Abstract LB113: Bio-printed 3D cell models and high-content imaging for testing anti-cancer compounds
Authors: Macha Prathyushakrishna , Huang John , Tong Zhisong , Hoying James B. , Sirenko Oksana 
Published: April 2023

Abstract
The discovery and screening of anti-cancer drugs is a vast field that is constantly evolving. More advanced cell-based models are needed to have an efficient way of studying cellular and subcellular effects of compound treatments, especially in early drug discovery. 3D cellular models that more accurately represent various microenvironments are very important for accurate drug screening and disease modeling. However, the complexity of those models is a limiting factor for the wider adoption of 3D models for research and screening. To address this, we have developed an automated workflow that includes a multitool 3D printing/automation robot enabling printing cells in a hydrogel-based matrix and high-content imaging for the characterization of phenotypic effects of compounds. We demonstrate examples of several bio-printed 3D cell models that can be used for testing anti-cancer drugs. For the development of 3D cellular models, we used VitroGel/VitroInk, xeno-free (animal origin-free) bio-functional hydrogel matrices. Hydrogel matrices with cells were used for printing 3D cellular patterns in 96 well plates, or dispensing cells in hydrogel “domes”. We have utilized a variety of widely used cancer lines HCT-116, HELA, HepG2, and MCF-7, as well as patient-derived triple-negative breast cancer cell line 4IC. Cells mixed with hydrogels/inks were dispensed/printed into a 96-well format using the multi-tool robotic platform, BioAssemblyBot. The platform enabled efficient dispensing/printing of cells into domes, lines, or other patterns. This assay was used for compound testing and evaluation of the anti-cancer effects of various drugs. The integrated system included a high-content confocal imager (IXM-C) and enabled automated seeding, bioprinting, liquid handling, plate transferring, as well as high-content imaging. The 3D bioprinted cells were monitored daily using imaging in transmitted light. In 2-3 days, cells formed spheroids in the matrix. Those were treated with a panel of anti-cancer drugs including doxorubicin, cytarabine, taxol, mitomycin, romidepsin, cisplatin, trametinib, and other compounds with various modes of action. Cells were treated with compounds for 72h, then stained and imaged for the endpoint measurements. 3D models were stained with viability dyes including Calcein AM, EthD, and Hoechst stain to evaluate numbers of live, dead, and total cells, then imaged using the IXM-C confocal imaging system. Image analysis allowed the characterization of cytostatic and cytotoxic effects of various compounds measuring the impact of compounds on cell proliferation, number of spheroids, spheroid size, cell death, and apoptosis. Different measurements, including spheroid size, and numbers of live and dead cells were used to determine EC50s for different compounds and cell types. The results showed the feasibility of 3D cellular models bioprinted with ECM matrices for anti-cancer drug screening workflows. An increase in throughput and ease of operation was achieved through automation. Also, imaging and data analysis methods provided valuable information about complex compound effects in 3D printed and cell-tissue-engineered cancer models.
Citation Format: Prathyushakrishna Macha, John Huang, Zhisong Tong, James B. Hoying, Oksana Sirenko. Bio-printed 3D cell models and high-content imaging for testing anti-cancer compounds [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB113.