2021 Building Biology in 3D Hybrid Symposium

These recordings address the successes and limitations of using 3D systems in discovery and applied research, while acknowledging the need for improvements to ensure widespread adoption.

Topics include:

current and near-future enabling technologies,
applications of such systems in high-throughput screening,
advances in imaging and analysis of data generated
and the expansion into novel model systems.

Randy D. Blakely, Ph.D.

Executive Director, FAU Brain Institute, Professor of Biomedical Science, Charles E. Schmidt College of Medicine

Florida Atlantic University

Joern Dengjel, Ph.D

University of Friborg

Hongjun Song, Ph.D.

University of Pennsylvania

James Hoying, Ph.D.

Advanced Solutions Life Sciences

Daniel Todd, Ph.D.

Charles River Laboratories

Joseph Kissil, Ph.D.

Moffitt Cancer Center

Kristin Bircsaka, Ph.D.

MIMETAS

Paul Johnston, Ph.D.

University of Pittsburgh Dept. Pharmaceutical Sci.

Dr. Johnston obtained a B.Sc. with Honors (2.1) (1978) and a Ph.D. (1983) in Biochemistry, from the University of East Anglia, Norwich, England. Postdoctoral positions at the University of North Carolina, Duke University, and the University of Texas Southwestern Howard Hughes Institute provided diverse experience in biochemistry, molecular biology, cell biology, immunology, protein purification and recombinant protein expression. Dr Johnston has twenty-nine years of drug discovery experience in the Pharmaceutical, Biotechnology, and Academic sectors. He is an innovator of cell based lead generation, and founding member of the Society for Biomolecular Imaging and Informatics. In 2005, he joined the University of Pittsburgh to design and build the Pittsburgh Molecular Library Screening Center where he led 21 HTS campaigns and the NCI 60 drug combination screen. In 2011, he established independent chemical biology laboratories to discover new drugs or drug combinations for prostate cancer, melanoma, and head and neck cancers.

Paul Kenny, Ph.D.

Mt. Sinai Icahn School of Medicine

Nikolay Samusik, Ph.D.

Genentech

Sven Fengler, Ph.D.

German Center for Neurodegenerative Diseases

Andrew Aguirre, M.D., Ph.D.

Harvard Medical School

Carla Grandori, M.D., Ph.D.

SEngine Precision Medicine

James Hickman, Ph.D.

University of Central Florida NanoScience Technology Center

Pouria Rafsanjani Nejad

The University of Akron

David Tuveson, M.D., Ph.D.

Cold Spring Harbor Laboratory

Sarah Moss

Linda Boekestijn

Evan Cromwell

Emma Ãkerlund

Oksana Sirenko

Key:

