Martinez Espuga, Magda
(2025)
Engineering biomimetic in vitro models for personalized cancer treatments.
PhD thesis, University of Nottingham.
Abstract
Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. Conventional therapies often fail due to the inability of current in vitro models to recapitulate the complexity and heterogeneity of the tumour microenvironment (TME). Most research still relies on 2D cultures, which poorly model tumour biology or on 3D models that use animal-derived matrices such as Matrigel, which suffer from high variability and limited tuneability. To address these limitations, this thesis presents the development and optimisation of peptide amphiphiles (PA)-based composite hydrogels co-assembled with tumourspecific CRC-ECM macromolecules (PA-ECM), including collagen, hyaluronan, fibronectin, and laminin. By engineering these nanofibrillar hydrogels with either random (rPA) or aligned (aPA) architectures, it is possible to closely mimic the CRCTME, enabling precise control over ECM composition, nanofibrillar organisation, and mechanical properties. Structural, biochemical, and mechanical analysis demonstrated that rPA-based hydrogels enable a more stable and homogenous ECM incorporation, maintaining a physiological soft stiffness. In contrast, aPA-based hydrogels were stiffer, less permissive to ECM incorporation, and failed to support long-term cell viability. Using CRC cell lines and patient-derived organoids (PDOs), we demonstrate that rPA-based hydrogels support long-term cell viability and growth, preserving tumour-specific morphology and gene expression profiles of primary colorectal tumours. Transcriptomic analysis revealed that ECM incorporation in rPA hydrogels modulates gene expression related to ECM remodelling, cell adhesion, and signalling pathways key in CRC progression, while maintaining lineage-specific differentiation crucial for disease modelling. In order to achieve a physiologically relevant model, rPA-based hydrogels were tested in immune-tumour co-culture models. These hydrogels maintained structural integrity, promoted CD8⁺ T-cell infiltration, and enabled the assessment of chemotherapeutic and immunotherapeutic responses. By integrating tumour-specific ECM macromolecules with architecturally defined PA nanofibres, this platform bridges the gap between simplistic 2D in vitro models and clinical 3D tumour complexity. This approach offers a complex, yet defined and reproducible platform with significant potential to not only recapitulate CRC-TME but also a wide range of different types of other TMEs.
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