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.
Expert: Absolutely. IBD is an umbrella term referring to 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 contribute to the disease progression. An important role is played by 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 the development of resistance to anti-TNF therapies.
Expert: The inFOCUS assay is a inflammatory 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 inflammation affects the fibroblast and epithelium homeostasis and, importantly, to determine which if there are pathways that 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?
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 by an inflammatory stimulus. This setup lets us observe how the phenotypic change in the fibroblasts influence the epithelial compartment.
Expert: We monitor several key indicators, some of which are soluble mediators released by either fibroblasts and/or organoids. Another crucial readout is cell death to measure epithelial erosion. We also assess organoid morphology—whether they appear enlarged or cystic in response to inflammatory fibroblasts—as well as changes in the expression of cell markers tied to proliferation and differentiation.
Expert: Sure. Interestingly, we’ve found that non-inflammatory fibroblasts can actually protect organoids from cytokine-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.
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.
Expert: Yes of course! We’re using the inFOCUS assay in a screening capacity to identify druggable targets. 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. The goal is to uncover specific fibroblast pathways that could be therapeutically targeted to manage or reverse IBD pathology.
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.
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.