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Keynote Presentations
Autism Spectrum Disorder - From Molecule(s) to Drug Candidate - A 30 Year Journey Propelled by a 60-Year-Old Biomarker
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Open to view video. Autism Spectrum Disorder (ASD) is recognized to be both much more prevalent that previously described and woefully deficient in therapeutics that can treat the core symptoms of the disorder. Our work in ASD has been a case of serendipity, surprise, and linkages, both at the genetic level and with respect to laboratory projects. In my lecture, I will describe our journey from cloning and study of the brain's major target for antidepressant medications, and an initial goal to develop new therapeutics for debilitating mood disorders, to new ideas, models and molecules potentially more relevant for ASD than depression.
3D Model Systems to Study and Fight Pancreatic Cancer
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Open to view video.
Enabling Technologies
A Patient-derived Glioblastoma Organoid Model That Recapitulates Inter- and Intra-tumoral Heterogeneity for Drug Testing
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Open to view video. Glioblastomas exhibit vast inter- and intra-tumoral heterogeneity, complicating the development of effective therapeutic strategies. Current in vitro models are limited in preserving the cellular and mutational diversity of parental tumors and require a prolonged generation time. We developed a method to generate patient-derived glioblastoma organoids (GBOs) that recapitulate the histological features, cellular diversity, gene expression, and mutational profiles of their corresponding parental tumors. GBOs can be generated quickly with high reliability, and exhibit rapid, aggressive infiltration when transplanted into adult rodent brains. We further demonstrate the utility of GBOs to test personalized therapies by correlating GBO mutational profiles with responses to specific drugs and by modeling chimeric antigen receptor T cell immunotherapy. In addition, we developed a re-aggregation assay for medium-throughput drug testing. Our studies show that GBOs maintain many key features of glioblastomas and can be rapidly deployed to investigate patient-specific treatment strategies.
A Versatile, Enabling Platform for Vascularizing Tissues and Tissue Models
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Open to view video. The ability to build or manipulate microvasculatures is critically important for both in vivo vascular regeneration and in vitro tissue fabrication. In the repair or construction of a functional microvasculature, it's important to consider that an effective microcirculation critically depends on the proper organization of stable microvessels into a perfusion-competent network. Therefore, successful neovascularizing strategies must address the construction of individual microvessel elements, and also post-angiogenesis assembly of these new elements into an organized and functionally matched vascular network. Microvascular form and function depend on a dynamic interplay between multiple vascular and perivascular cell types. It has become clear that angiogenesis, vascular remodeling, and vascular stability depend not only endothelial cells, but proper vessel architecture, mature matrix elements, and perivascular cells including smooth muscle cells, pericytes and vascular-niche stromal cells. These cells not only establish proper structure and stability, they impart an intrinsic phenotypic plasticity that enables the microvasculature to meet tissue-specific needs. Furthermore, the cellular complexity of a microvessel drives the microvessel remodeling and adaptation necessary for the evolution of an effective perfusion network. With these considerations in mind, we developed a versatile, enabling platform for vascularizing tissues and tissue models (for in vitro and in vivo applications) utilizing intact, isolated human microvessels. Harvested from discarded adipose, the microvessel isolate is a collection of intact arterioles, capillaries, and venules. Importantly, the microvessels contain all the varied cell types normally comprising the microvessels, including vascular niche cells such as MSCs. When cultured in 3D collagen gels, the microvessels spontaneously give rise to an interconnected network of immature neovessels with formed lumens (or neovasculature). Upon implantation, this neovasculature forms a mature microcirculation and spontaneously inosculates with the host circulation, perfusing of the implant. We highlighted the utility of this platform by accurately recapitulating native angiogenic sprouting and neovessel growth from isolated, intact parent microvessels that retain all intrinsic vascular and perivascular cells within a 3-D matrix environment. This system is uniquely positioned to enable the assessment of an integrated angiogenesis response. This model has been effectively used to identify and characterize angiogenic factors and inhibitors, evaluate microvascular instability, and build functional tissue models (e.g. liver, pancreas, muscle, etc.). Importantly, this versatile microvessel system is compatible with high-content