Systematic optimization and evaluation of culture conditions for the construction of circulating tumor cell clusters using breast cancer cell lines

Circulating tumor cell (CTC) clusters play a critical role in carcinoma metastasis. However, the rarity of CTC clusters and the limitations of capture techniques have retarded the research progress. In vitro CTC clusters model can help to further understand the biological properties of CTC clusters and their clinical significance. Therefore, it is necessary to establish reliable in vitro methodological models to form CTC clusters whose biological characteristics are very similar to clinical CTC clusters. The assays of immunofluorescence, transmission electron microscopy, EdU incorporation, cell adhension and microfluidic chips were used. The experimental metastasis model in mice was used. We systematically optimized the culture methods to form in vitro CTC clusters model, and more importantly, evaluated it with reference to the biological capabilities of reported clinical CTC clusters. In vitro CTC clusters exhibited a high degree of similarity to the reported pathological characteristics of CTC clusters isolated from patients at different stages of tumor metastasis, including the appearance morphology, size, adhesive and tight junctions-associated proteins, and other indicators of CTC clusters. Furthermore, in vivo experiments also demonstrated that the CTC clusters had an enhanced ability to grow and metastasize compared to single CTC. The study provides a reliable model to help to obtain comparatively stable and qualified CTC clusters in vitro, propelling the studies on tumor metastasis.