Introduction: Pancreas cancer remains one the most lethal malignancies in the world and the high rate of metastatic progression contributes to its lethality. Up to 80% of patients with no clinical evidence of distant metastasis upon initial diagnosis recur with distant metastatic lesions even after surgical resection of their pancreas tumor. The critical drivers of metastatic dissemination and immune escape are not yet well understood, and a model to recapitulate the progression of pancreas cancer to its metastatic spread to the liver is not available. Understanding the biology of this and being able to predict which patients are at risk for metastatic reoccurrence would be a great treatment advance. Here we present a murine model to reflect human pancreas cancer disease progression. Following resection of the primary pancreas tumor, we observe arrival of early micrometastatic tumor cell spread in liver and monitor its progression to full metastatic lesion development. During this tumor progression, we are able to monitor the tumor microenvironment.
Methods: A murine model was developed to recapitulate early micrometastatic occurrence in the liver after primary tumor resection. The murine pancreatic adenocarcinoma cell line KCKO was retro-virally transfected with a LacZ gene, containing the genetic expression for Green Fluorescent Protein (GFP) and β-Galactosidase. Clonal selection for a stable GFP expression was done by flow cytometry. For experiments, eight to ten week-old C57BL/6 mice underwent orthotopic primary tumor implantation via injection into the tail of the pancreas with 1×105 luciferase-labeled KCKO cells suspended in a 1:1 PBS to Matrigel (Corning) mixture. Following tumor implantation, mice were monitored for primary tumor growth with bioluminescent imaging. After six days from the date of orthotopic implantation, the mice were anesthetized and underwent the second stage surgery involving primary tumor resection, injection of 1×105 LacZ-transfected KCKO cells into the lower pole of the spleen, followed by hemi-splenectomy. At varying time points thereafter (i.e. 24 hours, 48 hours, 72 hours, 96 hours), the mice were sacrificed following intracardiac perfusion with PBS, and individual liver lobes were taken for processing for flow cytometry, formalin fixation-paraffin embedment, and frozen section medium embedment.
Results: Analysis of the liver tumor microenvironment (TME) was performed to elucidate the spatial-temporal relationship between the tumor cells and stromal cells, which we determined were predominantly immune cells. β-Galactosidase expressing micrometastatic tumor cells could be clearly delineated in the liver by X-gal staining of frozen sections. Immunohistochemistry staining with anti-GFP and anti β-Galactosidase further confirmed the presence of the micrometastatic tumor cells. Furthermore, IHC staining was used to localize immune (CD45, CD11b, GR1, Ly6C, Ly6G, FoxP3, CD4, CD8) and non-immune cells (CD31, Collagen, SMA). Flow cytometry was used to characterize tumor and non-tumor cell presence across the time of metastatic progression.
Conclusion: We developed a clinically relevant murine model of micrometastatic pancreas cancer that we used to assess the formation of liver metastasis and associated changes in the liver TME, many of which promote tumor progression. Understanding the complex biology behind this process will lead to improved understanding of how tumor-associated changes in the subsequent site of metastasis promote metastasis formation.
Citation Format: Ankit Patel, Brian Belt, Nathania Figueroa, Sapna Patel, Aditi Murthy, Kelli Connolly, Scott Gerber, David Linehan. Micrometastatic progression of pancreas cancer: Murine model [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A133.
- ©2016 American Association for Cancer Research.