Ask the Expert: Inside the inFOCUS Assay for IBD Drug Discovery

In this "Ask the Expert" interview, Dr. Javier Frias Aldeguer, Senior Scientist at HUB Organoids shares insights into the inFOCUS assay—an innovative fibroblast-organoid co-culture model developed to explore the role of inflammatory fibroblasts in Inflammatory Bowel Disease (IBD). Learn how this assay uncovers key disease mechanisms, supports drug target discovery, and opens new doors for collaborative IBD drug development.

Could you briefly explain the context of this assay in relation to Inflammatory Bowel Disease (IBD)?

Expert: Absolutely. IBD includes chronic conditions like Crohn’s disease and Ulcerative Colitis, both characterized by long-term inflammation in the gastrointestinal tract. While the exact causes are still unclear, it’s widely accepted that a combination of genetics, environmental triggers, gut microbiota, and immune dysfunction play a role. An important component of this immune response involves the stromal compartment—particularly fibroblasts. In IBD, fibroblasts become activated and contribute to chronic inflammation. In fact, a specific subset known as inflammatory fibroblasts has been linked to resistance to anti-TNF therapies.

Thanks for the layman explanation. So, what exactly is the inFOCUS assay, and what is it designed to achieve?

Expert: The inFOCUS assay is a fibroblast-organoid co-culture model developed here at HUB. It was created to help us better understand the role of inflammatory fibroblasts in IBD. This assay allows us to dissect how specific fibroblast pathways contribute to the inflammatory phenotype and, importantly, to determine which of these are therapeutically targetable. We're also interested in identifying the fibroblast-derived factors that directly affect epithelial cells—essentially asking, which signals from fibroblasts are pathogenic?

That leads perfectly to my next question. The assay involves both fibroblasts and organoids—how are they used together?

Expert: In the inFOCUS assay, we co-culture intestinal organoids with fibroblasts under various conditions. For example, we might use different concentrations of fibroblasts per well, either in a resting state or activated with cytokines to simulate an inflammatory environment. This setup lets us observe how the fibroblasts influence the epithelial compartment.

And what kinds of readouts do you get from this model?

Expert: We monitor several key indicators. One is the release of soluble mediators by fibroblasts—cytokines like IL6 and CXCL2, the latter of which is produced by both cell types. Another crucial readout is barrier integrity, measured through caspase activity in organoids exposed to TNF/IFNγ. We also assess organoid morphology—whether they appear enlarged or cystic in response to inflammatory fibroblasts—as well as changes in cell markers tied to proliferation and differentiation.

You mentioned barrier damage—can you elaborate on how the fibroblast state influences that?

Expert: Sure. Interestingly, we’ve found that non-inflammatory fibroblasts can actually protect organoids from TNF/IFNγ-induced damage. However, once these fibroblasts become pro-inflammatory, that protective effect disappears. This suggests that the fibroblast’s inflammatory state is a major factor in how the epithelial barrier responds to inflammatory stress—a key feature of IBD.

How well does this in vitro model reflect what actually happens in IBD patients?

Expert: Quite well. The assay recapitulates multiple epithelial hallmarks of IBD, including inflammation-induced apoptosis and compromised barrier function. Organoids co-cultured with inflammatory fibroblasts exhibit signs of inflammation, altered morphology, and a shift in expression of markers like OLFM4 (a stem cell marker), KI67 (proliferation), and ALPI (differentiation). These changes are consistent with the disrupted epithelial homeostasis observed in IBD tissue.

That is so interesting! Can you apply this assay to screen compounds for IBD?

Expert: Yes ofcourse! We’re using the inFOCUS assay in a screening capacity to identify druggable targets within fibroblasts. One approach involves a CRISPR knockout library in fibroblasts to discover genes that, when silenced, reduce their inflammatory behavior. We track this through multiple readouts: imaging, caspase activity, and cytokine levels like CXCL2. The goal is to uncover specific fibroblast pathways that could be therapeutically targeted to manage or reverse IBD pathology.

Sounds promising. What’s next for the inFOCUS assay within HUB’s research pipeline?

Expert: We’re aiming to integrate the inFOCUS assay into HUB’s standard assay portfolio. That includes ongoing optimization to improve reproducibility across various donor-derived fibroblasts, fine-tuning of media and timepoints, and expanding our mechanistic understanding of the assay’s readouts. Longer-term, we’re looking at possibilities for patenting, publishing the work, and developing partnerships around the assay’s application in IBD drug discovery.

For anyone with a novel hypothesis—like the inFOCUS assay was at the start—how can they co-develop with HUB Organoids?

Expert: That’s a great question. HUB is always open to collaboration, especially when it comes to developing innovative models that address unmet needs in disease biology. Partners can engage with us through co-development projects, where we bring together our organoid expertise with their scientific hypothesis or therapeutic angle. This could mean working jointly on assay development, optimizing readouts, or applying our models to their compound screens. It’s a collaborative process, tailored to the scientific question at hand and driven by mutual innovation.