Organoids are three‐dimensional self‐renewing and organizing clusters of cells that recapitulate the behavior and functionality of developed organs. Referred to as “organs in a dish,” organoids are invaluable biological models for disease modeling or drug screening. Currently, organoid culture commonly relies on an expensive and undefined tumor‐derived reconstituted basal membrane which hinders its application in high‐throughput screening, regenerative medicine, and diagnostics. Here, we introduce a novel engineered plant‐based nanocellulose hydrogel is introduced as a well‐defined and low‐cost matrix that supports organoid growth. Gels containing 0.1% nanocellulose fibers (99.9% water) are ionically crosslinked and present mechanical properties similar to the standard animal‐based matrix. The regulation of the osmotic pressure is performed by a salt‐free strategy, offering conditions for cell survival and proliferation. Cellulose nanofibers are functionalized with fibronectin‐derived adhesive sites to provide the required microenvironment for small intestinal organoid growth and budding. Comparative transcriptomic profiling reveals a good correlation with transcriptome‐wide gene expression pattern between organoids cultured in both materials, while differences are observed in stem cells‐specific marker genes. These hydrogels are tunable and can be combined with laminin‐1 and supplemented with insulin‐like growth factor (IGF‐1) to optimize the culture conditions. Nanocellulose hydrogel emerges as a promising matrix for the growth of organoids.

Pancreatic cancer tissues are made of different cell populations surrounded by a dense extracellular matrix. The stiff cancerous matrix impairs the diffusion of cytotoxic drugs, contributing to poor outcomes for patients. Matrix-targeting therapies normalise the extracellular matrix and, therefore, cell-matrix interactions. However, our knowledge of how matrix stiffness influences cancer cell and transcriptional dynamics and responses to anti-cancer compounds is incomplete. Here we developed a 3D cancer model to replicate the stiffness of patient-derived tissues and evaluated the effects of matrix-targeting compounds. Transcriptomic analyses showed that matrix stiffness regulated matrisome-related genes, cytokines and chemokines. The incusion of stromal cells further increased the mechanical properties of our 3D cancer model. Treatment with the ROCK inhibitor fasudil induced matrix softening and improved sensitivity of cancer and stromal cells to cytotoxic treatment, whereas inhibition of matrix metalloproteinases disrupted cancer cell invasion. Our results indicate that matrix stiffness impacts cancer cell profiles and targeting the cancerous matrix may lead to improved combination therapies for pancreatic cancer. STATEMENT OF SIGNIFICANCE: Pancreatic cancers have a dense extracellular matrix that drives resistance to therapy and disease progression, yet the mechanistic links between matrix stiffness and cancer cell behaviour are unclear. We engineered a 3D cancer model based on mechanical profiling of patient tissues to investigate how matrix stiffness influences gene expression and drug response. We found that increased stiffness regulates matrisome and inflammatory genes, and that stromal-induced stiffening affects sensitivity to matrix-targeting and cytotoxic drugs. The inhibition of Rho-kinase signalling enhanced the efficacy of cytotoxic drugs. These findings establish matrix stiffness as a key factor in tumour biology and therapy response, supporting the use of our preclinical model to guide new combination treatments for pancreatic cancer, aimed at improving outcomes for patients.