Nucleoside analogue labelling to study the cell cycle of pancreatic cancer xenografts in the chicken embryo model

The chorioallantoic membrane (CAM) model is an underutilised alternative model for the research into tumour xenografts. We demonstrate that in the pancreatic cancer cell lines BxPC-3 and AsPC-1, nucleoside labelling with 5-ethynyl-2’-deoxyuridine (EdU) can be multiplexed with other human cell cycle markers, (e.g., cyclin B1 and Ki-67), particularly when combined with digital image analysis, allowing human versus chicken cell classification. Furthermore, starting from the embryonic day of development 14 (ED14), we observe embryonic cells exhibiting substantial apparent cytoplasmic accumulation of synthetic nucleosides. This was observed for (F-ara-)EdU as well as for the halogenated analogues BrdU and IdU. Characterisation of these cells suggests that they are non-proliferating and possess granules and can be found in the embryonic liver of (non-)grafted embryos, as well as in the CAM itself and within xenograft sections. Importantly, these cells are unlikely to represent activated professional antigen presenting cells as they lack MHC II expression, nor do they express CD41/CD61, ruling out thrombocyte origin. Together, our findings demonstrate that the CAM model constitutes a valuable platform for studying tumour cell cycle dynamics in vivo. However, nucleoside labelling applications beyond ED14 may be confounded by a previously unidentified phenomenon, namely cytoplasmic nucleoside accumulation in certain chicken cells.