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The prognosis of patients with advanced diffuse-type gastric cancer (GC), especially scirrhous gastric cancer (SGC) remains extremely poor. Peritoneal carcinomatosis is a frequent form of metastasis of SGC. With survival rates of patients with peritoneal metastasis at 3 and 5 years being only 9.8% and 0%, respectively, development of a new treatment is urgently crucial. For such development, the establishment of a therapeutic mouse model is required. Among the 11 GC cell lines we examined, HSC-60 showed the most well-preserved expression profiles of the Hedgehog and epithelial-mesenchymal transition pathways found in primary SGCs. After six cycles of harvest of ascitic tumor cells and their orthotopic inoculation in scid mice, a highly metastatic subclone of HSC-60, 60As6 was obtained, by means of which we successfully developed peritoneal metastasis model mice. The mice treated with small interfering (si) RNA targeting NEDD1, which encodes a gamma-tubulin ring complex-binding protein, by the atelocollagen-mediated delivery system showed a significantly prolonged survival. Our mouse model could thus be useful for the development of a new therapeutic modality. Intraperitoneal administration of siRNAs of targeted genes such as NEDD1 could provide a new opportunity in the treatment of the peritoneal metastasis of SGC.
Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide.[1, 2] Histopathological research has long suggested that gastric cancer is not a single disease and recognizes two major categories: intestinal and diffuse. Intestinal-type GC develops through some sequential stages including Helicobacter pylori (H. pylori)-associated gastritis, intestinal metaplasia (IM), and dysplasia. This type predominates in high-risk geographic areas, such as East Asia, showing a correlation with the prevalence there of H. pylori infection among elderly people. Diffuse-type GC, however, is more uniformly distributed geographically, is apparently unrelated to H. pylori prevalence and typically develops from H. pylori-free, morphologically normal gastric mucosa without atrophic gastritis, or IM. Unlike the decreasing incidence of the intestinal-type, the prevalence of the diffuse-type is reportedly increasing worldwide. Although therapeutic results for GC have recently improved, the prognosis for patients with advanced diffuse-type GC, especially scirrhous gastric cancer (SGC, Borrmann's type IV carcinoma or the linitis plastic type) remains extremely poor. The 5-year overall survival rate of SGC is approximately 10%, and ranges from 18% to 29% even after curative surgery.[5-7] Histopathologically, SGC does not form glands; instead, it causes diffuse infiltration of a broad region of the gastric wall rather than a well-defined mass, resulting in a fibrous-like thickening of the wall. Such pathological features make an early clinical diagnosis of SGC difficult, and in approximately half of the cases, by the time the diagnosis is made, peritoneal dissemination has, unfortunately, already occurred.[5, 8] Peritoneal dissemination, known to be a frequent form of metastasis and recurrence of SGC, serves as a major factor determining patient prognosis. Currently, no effective therapy exists for this condition. For SGC patients with peritoneal metastasis, the survival rates at 3 and 5 years are only 9.8% and 0%, respectively, even if the patients received multidisciplinary treatment.
It has been suggested that peritoneal dissemination is a consequence of free cancer cells that are shed from the serosa of the primary lesion and/or may leak out from the lymphatics to the peritoneal cavity; however, no detailed mechanism of peritoneal dissemination has been fully elucidated. In either situation, it is assumed that free cancer cells detached from a primary lesion must have a predilection for the peritoneum. Efficacious control of invisible free cancer cells in the peritoneal cavity should help suppress the progression of carcinomatous peritonitis, and could ultimately yield a survival benefit. Some investigators have reported good, but limited, outcomes with new treatment strategies for peritoneal dissemination, including systemic chemotherapy, intraperitoneal (i.p.) chemotherapy and/or hyperthermia, and peritonectomy. Therefore, to improve patient outcome, the development of a new therapeutic strategy for peritoneal dissemination of SGC is urgently crucial.
In this study, we developed peritoneal metastasis model mice of SGC and an atelocollagen-mediated delivery system for i.p. administration of small interfering (si) RNA, and also reported that the i.p. delivery of an siRNA targeting NEDD1, which functions in the metaphase regulation of the cell cycle, was able to regress the tumor and prolong, without toxicity, the survival of the mice.
