Application of base editors in organoids opens new doors for cancer research

Cancer is one of the leading causes of death worldwide. For the development of new treatments, it is essential to be able to mimic the disease as closely as possible in the laboratory. One way to model tumors is with organoids: miniature organs that can be cultured in the laboratory to mimic an actual organ. Tumor organoids can be grown from patient tumor tissue, but it is also possible to use healthy tissue as a basis. In order to simulate a specific tumor type, mutations must be introduced into the DNA of the cells that are characteristic of that type of cancer. Using the CRISPR/Cas9 technique, researcher Maarten Geurts and his colleagues have succeeded in doing this and have created models for liver, colorectal and endometrial cancer. These tumor organoids can be used for further research, in order to better understand the development of tumors and to test new drugs.

Introducing point mutations

In order to incorporate the necessary mutations in the DNA of the organoids, the researchers used CRISPR/Cas9. Geurts explains how this works: “In the lab we usually use CRISPR/Cas9 to cut out specific parts of a cell’s DNA and thus render a gene inactive. However, not every cancer is caused by such an inactivating mutation, sometimes genes actually become more active. A few years ago, the CRISPR/Cas9 technique was further developed by scientists, which means that we can now also make these activating mutations in the DNA. We now have protein complexes at our disposal, so-called base editors, which can make very targeted point mutations in the DNA. This means that we can modify individual ‘letters’ in the DNA code, for example changing an A into a G. What we have done now is to apply this technique to normal, healthy organoids in order to incorporate specific combinations of both activating and inactivating mutations. We also succeeded in introducing several mutations at the same time.”

Base editing in liver organoids

The researchers first applied the technique in liver organoids, where they introduced several activating mutations at specific locations in the CTNNB1 gene. This gene is mutated in multiple types of cancer, including liver cancer. “For this we used three different base editors, hereby showing that they can all be used in human organoids,” says Geurts. When the researchers then looked at the effect of these mutations on the cells, they saw the CTNNB1 protein at locations in the cells where it does not belong. “Normally, this protein is located at the cell membrane, but in the organoids with mutations, we saw it in the nucleus and cytoplasm. This is because the protein is no longer broken down properly, so there is too much of it in the cells,” Geurts explains. “What surprised us was that the exact effect was not the same for all CTNNB1 mutations. This gives us starting points to further investigate how exactly these mutations contribute to the development of liver cancer.”

Higher efficiency

Cancer is often caused by a combination of mutations in several genes. If researchers were to use conventional CRISPR/Cas9 to mimic this in organoids, each individual mutation would have to be added in a separate reaction: a process that takes up to three months extra per mutation. Geurts and his team therefore investigated whether base editors could be used to more efficiently incorporate multiple mutations into intestinal organoids. “We managed to create overactivation of one gene, PIK3CA, while simultaneously knocking out two other genes, APC and TP53, all in one reaction. In the past this would have taken us much longer to accomplish. It therefore saves us a lot of valuable time,” says Geurts.

Development of colorectal cancer in a dish

The researchers then created a biobank of intestinal organoids, which they can use to simulate different stages of colorectal cancer. Any combination of mutations in five different genes can be found in this mini-biobank of 128 organoids. “During the development of a tumor, including colorectal cancer, the number of mutations often builds up over time. Therefore, we introduced different combinations of common mutations in the organoids to mimic the development of an intestinal tumor. For example, we had organoids with only one mutation, in the APC gene, but also specimens with one or more additional mutations. We clearly saw that the appearance of the organoids changes as the number of mutations increases: from spherical to an increasingly chaotic shape. This is consistent with the fact that the organoids represent an increasingly advanced stage of colorectal cancer. Creating this biobank was now feasible because we could introduce multiple mutations at the same time through base editing and it allows us to further investigate the individual stages of colorectal cancer,” says Geurts.

This video shows the difference in appearance between a healthy intestinal organoid without mutations (first video) and an intestinal organoid with mutations in APC, PIK3CA, SMAD4 and TP53 (second video). Credit: Maarten Geurts. Copyright: Hubrecht Institute.

Opportunities for future research

Geurts sees many applications for tumor organoids created through base editing. “Taken together, we made tumor organoids for three different organs in this study. That is, in addition to modeling liver and colorectal cancer, we have also succeeded in mimicking the early stages of endometrial cancer and gaining more insight into this. We have thus shown that base editing makes it possible to make tumor models of different cancer types in a very efficient way. We have also shown that it is possible to incorporate up to five different mutations in one reaction. Since the efficiency of the reaction did not decrease, we may be able to increase this even further in the future. Our group has now also applied this technique to organoids from other organs. We are convinced that this provides us with a valuable tool for further research into the onset, development and treatment of various tumor types.”

Publication

One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids. Maarten H. Geurts*^＋, Shashank Gandhi*, Matteo G. Boretto*, Ninouk Akkerman, Lucca L. M. Derks, Gijs van Son, Martina Celotti, Sarina Harshuk-Shabso, Flavia Peci, Harry Begthel, Delilah Hendriks, Paul Schürmann, Amanda Andersson-Rolf, Susana M. Chuva de Sousa Lopes, Johan H. van Es, Ruben van Boxtel and Hans Clevers^＋. Nature Communications, 2023.

* Authors contributed equally
^＋ Shared corresponding authors