324 (PB104) - Quantitative analysis of EGFR expression and distribution upon drug treatment in colorectal organoid models

Abstract

Background: Human organoids are patient-derived tissues that can be expanded in vitro by stimulating the (cancer) stem cells with WNT pathway activators, treatment with differentiation inhibitors and by providing growth factors in an extracellular matrix environment. These organoids form three-dimensional (3D) multicellular structures that polarize and self-organize and express relevant disease-related markers, which makes an organoid biobank ideal for testing the efficacy and mode of action of compounds and antibodies across a diverse patient population. Here, our colorectal organoid biobank and high content imaging platform were used to characterize EGFR dynamics upon drug treatment. Methods: An established biobank of normal colon and colorectal cancer organoid models was characterized for its sensitivity to the growth factor EGF, EGFR-inhibitors (Cetuximab), and standard-of-care compounds. To study the distribution of the EGF receptor, antibodies followed by immunofluorescence staining were used to detect the localization and distribution upon growth factor stimulation. (Inducible) knockdown organoid models with shRNA constructs targeting EGFR were generated to validate the findings. Results: The 3D organoid screening and our proprietary image analysis platform allows the in-depth detection and distinction of growth factor responses that not necessarily only relate to proliferation. By making use of this system, the sensitivity of organoid models to EGF stimulation that are independent of EGF for their outgrowth could be determined. By combining the detection of the target, EGFR, with the detection of targeting EGFR-inhibitory antibodies by immunofluorescence followed by image analysis, the redistribution of the receptor upon EGF-stimulation and in response to antibody exposure could be visualized and quantified. This revealed that EGFR is internalized in the first hours upon EGF stimulation. Along with making use of (inducible) knockdown organoids, it also revealed that different EGFR-targeting antibodies have different effects on the redistribution, internalization, and degradation of EGFR. Conclusions: The high content imaging platform in combination with high throughput screening in 3D-cultured organoid models allows compound and antibody testing in physiologically relevant patient-derived models to not only study the sensitivity to novel compounds, but also for patient stratification and to study the mode of action or understand the underlying relevant biology. The outcome of this work demonstrated that organoids are versatile, multicellular structures that are highly suitable for studying the expression and redistribution of disease-relevant targets. No conflict of interest.