Complex Tumor Spheroids, a Tissue-Mimicking Tumor Model, for Drug Discovery and Precision Medicine

Abstract

Malignant tumors are complex tissues composed of malignant cells, vascular cells, structural mesenchymal cells including pericytes and carcinoma-associated fibroblasts, infiltrating immune cells, and others, collectively called the tumor stroma. The number of stromal cells in a tumor is often much greater than the number of malignant cells. The physical associations among all these cell types are critical to tumor growth, survival, and response to therapy. Most cell-based screens for cancer drug discovery and precision medicine validation use malignant cells in isolation as monolayers, embedded in a matrix, or as spheroids in suspension. Medium- and high-throughput screening with multiple cell lines requires a scalable, reproducible, robust cell-based assay. Complex spheroids include malignant cells and two normal cell types, human umbilical vein endothelial cells and highly plastic mesenchymal stem cells, which rapidly adapt to the malignant cell microenvironment. The patient-derived pancreatic adenocarcinoma cell line, K24384-001-R, was used to explore complex spheroid structure and response to anticancer agents in a 96-well format. We describe the development of the complex spheroid assay as well as the growth and structure of complex spheroids over time. Subsequently, we demonstrate successful assay miniaturization to a 384-well format and robust performance in a high-throughput screen. Implementation of the complex spheroid assay was further demonstrated with 10 well-established pancreatic cell lines. By incorporating both human stromal and tumor components, complex spheroids might provide an improved model for tumor response in vivo.

Optimization of 3D-aggregated spheroid model (3D-ASM) for selecting high efficacy drugs

“…To overcome these limitations, efforts to introduce a 3D-HTS method using spheroids are ongoing 2 , 3 . Till now, 3D-HTS has mainly been performed in matrix-free spheroids, such as hanging drop or ultra-low attachment (ULA) methods 4 , 5 . These methods show low throughput and low reproducibility due to the difficulty of changing clean culture media and the problem of spheroid loss.…”

Multicellular Complex Tumor Spheroid Response to DNA Repair Inhibitors in Combination with DNA-damaging Drugs

“…All complex tumor spheroids were grown from a fixed ratio of 60% malignant cells, 25% HUVECs, and 15% hMSCs according to a standardized protocol. Spheroids grown by this method from the patient-derived pancreatic adenocarcinoma cell line K24384-001-R were characterized extensively ( 32 ). However, complex tumor spheroid morphologies varied among malignant cell lines ( Supplementary Fig.…”

“…Following removal of the supernatant, the cells were resuspended in fresh medium and counted using a Cellometer auto T4 bright field cell counter (Nexcelom) and trypan blue to distinguish viable cells. Complex tumor spheroids were grown from the mixture of three cell types: 60% malignant cells, 25% HUVECs, and 15% hMSCs as described previously ( 32 ). Mixed cell suspensions of 42 μL were dispensed into the wells of 384-well black/clear round-bottom ultra-low attachment (ULA) spheroid microplates (Corning Inc., catalog no.…”

“…This study explores the anticancer activities of DNA-damaging drugs and inhibitors of DNA damage repair, both as single agents and in combinations ( Table 1 ). Complex, multicellular tumor spheroids comprised of malignant cells, endothelial cells, and mesenchymal stem cells served as an in vitro model for human solid tumors ( 32 ). This model system incorporates physiologically relevant features and patient-derived or established malignant cell lines with clinically relevant genetic alterations from many tumor types.…”

Deciphering the underlying mechanism of liver diseases through utilization of multicellular hepatic spheroid models

“…The three 3D cell lines that form compact multicellular spheroids have increased resistance to doxorubicin and paclitaxel than the 2D cell lines (61). Kaur G. et al developed a pancreatic adenocarcinoma cell line that was derived from a patient to study the complicated structure of the spheroid and the response of the model to therapeutic drugs in a 96-or 384-well format appropriate for robust performance in an HTS (62).…”