The ability to produce and collect Circulating Tumor Cells (CTCs) from cultured metastatic tumor tissue and established pancreatic cancer cell lines using a 3D perfusion culture system is demonstrated. The culture system used, the RealBio D4 ™ Culture System, incorporates low-sheer, tangential flow of nutrient medium above and below an open synthetic 3D cell scaffold, and gas-permeable membranes above and below the nutrient flow compartments to facilitate creation of in vivo-like gradients of nutrients, growth factors, and gasses that promote the recreation of an in vivo-like environment. This design allows CTCs generated within the cultures to migrate out of the cultured tissue or cell mass into the circulating nutrient medium in a manner reminiscent of the migration of CTCs out of tumors and into the blood stream in vivo. Once in the medium, the CTCs can be easily collected for characterization and further study.
Human metastatic pancreatic (to liver) tumor tissue from a mouse xenograft, and established human pancreatic cancer cell lines were cultured in the RealBio D4™ Culture System for at least 30 days. Cells migrating from the cultured tissues into the circulating culture medium were collected periodically throughout the study and characterized with respect to functional and cell surface CTC markers using a commercially available CTC technology designed for detecting CTCs in whole human blood (Vita-Assay™, Vitatex Inc., Stony Brook, NY). CTC's were positively identified in the circulating medium at all time points examined.
CTCs were produced by the cultured primary tumor tissue at a rate of more than 100 CTCs per culture per day after 30 days accounting for nearly 10% of all viable cells shed into the medium. Highly metastatic cell lines (MIA PaCa-2 and AsPC-1) produced as many as 6,000 CTCs per day accounting for 22% of the viable cells migrating from the cultures. In contrast, poorly metastatic cell lines (PL45 and Capan-2) produced no more than 20 CTCs per day representing no more than about 3% of the viable cells.
The results presented here demonstrate that CTCs can be produced in vitro by heterogeneous tumor tissue cultures. The results also show that CTCs can be produced in large numbers by highly metastatic cancer cell lines grown in vitro, but that poorly metastatic cancer cell lines produce far fewer CTCs when cultured under the same conditions. The methods described here represent a valuable new approach for the study of CTCs in vitro with direct applicability for developing improved diagnostic assays for CTCs, the study of factors that affect their formation and migration from tumor tissue, and development of therapies targeting CTCs.
Citation Format: William P. Pfund, George Martin, Paul A. Neeb. In vitro production of circulating tumor cells (CTCs) using 3D cultures of human tumor tissues and established tumor cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1456. doi:10.1158/1538-7445.AM2013-1456