High-Resolution In Situ High-Content Imaging of 3D-Bioprinted Single Breast Cancer Spheroids for Advanced Quantification of Benzo( a )pyrene Carcinogen-Induced Breast Cancer Stem Cells.

Cancer stem cells (CSCs), also known as tumor-initiating cells, are critically correlated with carcinogenesis and are strongly affected by the environmental factors. Environmental carcinogens, such as benzo()pyrene (BaP), are associated with the overproduction of CSCs in various types of cancers, including breast cancer. In this report, we present a sophisticated 3D breast cancer spheroid model for the direct identification and quantitative determination of CSCs induced by carcinogens within intact 3D spheroids. To this end, hydrogel microconstructs containing MCF-7 breast cancer cells were bioprinted within direct-made diminutive multi-well chambers, which were utilized for the mass cultivation of spheroids and in situ detection of CSCs. We found that the breast CSCs caused by BaP-induced mutations were higher in the biomimetic MCF-7 breast cancer spheroids than that in standard 2D monolayer cultures. Precisely controlled MCF-7 cancer spheroids could be generated by serially cultivating MCF-7 cells within the printed hydrogel microconstructs, which could be further utilized for high-resolution in situ high-content 3D imaging analysis to spatially identify the emergence of CSCs at the single spheroid level. Additionally, potential therapeutic agents specific to breast CSCs were successfully evaluated to verify the effectiveness of this model. This bioengineered 3D cancer spheroid system provides a novel approach to investigating the emergence of CSC induced by a carcinogen for environmental hazard assessment in a reproducible and scalable format.