Compared with traditional monolayer cell culture, the three-dimensional tumor spheroid has emerged as an essential in vitro model for cancer research due to the recapitulation of the architecture and physiology of solid human tumors. Herein, by implementing the rapid prototyping of a benchtop 3D printer, we developed a new strategy to generate and analyze tumor spheroids on a commonly used multi-well plate. In this method, the printed artifact can be directly mounted on a 96/384-well plate, enables hanging drop-based spheroid formation, avoiding the tedious fabrication process from micromechanical systems. Besides long-term spheroid culture (20 days), this method supports subsequent analysis of tumor spheroid by seamlessly dripping from the printed array, thereby eliminating the need for spheroids retrieval for downstream characterization. We demonstrated several tumor spheroid-based assays, including tumoroid drug testing, metastasis on or inside extracellular matrix gel, and tumor transendothelial (TEM) assay. Based on quantitative phenotypical and molecular analysis without any precarious retrieval and transfer, we found that the malignant breast cancer (MDA-MB-231) cell aggregate presents a more metastatic morphological phenotype than the non-malignant breast cancer (MCF-7) and colonial cancer (HCT-116) cell spheroid, and shows an up-regulation of epithelial-mesenchymal transition (EMT) relevant genes (fold change > 2). Finally, we validated this tumor malignancy by the TEM assay, which could be easily performed using our approach. This methodology could provide a useful workflow for expediting tumoroid modeled in vitro assay, allowing the “Lab-on-a-Cloud” scenario for routine study.

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Single-atom catalysts with extraordinary catalytic activity have been receiving great attention in tumor therapy. However, most single-atom catalysts lack self-propulsion properties, restricting them from actively approaching cancer cells or penetrating the interior of tumors. Herein, we design N-doped jellyfish-like mesoporous carbon nanomotors coordinated with single-atom copper (Cu-JMCNs). It is a combination of singleatom nanocatalytic medicine and nanomotor self-propulsion for cancer therapy. The Cu single atom can catalyze H 2 O 2 into toxic hydroxyl radical ( • OH) for chemodynamic therapy (CDT). Nearinfrared light triggers Cu-JMCNs to achieve self-thermophoretic motion because of the jellyfish-like asymmetric structure and photothermal property of carbon, which significantly improves the cellular uptake and the penetration of three-dimensional tumors. In vivo experiments indicate that the combination of single-atom Cu for CDT and near-infrared light propulsion can achieve over 85% tumor inhibition rate. This work sheds light on the development of advanced nanomotors with single-atom catalysts for biomedical applications.

The 3D cell spheroid is an emerging tool that allows better recapitulating of in vivo scenarios with multiple factors such as tissue-like morphology and membrane protein expression that intimately coordinates with enzyme activity, thus providing a psychological environment for tumorigenesis study. For analyzing different spheroids, conventional optical imaging may be hampered by the need for fluorescent labeling, which could cause toxicity side effects. As an alternative approach, scanning electrochemical microscopy (SECM) enables label-free imaging. However, SECM for cell spheroid imaging is currently suffering from incapability of systematically analyzing the cell aggregates from spheroid generation, electrochemical signal gaining, and the gene expression on different individual cell spheroids. Herein, we developed a top-removable microfluidic device for cell aggregate yielding and SECM imaging methodology to analyze heterotypic 3D cell spheroids on a single device. This technique allows not only on-chip culturing of cell aggregates but also SECM imaging of the spheroids after opening the chip and subsequent qPCR assay of corresponding clusters. Through employment of the micropit arrays (85 × 4) with a top withdrawable microfluidic layer, uniformly sized breast tumor cell and fibroblast spheroids can be simultaneously produced on a single device. By leveraging voltage-switching mode SECM at different potentials of dual mediators, we evaluated alkaline phosphatase without disturbance of substrate morphology for distinguishing the tumor aggregates from stroma. Moreover, this method also enables gene expression profiling on individual tumor or stromal spheroids. Therefore, this new strategy can seamlessly bridge SECM measurements and molecular biological analysis.

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