| The loco-regional progression of gastric cancer usually results in peritoneal carcinomatosis (PC), characterized by the presence of tumor nodules of various size, number, and distribution on the peritoneal surface as well as malignant ascites, with very poor prognosis and a median survival of less than6months.The most widely accepted therapies for such PC are surgery alone plus chemotherapy. Surgery alone, however, can only remove the bulky visible tumor nodules. For the micrometastases, invisible free cancer cells and those tumor masses not suitable for resection, surgery can not achieve any effect. Therefore, the traditional therapy can not make obvious positive impact on the survival and quality of life in patients with PC.In order to tackle this difficult problem, a new treatment strategy called cytoreductive surgery (CRS) plus hyperthermic intraperitoneal chemotherapy (HIPEC) has been developed over the past three decades, which has the advantages of surgery to reduce the visible tumor burden and regional hyperthermic chemotherapy to eradicate micrometastses and free cancer cells.Although many clinical studies have been performed to test and confirm the efficacy of this combined treatment approach, there is a lack of high quality evidence from phase Ⅲ randomized prospective studies. In order to more objectively evaluate such treatment, it is necessary to study this treatment modality under experimental conditions, in which most of the confounding factors could be well controlled. In this respect, suitable animal models of PC are indispensable platforms. Small animal models of PC have been established, including nude mouse PC models, mouse PC models and rat PC models. In most of these animal models, cancer cells are injected directly into the peritoneum, which will result in widespread PC in due time. Such models have been used to test various treatment modalities, including CRS and HIPEC, either alone or in combination, producing valuable information on the validity of different therapies. The small body size and delicate hemodynamic conditions are limiting factors for complex surgical interventions. Large animal models of PC might be more suitable for extensive surgical treatment. Therefore, it is necessary to establish large animal model of gastric cancer with PC for experimental studies to test extensive CRS and HIPEC.During the development of peritoneal carcinomatosis, GC cells secret enzymes to facilitate cancer cells seeding and colonization on the peritoneum. Cathepsin B is among the key enzymes in this critical process, overexpressed in GC as well as other cancers,10-12and actively involved in cancer invasion. Conversely, its expression is extremely low in normal cells, and it is inactive or loses activity as soon as it is dispersed in aqueous media away from cells. Thus, cathepsin B has long been considered a candidate target in cancer therapy [16].Doxorubicin (DOX) is a typical representative of anthracyclines. Although doxorubicin is an important drug in chemotherapy, its toxicities are also well known, such as cardiac toxicities and bone marrow suppression [17]. To retain the therapeutic effect while reducing the side effects, we designed a smart prodrug of doxorubicin, PDOX (Ac-Phe-Lys-PABC-DOX)[18-20]. In this modified doxorubicin, Phe-Lys is a dipeptide specific for cathepsin B, and PABC (para-aminobenzyloxycarbonyl) is a self-immolative spacer [21]. The prodrug is inactive when there is little cathepsin B activity, such as in normal tissues and peripheral blood, thus avoiding the side effects on normal tissue. During cancer invasion, activated cathepsin B is overexpressed on the exterior membrane of the invading cancer cells, which cleaves the Phe-Lys dipeptide at the Lys-PABC bond [22]. Then the exposed PABC spacer can self-hydrolyze upon deacylation[23], and free doxorubicin molecules are released, resulting in direct killing of the invading cancer cells[16].This study was divided into three parts as below:Part One:Establishment and Identification of Rabbit Model of Gastric Cancer with Peritoneal CarcinomatosisObjective To establish a stable model of peritoneal carcinomatosis in rabbit using VX2tumor and analyzed the features of metastasis.Method VX2tumor was implanted into36New Zealand rabbits by3methods:laparotomic orthotopic injection of cancer cells into the submucosal layer of the stomach (Group A), laparotomic implantation of tumor tissue into the greater omentum immediately beneath the gastric antrum (Group B), and percutaneous injetion of tumor cells directly into the peritoneal cavity (Group C),12rabbits were randamized aranged in each group. The animals wereclosely observed and detailed clinico-pathological studies were conducted. Result The success rates of peritoneal carcinomatosis formation were100%(12/12),91.7%(11/12) and58.3%(7/12), respectively, for Groups A, B and C (P=0.019, A vs. C; P=0.077, B vs. C; P=0.500, A vs. B, Fisher’s exact test). Two weeks after submucosal cancer cells injection in Group A, ulcerative gastric cancer with peritoneal carcinomatosis showed typical VX2tumor