Bridging systems biology and tissue engineering: Unleashing the full potential of complex 3D in vitro tissue models of disease.

Rapid advances in tissue engineering have resulted in more complex and physiologically relevant 3D in vitro tissue models with applications in fundamental biology and therapeutic development. However, the complexity provided by these models is often not leveraged fully due to the reductionist methods used to analyze them. Computational and mathematical models developed in the field of systems biology can address this issue. Yet, traditional systems biology has been mostly applied to simpler in vitro models with little physiological relevance and limited cellular complexity. Therefore, integrating these two inherently interdisciplinary fields can result in new insights and move both disciplines forward. In this review, we provide a systematic overview of how systems biology has been integrated with 3D in vitro tissue models and discuss key application areas where the synergies between both fields have led to important advances with potential translational impact. We then outline key directions for future research and discuss a framework for further integration between fields.

An Automation Workflow for High-Throughput Manufacturing and Analysis of Scaffold-Supported 3D Tissue Arrays.

Patient-derived organoids have emerged as a useful tool to model tumour heterogeneity. Scaling these complex culture models while enabling stratified analysis of different cellular sub-populations, however, remains a challenge. One strategy to enable higher throughput organoid cultures is the scaffold-supported platform for organoid-based tissues (SPOT). SPOT allows the generation of flat, thin, and dimensionally-defined microtissues in both 96- and 384-well plate footprints that are compatible with longitudinal image-based readouts. SPOT is currently manufactured manually, however, limiting scalability. In this study, an automation approach to engineer tumour-mimetic 3D microtissues in SPOT using a liquid handler is optimized and comparable within- and between-sample variation to standard manual manufacturing is shown. Further, a liquid handler-supported cell extraction protocol to support single-cell-based end-point analysis using high-throughput flow cytometry and multiplexed cytometry by time of flight is developed. As a proof-of-value demonstration, 3D complex tissues containing different proportions of tumour and stromal cells are generated to probe the reciprocal impact of co-culture. It is also demonstrated that primary patient-derived organoids can be incorporated into the pipeline to capture patient-level tumour heterogeneity. It is envisioned that this automated 96/384-SPOT workflow will provide opportunities for future applications in high-throughput screening for novel personalized therapeutic targets.

An Engineered Paper-Based 3D Coculture Model of Pancreatic Cancer to Study the Impact of Tissue Architecture and Microenvironmental Gradients on Cell Phenotype.

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.

An off-the-shelf multi-well scaffold-supported platform for tumour organoid-based tissues.

Complex 3D bioengineered tumour models provide the opportunity to better capture the heterogeneity of patient tumours. Patient-derived organoids are emerging as a useful tool to study tumour heterogeneity and variation in patient responses. Organoid cultures typically require a 3D microenvironment that can be manufactured easily to facilitate screening. Here we set out to create a high-throughput, "off-the-shelf" platform which permits the generation of organoid-containing engineered microtissues for standard phenotypic bioassays and image-based readings. To achieve this, we developed the Scaffold-supported Platform for Organoid-based Tissues (SPOT) platform. SPOT is a 3D gel-embedded in vitro platform that can be produced in a 96- or 384-well plate format and enables the generation of flat, thin, and dimensionally-defined microgels. SPOT has high potential for adoption due to its reproducible manufacturing methodology, compatibility with existing instrumentation, and reduced within-sample and between-sample variation, which can pose challenges to both data analysis and interpretation. Using SPOT, we generate cultures from patient derived pancreatic ductal adenocarcinoma organoids and assess the cellular response to standard-of-care chemotherapeutic compounds, demonstrating our platform's usability for drug screening. We envision 96/384-SPOT will provide a useful tool to assess drug sensitivity of patient-derived organoids and easily integrate into the drug discovery pipeline.

REVOLVER: A low-cost automated protein purifier based on parallel preparative gravity column workflows.

Protein purification is a ubiquitous procedure in biochemistry and the life sciences, and represents a key step in the protein production pipeline. The need for scalable and parallel protein purification systems is driven by the demands for increasing the throughput of recombinant protein characterization. Therefore, automating the process to simultaneously handle multiple samples with minimal human intervention is highly desirable, yet there are only a handful of such systems that have been developed, all of which are closed source and expensive. To address this challenge, we present REVOLVER, a 3D-printed programmable protein purification system based on gravity-column workflows and controlled by Arduino boards that can be built for under $130 USD. REVOLVER takes a cell lysate sample and completes a full protein purification process with almost no human intervention and yields results indistinguishable from those obtained by an experienced biochemist when purifying a real-world protein sample. We further present and describe MULTI-VOLVER, a scalable version of the REVOLVER that allows for parallel purification of up to six samples and can be built for under $250 USD. Both systems can help accelerate protein purification and ultimately link them to bio-foundries for protein characterization and engineering.

3D microgels to quantify tumor cell properties and therapy response dynamics.

