Pancreatic ductal adenocarcinoma (PDAC) tumors exhibit pronounced phenotypic plasticity, alternating between a treatment-sensitive classical phenotype and a more aggressive basal-like state associated with drug resistance and poor prognosis. The frequent coexistence of these phenotypes complicates patient stratification and the selection of effective therapies. Tumor-derived organoids are valuable tools for drug screening; however, their clinical relevance relies on how accurately they recapitulate the phenotypic and functional characteristics of the original tumors. In this study, we present a quantitative analysis of how hydrogel composition influences the phenotype, tissue remodeling, metabolism, and drug resistance of PDAC organoids. Organoids were cultured within three types of hydrogels: Matrigel, collagen-I, and a mixture of collagen-I and Matrigel. Our results demonstrate that: (i) PDAC organoids grown in Matrigel exhibit a classical phenotype, with metabolic and drug response profiles similar to those of low-physiological two-dimensional cultures; (ii) Organoids grown in collagen-containing hydrogels, particularly those in collagen-Matrigel composites, faithfully recapitulate basal-like tumors, characterized by epithelial-to-mesenchymal transition, tissue remodeling, metabolic activity, and drug resistance; (iii) TGFβ induces an exacerbated, highly invasive basal-like phenotype. Summarizing, our findings highlight the importance of 3D hydrogel composition in modulating PDAC organoid phenotype and behavior and suggest collagen-Matrigel hydrogels as the most suitable matrix for modeling PDAC biology.

Intravital microscopy (IVM) using dorsal skinfold chambers (DSCs) enables real‐time, high‐resolution imaging of the tumor microenvironment. Conventional, metal‐based DSC systems often cause animal distress and require technically demanding surgical implantation. To address these challenges and adhere to the 3Rs principles of animal welfare, this study presents a novel, stereolithography (SLA)‐based, 3D‐printed DSC featuring a lightweight (0.69 g) biocompatible resin structure and a suture‐free “one‐click” fixation system. This affordable device simplifies surgical installation and minimizes postoperative inflammation, allowing continuous multimodal imaging—IVM, micro‐CT, ultrasound, and in vivo fluorescence—for up to four weeks. The system is validated in mice bearing subcutaneous tumors generated from PDAC93‐GFP pancreatic tumor organoids implanted in the DSC. Longitudinal, high‐resolution imaging successfully tracks tumor expansion, active stromal remodeling characterized by progressive collagen fiber compaction and alignment, and the emergence of a highly tortuous peritumoral vascular network. Furthermore, in vivo tracking in Catchup mice reveals a distinct transition of tumor‐associated neutrophils from random acute inflammatory migration to directed tumor‐driven chemotaxis. Overall, this ergonomic and robust DSC design provides a highly reliable platform for sustained, high‐quality assessment of tumor‐stroma‐immune interactions and therapeutic responses, while notably improving animal welfare.