Prospective Derivation of a Living Organoid Biobank of Colorectal Cancer Patients

Abstract

In Rspondin-based 3D cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. We report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely recapitulate several properties of the original tumor. The spectrum of genetic changes within the "living biobank" agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell-line- and xenograft-based drug studies, and allow personalized therapy design. PAPERCLIP.

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

“…We thus designed a strategy based on CRISPR/Cas9‐mediated homologous recombination to mark LGR5 + cells in human CRCs. We made use of CRC PDOs, which are good surrogates of the disease in vitro and in vivo (Calon et al , 2015; van de Wetering et al , 2015). For these experiments, we initially selected a PDO derived from a stage IV 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 study followed the guidelines of the Declaration of Helsinki, and patient identity for pathological specimens remained anonymous in the context of this study. Tumor cells were grown as organoids embedded in BME2 (basement membrane extract 2, AMSbio) using a modification of the media described by the Clevers laboratory (van de Wetering et al , 2015) (Advanced DMEM/F12, 10 mM HEPES, 1× Glutamax; 1× B‐27 without retinoic acid, 20 ng/ml bFGF (basic fibroblast growth factor); 50 ng/ml EGF (epidermal growth factor), 1 μM LY2157299, 10 μM Y27632, and recombinant Noggin (100 ng/ml). PDO#7, a kind gift from G. Stassi (University of Palermo), was obtained from the dissociation of whole CRCs in suspension as described elsewhere (Lombardo et al , 2011).…”

Expansion of airway basal epithelial cells from primary human non‐small cell lung cancer tumors

“…Normal cells also outcompete tumor‐derived cells during cancer organoid derivation. Here, it has been possible to develop tumor‐specific media to select tumor organoids over normal colonic25 and hepatic26 epithelial organoids. For example, intestinal epithelial cells are dependent on exogenous Wnt in culture but constitutively active Wnt signaling, due to genomic aberrations in genes such as APC and/or CTNNB1 , often confers tumor cells with the ability to survive culture in Wnt‐free culture media.…”

“…For example, intestinal epithelial cells are dependent on exogenous Wnt in culture but constitutively active Wnt signaling, due to genomic aberrations in genes such as APC and/or CTNNB1 , often confers tumor cells with the ability to survive culture in Wnt‐free culture media. Such strategies might be relevant in NSCLC culture: for example, EGF could be withdrawn in cases where tumors have EGFR ‐activating mutations or nutlin‐3 could be added to P53 ‐mutant tumor cultures 25. These selection strategies could be effective in cases where the tumor genotype is known at the time of surgery or in lung squamous cell carcinoma cell culture where the vast majority of tumors are P53 mutant.…”

Modeling infectious diseases and host-microbe interactions in gastrointestinal organoids

“…Genome wide association studies (GWAS) have identified risk factors such as cytokines and pattern recognition receptors (reviewed in Wroblewski et al, 2010), but the mechanism underlying susceptibility is still unclear. Uniquely, organoids can be grown from seemingly every patient, which has allowed the generation of "living biobanks" amenable to phenotypic or drug screens (Fujii et al, 2016;van de Wetering et al, 2015;VanDussen et al, 2015). Such biobanks will enable researchers to identify and better understand risk factors in infection-associated pathologies such as gastric cancer and IBD, eventually paving the way for personalized treatments.…”