70.21 Murine Breast cancer cells eliminated in non-derived strain mice; using an improved breast cancer model

Posted on January 18, 2016

E. Katsuta1, S. DeMasi1, K. P. Terracina1, H. Aoki1, M. Aoki1, P. Mukhopadhyay1, K. Takabe1 1Virginia Commonwealth University School Of Medicine And Massey Cancer Center,Division Of Surgical Oncology, Department Of Surgery,Richmond, VA, USA

Introduction: We have previously established a murine syngeneic breast cancer model utilizing cell implantation under direct vision technique, which mimic human cancer progression (Rashid, Takabe et al. Breast Cancer Research and Treatment 2014). Other groups have reported that cell implantation using Matrigel produced stable results in xenograft models. Here, we report the establishment of improved syngeneic orthotopic murine breast cancer model using Matrigel. In this study, we determined the maximum amount of Matrigel to be implanted without spillage, the tumor growth with various number of cells, and utilizing this new model, we investigated the growth of murine cancer cell derived from different strain mice.

Methods: Matrigel was injected to #2 and #4 mammary glands. Various number of murine breast cancer E0771cells in Matrigel were implanted into bilateral #2 and #4 mammary gland of C57Blk6 mice. Three weeks after inoculation, tumorigenesis were compared. 1 x 104 of murine breast cancer 4T1-luc2 cells, derived from Balb/C mice, were implanted into the Right side #2 gland of Balb/C or C57Blk6 mice. Tumor growth was monitored by bioluminescence (IVIS) imaging.

Results: We found that implantation of the cells will be more efficient with less variability when the cells are suspended in Matrigel compared with PBS, which was the technique we reported previously. Maximum volume of Matrigel inoculated without spillage was 20 μl in #2 gland, 30 μl in #4 gland, respectively. Therefore, we implanted 20 μl of Matrigel in #2 gland, and 30 μl in #4 gland in the subsequent experiments. In order to determine the difference of tumor development, 504, 105, 505, 106 E0771 cells suspended in 20µl Matrigel were inoculated. Three weeks after inoculation, ‘the take rates’ (tumorigenesis) were 0%, 12.5%, 75%, 75%, 100%, respectively. Utilizing 4T1-luc2 cells in Matrigel suspended cell implantation method, we investigated how long the mouse-derived cancer cells survive in mice from a different background. The fold increase in tumor growth in both backgrounds were nearly identical 24 h after inoculation at 5-fold increase measured by bioluminescence imaging. By 7 days after inoculation, tumor in C57Blk6 reached a maximum increase of approximately 720-fold their Day 0 size, whereas the tumor in Balb/C had almost a 2000-fold increase in tumor size. The Balb/C tumor continued to increase rapidly to reach an almost 3000-fold increase in size, while the C57Blk/6 mice tumors swiftly decreased from Day 7 and was eliminated by Day 14.

Conclusion: We identified the maximum amount of Matrigel that can be implanted into #2 or #4 mammary gland without spillage, and the difference in take rates with various number of cells for murine orthotopic breast cancer model. Utilizing Matrigel implantation method, we found that cancer cells will continue to grow until one week, then it will eliminated by 2 weeks when implanted into different background strain mice.

02.07 Role of Sphingosine-1-phoshate of the Host in Progression of Pancreatic Cancer Carcinomatosis

Posted on January 18, 2016

H. Aoki1,2, M. Aoki1,2, P. Mukhopadhyay1,2, C. C. Barnett3, S. Spiegel1,2, K. Takabe1,2 1Virginia Commonwealth University,Division Of Surgical Oncology, Department Of Surgery,Richmond, VA, USA 2Virginia Commonwealth University,Department Of Biochemistry & Molecular Biology,Richmond, VA, USA 3University Of Colorado Denver,Department Of Surgery,Aurora, CO, USA

Introduction: In the U.S., pancreatic cancer is the 4th leading cause of cancer death in both gender. The 5-year relative survival rate is approximaly 6% for all stages. Up to 60% of the patients are found to have distant metastatic disease at the time of diagnosis, at which median survival is around 6 months. The most common sites for distant metastases are the liver (80%), peritoneum (60%), lung and pleura (50-70%), and adrenal glands (25%). Prognosis of patients with peritoneal carcinomatosis (PC), dissemination of cancer cells throughout the abdominal cavity, are particularly poor with median survival of only 6 weeks. This poor overall 5-year survival rate has not significantly changed over the past 5 decades, which reflects the fact that there is no effective treatment available for this condition. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator produced by sphingosine kinase 1 (SphK1) and sphingosine kinase 2 (SphK2), plays critical roles in many aspects of cancer progression, such as cell proliferation, migration, angiogenesis and lymphangiogenesis. We have recently published that S1P link inflammation and cancer in colitis-associated cancer progression (Cancer Cell 2013). Given the fact that inflammation is known to be essential for establishment and progression of PC, where cancer cells need to adhere to the peritoneum and form a nodule, we hypothesized that S1P levels regulated by SphK1 and SphK2 in the host animal may have different mechanism in promoting progression of pancreatic cancer PC depending upon its levels.