analysis modalities, 3D bioprinting, and sacrificial molding approaches. Finally, in a 3D tissue model involving astrocyte precursors, important in establishing the blood brain barrier, we show that organotypic tissue environments induce phenotypic changes in the isolated microvessel-derived vasculatures to match tissue function. By isolating intact microvessels, instead of single cells, we retain the full complement of cells intrinsic to the native microcirculation. Consequently, the isolated microvessels exhibit the native-like plasticity and adaptability necessary to form effective, organotypic microvasculatures. We envision this tissue vascularizing system, based on isolates of native, human microvessels, will prove essential in fabricating tissues and tissue models for research, pharmaceutical, and autologous therapeutic applications.
Regulation of the Proteome in 3D
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Open to view video. The extracellular matrix (ECM) of human skin is highly developed and vital for proper skin functioning and homeostasis. Its 3D architecture plays a pivotal role in supporting and guiding resident as well as invading cells. We establish scaffold-based and scaffold-free 3D in vitro cell culture systems to mimic the complex design of skin observed in vivo. Especially scaffold-free systems are well suited for proteomic analyses of ECM protein regulation in 3D using mass spectrometry-based approaches. Transferring cells from 2D to 3D induces profound upregulation of matrisome proteins indicating the generation of a complex cell-ECM interface that mimics tissue architecture.
High Throughput Advanced Cellular Models
Use of 3-dimensional High Throughput Screening Approaches to Identify Oncogenic KRAS Selective Inhibitors
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Open to view video. The RAS proteins are the most frequently mutated oncogenes in cancer, with the highest frequency of mutations found in cancers of the pancreas and lung. Since activity of RAS is required for the proliferation and/or survival of these tumor cells it represents a high-value target for therapeutic development. Although direct targeting of RAS has proven challenging for multiple reasons stemming from the biology of the protein, the complexity of downstream effector pathways and upstream regulatory networks, recent progress has rekindled efforts in this area. To overcome challenges faced by previous screening efforts we implemented a number of screening approaches that include: The use of spheroid-based 3-dimensional culture formats, thought to more closely reflect conditions experienced by cells in vivo. The use of isogenic cells, differing only in the status of KRAS, thus facilitating identification of highly selective hits. The implementation of these approaches, in combination with functionalized small-molecule libraries that enable direct progression from phenotypic screening to target identification in living cells, will be discussed.
A High-throughput Liver-on-a-chip Model for Hepatotoxicity Detection
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Open to view video. Drug-induced liver injury (DILI) is the leading cause of market withdrawal in the pharmaceutical industry and poses a serious health risk to affected patients. Identification of hepatotoxic compounds in the preclinical phase of drug development is key to preventing DILI, however currently employed animal and two-dimensional (2D) in vitro models do not adequately predict human hepatotoxicity. Existing models suffer from many challenges including species differences, throughput limitations, and lot-to-lot variability of primary human hepatocytes. Here, we developed a 3D in vitro model of the human liver and predictive hepatotoxicity assays in MIMETAS’ high-throughput organ-on-a-chip platform, the OrganoPlate®. To build the model, up to 96 independent 3D perfused cultures are established on a 2lane OrganoPlate by adding induced pluripotent stem cell-derived (iPSC) hepatocytes and extracellular matrix to a microfluidic channel, alongside an endothelial and Kupffer cell-lined liver sinusoid mimic channel. Characterization of the model revealed long-term hepatocyte function including CYP3A4 activity, as well as albumin and urea production (both up to 20 µg/day/106 cells) for up to 21 days of culture. Fetal hepatocyte marker alpha-fetoprotein (AFP) dramatically declined over the 21-day culture, supporting iPSC hepatocyte maturation in the OrganoPlate co-culture. Importantly, we demonstrated the compatibility of the 3D liver model with automated liquid handling and high content imaging platforms, enabling the use of the complex co-culture in a screening capacity. Assay validation studies using troglitazone as a positive, hepatotoxic control revealed robust Z-factors ≥ 0.2 for four assay readouts. Using these assays, 159 compounds of known hepatotoxicity (50 µM, 72 h) were screened and ranked by a composite score combining the assay readouts. The ranking system aided in compound prioritization for detailed dose response and time course studies. Taken together, our iPSC-derived liver-on-a-chip model is a promising platform for hepatotoxicity screening and has the potential to improve DILI prediction in preclinical drug development.
