Application of nucleoside analogue labelling to study the cell cycle of xenografted PDAC cell lines in the chorioallantoic membrane model

The chorioallantoic membrane (CAM) model is an underutilised alternative animal model within the scope of cell cycle-related research of tumour xenografts. The usefulness of nucleoside labelling in standard rodent xenograft models is limited due to the extended labelling durations as a result of intraperitoneal route of administration. Due to its easy accessibility, the CAM allows xenograft S-phase nuclei to be labelled in as little as 30 minutes in vivo for a large number of biological replicates in parallel. We show that for the BxPC-3 and AsPC-1 cell lines, nucleoside labelling with 5-ethynyl-2’-deoxyuridine (EdU) can be multiplexed successfully with other cell-cycle markers such as cyclin B1 and Ki67, especially when combined with digital image analysis techniques. The latter also allows for accurate human versus chicken cell segmentation. Moreover, starting from embryonic day of development 14 (ED14), we observe the presence of a chicken embryonic cell type that appears to possess a high-quantity of extranuclear accumulation of EdU. Initial assessment of these cells showed that they are likely (non-proliferating) granulocytes which can be found in the embryonic liver of grafted and non-grafted embryos, as well as in xenograft sections. Importantly, these cells do not express chicken MHC II, in turn making it less likely that they represent professional antigen presenting cells. Our findings demonstrate that the CAM xenograft model can be used as a valuable tool to study the cell cycle of tumour cells in vivo . Lastly, we potentially revealed a novel biological phenomenon, namely that of extranuclear nucleoside accumulation in certain chicken embryonic cells.