Understanding cancer biology requires a clear view into the tumor microenvironment (TME)—a highly dynamic and complex biological niche shaped by cell–extracellular signaling, matrix interactions, immune infiltration, and fibroblast remodeling. These elements actively shape disease progression, therapeutic sensitivity, and immune evasion in ways that remain incompletely understood. To unravel these relationships, researchers need experimental models that reflect the complexity, heterogeneity, and spatial organization of real tumor tissues and their TME.
Traditional 2D cultures lack dimensional relevance and fail to capture key disease features. Animal-derived matrix options such as collagen, or basement membrane extracts, offer modest physiological relevance, however users are still hindered by poor reproducibility, undefined composition, and limited control over key biophysical properties. These constraints pose major challenges across all discovery research and translational efforts, including immuno-oncology and drug screening.
The challenge is not only to mimic the biochemical and mechanical properties of tumor tissues, but to do so in a way that both reflects the inherent heterogeneity of the TME and supports cells in a manner that facilitates their expression of relevant phenotypic behaviour. This includes the ability to co-culture cancer cells, fibroblasts, and immune cells within defined 3D environments that facilitate biologically meaningful interactions. Accurately modeling these dynamics is essential for building disease-relevant tumor models and generating predictive data for therapeutic development.
To meet this need, researchers are turning to 3D culture platforms that offer the tunability, reproducibility, and throughput required to study dynamic tumor microenvironments. These platforms must balance biological relevance with experimental scalability—empowering scientists to generate phenotypic data that are not only reproducible, but also predictive of in vivo responses.
The RASTRUM platform addresses these challenges directly. Built around a drop-on-demand bioprinting engine and a suite of synthetic, xeno-free matrices, the platform enables researchers to reconstruct tumor microenvironments with tuned biochemical and mechanical properties. Multiple cell types can be embedded within customizable 3D matrices in standard culture plate formats—making it possible to model immune infiltration, stromal remodeling, and multicellular invasion with high fidelity.
Whether replicating the dense fibrotic stroma of pancreatic cancer, studying matrix-driven phenotypes in renal cell carcinoma, or assessing T cell-mediated cytotoxicity in lung adenocarcinoma, RASTRUM provides researchers with the tools to ask and answer more sophisticated questions. By combining architectural precision with biological relevance, the platform is redefining what’s possible in cancer modeling and bringing us closer to in vitro systems that reflect the complexity of living tumors.
Key components of a functional tumor model
The tumor microenvironment is more than just a backdrop for cancer cells—it actively contributes to tumor growth, progression, and therapeutic resistance. Accurately modeling the TME in vitro requires attention to three interdependent components:
- Extracellular matrix (ECM): Provides structural support and both creates and transmits biochemical and biophysical signals that regulate cell behavior. In tumors, the ECM can be degraded, remodeled, and in some cases stiffened, by fibroblasts. This influences cell migration, survival, and drug response.
- Cellular players: The TME comprises a variety of stromal cell types including fibroblasts, adaptive and innate immune cells, and endothelial cells. All cell types in the TME participate in the complex signaling interplay with tumor cells. Assessing and understanding these dynamics is critical for studying immune evasion, stromal reprogramming, and therapeutic resistance.
- Spatial organization: The relative positioning of cell types within a 3D environment shapes how they interact and is therefore a critical element to successful in vitro modelling of disease. Co-culture systems that support adjacent placement of different cell populations and biological niches facilitate the modelling of immune cell infiltration, localized invasion, and matrix remodeling.
Traditional models—particularly basement membrane extract (BME)-based systems like Matrigel—are limited by batch variability, undefined composition, low tunability of stiffness, and poor imaging quality due to autofluorescence and opacity.
RASTRUM addresses these challenges with a library of synthetic, biofunctional matrices. Researchers can select from a range of potential substrate stiffness options, and incorporate specific adhesion motifs (e.g., laminin, fibronectin, various collagens) to match disease-relevant conditions and support diverse cell types. This modularity enables consistent, customizable construction of tumor models tailored to specific research questions—with guided cloud software and technical support available to help optimize matrix selection and model design for each application.
