A 3D Heterotypic Multicellular Tumor Spheroid Assay Platform to Discriminate Drug Effects on Stroma versus Cancer Cells.

Authors:
Weydert Z, Lal-Nag M, Mathews-Greiner L, Thiel C, Cordes H, Küpfer L, Guye P, Kelm JM, Ferrer M.
In:
Source: SLAS Discov
Publication Date: (2019)
Issue: 1: 12
Research Area:
Cancer Research/Cell Biology
Toxicology
Drug Discovery
Cells used in publication:
NIH/3T3
Species: mouse
Tissue Origin: embryo
Platform:
4D-Nucleofector™ X-Unit
Experiment

Transfection of the NIH3T3 fibroblasts by Nucleofection in order to secrete NanoLUC luciferase (pNL1.3CMV [secNluc] luciferase reporter vector; Promega, Madison, WI) was performed according to Lonza’s manual for the Amaxa 4D-Nucleofector Protocol for NIH/3T3 (Lonza Cologne GmbH, Cologne, Germany) using a Single Nucleocuvette (100 µL). Solution SG The cells were trypsinized and an aliquot of 106 cells was resuspended in 100 µL of 4D-Nucleofector Solution (Lonza Cologne). One microgram of pNL1.3CMV [secNluc] luciferase reporter vector was added, and the suspension was transferred into a Nucleocuvette. The Nucleocuvette was placed into the 4D-Nucleofector X Unit. The cells were transfected with program EN-158. After transfection, the cells were plated in cell culture medium into a T flask. For separation of the dead cells, the flask was incubated overnight, and vital cells were allowed to adhere to the bottom. The vital cells were subsequently used for the microtissue production process.

Abstract

Three-dimensional (3D) cell culture models are thought to mimic the physiological and pharmacological properties of tissues in vivo more accurately than two-dimensional cultures on plastic dishes. For the development of cancer therapies, 3D spheroid models are being created to reflect the complex histology and physiology of primary tumors with the hopes that drug responses will be more similar to and as predictive as those obtained in vivo. The effect of additional cell types in tumors, such as stromal cells, and the resulting heterotypic cell-cell crosstalk can be investigated in these heterotypic 3D cell cultures. Here, a high-throughput screening-compatible drug testing platform based on 3D multicellular spheroid models is described that enables the parallel assessment of toxicity on stromal cells and efficacy on cancer cells by drug candidates. These heterotypic microtissue tumor models incorporate NIH3T3 fibroblasts as stromal cells that are engineered with a reporter gene encoding secreted NanoLUC luciferase. By tracking the NanoLUC signal in the media over time, a time-related measurement of the cytotoxic effects of drugs on stromal cells over the cancer cells was possible, thus enabling the identification of a therapeutic window. An in vitro therapeutic index parameter is proposed to help distinguish and classify those compounds with broad cytotoxic effects versus those that are more selective at targeting cancer cells.