Tumors contain heterogeneous and dynamic populations of cells that do not all display the fast-proliferating properties that traditional chemotherapies target. There is a need therefore, to develop novel treatment strategies that target diverse tumor cell properties. Identifying therapy combinations is challenging however. Current approaches have relied on cell lines cultured in monolayers with treatment response being assessed using endpoint metabolic assays, which although enable large-scale throughput, do not capture tumor heterogeneity. Here, a 3D in vitro tumor model using micro-molded hydrogels (microgels), the Gels for Live Analysis of Compartmentalized Environments (GLAnCE) platform, is adapted into a 96-well plate format (96-GLAnCE) that integrates patient-derived organoids (PDOs) and is combined with longitudinal automated imaging to address these limitations. Using 96-GLAnCE, two measures of tumor aggressiveness are quantified, tumor cell growth and in situ regrowth after drug treatment, in both cell lines and PDOs. The use of longitudinal image-based readouts enables the identification of tumor cell phenotypes with cell population and subpopulation resolution that cannot be detected by standard bulk-soluble assays. 96-GLAnCE is a versatile and robust platform that combines 3D-ECM based models, PDOs, and real-time assay readouts, to provide an additional tool for pre-clinical anti-cancer drug discovery for the identification of novel targets with translatable clinical significance.

A TRACER culture invasion assay to probe the impact of cancer associated fibroblasts on head and neck squamous cell carcinoma cell invasiveness.

Cancer associated fibroblasts (CAFs) are a major cellular component of the tumour stroma and have been shown to promote tumour cell invasion and disease progression. CAF-cancer cell interactions are bi-directional and occur via both soluble factor dependent and extracellular matrix (ECM) remodelling mechanisms, which are incompletely understood. Previously we developed the Tissue Roll for Analysis of Cellular Environment and Response (TRACER), a novel stacked paper tumour model in which cells embedded in a hydrogel are infiltrated into a porous cellulose scaffold that is then rolled around an aluminum core to generate a multi-layered 3D tissue. Here, we use the TRACER platform to explore the impact of CAFs derived from three different patients on the invasion of two head and neck squamous cell carcinoma (HNSCC) cell lines (CAL33 and FaDu). We find that co-culture with CAFs enhances HNSCC tumour cell invasion into an acellular collagen layer in TRACER and this enhanced migration occurs independently of proliferation. We show that CAF-enhanced invasion of CAL33 cells is driven by a soluble factor independent mechanism, likely involving CAF mediated ECM remodelling via matrix metalloprotenases (MMPs). Furthermore, we find that CAF-enhanced tumour cell invasion is dependent on the spatial pattern of collagen density within the culture. Our results highlight the utility of the co-culture TRACER platform to explore soluble factor independent interactions between CAFs and tumour cells that drive increased tumour cell invasion.

The life cycle of cancer-associated fibroblasts within the tumour stroma and its importance in disease outcome.

The tumour microenvironment (TME) determines vital aspects of tumour development, such as tumour growth, metastases and response to therapy. Cancer-associated fibroblasts (CAFs) are abundant and extremely influential in this process and interact with cellular and matrix TME constituents such as endothelial and immune cells and collagens, fibronectin and elastin, respectively. However, CAFs are also the recipients of signals-both chemical and physical-that are generated by the TME, and their phenotype effectively evolves alongside the tumour mass during tumour progression. Amid a rising clinical interest in CAFs as a crucial force for disease progression, this review aims to contextualise the CAF phenotype using the chronological framework of the CAF life cycle within the evolving tumour stroma, ranging from quiescent fibroblasts to highly proliferative and secretory CAFs. The emergence, properties and clinical implications of CAF activation are discussed, as well as research strategies used to characterise CAFs and current clinical efforts to alter CAF function as a therapeutic strategy.

Gels for Live Analysis of Compartmentalized Environments (GLAnCE): A tissue model to probe tumour phenotypes at tumour-stroma interfaces.

The interface between a tumour and the adjacent stroma is a site of great importance for tumour development. At this site, carcinoma cells are highly proliferative, undergo invasive phenotypic changes, and directly interact with surrounding stromal cells, such as cancer-associated fibroblasts (CAFs) which further exert pro-tumorigenic effects. Here we describe the development of GLAnCE (Gels for Live Analysis of Compartmentalized Environments), an easy-to-use hydrogel-culture platform for investigating CAF-tumour cell interaction dynamics in vitro at a tumour-stroma interface. GLAnCE enables observation of CAF-mediated enhancement of both tumour cell proliferation and invasion at the tumour-stroma interface in real time, as well as stratification between phenotypes at the interface versus in the bulk tumour tissue compartment. We found that CAF presence resulted in the establishment of an invasion-permissive, interface-specific matrix environment, that leads to carcinoma cell movement outwards from the tumour edge and tumour cell invasion. Furthermore, the spatial stratification capability of GLAnCE was leveraged to discern differences between tumour cell epithelial-to-mesenchymal (EMT) transition genes induced by paracrine signalling from CAFs versus genes induced by interface-specific, CAF-mediated microenvironment. GLAnCE combines high usability and tissue complexity, to provide a powerful in vitro platform to probe mechanisms of tumour cell movement specific to the microenvironment at the tumour-stroma interface.