Product Guide: The Tumour Microenvironment in Phenotypic Drug Discovery
Exploring immune cell infiltration with 3D cell models
A key challenge in immuno-oncology is modeling how immune cells interact with, and are affected by, the tumor microenvironment. Dense matrices and stiff tissues in solid tumors can restrict immune cell access, while non-physiological matrices, used in vitro, can non-specifically activate immune cells, confounding interpretation of experimental readouts.
Two key immune-tumor interaction formats supported by RASTRUM are:
- Direct co-culture models: Tumor and immune cells are printed together into a 3D matrix to observe activation, persistence, and T-cell mediated cytotoxicity.
- Infiltration models: Immune cells are added to cell culture medium after tumor model formation to assess migration, infiltration, and engagement with tumor structures.
In a study using A549 lung adenocarcinoma cells, researchers introduced CD8+ T lymphocytes into wells containing 3D cultures of established tumor spheroids and observed their infiltration over four days. Imaging revealed cell concentration-dependent T cell penetration and contact with tumor cells, modulated by matrix stiffness. Higher stiffness was associated with reduced motility and engagement.
Read more in this poster: Tunable 3D cell models recapitulating the tumour microenvironment for in vitro immuno-oncology assays
These models enable detailed study of immune exclusion, checkpoint activity, and the impact of matrix conditions on immunotherapy response—supported by high-content imaging and transcriptomic analysis.
Modeling fibrotic tumor microenvironments
Fibroblasts are key regulators of the tumor microenvironment, playing a central role in ECM remodeling, immune modulation, and the formation of fibrotic barriers that can restrict drug penetration. Accurately modeling fibroblast behavior in vitro is challenging, as primary fibroblasts often undergo spontaneous activation under non-physiological conditions.
Using RASTRUM Matrices that reflect a lung-relevant stiffness and molecular composition, researchers have successfully maintained primary human lung fibroblasts (NHLFs) in a low-activation state over extended culture. Upon stimulation with TGF-β, a known pro-fibrotic cytokine, fibroblasts exhibited robust collagen I upregulation, consistent with myofibroblast activation. This phenotypic shift was clearly visualized via immunofluorescence imaging, showing dramatic differences in collagen between untreated and stimulated conditions.
These results demonstrate RASTRUM’s ability to support controlled fibroblast activation within a reproducible 3D environment—ideal for studying stromal remodeling, fibrosis-driven resistance mechanisms, and screening anti-fibrotic compounds. Additional data, including qPCR validation, fibroblast morphology, and matrix transparency comparisons with BME, are detailed in the full application note:
Immuno-responsive pancreatic cancer models
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive and treatment-resistant cancers, in large part due to its dense fibrotic stroma and immunosuppressive microenvironment. These features limit both immune cell infiltration and drug accessibility, making PDAC notoriously difficult to treat—and to model effectively in vitro.
To address this, researchers at the Garvan Institute of Medical Research used RASTRUM to develop a 3D PDAC model. The model incorporated KPC tumor cells overexpressing OVA albumin. Following tumor culture establishment, OT-I transgenic CD8+ T cells were introduced to assess immune infiltration, tumor cell engagement and T cell-mediated killing. Using high-resolution, time-lapse confocal imaging, researchers observed:
- Efficient infiltration of T cells into the stiff matrix over a 72-hour period
- Sustained contact and clustering of T cells around tumor spheroids
- Elevated cleaved caspase-3 expression in tumor cells, confirming immune-mediated apoptosis
These findings demonstrate not only the ability of T cells to engage with tumor targets in a fibrotic context, but also the importance of matrix selection in the creation of a relevant in vitro 3D model. RASTRUM- generated 3D models supported the downstream quantitative tracking of T cell behavior, including interaction frequency, depth of penetration, and cytotoxic effects, all within a reproducible 3D environment.
The model provides a powerful tool for evaluating T cell activity in solid tumors, validating immunotherapy targets, and probing the role of stromal stiffness in limiting immune efficacy—delivering insights that are difficult to obtain in traditional systems.
Read the full study here: Building better models of pancreatic cancer: development of a scalable 3D cell culture framework to evaluate novel immuno-modulatory agents
High-throughput 3D cell models for drug screening
As phenotypic screening becomes increasingly central to oncology drug discovery, there is a growing need for 3D tumor models that combine biological relevance with operational scalability. Models must not only reflect the complexity of the tumor microenvironment, but also be reproducible, automatable, and compatible with high-throughput analysis strategies.
