Phenotypic heterogeneity of stem cell-like cancer cells isolated with microfluidic-enabled one-cell culture

Abstract

Cancer metastasis is a complex process and remains the leading cause of cancer-related mortality. This is posited to be driven by a subpopulation of stem cell-like cancer cells, which possess self-renewal potential, differentiation capacity, and resistance to conventional therapies. Effective isolation and culture of these cells are the foremost steps for understanding their biology and developing targeted treatments. However, conventional methods, such as surface marker-based isolation and suspension culture, are limited due to cancer cell heterogeneity and the difficulty in maintaining stemness. To overcome these issues, we employ a bioinspired one-cell culture approach using hyaluronic acid (HA)-enriched alginate core-shell microcapsules to create a microenvironment that selectively promotes the survival and proliferation of stem cell-like cancer cells while inducing cell death in non-stem cell-like cancer cells. Furthermore, despite significant progress in cancer research, stem cell-like cancer cells in metastatic lesions and circulating tumor cell (CTC) populations remain underexplored. These cells may differ from stem cell-like cancer cells within primary tumors. In this study, we extend the application of the one-cell culture to patient-derived CTCs and cells from metastatic lesions, allowing for a more clinically relevant investigation of these stem cell-like cells. To identify and characterize these distinct subpopulations from both CTC populations and metastatic sites, we characterize multiple phenotypic properties. First, we assess cellular morphology and cancer stemness in terms of self-renewal ability, and protein expression of cells isolated using the one-cell culture method. In addition, we evaluate the mechanophenotypes of stem cell-like CTCs using real-time deformability cytometry and explore the role of YAP (Yes-associated protein), as it is a key regulator that controls the mechanical phenotypes in stem cell-like behavior. Lastly, we examine the dissemination behaviors of the stem cell-like CTCs using intravital imaging in a zebrafish xenograft model, which offers a dynamic view of metastatic potential in vivo. Together, this dissertation presents a comprehensive phenotypic analysis of stem cell-like cancer cells isolated using the one-cell culture method across diverse cancer types and patient samples. The phenotypic features, including cellular morphology, self-renewal capacity, protein expression patterns, mechanophenotypes, and metastatic dissemination behavior in zebrafish may contribute to the development of therapeutic strategies targeting stem cell-like cancer cells.