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Patient-derived organoids (PDOs) are emerging as powerful models to capture the genetic heterogeneity of human tumors. However, the self-assembling nature of PDOs limits their use in studies of the impact of microenvironmental heterogeneity on tumor cell function. Here, a paper-based model, the Tissue Roll for Analysis of Cellular Environment and Response (TRACER) is adapted, using patterned polymer infiltration, to enable controlled assembly and disassembly of organoid structures to study the impact of both genetic and microenvironmental heterogeneity on tumor cell behavior. In the adapted platform (TRACER2), pancreatic cancer PDOs establish oxygen gradients across the tissue and in response exhibit graded cell viability, proliferation, hypoxiaresponse gene transcription, and response to gemcitabine therapy. Further, PDOs retrieved from the hypoxic regions of the TRACER2 cultures show graded transcriptional changes in immunosuppression-related genes and upon co-culture, after TRACER2 disassembly, induce graded functional changes in Jurkat cells and macrophage cells. Therefore, TRACER2 offers a novel platform to dissect the effects of microenvironmental parameters on tumor cell function.

The spatial configuration of cells in the tumor microenvironment (TME) affects both cancer and fibroblast cell phenotypes contributing to the clinical challenge of tumor heterogeneity and therapeutic resistance. This is a particular challenge in stroma‐rich pancreatic ductal adenocarcinoma (PDAC). Here, a versatile system is described to study the impact of tissue architecture on cell phenotype using PDAC as a model system. This fully human system encompassing both primary pancreatic stellate cells and primary organoid cells using the TRACER platform to allow the creation of user‐defined TME architectures that have been inferred from clinical PDAC samples and are analyzed by CyTOF to characterize cells extracted from the system. High dimensional characterization using CyTOF demonstrates that tissue architecture leads to distinct hypoxia and proliferation gradients. Furthermore, phenotypic markers for both cell types are also graded in ways that cannot be explained by either hypoxia or coculture alone. This demonstrates the importance of using complex models encompassing cancer cells, stromal cells, and allowing control over architecture to explore the impact of tissue architecture on cell phenotype. It is anticipated that this model will help decipher how tissue architecture and cell interactions regulate cell phenotype and hence cellular and tissue heterogeneity.

Hydrogel models of metastasis traditionally focus on the invasion of cancer cells; however, other cells in the tumor microenvironment that are associated with metastasis also have the ability to migrate. Macrophage phenotype plays a key role in the tumor microenvironment, yet understanding their migration within tunable 3D in vitro models has been limited. To gain a greater understanding of macrophage invasive behavior, stable and transparent oxime‐crosslinked cryogels comprised of click‐crosslinked gelatin‐oxyamine and hyaluronan‐aldehyde (GELox‐HAa) are synthesized. Fibronectin‐derived, oxyamine‐modified PHSRN‐RGDSP peptides are incorporated to further mimic the tumor extracellular matrix without impacting cryogel mechanics. It is found that primary human macrophages migrate to a greater depth in cryogels with greater porosity and pore size. To better understand the mechanism of migration, cells are treated with either inhibitors of matrix metalloproteinases (MMPs) or rho‐associated protein kinase (ROCK) and a predominantly MMP‐mediated mechanism of invasion is found. Macrophage polarization studies reveal that anti‐inflammatory, interleukin‐4/13 (IL4/IL13)‐treated macrophages migrate through cryogels to a greater extent than pro‐inflammatory, interferon‐gamma/lipopolysaccharide (IFNγ/LPS)‐treated cells. Interestingly, polarized macrophages move through cryogels using a combination of amoeboid and mesenchymal migration. These findings of macrophage invasion in this cryogel platform set the stage for their further study in a biomimetic tumor microenvironment.

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