Abstract
Understanding tumor heterogeneity is an urgent and unmet need in cancer research. In this study, we used a morphology-based optical cell separation process to classify a heterogeneous cancer cell population into characteristic subpopulations. To classify the cell subpopulations, we assessed their morphology in hydrogel, a three-dimensional culture environment that induces morphological changes according to the characteristics of the cells (i.e., growth, migration, and invasion). We encapsulated the murine breast cancer cell line 4T1E, as a heterogeneous population that includes highly metastatic cells, in click-crosslinkable and photodegradable gelatin hydrogels, which we developed previously. We observed morphological changes within 3 days of encapsulating the cells in the hydrogel. We separated the 4T1E cell population into colony- and granular-type cells by optical separation, in which local UV-induced degradation of the photodegradable hydrogel around the target cells enabled us to collect those cells. The obtained colony- and granular-type cells were evaluated in vitro by using a spheroid assay and in vivo by means of a tumor growth and metastasis assay. The spheroid assay showed that the colony-type cells formed compact spheroids in 2 days, whereas the granular-type cells did not form spheroids. The tumor growth assay in mice revealed that the granular-type cells exhibited lower tumor growth and a different metastasis behavior compared with the colony-type cells. These results suggest that morphology-based optical cell separation is a useful technique to classify a heterogeneous cancer cell population according to its cellular characteristics.
Highlights
Most tumors are composed of different types of cells, including cancer cells, fibroblasts, vascular endothelial cells, and immune cells [1]
We investigated the relationship between the Morphology-based optical cancer cell separation morphology of the cells in the PD-gelatin hydrogel and their characteristics by assessing the cells in terms of spheroid formation in vitro, and tumor growth and metastasis in vivo
Under 2D culture conditions, the C1 and C2 cells attached to the surface of the culture dish strongly, whereas the G1 and G2 cells attached weakly
Summary
4T1E cells, which are the parent line of 4T1E/M3 cells, were established in our previous study [16]. RPMI-1640 supplemented with 4,500 mg/L glucose, L-glutamine, phenol red, HEPES, and sodium pyruvate was obtained from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan) for culturing the 4T1E cells. Cell culture medium was prepared by adding 10 vol% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, UT, USA) and 1 vol% penicillin/ streptomycin (Nacalai Tesque, Inc., Kyoto, Japan) to the RPMI-1640. SU-8 2075 negative photoresist was obtained from MicroChem (Newton, MA, USA). 1H,1H,2H,2H-Perfluorooctyltrichlorosilane was obtained from Wako Pure Chemical Industries, Ltd. Preparation of a sandwich culture chamber SU-8 2075 negative photoresist was obtained from MicroChem (Newton, MA, USA). 1H,1H,2H,2H-Perfluorooctyltrichlorosilane was obtained from Wako Pure Chemical Industries, Ltd.
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