CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities
Open accessAbstract
Cancer genomics studies have identified thousands of putative cancer driver genes 1. Development of high-throughput and accurate models to define the functions of these genes is a major challenge. Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif 2 from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.
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Recent studies have shown that phenotypes associated with CRISPR-mediated knockout of putative oncogenic loci are more pronounced when cancer cells are grown as 3D spheroids than 2D monolayers.
“Recent studies have shown that phenotypes associated with CRISPR-mediated knockout of putative oncogenic loci are more pronounced when cancer cells are grown as 3D spheroids than 2D monolayers, suggesting that 3D culture systems may be particularly useful for uncovering cancer vulnerabilities55.”
From Large-scale CRISPR screening in primary human 3D gastric organoids enables comprehensive dissection of gene-drug interactions (2025) · cites this paper as [55]
Earlier work links the YAP/TAZ/TEAD pathway to resistance to other targeted therapies, including MEK and BRAF V600E inhibitors.
“These findings comport with previous results implicating the YAP/TAZ/TEAD pathway in resistance to other targeted therapies (40,41) including MEKi and BRAF V600E inhibitors (36,55).”
From Genome-Wide CRISPR Screens Identify Multiple Synthetic Lethal Targets That Enhance KRASG12C Inhibitor Efficacy (2023) · cites this paper as [55]
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References 59 · Cited by 311
- Authors
- Han, K., Pierce, S. E., Li, A., Spees, K., Anderson, G. R., Seoane, J. A., Lo, Y.-H., Dubreuil, M., Olivas, M., Kamber, R. A., Wainberg, M., Kostyrko, K., Kelly, M. R., Yousefi, M., Simpkins, S. W., Yao, D., Lee, K., Kuo, C. J., Jackson, P. K., Sweet-Cordero, A., Kundaje, A., Gentles, A. J., Curtis, C., Winslow, M. M., & Bassik, M. C.
- Affiliations
- Stanford Medicine (US)
- Stanford University (US)
- University of California, San Francisco (US)
- Corresponding author
- Han, Kyuho
- Bassik, Michael C.
- ORCID
- Han, Kyuho
- Pierce, Sarah E.
- Li, Amy
- Seoane, Jose A.
- Lo, Yuan-Hung
- Olivas, Micah
- Kamber, Roarke A.
- Wainberg, Michael
- Kostyrko, Kaja
- Kelly, Marcus R.
- Yousefi, Maryam
- Simpkins, Scott W.
- Yao, David
- Kuo, Calvin J.
- Jackson, Peter K.
- Sweet-Cordero, Alejandro
- Kundaje, Anshul
- Gentles, Andrew J.
- Curtis, Christina
- Winslow, Monte M.
- Bassik, Michael C.
- Venue
- Nature
- Published
- 2020-03-11
- Volume
- 580
- Issue
- 7801
- Pages
- 136-141
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC7368463
- OpenAlex
- W3011410517
- Cited by
- 311
- References
- 59
- Citations by year
- 2026: 28 · 2025: 48 · 2024: 36 · 2023: 48 · 2022: 55 · 2021: 65 · 2020: 31
- FWCI
- 16.87
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- Funders
- National Institutes of Health (1dp2hd084069-01)
- National Human Genome Research Institute (UM1HG009436)
- National Cancer Institute (K00CA212433, U01CA199216, U01CA217851, T32CA009302)
- Eunice Kennedy Shriver National Institute of Child Health and Human Development (DP2 HD084069)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer Genomics and Diagnostics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
- Keywords
- CRISPR screen, tumor spheroids, 3D tumor models, lung cancer, cancer driver genes, carboxypeptidase D, IGF-1 receptor, patient outcomes
- MeSH terms
- Adenocarcinoma, Animals, Carboxypeptidases, Female, Humans, Lung Neoplasms, Mutation, Phenotype, Signal Transduction, Genome, Human, Receptor, IGF Type 1, Spheroids, Cellular, Cell Culture Techniques, Amino Acid Motifs, Xenograft Model Antitumor Assays, Cell Proliferation, Mice, Molecular Targeted Therapy, CRISPR-Cas Systems
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