In a recent study presented at SLAS, researchers addressed this challenge by leveraging the RASTRUM Allegro platform to develop a high-throughput PDAC screening model. The model incorporated patient-derived organoids (PDOs) co-cultured with cancer-associated fibroblasts (CAFs) in RASTRUM matrices selected to mimic key pancreatic tumour disease features.
Using RASTRUM Allegro’s the team successfully:
- Printed over 9,000 3D tumor models, in a single day, across 30 plates
- Achieved industry-leading intra- and inter-plate coefficient of variation results (CVs), demonstrating strong reproducibility
- Generated robust dose–response curves to gemcitabine and internal pipeline compounds, with performance aligning with existing PDAC data and compound benchmarks
- Reduced cell input by ~40% when moving from 96- to 384-well formats, making the system suitable for rare or precious samples such as PDOs or stem cell-derived models
The model was fully compatible with CellTiter-Glo viability assays and high-content imaging, supporting its seamless integration into screening pipelines.
This case study highlights how RASTRUM Allegro bridges the gap between model complexity and screening throughput, empowering research teams to generate rich, reproducible data at scale—without compromising on biological fidelity.
Read the full study here: Development of a reproducible, high-throughput, and screenable 3D PDAC model using the RASTRUM™ platform
Modeling tumor invasion and metastatic progression
Metastasis accounts for over 90% of cancer-related mortality, yet remains one of the most complex and poorly understood aspects of cancer biology. Tumor cell dissemination is governed not only by genetic changes within cancer cells, but also by dynamic interactions with the surrounding microenvironment—including the ECM, stromal cells, and immune components. Recreating these multifaceted processes in vitro has long posed a challenge, particularly when using traditional 2D systems or static 3D cultures.
RASTRUM enables researchers to build spatially organized, biologically relevant models of tumor invasion and metastasis using synthetic, tunable hydrogels and drop-on-demand bioprinting. The platform supports a range of model architectures that reflect key steps in metastatic progression:
- Invasion assays with cell-free zones, where tumor cells are printed adjacent to an unpopulated matrix region. Over time, invasive cells migrate into the cell-free zone, mimicking local invasion into surrounding stromal tissue or vasculature.
- Compartmentalized co-cultures, in which tumor cells are printed in proximity to fibroblasts, endothelial cells, or immune cells. These spatially defined models allow researchers to study how tumor–stroma interactions shape invasion, migration, matrix remodeling, and immune modulation.
These models enable high-resolution analysis of:
- Cell invasion dynamics, including changes in morphology, migratory behavior, and gene expression associated with increased plasticity and invasiveness
- Stromal influence, such as CAF-driven ECM remodeling or endothelial cell-mediated pathways that promote tumor escape
- Anti-metastatic drug effects, through live-cell imaging, viability assays, and transcriptomic profiling in a controlled, reproducible setting
The figure depicts a model of the tumor-stroma interface in a Triple Matrix Model made with RASTRUM. Fibroblasts (primary human lung fibroblasts) and endothelial cells (primary HUVECs) were grown next to lung adenocarcinoma cancer cells (A549) and fibroblasts (HUVECs) were cultured for 10 days. Endothelial cells were stained with CD31 (yellow) and fibroblasts and endothelial cells were stained with Phalloidin (blue).
Crucially, these models maintain compatibility with high-content imaging, in-well RNA extraction, and 96- or 384-well formats, making them suitable for both mechanistic studies and screening campaigns targeting metastatic potential.
By enabling researchers to visualize and quantify the cellular and microenvironmental drivers of metastasis, RASTRUM delivers a powerful toolset for advancing the study of cancer dissemination and evaluating therapeutic strategies aimed at halting tumor spread.
Read the Technical Note: Beyond Simple Co-Cultures: Development of complex tissue architectures in 3D
Conclusion
In vitro cancer modeling no longer requires a trade-off between biological complexity and experimental scalability. RASTRUM enables researchers to build reproducible, tunable 3D tumor microenvironments that support meaningful biological interactions and downstream analysis.
By facilitating multicellular co-culture, matrix customization, and spatial control within standard workflows, RASTRUM provides a robust platform for addressing key questions in cancer biology—from immune infiltration to stromal remodeling and metastasis.
As cancer research advances toward more functional and personalized approaches, platforms like RASTRUM will play a central role in bridging basic discovery and translational application.