of ascites formation and peritoneal dissemination of ovarian cancer, we examined the expression of VEGF and E‐cadherin in different ovarian cancer cell lines and utilized nude mice to compare the biological characteristics of ovarian cancer cells. Three human ovarian cancer cell lines (AMOC‐2, HNOA and HTBOA) were used in this study. Expression of genes was analyzed by northern blotting and RT‐PCR methods. AMOC‐2 expressed E‐cadherin, but not VEGF. HNOA expressed VEGF without E‐cadherin expression. HTBOA expressed both VEGF and E‐cadherin. Each human ovarian cancer model revealed a specific feature. The AMOC‐2 mouse had a single large peritoneal tumor without ascites or remarkable peritoneal dissemination. HTBOA and HNOA mice had bloody ascites and marked peritoneal dissemination. Introduction of VEGF antisense into HTBOA cells could inhibit the ascites formation. It is suggested that VEGF is important for the ascites formation via the increased vascular permeability effect. The deregulation of E‐cadherin expression might be involved in the peritoneal dissemination. These molecules are important for the formation of specific features of advanced ovarian cancer. Ovarian cancer cell lines that had different gene expression patterns produced nude mouse human ovarian cancer models with different characteristics.

Significance of integrin α2/β1 in peritoneal dissemination of a human gastric cancer xenograft model

In the present study, the role of integrin alpha2/beta1 in peritoneal dissemination of gastric cancer was investigated using an in vivo xenograft model for the highly metastatic MKN-45-P gastric cancer cells. Metastatic ability of MKN-45-P cells was significantly associated with the simultaneous expression of integrin alpha2 and alpha3 subunits. In an in vitro adhesion assay, neutralizing antibody for integrin alpha2 or beta1 subunit inhibited the adhesion of MKN-45-P cells to collagen type I and type IV. Moreover, the injection of anti-beta1 monoclonal antibody reduced the number of cancer cells on the peritoneum in nude mice that had been inoculated with MKN-45-P cells. These results suggest that integrin alpha2/beta1 represents a candidate target molecule available for the prevention of gastric cancer peritoneal dissemination.

Polymerase chain reaction for detection of carcinoembryonic antigen-expressing tumor cells on milky spots of the greater omentum in gastric cancer patients: a pilot study.

Our recent studies indicate that omental milky spots are frequently involved in the early stage of peritoneal cancer dissemination. We have used carcinoembryonic antigen (CEA)-specific RT-PCR for omental milky spots to predict peritoneal recurrence in gastric cancer patients. CEA mRNA was found to be positive in both 10 peritoneal washes and 16 greater omenta of 30 gastric cancer patients, including all 6 patients who showed positive results for both cytology and RT-PCR of peritoneal wash and omentum. Three of the 6 cases with positive RT-PCR in the greater omentum but not in the peritoneal wash showed recurrence of peritoneal carcinomatosa within 2 years after operation. Micrometastasis on omental milky spots was histologically confirmed in 6 of 30 gastric cancer cases. Non-specific band was detected only in the omentum of 1 case of 15 benign disease (7%), but not in peritoneal washes (0%), probably due to weak expression of CEA in mesothelial cells. Our results show that CEA-specific RT-PCR targeting micro-metastases on omental milky spots is more sensitive than targeting the peritoneal wash or conventional cytology, and suggest that this method is useful for the prediction of peritoneal recurrence in gastric cancer patients.

Antitumor efficacy of regional oncolytic viral therapy for peritoneally disseminated cancer.