Methods: Murine pancreatic adenocarcinoma panc02-luc cells were intraperitoneally injected into SphK1 wild type (WT) or knockout (KO), or SphK2 WT or KO mice to generate PC model. Tumor burden was quantified using bioluminescence imaging. Survival was assessed by Kaplan-Meier analysis. PC nodules were harvested 14 days after injection and analyzed. The proliferation was assessed by Ki-67 staining and apoptosis was evaluated by TUNEL assay. We also compared mRNA expression by RT-PCR.

Results: Circulating S1P levels were lower in SphK1 KO mice and higher in SphK2 KO mice compared with respective littermate WTs probably due to compensatory elevation of SphK1. Panc02-luc cells developed significantly less tumor burden, less inflammatory cell infiltration, and less cancer cell proliferation, but with no difference in apoptosis in PC of SphK1 KO mice. These results suggest that host S1P promotes PC progression by stimulation of proliferation of cancer cells. Interestingly, SphK2 KO mice developed less tumor burden, longer survival with elevated CD4 and CD8 lymphocyte infiltrates in PC.

Conclusion: Our results implicate an intriguing possibility that S1P levels in the host may have different mechanisms in promoting progression of pancreatic cancer PC depending upon its levels.

02.08 Obstructive Jaundice Aggravate Pancreatic Cancer via Sphingosine 1 Phosphate Receptors.

Posted on January 18, 2016

H. Aoki1,2, M. Aoki1,2, E. Katsuta1,2, L. J. Fernandez1, P. Mukhopadhyay1,2, J. Yang3, H. Zhou3, S. Spiegel2, K. Takabe1,2 1Virginia Commonwealth University,Division Of Surgical Oncology, Department Of Surgery,Richmond, VA, USA 2Virginia Commonwealth University,Department Of Biochemistry & Molecular Biology,Richmond, VA, USA 3Virginia Commonwealth University,Department Of Microbiology And Immunology,Richmond, VA, USA

Introduction: Pancreatic cancer (PC) remains one of the deadliest types of cancers. Despite the recent improvement in survival of patients with small resectable tumors, prognosis of unresectable PC that collectively represents over 80% of individuals remains dismal with the 5-year survival of 5% and median survival is within ten months. The classical clinical sign of pancreatic head cancer is painless jaundice. There have been numerous publications regarding the clinical benefit of bile drainage in obstructive jaundice, however, the effect of it on pancreatic cancer biology, such as tumor growth, has never been thoroughly investigated. We have recently published that conjugated bile acids (CBAs) binds to sphingosine 1-phosphate receptor 2 (S1PR2), and it activate nuclear SphK2 that epigentically regulate gene expression (Nagahashi, Takabe, Zhou et al, Hepatology 2015). Indeed, it was reported that CBAs promote growth of cholangiocarcinoma through S1PR2. Thus, we hypothesized that CBAs from obstructive jaundice aggravate the pancreatic cancer progression via S1PRs.

Method: Expression of S1P receptors (S1PR1-5) in murine (panc02-luc) and human (MiaPaca-2) pancreatic cancer cell lines were determined by real-time RT-PCR. Cells were treated with CBAs with or without JTE-013 (S1PR2 antagonist) and viable cells were quantified using WST-8. Panc02-luc cells were implanted in the left lobe of the liver of C57/Bl6 mice with or without obstructive jaundice, created by left and middle bile duct ligation with cholecystectomy. Tumor burden was quantified using bioluminescence imaging on the indicated days and tumors were harvested on day 18.

Result: Hielarchial cluster analysis of pancreatic adenocarcinoma in The Cancer Genome Atlas (N=183) demonstrated that S1PR2, S1PR5 and SphK1 gene expressions have strong positive correlations. Among 5 S1P receptors, S1PR2 is the only one expressed in panc02-luc cells and S1PR2 and S1PR5 are expressed in MiaPaca-2 cells. CBA-mediated cell growth was inhibited by JTE-013, but JTE-013 alone was also found to be growth inhibitory even in the absence of CBA. In obstructive jaundice mice group, significant increase in tumor burden was observed by bioluminescence. Left lobe+tumor / body weight ratio was significantly larger in obstructive jaundice group.

Conclusion: Conjugated bile acid signaling via S1PR2 promotes pancreatic cancer cell growth. Obstructive jaundice aggravated pancreatic cancer, and further study is warranted to investigate the possibility of targeting S1PR2 as a potential new therapeutic for pancreatic cancer treatment.