Utilizing (iPSC)-Based Tumor Modeling and Genetic Engineering in Sarcoma Research

By Miranda Mead

Beau Webber, PhD
Beau Webber, PhD – Assistant Professor, Department of Pediatrics, University of Minnesota

Beau Webber, a 2018 RIS research grant recipient, is an Assistant Professor, Department of Pediatrics at the University of Minnesota, as well as Faculty Member of the Division of Pediatric Hematology and Oncology and Stem Cell Institute. While getting his PhD in stem-cell biology and genetic engineering at the University of Minnesota, Webber used genetic tools to help correct mutations that cause disease. He worked primarily with cells called Induced Pluripotent Stem Cells (iPSCs). Basically, iPSCs are adult cells that have been genetically reprogrammed into embryonic stem cells, helping eliminate the ethical concern with using real embryonic cells.

While working with a colleague, Webber was introduced to cancer genetics, which included understanding which genes go wrong in a cell that leads to cancer. Webber’s colleagues created mouse models to do forward genetic screens by using a transposon system, or something called a jumping gene that would jump around and land in genes, causing the cell to become cancerous. Working backwards, Webber’s team studied these genes in the mice to determine which genes became broken, causing cancer. Then his team would identify cells that could be targeted with specific therapies. However, mice and humans are very different from one another, limiting the usefulness of mice studies.
Another way to study cancer is to put tissue samples from human tumors in a culture dish and see which cells stick and grow with the right conditions. This would show which lines were cancer. While this works, it’s a poor representation of what tumors look like considering tumors are three-dimensional and complex. So, Webber’s goal became how can we make these tumors more realistic and “better predictors” to help with treatments. By using what he was familiar with, Webber hoped to make models of cancer using iPSCs cells. He wanted to combine what his colleagues were doing in the mice with the iPSCs cells, to create a controlled way to make the iPSCs cells into cancer. Through this process, Webber wants to understand all the different pieces that need to be broken in order to make the iPSCs cells into cancer.

By using a ground up method, Webber starts with a normal iPSCs cell and then breaks it piece by piece until he gets something that resembles cancer. There’s a lot of potential with this method in that they can observe the cell becoming cancer in a dish. The next step involves genetic engineering. Currently there are a lot of techniques to help make 3D models resembling cubes or a circular shape. These are known as organoids. These organoids can take on all the complex properties associated with organs. By using genetic engineering to help create 3D shapes and by using iPSCs cells, Webber is hoping to create mini 3D tumors that can be easily monitored. By creating these through a controlled process, Webber’s’ team will know everything that “went wrong” (each step required to create the cancerous tumor).

Webber is currently working on two main projects involving Osteosarcoma and Ewing Sarcoma. While the two cancers differ from one another, they start off virtually the same—a bone progenitor cell. First, Webber and his team direct the iPSCs cells to become mesenchymal progenitor cells, which then can become cartilage and bone. Webber created his own serum to help ensure that every step was as controlled as possible. After, “getting those systems to work really well” the next steps consisted of genetic engineering. There are well known proteins called tumor suppressors – think of these proteins as breaks. Webber and his team use CRISPR technology, a system that was taken from bacteria that help fight off viruses. CRISPR is used as a way to cut DNA wherever needed. Webber and his team can cut these tumor suppressors in the iPSCs cells. Then they add oncogenes, genes that have the potential to turn tumorous. By cutting the breaks, the tumor suppressors, and by applying the gas, adding the oncogenes, Webber’s team is modifying the bone IPS cells to try to replicate Osteosarcoma. They are getting pretty close. In the mice screening, Webber’s colleagues believe they found genes that could be linked to Osteosarcoma metastasis. Once the model is created, Webber can put those genes in the 3D Osteosarcoma model and see how it influences the cell.

With Ewing Sarcoma it is a bit different. A unique characteristic associated with Ewing Sarcoma is nearly every diagnosed patient is of European dissent. A common mutation that occurs in Ewing Sarcoma is called the EWS-FLI-1 fusion translocation. This is present in 90% of Ewing cases. There is only one driving fusion protein. Webber describes this as, “[The protein] scrambles the computer programming of a cell. It jumps around and turns on genes that shouldn’t be on and shuts genes off that shouldn’t be off.” By using iPSCs cells, Webber’s team used CRISPR to cut at EWS and at FLI to break them and then get them to come together in the cell, creating that EWS-FLI-1 translocation, leading to Ewing Sarcoma. The EWS-FLI-1 protein when injected into most cells kills them. Webber and his team are working on finding the perfect balance between turning the protein on at a high level while keeping the cell alive. They are aiming to hit that fine line in a specific cell type in a given situation where the cell can respond to the protein being turned on. For all this to occur the perfect condition must be present.

Currently with Ewing Sarcoma, Webber’s team is working on inserting these EWS-FLI-1 translocations into the different cell lines to see which lines stick and start forming Ewing Sarcoma in the bone cells made from iPSCs cells. They are looking to answer, “What is the exact dose that is required to get these cells to transform?” Then by utilizing the system, they can determine the origin of Ewing Sarcoma. Using targeted therapies, Webber and his team can then see how the tumor responds to the given therapy.

Using the Rein in Sarcoma research grant, Webber went to the WiCell lab in Madison, Wisconsin. There, they pulled out 10 iPSCs lines ranging from 100% European to 100% African ancestry and many degrees in between. Webber and his team are hoping to eventually convert those lines to Ewing Sarcoma. What most likely will happen is the European cell lines will turn into Ewing Sarcoma and those of African ancestry won’t. This may or may not occur, but the goal is to monitor what happens when the EWS-FLI 1 protein is turned on in the differing ancestry lines. Webber’s team believe that a comparison of the two ancestry lines will help point to the important genes that potentially cause Ewing Sarcoma
Ultimately, using his prior knowledge of iPSCs cells, Webber is hoping to learn more about the formation of sarcoma cancers to determine the most beneficial treatments. By striving to create artificial sarcoma tumors, more research can be done, since patient samples are no longer the only way to obtain tumor samples.

Currently, Webber and his team are satisfied with the progress being made towards the final goal of changing these iPSCs cells into tumors. While they are not completely there, Webber and his team are well on the way to providing new tools and understanding towards the fight against sarcoma cancer.

Miranda Mead is a Ewing Sarcoma survivor and Journalism student at the University of St. Thomas. As an RIS volunteer, Miranda is interviewing RIS research grant awardees, such as Dr. Webber, to bring that work to life for the Rein in Sarcoma community. Read Miranda’s sarcoma story

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