PDX Tumor Organoids: A 3D Platform that is also Fully Human

Charles River is among the first in the race developing commercially viable 3D models that marries PDX technology and tumor organoids. Will the match improve translation?

In 1969, two Danish scientists reported the first patient-derived xenograft when they removed a malignant colon tumor from a patient and planted the tumor fragments in a nude mouse. Since then, PDX mice-what the industry has dubbed mouse avatars-have been stand-ins in the hunt for cancer cures for people, and today PDX models are an increasingly common tool that drug developers reach for when evaluating their new therapies, particularly immunotherapies.

But what if you did not need the mouse at all. What if you could replace animals with a 3D model that is fully human. That's the idea behind a bold project initiated last year by Charles River's Discovery Oncology site in Freiburg, Germany, a pioneer in patient-derived xenograft studies. In collaboration with the Institute for Clinical Pharmacology in Stuttgart and Freiburg's sister site in North Carolina, they are developing PDX tumor organoids-a 3D in vitro model of a patient's tumor derived from a patient-derived xenograft-to give biopharma clients a more effective, translatable way of identifying human-specific drug targets and disease mechanisms, and even treatments for individual patients.

As a new research model and platform, the organoid star is rising fast. Insight Partners, a leading market research provider, estimates the market was valued at $2.5 billion in 2022 and will grow to $12 billion by 2030, with North America the largest contributor to market growth and Asia Pacific the fastest-growing region. Since the development of the first organoids in 1987 their use has moved beyond academic labs and into the commercial arena. Today, organoids are a valid substitution for two-dimensional models, and a number of large pharma companies, such as AstraZeneca, Roche and GlaxoSmithKline are investing in organoid research.

More translation, fewer animals

What makes the Freiburg project stand out is that it is among the first commercially viable ventures to marry the PDX model and the organoid system. Will it be a successful match?

Julia Schueler, therapeutic area lead for oncology at Charles River Laboratories thinks it will. She said the Freiburg lab has developed organoids from four well-characterized PDX-derived breast cancer cell lines and eight PDX-derived lung cancer cell lines, while their sister facility in North Carolina is exploring organoids based on two PDX-derived colon cancer lines. Schueler says the idea is to have an in vitro tool that is as heterogeneous as the PDX, yet still animal-free.

To be truly translational, the project is looking at organoid panels of both cold and hot tumors-those unlikely to trigger a strong immune response vs. those whose behavioral patterns in vivo are well-understood-to increase the predictive strength of which population might benefit most from selected drugs. Since the patients who have hot or cold tumors vary, it is important to have models for both, says Schueler.

"Our ultimate goal is to be able to use organoids on client compounds instead of relying on a mouse, or afterwards doing a smaller study in the mouse," says Schueler, adding that she hopes to have data on the breast cancer organoids by the end of the year, and lung cancer data in early 2025.

PDX Tumor Organoids: Cheaper and faster to establish

Organoids are self-organized 3D cell cultures that aim to mimic the structure, function, and cellular complexity of human organs, making them, in some ways, more translatable than animals.

The process of making an organoid begins with isolated embryonic, or in the case of Charles River, pluripotent stem cells. Schueler says Freiburg cultures the cells in a gelatinous mixture of proteins that support three-dimensional growth and banks them, similar to what they do with PDX cell lines. After the organoids mature, they are harvested and sliced for immunohistochemistry (IHC) analysis -a staining technique that uses antibodies to detect antigens in tissue samples-to characterize their genetic makeup. The "organoid slices" are funneled through a fluorescent microscope to study their cell surface markers or blended for PCR.

The IHC staining and other analysis is being done by their collaborator in Stuttgart using a multiplex that allows up to 60 cell markers to be evaluate in each organoid. "You can see protein expression in these markers, but also local colonization," says Schueler. Freiburg meanwhile uses multiplex gene expression, a high-throughput technology that measures the expression of multiple genes simultaneously, to prove that the organoids express the same markers as the PDX. This is extremely important, says Schueler, because if they don't the PDX organoid won't function properly.

Schueler says PDX tumor organoids are faster, easier, and cheaper to establish because all you need is the protein mixture, such as Matrigel, to allow the organoids to develop into more complex tissue, and equipment and a bioreactor system that helps maintain the appropriate tumor phenotype (a tumor's correct features) and response to therapy.

Challenges of PDX organoids

However, these 3D models are not without challenges. Success rates are low for some organs, and there is limited standardization across the structure of different tissues. Because of their complexity, the readouts are much more sophisticated than they are for a typical in vitro model. Another challenge is that certain conditions related to the various growth factors can affect the interpretation of the drug screenings. Lastly, most tumor-derived organoid technology only applies to epithelial cells, leaving two critical components of the tumor microenvironment-stromal cells and immune cells-under-represented. (In contrast some PDX mouse models include human immune cells and murine stromal cells). Schueler said the organoid field is partially starting to close this last gap by co-culturing tumor organoids with immune cells. Stromal cells are a bigger challenge, though.

"I'm excited about where organoids will take cancer drug development," said Schueler. "Being able to move to the next level and have a completely human model give us the best chance at translatability. … Having an animal-free 3D in vitro system like this could enable us to identify better candidates more quickly because at the end of the day, it's all about the patients."

References:
1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9585375/

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