Oncolytic viral therapy is a promising new method of cancer treatment. Peritoneal dissemination of cancer is a common and fatal clinical condition seen in many malignancies, with few effective therapies available. G207, a multimutated replication-competent herpes simplex virus type-1, effectively treats disseminated peritoneal cancer. This study evaluates viral proliferation and subsequent tumoricidal effects in vitro and in vivo after regional viral delivery. In vitro studies demonstrate that G207 efficiently kills five human gastric cancer cell lines, and that permissiveness to viral replication is correlated with cytotoxicity. In a murine xenograft model of human gastric carcinomatosis, peritoneal delivery of G207 effectively kills tumor and prolongs survival. Data from quantitative PCR characterizes peritoneal clearance of virus after intraperitoneal injection, and identifies G207 replication within tumor cells in vivo, similar to in vitro proliferation. Further analysis of various organs confirms that G207 does not replicate within normal tissue after peritoneal delivery. Wild-type KOS viral replication was also demonstrated in vivo, with significant toxicity secondary to dissemination and encephalitis. In vivo viral proliferation of G207 is restricted to tumor cells, is correlated with in vitro assays, and is an important mechanism of anticancer efficacy.

ROLE OF PERITONEAL LAVAGE CYTOLOGY IN SURGERY FOR GASTRIC CANCER

The role of peritoneal lavage cytology (PLC) in surgery for gastric cancer was investigated in terms of the correlations between PLC and clinicopathological findings of advanced gastric cancers. Subjects were 49 patients who were diagnosed as having gastric cancer with the depth of tumor invasion of more than T_??_on imaging methods preoperatively and underwent peritoneal lavage at laparotomy. Positive carcinoma cells determined by PLC was found in 38.8% of the patients and the rate of positive cytology was significantly correlated with the following factors: macroscopic peritoneal dissemination; the depth of tumor invasion, especially serosal invasion; and the infiltrating growth pattern of the tumor. Among patients without macroscopic peritoneal dissemination, five were found as positive in PLC. None of these patients survived from peritoneal recurrence within 2 years after surgery. These results suggest that PLC are useful for predicting the peritoneal cancer dissemination. For future treatment of gastric cancer, patients with positive PLC are expected to be targeted for intensive adjuvant therapies during and/or after surgery.

Colorectal carcinoma and the relationship of peritoneal cytology

Background This study was designed to investigate the frequency of the association between colorectal cancer and peritoneal cytology as well as the impact of surgical resection on conversion of cytology from benign to malignant cells being present. Furthermore, increasingly frequent reports of port site recurrences with laparoscopic colectomy for limited stage colon carcinoma prompted us to evaluate the incidence of tumor cell spillage with traditional “open” colonic surgery. Methods Fifty random patients undergoing surgery for colorectal carcinoma were prospectively evaluated with peritoneal washings prior to and following colon resection. Results Five patients (10%) were found to have malignant cytology, with no patients converting from negative to positive cytology. All five positive cytologies were associated with stage IV disease and poorly differentiated colon cancer. Conclusion Intraoperative peritoneal cancer dissemination could not be demonstrated by cytologic washings of the abdominal cavity before and after colorectal resection utilizing standard cytomorphological criteria.

EVALUATION OF INTRAOPERATIVE PERITONEAL LABAGE CYTOLOGY IN PATIENTS WITH GASTRIC CANCER

Cytologic examination in the lavaged peritoneal fluid during surgery was performed in 125 patients with gastric cancer. Immediately after laparotomy, 50ml of saline solution was poured into the Douglas pouch and the left subphrenic cavity. Precipitation of collected fluid was stained by Papanicolaou's procedure, and according to Papanicolou's classification class N and V were judged as positive cytology. The incidence of the positive cytology was 28.8% in all patients, and 50.8% in stage N patients. It was closely correlated with such factors as peritoneal dissemination (P), histological depth of wall invasion, serosal invasion (S), area and gross findings of serosal invasion. The number of cases with microscopic intraperitoneal free cancer cells without peritoneal dissemination were 10 (11.8%), and their survival curves were lower than those without free cancer cells nor peritoneal dissemination. The incidence of the positive cytology was higher (60.5%) in patients whose areas of serosal invasion were over 10cm2 and macroscopically granular, tendon like or mossy like, and in patients whose areas of serosal invasion were over 25cm2 and scar like. It was suggested that peritoneal lavage cytology was useful to reveal the potential cases of disseminated peritoneal metastaiss and might be helpful for prophylaxis against peritoneal dissemination and forcast of recurrece.

癌の腹膜播種の成立機序に関する実験的研究

癌の腹膜播種の成立機序に関する実験的研究