pathology, with widespread intraperitoneal metastatic nodules, bloody asites and perspicuous pulmonary metastases. The clinicalopathological progression pattern was very similar to patients of advanced gastric cancer with peritoneal carcinomatosis.Conclusion The orthotopic tissue implantation to establish a peritoneal carcinomatosis modelis convenient, feasible and timesaving. The clinicalpathological features of the model are similar to those of peritoneal carcinomatosis. Part Two:Experimental Animal Study on The Efficacy and Safty of Cytoreductive Surgery plus Hyperthermic Intraperitoneal Chemotherapy to Treat Peritoneal Carcinomatosis from Gastric CancerObjective To study the efficacy and safety of cytoreductive surgery (CRS) with hyperthermic introperitoneal chemotherapy (HIPEC) to treat rabbit model of gastric cancer with peritoneal carcinomatosis (PC), so as to provide support to clinical application.Method VX2tumor cells were injected into the gastric submucosa of42adult male New Zealand rabbits using a laparotomic implantation technique, to construct rabbit model of gastric cancer with PC. The rabbits were randomly divided into three groups:Control group (n=14), CRS group (n=14), and CRS+HIPEC group (n=14). The Control group was observed for natural course of disease progression. Treatment was initiated8ro9days after tumor cells inoculation, including optimal removal of tumor nodules in CRS group, and maximal removal of tumor nodules and heperthermic chemoperfusion in the CRS+HIPEC group with docetaxel (10mg/rabbit) and carboplatin (40mg/rabbit) at42℃for30minutes. The primary endpoint was overall survival in each group. The secondary endpoints were body weight, biochemistry, major organ functions and serious adverse events.Result The success rates of rabbit PC model were100%(42/42). The clinicopathological feature of the model is similar to peritoneal carcinomatosis in human. Overall survival was18-30days (median24days) in Control group,20~40days (median27days) in CRS group; and23~55days (median46days) in CRS plus HIPEC group (CRS alone group vs. control group P=0.1133; CRS+HIPEC group vs. control group P=2.45x10-6; CRS+HIPEC group vs. pure CRS group P=0.0012). Compared with CRS only or Control group, HIPEC could extend the overall survival by at least60%. At the baseline, on the day of surgery and7days after surgery, the peripheral blood cells counts, liver and renal functions, and biochemistry parameters were all comparable. Serious adverse events occurred in0animal in Control group, 2animals in CRS alone group including1animal died of anesthesia overdose and another1died of postoperative hemorrhage, and3animlas in CRS+HIPEC group including1animal died of anesthesia overdose, and2died of diarrhea23and27days after operation.Conclusion For rabbit model of gastric cancer PC, CRS alone could not bring benefit while CRS+HIPEC with docetaxel and cisplatin could significantly prolong the survival with a acceptable safety. Part III:Synthesis, Identification and In Vivo Studies of Tumor-Targeting Agent Peptide Doxorubicin (PDOX) to Treat Peritoneal Carcinomatosis of Gastric Cancer with Similar Efficacy and Reduced ToxicityObjective This work aimed to synthesize a cathepsin B (Cat B)-cleavable tumor-targeting prodrug peptide doxorubicin (PDOX) and study the in vivo efficacy and toxicities on an animal model of gastric cancer (GC) with peritoneal carcinomatosis (PC).Methods PDOX was synthesized using doxorubicin (DOX) attaching to a Cat B-cleavable dipeptide Ac-Phe-Lys and a para-amino-benzyloxycarbonyl (PABC) spacer. PC model was established by injecting VX2tumor cells into the gastric sub-mucosa of40rabbits, which then were randomized into4groups:the Control (n=10) without treatment, the HIPEC (n=10) receiving cytoreductive surgery (CRS) plus hyperthermic intraperitoneal chemotherapy (HIPEC), the PDOX (n=10) and the DOX (n=10) receiving systemic chemotherapy with PDOX50.0mg/kg or DOX5.0mg/kg after CRS+HIPEC respectively.Results The median overall survivals (OS) were23.0d (95%CI:19.9d-26.1d) in the Control,41.0d (36.9d-45.1d) in the HIPEC,65.0d (44.1d-71.9d) in the PDOX, and58.0d (39.6d-54.4d) in the DOX. Compared with the Control, the OS was extended by70%in the HIPEC (P<0.001) and further extended by40%in the DOX (P=0.029) and by58%in the PDOX (P=0.021), the PC severity was decreased in the HIPEC and further decreased in the PDOX and DOX. Animals receiving DOX treatment showed hematological toxicities with marked reduction of white blood cells and platelets, as well as cardiac toxicities with significant increases in creatine kinase mb isoenzyme, evident myocardium coagulation necrosis, significant nuclear degeneration, peri-nucleus mitochondria deletion, mitochondria-pyknosis, and abnormal intercalated discs. But these toxicities were not evident in the PDOX.Conclusions PDOX is a newly synthesized tumor-targeting prodrug of DOX. Compared with DOX, PDOX has similar efficacy and reduced hematological and cardiac toxicities in treating rabbit model of GC with PC. |
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