Implementing 3D Models for Cancer Drug Discovery
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Open to view video.
Advances in Imaging and Analysis
In vivo Brain Imaging and Circuit-based Therapeutics
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Open to view video. Neuropsychiatric disorders are increasing recognized as disorders in the wiring and function of brain circuits. Indeed, abnormalities in how neurons in the brain communicate within and between different functional domains gives rise to symptoms associated with neurodegenerative neurodevelopmental and psychiatric disorders. Hence, the ability to monitor the activity of brain circuits in freely moving animals as they engage in behaviors relevant to the symptoms of neuropsychiatric disorders and determining the impact of disease associated gene mutations on circuit function, is likely to reveal important new insights into the origins of such disorders. Moreover, in vivo circuit monitoring is likely to serve as an important tool for the identification and validation of novel therapeutic agents for neuropsychiatric disorders. Here, I will present data on the use of in vivo imaging technologies to monitor brain circuit function in behaving rodents and present examples of the utility of these technologies to support new medications development in the context of substance use disorders.
Spatial Multiomics Analysis of Tumor-immune Interactions in HNSCC with CODEX
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Open to view video. Advances in multiplexed immunofluorescent in situ imaging are revealing single-cell resolution of tissue architecture and generating new insights in spatial biology. CODEX is an emerging multiplexed immunofluorescence technology that allows simultaneous imaging of 40+ proteins in fresh-frozen and FFPE tissues. We created a 54-marker CODEX panel aimed at cancer, stroma and immune cell populations in head and neck squamous cell carcinoma and applied it to a study cohort of ten primary HNSCC tumors, comprised of five node-positive (N+) and five node-negative (N0) samples. We used 3D segmentation, single-cell expression profile deconvolution and cluster analysis to identify common cell populations within the CODEX images. By analyzing spatial interactions between cell types, we found a striking and significant difference in CD4+ T-reg distributions between the N+ and N0 samples, suggesting a mechanical link between T-regs and the node-metastasis-induced systemic tumor immunotolerance. By coupling our spatial analysis results with single-cell RNA-sequencing data, we identified cytokines implicated in cell-cell crosstalk associated with lymph node metastasis, demonstrating the ability of spatial biology to drive clinical discovery.
Human iPSC-derived brain endothelial microvessels in a multi-well format enable permeability screens of anti-inflammatory drugs
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Open to view video. Optimizing drug candidates for blood-brain barrier (BBB) penetration remains one of the key challenges in drug discovery to finally target brain disorders including neurodegenerative diseases which do not have adequate treatments so far. It has been difficult to establish state-of-the-art stem cell derived in vitro models that mimic physiological barrier properties including a 3D microvasculature in a format that is scalable to screen drugs for BBB penetration in early drug development phases. To address this challenge, we established human induced pluripotent stem cell (iPSC)-derived brain endothelial microvessels in a standardized and scalable multi-well plate format. iPSC-derived brain microvascular endothelial cells (BMECs) were supplemented with primary cell conditioned media and grew to intact microvessels in 10 days of culturing. Produced microvessels show a typical BBB phenotype including endothelial protein expression, tight-junctions and polarized localization of efflux transporter. Microvessels exhibited physiological relevant trans-endothelial electrical resistance (TEER), were leak-tight for 10 kDa dextran-Alexa 647 and strongly limited the permeability of sodium fluorescein (NaF). Permeability tests with reference compounds confirmed the suitability of our model as platform to identify potential BBB penetrating anti-inflammatory drugs. In summary, the here presented brain microvessel platform recapitulates physiological properties and allows rapid screening of BBB permeable anti-inflammatory compounds that has been suggested as promising substances to cure so far untreatable neurodegenerative diseases.
Complex Transitional Models
Drug Efficacy and Safety Determination in Drug-dosed Human-on-a-chip Systems
