A living biobank? Well, not quite a living, breathing organism, but van de Wetering et al. (2015) present the case for establishing a colorectal carcinoma (CRC) organoid biobank that could help researchers screen drugs and thereby develop personalized therapeutic regimes for patients. Using tissue explants from surgical resections, the research team developed a protocol for developing three-dimensional (3-D) cultures that maintain the physical characteristics of the primary tissue. Once derived, the cells are used to establish drug-screening arrays in addition to setting up frozen stocks for future examination.
Organoids are 3-D mini organs grown in culture dishes. Selective and specialized culture conditions coax cells from primary tissue into organoid form in vitro, where they maintain many physical and physiological characteristics of the original itself. In the case of intestinal organoids, this includes crypt and epithelial structure and function. Van de Wetering et al. established a method for harvesting and long-term culture that maintains not only the intestinal crypt physiology but also the genetic changes and mutations that are hallmarks of the primary colorectal carcinoma from which it is collected.
The researchers took tumor and adjacent healthy tissue from surgical resections carried out in untreated patients. Exploiting various pathways, including Wnt signaling, the team developed a protocol that promoted organoid development in both tumor and healthy adjacent tissue samples. Following several instances of microbial contamination and subsequent loss of cultures, the researchers used routine antibiotics in growth media; this improved the success rate, with initial establishment of cultures from 27 tumors.
Once fully established, the researchers found that most organoid cultures showed good growth rates, although two did slow down. They discarded these preparations and concentrated on the remaining 22 tumor and 19 normal adjacent organoid cultures. The team established master cell banks by freezing down populations from each culture. On thawing, the cells generally grew well with good viability.
Physical examination of the organoid cultures showed maintenance of intestinal crypt anatomy for normal adjacent cells. The researchers noted a variable appearance for those established from tumor cells, with the organoid cultures derived from them showing morphologies ranging from solid to cystic that correlated somewhat with marker expression.
The team then examined the genomic DNA using whole exome sequencing. They found that the organoid cultures expressed the common CRC mutations and microsatellite instabilities expressed in the primary tumors, including alterations in the Wnt pathway and MLH1 deletion. Moreover, with subsequent cultures, the organoids continued to express these genomic alterations, thus providing researchers with representative copies of each patient’s tumor to study. Overall, van de Wetering et al. found that the organoid establishment protocols maintained the mutation heterogeneity seen in the primary tumors without bias according to culture method.
As a final step, the researchers used the organoid cultures to screen sensitivity to chemotherapeutic drugs. After disrupting the organoids to break up the architecture, the team cultured the cells in multi-well plates before exposing them to various chemotherapeutic drugs delivered over several days using robotics liquid handling. Following exposure, they examined the cell morphology and assessed viability, screening the cultures for resistance and sensitivity to the agents. Van de Wetering et al. then compared the results obtained with those reported in the literature for known genomic-drug response interactions. They found agreement with known genomic-drug sensitivity pairs, in addition to demonstrating novel sensitivities and resistance patterns among organoid cultures. Noting that most of the primary tumors from which the organoids were derived were polyclonal, the researchers suggest that the drug screening results could explain resistance patterns encountered in individual patients.
Van de Wetering et al. suggest that patient-derived tumor organoids could be a valuable tool in examining the effect of tumor genetic profile on drug response, as an adjunct to patient trials. They advise developing and screening a larger population of tumor-derived organoids to increase knowledge of drug therapies suitable for rare genotypes. Although they feel that the protocol would benefit from faster culture methods, the researchers strongly suggest that establishing and maintaining a living organoid biobank could benefit individual patients for development of tailored therapy, since high-throughput drug screening results would be available within weeks of initial tissue derivation.
Reference
1. van de Wtering, M. et al. (2015) “Prospective derivation of a living organoid biobank of colorectal cancer patients,” Cell 161(4) (pp.933–45).
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