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- Materials and Methods
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For gene therapy, one of the most dramatic events of the past 5 years in this field has been the discovery of RNA interference (RNAi). The success of cancer therapeutic use of RNAi relies on the development of safe and efficacious delivery systems that introduce siRNA and shRNA expression vectors into target tumor cells. However, such delivery systems into peritoneal metastatic tumor cells have not been established well. The atelocollagen-mediated gene delivery system was originally developed for a adenovirus vector by a collaborator. An atelocollagen is a highly purified type I collagen of calf dermis with pepsin treatment, which allows nuclease resistance, prolonged release of genes and reduction of cellular immune responses. In mice, this delivery system has been reported to be useful for gene delivery into some body sites including metastatic tumors and also for systemic gene delivery. As mentioned in the introduction, diffuse-type GCs including scirrhous GC frequently show peritoneal dissemination even if tumor cells are circulating systemically. Therefore, in this type of GCs, peritoneal metastasis control is urgently crucial for improving the quality of life and patient outcome. Here we provided peritoneal metastasis model mice and an effective delivery system for i.p. administration of siRNA. Figure 2 showed that the 0.5% atelocollagen/DharmaFECT1/siRNA complex rather than the 0.05% atelocollagen/DharmaFECT1/siRNA complex reduced luciferase activity and that the DharmaFECT1 free complex did not reduce it. To date, a collaborator usually uses DharmaFECT1 in the atelocollagen-mediated systemic gene delivery by i.v. administration, because this reagent improves it (Takeshita F, unpublished observation, 2010).
In peritoneal metastasis model mice, the i.p. administration of NEDD1 siRNA was able to inhibit tumor growth and prolong survival even without any side effects (Figs 5, 7). If targets such as NEDD1 function in the cell cycle regulation, the slow gene release arising from protection from nucleases by atelocollagen may provide an advantage for long acting and for reducing the number of administrations. As shown in Figure 7, we administered the NEDD1 siRNA complex five times every 3 days for 15 days in this study; however, that number may possibly be reduced. In another report for intraperitoneal administration of siRNA targeting nuclear factor-κB with only DharmaFECT1, the siRNA prolonged the survival of mice only by the administration of paclitaxel, whereas the siRNA/DharmaFECT1 complex alone could not succeed. Taken together, the atelocollagen/DharmaFECT1/siRNA complex is very useful for gene delivery to the peritoneal cavity.
In the mouse model used, the number of tumor cells (1 × 105 cells) implanted into the mouse peritoneal cavity was estimated to be still very large compared with the number of tumor cells in the peritoneal cavity in human GC patients with cytology positive, who often showed peritoneal metastasis within 2 years. Therefore, the present i.p. delivery system of siRNA has a great potential for treatment of such GC patients.
Although in vitro cell growth inhibition was observed by the double siRNA treatment of ELK1 and MSX2 (Fig. 3b), no significant difference on in vivo tumor growth and mouse survival was found (Fig. 3c and data not shown). Investigation of other hedgehog components (SMO, GLIs, ISL1, BMP4, FOXM1, and FOXA2) and EMT-regulators (SIP1/ZEB2, TWIST2) remains for a future study, because cross-talk between hedgehog and EMT signals is specific to diffuse-type GC.
Atelocollagen has also been reported to efficiently deliver microRNA. Recently, genome-wide microRNA expression profiles of 353 GC samples have shown that some microRNAs including let-7b, miR-214, and miR-433 are expressed aberrantly and correlate with tumorigenesis, progression, and prognosis of diffuse-type GC. Thus, these microRNAs may be candidates for SGC therapy. In addition, transforming growth factor-β (TGF-β) has been reported to induce apoptosis of a subset of diffuse-type GCs whose receptor is not inactivated.[28, 29] Therefore, adenovirus-mediated TGF-β or the downstream targets such as Gasdermin/GasderminA delivery also have great potential for SGC therapy.
In conclusion, we developed a novel i.p. delivery system of siRNA to disseminated tumor cells in the peritoneal cavity that successfully prolongs the survival of model mice. The present mouse model is for an adjuvant therapy after surgical resection. The ability of atelocollagen/DharmaFECT complex is keeping siRNA from nucleases, leading slow gene release and reducing the amount of administration that results in effective eradication of residual tumor cells in the peritoneal cavity.
Thus, considering other potential targets of the diffuse-type GCs, this system is a highly flexible therapeutic platform for the treatment of peritoneal dissemination.