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Open to view video. The current drug development process is inefficient and costly, especially for pain therapeutics, taking years from compound identification to marketable drug, with costs up to 2.5 billion dollars per drug compared to less than half that the previous decade. One reason for the lack of novel analgesics is that preclinical animal models of pain do not simulate multidimensional clinical pain conditions leading to poor performance in human clinical trials of efficacy and safety. Consequently, human-based in vitro systems capable of measuring “organ” physiology, biomarker generation, and the interactions between organs would provide an improved platform for analgesic drug testing for efficacy and off-target toxicity. Optimally configured human-on-a-chip (HoaC) systems integrate micro-electro-mechanical systems (bio-MEMS) devices in a housing with recirculating medium supporting long-term survivability. Scaled accurately, these systems can capture and predict organ physiology, organ-organ interactions, and PKPD in response to analgesic dosing. Taken together, these data can generate predictive models regarding pain drug efficacy and safety for clinical testing, reducing the cost of drug development. Hesperos has constructed stem cell-based, human-on-a-chip systems demonstrating long-term physiology (>28 days) in configurations of up to five organs [1]. Acute and chronic compound testing in systems has generated drug efficacy and safety responses similar to those seen in clinical data or reports from literature [2]. Here we describe HoaC systems composed of liver, cardiomyocytes, skeletal muscle myotubes, motoneurons and a functional, rudimentary kidney module in configurations relevant for parent compound and metabolite efficacy and safety testing. The organ modules functioned for 28 days in a recirculating serum-free medium providing efficacy and safety data generated by noninvasive measurements in acute-dose, chronic-dose and control systems [3]. In dosed systems, we have observed clinically-relevant responses to over 20 compounds in all modules indicating the predictive capabilities of this system for drug testing. This HoaC model exhibits a multi-organ toxicity response to chronic drug dosing which can be integrated with PKPD models and represents a step towards an in vitro platform for analgesic efficacy and safety testing. References 1.Oleaga C. et al. Multi-Organ toxicity demonstration in a functional human in vitro system composed of four organs. 2016 Nature Scientific Reports 2.Oleaga C. et al. Investigation of the effect of hepatic metabolism on off-target cardiotoxicity in a multi-organ human-on-a-chip system. 2018. Biomaterials 3.Oleaga C. et al. Long‐Term Electrical and Mechanical Function Monitoring of a Human‐on‐a‐Chip System. 2018. Advanced Functional Materials
Targeting of Tumor-stromal Interactions in an Organotypic Breast Tumor Model
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Open to view video. The tumor microenvironment (TME) promotes proliferation, drug resistance, and invasiveness of cancer cells and metastatic disease progression. Therapeutic targeting of the TME is an attractive strategy to improve outcomes for patients, particularly in aggressive cancers such as triple negative breast cancer (TNBC) that have a rich stroma and currently limited targeted therapies. However, lack of preclinical human tumor models that enable mechanistic understanding of tumor-stromal interactions has been an impediment to identify effective treatments against the TME. To address this need, we developed a three-dimensional (3D) organotypic tumor model to study interactions of patient-derived cancer-associated fibroblasts (CAFs) with TNBC cells and explore potential therapy targets. Our mechanistic studies showed that CAFs predominantly secreted hepatocyte growth factor (HGF) and activated MET receptor tyrosine kinase in TNBC cells. This tumor-stromal interaction promoted proliferation, invasiveness, and epithelial-to-mesenchymal transition (EMT) of TNBC cells and activated multiple oncogenic pathways in TNBC cells. Importantly, we established that TNBC cells become resistant to single-agent targeted inhibition and demonstrated a design-driven approach to select drug combinations that effectively inhibit several pro-metastatic functions of TNBC cells. Our study showed that HGF-MET is a critical signaling axis that also promotes colony formation by TNBC cells in lung stroma, suggesting that blocking HGF-MET signaling potentially could target both primary TNBC tumorigenesis and lung metastasis. Overall, this work established the utility of our 3D organotypic tumor model to identify and therapeutically target specific mechanisms of tumor-stromal interactions in TNBC toward developing targeted therapies against the TME.
Flash Talks on Disruptive Technologies
Automated manufacture of functional, vascularized human liver tissue
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Open to view video.
The development of a high throughput 3D in vitro wound healing model
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Open to view video.
Disease modeling of patient-derived tumoroids using multifunctional profiling with an automated microfluidic-based assay platform
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Open to view video.
Next Generation Drug Testing in Ovarian Cancer Patient-derived 3D Cultures
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Open to view video.
Automation and high content imaging of 3D triple-negative breast cancer patient-derived tumoroids assay for compound screening
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Open to view video.