A genome editing approach to study cancer stem cells in human tumors

Marta Sevillano,Xavier Hernando‐Momblona,Carme Cortina,Camille Stephan‐Otto Attolini,Sébastien Tosi,Anna Merlos‐Suárez,Diana Stork,Mònica Aguilera,Eduard Batlle,Gemma Turon,Elena Sancho

doi:10.15252/emmm.201707550

Abstract

The analysis of stem cell hierarchies in human cancers has been hampered by the impossibility of identifying or tracking tumor cell populations in an intact environment. To overcome this limitation, we devised a strategy based on editing the genomes of patient‐derived tumor organoids using CRISPR/Cas9 technology to integrate reporter cassettes at desired marker genes. As proof of concept, we engineered human colorectal cancer (CRC) organoids that carry EGFP and lineage‐tracing cassettes knocked in the LGR5 locus. Analysis of LGR5‐EGFP + cells isolated from organoid‐derived xenografts demonstrated that these cells express a gene program similar to that of normal intestinal stem cells and that they propagate the disease to recipient mice very efficiently. Lineage‐tracing experiments showed that LGR5+ CRC cells self‐renew and generate progeny over long time periods that undergo differentiation toward mucosecreting‐ and absorptive‐like phenotypes. These genetic experiments confirm that human CRCs adopt a hierarchical organization reminiscent of that of the normal colonic epithelium. The strategy described herein may have broad applications to study cell heterogeneity in human tumors.

Highlights

Most cancers are amalgams of phenotypically distinct tumor cell populations, which display marked differences in their behaviors and fates
We initially selected a PDO derived from a stage IV colorectal cancer (CRC) that displayed a prototypical combination of genetic alterations in major driver pathways including activation of the WNT pathway by APC loss of function, activation of EGFR signaling by KRAS G13D mutations, and loss of TGF-beta-mediated tumor suppression by inactivating mutations in SMAD4 (PDO#7 in Appendix Table S1)
The combination of organoid and CRISPR/Cas9 technology described opens up the study of human tumors through genetic approaches that had only been feasible in animal models

Summary

Introduction

Most cancers are amalgams of phenotypically distinct tumor cell populations, which display marked differences in their behaviors and fates. We generated single cell-derived organoid cultures and assessed integration of the EGFP reporter cassette by PCR (examples in Appendix Fig S1A and B) and Southern blot (examples in Appendix Fig S1C and D). These analyses showed that 41.7% of the clones had correctly integrated the EGFP reporter in the LGR5 locus (Appendix Table S2). Equivalent LGR5-EGFP knock-in experiments in a PDO grown from a different patient sample (PDO#6) (Fig EV1A) rendered a frequency of correct integrations of 84.6% (Appendix Table S2) In these single cell-derived knock-in PDO cultures, every organoid was composed by an admixture of cells expressing distinct EGFP levels (Figs 1C and D, and EV1B and C). Staining with KRT20 or MUC2 antibodies revealed complementary expression patterns of these differentiation markers with EGFP implying that LGR5+ CRC cells generated differentiated progeny in vitro (Fig 1C)

Methods

Results

Conclusion