The Chick Embryo Chorioallantoic Membrane (CAM) model for Circulating Tumor Cell Culturing for Precision Oncology in Patients with Pancreatic Cancer. A proof of concept.

The Chick Embryo Chorioallantoic Membrane (CAM) model for Circulating Tumor Cell Culturing for Precision Oncology in Patients with Pancreatic Cancer. A proof of concept. Objective The current proposal is aimed at developing a novel technique for culturing of Circulating Tumor Cells (CTCs) in pancreatic ductal adenocarcinoma (PDAC) that will allow for expansion of the population of isolated CTCs resulting in the ability to further study them. Background The underlying biological feature of PDAC that drive poor outcomes are the propensity for early systemic spread and only a modest impact of current systemic treatment paradigms. Despite improvements in systemic therapies their impact on survival has been modest at best. A majority of patients experience treatment failure though systemic progression of the so called “minimal residual disease”. CTCs have been identified as the seeds of metastasis and they have been proposed as biomarkers for non-invasive near “real-time” assessment of disease and predicting outcomes. Methods Prior to testing on human sample, we aim to optimize the chicken chorioallantoic membrane (CAM) assay, which supports the cellular growth even at low concentrations. We implanted a decreasing concentration of human pancreatic cell lines (MIA PaCa-2) on the CAM of 100 eggs, starting with 106 cells/egg, to 102 cells/egg. A small window was created in the egg shell at day 10 of fertilization, making the CAM to collapse. The region for cell implantation was carefully selected close to a vessel bifurcation to augment the blood supply. The implanted eggs rested in the incubator in a defined temperature and humidity till day 18 of embryo development. At day-18 of fertilization, prior to hatching, we harvested the CAM and the liver of each embryo. The tissues were fixed in paraffin and sliced to confirm the presence of the MIA PaCa-2 and enumeration through H&E and immunohistochemistry. Results Considering the final technique, we were able to see macroscopic tumor growth in 90% of the eggs . Upon H&E and IHC, even in macroscopically negative membrane we were able to observe growth of tumor cells. CK8 was specific to the cell line that was implanted and as demonstrated in the figure CK8 positive cells were observed on the membrane. Conclusion A new ISET (Isolation by SizE of Tumor cells) device now allows for live cell isolation of CTCs and novel techniques have shown promise in culturing them in other cancer types. Expansion of CTC populations would allow for studies to identify aggressive clones and potentially result in the development of novel therapies that specifically target these cells.