Gene Expression Changes Induced by HIPEC in a Murine Model of Gastric Cancer

Materials and Methods

Cell line. The human gastric cancer cell line, MKN45 and MKN74, were purchased from the Japanese Collection of Research Bioresources, Human Science Research Resources Bank (Osaka, Japan). The gastric cell lines, were maintained in RPMI medium with 10% fetal bovine serum (FBS) and penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO₂ in air. Cells were regularly passaged to maintain exponential growth.

Study protocol. Several eight-week-old male NOD-SCID mice supplied by the Animal Center of the University of Perugia were housed under pathogen-free conditions. The care and the use of the animals were approved by the Institutional Animal Care and Use Committee of the University of Perugia and were in accordance with European guidelines for the care of experimental animals. Protocols were approved by the Instituto Superiore di Sanità. To examine the potential for peritoneal metastasis of carcinoma cell lines, a single cell suspension of MKN45 and MKN74 cells of 1×10⁷ cells in a total volume of 0.2 ml of medium without serum was injected into the peritoneal cavity of each mouse using a 23-gauge needle. The extent of PC was evaluated on the 7th, 14th and 21st day by necroscopy (Figure 1). At given time points, the mice were sacrificed under penthotal (50 mg/kg) anesthesia and the peritoneal and mesenteric nodules counted and removed from each mouse.

At sacrifice, all the tissues were immediately snap frozen in liquid nitrogen and stored at −80°C until used or fixed in formalin. Tissue sections (5 μm thick) were then stained with hematoxylin and eosin (H&E).

Experimental design. We utilized MKN45 cell lne for this experimental study due to its strong carcinogenesis behaviour compared to that of MKN74 cell line.

At day 1 from MKN45 cells inoculation, the mice were randomly put into five groups of ten animals: HIPEC with mitomycin (8.25 μg/l of perfusate each mouse) and cisplatin (62.5 μg/l of perfusate each mouse); normothermic intraperitoneal chemotherapy with the same chemotherapy solution (NIPEC); normothermic intraperitoneal saline solution (NIPES); hyperthermic intraperitoneal saline solution (HIPES); and no treatment. After 10 days from the intraperitoneal inoculation, the mice were sacrificed under penthotal (50 mg/kg) anesthesia and the peritoneal and mesenteric nodules were counted and removed from each mouse. The primary outcome parameter was the number of peritoneal disseminated nodules. In another set of experiments the animals were again randomly selected and treated as described above and the survival time was assessed.

Intraperitoneal treatments. All animals were anesthetized with an intraperitoneal injection of ketamine (Ketalar; Parke-Davis) 80 mg/kg and xilazine (Rompun; Bayer AG) at 5 mg/kg. Two catheters were introduced into the abdominal cavity through the upper and lower quadrants of the abdomen. The catheters were connected to a closed perfusion system containing 1 l of physiologic solution. The perfusion was performed according to the Coliseum technique at the open abdomen (7). The peritoneal perfusate was warmed in a tube coil using a thermostatically regulated water- bath. The perfusate was intraperitoneally introduced at a temperature of 40°C in the two groups of animalsfor HIPEC and NIPEC. Perfusion of the peritoneal cavity was performed for 50 min at an infusion speed of 4 ml/min. Mitomycin (8.25 μg/l of perfusate for each mouse) and cisplatin (62.5 μg/l of perfusate for each mouse) were dissolved in 0.9 % sodium chloride and then added to the perfusate in the HIPEC and NIPEC groups. During the intraperitoneal perfusion, the temperature of the abdominal cavity was highly controlled and maintained at the value of 40°C; the abdomen was massaged gently to achieve a uniform perfusate distribution. After completing the procedure, the abdominal cavity was irrigated with saline for 5 min and then the catheters were removed and the abdominal laparotomy was closed in a double layer using continuous sutures. Immediately after the perfusion, the mice were placed in a warmed cage to limit body heat loss and were given 1 ml of saline subcutaneously to rehydrate them .

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Figure 1.

To induce peritoneal carcinogenesis, a suspension of MKN45 and MKN74 cells were injected into the peritoneal cavity of the mice. The extent of peritoneal carcinogenesis was evaluated on the 7th, 14th and 21st day by necroscopy the peritoneal nodules and visceral metastases were counted and removed from each mouse.

Microarray analysis. Total RNA was prepared from each specimen using Trizol kit (Invitroge) to derive total RNA from cancer peritoneal nodules obtained in the mice inoculated with MKN45 alone or in a combination with HIPEC treatment. The RNA reverse was transcribed with Superscript-II reverse transcriptase (Invitrogen) following the manufacturer's instructions. A total of 100 ng cDNA was pipetted into each well of a 96-well gene array plate (Human Tumor Metastasis RT² Profiler™ PCR Array; - http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-028A.html - Superarray Bioscence, Frederick, MD, USA) and amplified following the manufacturer's instructions. This gene array is designed to assess 84 genes known to be involved in metastasis (Figure 2). Genes selected for this array encode several classes of protein factors including those for cell adhesion, extracellular matrix components, cell cycle, cell growth and proliferation, apoptosis, transcription factors and regulators and other genes related to tumour metastasis (Figure 3). Array analysis was carried out with the on-line software RT2 Profiler PCR Array Data Analysis (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php). The signal detected for each gene was normalized to the signal obtained for β-actin or gliceraldehyde 3-phosphate dehydrogenase (GAPDH) on the same gene array to derive gene expression values for each gene.

Up/down regulated genes were whose expression had altered by more than 2.

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Figure 2.

Results of Human Tumor Metastasis RT² Profiler™ PCR Array (http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-028A.html). The gene array was carried out using mRNA isolated from peritoneal gastric cancer nodules obtained from mice treated with and without HIPEC. A: Complete list of analyzed genes divided for pathways. Red: genes up-regulated by HIPEC; green: genes down-regulated by HIPEC; grey: genes not detected or unchanged following HIPEC treatment. B: Pie chart showing the percentage of genes down-regulated (in green), up-regulated (in red) and not detected or unchanged after HIPEC administration.

Statistical analysis. All values are expressed as the mean±SE of n experiments. The statistical analysis was carried out by GraphPad Prism software The variation between data sets was tested by Student's t-test for unpaired samples when we compared two groups. Comparisons of more than two groups were made with a one-way analysis of variance with post hoc Tukey tests. Differences were considered statistically significant if p was <0.05.