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Updated: May 7, 2026
3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening
Published on: October 4, 2017
A high-throughput-compatible 3D microtissue co-culture system for phenotypic RNAi screening applications
Claudio R Thoma1, Simon Stroebel, Nora Rösch
11Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland.
Abstract:
Cancer cells in vivo are coordinately influenced by an interactive 3D microenvironment. However, identification of drug targets and initial target validations are usually performed in 2D cell culture systems. The opportunity to design 3D co-culture models that reflect, at least in part, these heterotypic interactions, when coupled with RNA interference, would enable investigations on the phenotypic impact of gene function in a model that more closely resembles tumor growth in vivo. Here we describe a high-throughput-compatible method to discover cancer gene functions in a co-culture 3D tumor microtissue model system composed of human DLD1 colon cancer cells together with murine fibroblasts. Strikingly, DLD1 cells in this model failed to expand upon siRNA-mediated depletion of Kif11/Eg5, a member of the mitotic kinesin-like motor protein family. In contrast, these cancer cells proved to be more resistant to Kif11/Eg5 depletion when grown as a 2D monolayer. These results suggest that growth of certain cancer cells in 3D versus 2D can unveil differential dependencies on specific genes for their survival. Moreover, they denote that the high-throughput-compatible, hanging drop technology-based 3D co-culture model will enable the discovery, characterization, and validation of gene functions in key biological and pathological processes.
Insights
Developing 3D tumor microtissues reveals differential gene dependencies for cancer cell survival. This 3D co-culture model, unlike 2D systems, identified Kif11/Eg5 as crucial for DLD1 colon cancer cell expansion.
Area of Science:
- Oncology
- Cell Biology
- Biotechnology
Background:
- Cancer cell behavior is influenced by the 3D tumor microenvironment.
- Traditional 2D cell cultures limit the study of in vivo cancer complexities.
- 3D co-culture models offer a more relevant system for drug target discovery.
Purpose of the Study:
- To develop a high-throughput 3D co-culture model for discovering cancer gene functions.
- To investigate differential gene dependencies in 3D versus 2D cancer cell models.
- To validate the utility of 3D tumor microtissues for functional genomics.
Main Methods:
- A high-throughput-compatible 3D co-culture system using hanging drop technology.
- Incorporation of human DLD1 colon cancer cells and murine fibroblasts.
- siRNA-mediated gene depletion to assess functional impacts on cancer cell growth.
Main Results:
- Depletion of Kif11/Eg5 (a mitotic motor protein) significantly inhibited DLD1 cell expansion in 3D microtissues.
- DLD1 cells showed increased resistance to Kif11/Eg5 depletion when cultured in 2D monolayers.
- This highlights context-dependent gene essentiality in cancer cell survival.
Conclusions:
- 3D co-culture models reveal distinct gene dependencies compared to 2D cultures.
- The developed 3D tumor microtissue system is effective for high-throughput functional gene discovery.
- This model system advances the study of gene function in cancer biology and pathology.
