Related Experiment Video
Updated: Mar 6, 2026
Generation of 3D Tumor Spheroids for Drug Evaluation Studies
Published on: February 24, 2023
Exploring Drug Dosing Regimens In Vitro Using Real-Time 3D Spheroid Tumor Growth Assays
Madhu Lal-Nag1, Lauren McGee1, Steven A Titus1
11 National Center for Advancing Translations Sciences, Division of Preclinical Innovation, National Institutes of Health, Bethesda, MD, USA.
Abstract:
Two-dimensional monolayer cell proliferation assays for cancer drug discovery have made the implementation of large-scale screens feasible but only seem to reflect a simplified view that oncogenes or tumor suppressor genes are the genetic drivers of cancer cell proliferation. However, there is now increased evidence that the cellular and physiological context in which these oncogenic events occur play a key role in how they drive tumor growth in vivo and, therefore, in how tumors respond to drug treatments. In vitro 3D spheroid tumor models are being developed to better mimic the physiology of tumors in vivo, in an attempt to improve the predictability and efficiency of drug discovery for the treatment of cancer. Here we describe the establishment of a real-time 3D spheroid growth, 384-well screening assay. The cells used in this study constitutively expressed green fluorescent protein (GFP), which enabled the real-time monitoring of spheroid formation and the effect of chemotherapeutic agents on spheroid size at different time points of sphere growth and drug treatment. This real-time 3D spheroid assay platform represents a first step toward the replication in vitro of drug dosing regimens being investigated in vivo. We hope that further development of this assay platform will allow the investigation of drug dosing regimens, efficacy, and resistance before preclinical and clinical studies.
Insights
This study introduces a real-time 3D spheroid assay for cancer drug discovery, offering a more realistic model than 2D cultures. This 3D model helps predict drug efficacy and resistance in cancer. Keywords: 3D spheroid assay, cancer drug discovery, drug efficacy, drug resistance.
Area of Science:
- Oncology
- Biotechnology
- Pharmacology
Background:
- Traditional 2D cell proliferation assays offer limited insight into cancer drug discovery.
- Tumor growth and drug response are significantly influenced by the in vivo cellular and physiological context.
- There is a need for more predictive in vitro models that mimic in vivo tumor physiology.
Purpose of the Study:
- To develop a real-time 3D spheroid growth assay for high-throughput cancer drug screening.
- To improve the predictability and efficiency of cancer drug discovery by better mimicking in vivo tumor environments.
- To establish a platform for investigating drug dosing, efficacy, and resistance in a 3D in vitro setting.
Main Methods:
- Established a 384-well, real-time 3D spheroid growth assay.
- Utilized cells constitutively expressing green fluorescent protein (GFP) for real-time monitoring.
- Assessed the effects of chemotherapeutic agents on spheroid size over time.
Main Results:
- Successfully established a real-time 3D spheroid screening assay.
- Demonstrated real-time monitoring of spheroid formation and drug treatment effects.
- The assay platform allows for the observation of chemotherapeutic agent impact on spheroid growth dynamics.
Conclusions:
- The developed real-time 3D spheroid assay is a significant advancement over 2D models for cancer drug discovery.
- This platform provides a more physiologically relevant in vitro model for evaluating drug efficacy and resistance.
- Further development aims to enable in vitro replication of in vivo drug dosing regimens for preclinical and clinical studies.
