Mechanochemical effects of growth factor gradients in colorectal cancer organoids

Understanding how colorectal cancer (CRC) cells respond to growth factor (GF) cues in addition to mechanical forces is essential for defining the mechanisms that regulate tissue organization and tumor progression. Here, we used a platform that co-registers mechanical outputs with biochemical readouts to investigate CRC organoid responses to well-defined spatiotemporal GF gradients produced by GF-loaded beads. Embedding the beads within a liquid-like solid microenvironment creates a diffusion-limited system, enabling stable and quantifiable gradient formation. GF-loaded beads were positioned at defined locations within the in vitro model to establish local concentration fields, measured through spatially distributed ELISA beads operating under non-depleting Langmuir adsorption conditions. Concurrently, 2D CRC organoids expressing Erk and Akt kinase translocation reporters were cultured on ECM-functionalized hydrogels embedded with fiduciary traction beads, enabling reconstruction of traction stress maps via particle-tracking-based traction force microscopy. Preliminary data shows that organoids exposed to steep GF gradients will exhibit elevated Erk activity along gradient-facing edges, while regions experiencing lower concentrations will display reduced Akt activation. This spatiotemporal response further demonstrates that traction forces will increase in regions with high local GF concentration, indicating a gradient-dependent mechanochemical response. These projected spatial correlations will reveal discrete concentration thresholds above which CRC cells transition from apical to focal contractility, a shift that reflects loss of epithelial organization, increased ECM engagement, and acquisition of a more motile, invasion-prone phenotype relevant to tumor progression. Collectively, these outcomes demonstrate that localized GF release from GF-loaded beads can be quantitatively mapped and directly linked to force generation and kinase dynamics within CRC organoids. This integrated approach will establish a unified framework for probing how GF gradients and mechanical cues interact to shape CRC cell behavior, providing a platform for subtype-specific studies and enabling engineering-driven strategies for targeted therapeutic intervention.