Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.

Fibrosis is an underlying cause of cirrhosis and hepatic failure resulting in end stage liver disease with limited pharmacological options. The beneficial effects of relaxin peptide treatment were demonstrated in clinically relevant animal models of liver fibrosis. However, the use of relaxin is problematic because of a short half-life. The aim of this study was to test the therapeutic effects of recently identified small molecule agonists of the human relaxin receptor, relaxin family peptide receptor 1 (RXFP1). The lead compound of this series, ML290, was selected based on its effects on the expression of fibrosis-related genes in primary human stellate cells. RNA sequencing analysis of TGF-ß1-activated LX-2 cells showed that ML290 treatment primarily affected extracellular matrix remodeling and cytokine signaling, with expression profiles indicating an antifibrotic effect of ML290. ML290 treatment in human liver organoids with LPS-induced fibrotic phenotype resulted in a significant reduction of type I collagen. The pharmacokinetics of ML290 in mice demonstrated its high stability in vivo, as evidenced by the sustained concentrations of compound in the liver. In mice expressing human RXFP1 gene treated with carbon tetrachloride, ML290 significantly reduced collagen content, α-smooth muscle actin expression, and cell proliferation around portal ducts. In conclusion, ML290 demonstrated antifibrotic effects in liver fibrosis.-Kaftanovskaya, E. M., Ng, H. H., Soula, M., Rivas, B., Myhr, C., Ho, B. A., Cervantes, B. A., Shupe, T. D., Devarasetty, M., Hu, X., Xu, X., Patnaik, S., Wilson, K. J., Barnaeva, E., Ferrer, M., Southall, N. T., Marugan, J. J., Bishop, C. E., Agoulnik, I. U., Agoulnik, A. I. Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.

Deconstructed Microfluidic Bone Marrow On-A-Chip to Study Normal and Malignant Hemopoietic Cell-Niche Interactions.

Human hematopoietic niches are complex specialized microenvironments that maintain and regulate hematopoietic stem and progenitor cells (HSPC). Thus far, most of the studies performed investigating alterations of HSPC-niche dynamic interactions are conducted in animal models. Herein, organ microengineering with microfluidics is combined to develop a human bone marrow (BM)-on-a-chip with an integrated recirculating perfusion system that consolidates a variety of important parameters such as 3D architecture, cell-cell/cell-matrix interactions, and circulation, allowing a better mimicry of in vivo conditions. The complex BM environment is deconvoluted to 4 major distinct, but integrated, tissue-engineered 3D niche constructs housed within a single, closed, recirculating microfluidic device system, and equipped with cell tracking technology. It is shown that this technology successfully enables the identification and quantification of preferential interactions-homing and retention-of circulating normal and malignant HSPC with distinct niches.

A reductionist metastasis-on-a-chip platform for in vitro tumor progression modeling and drug screening.

Current animal and 2-D cell culture models employed in metastasis research and drug discovery remain poor mimics of human cancer physiology. Here we describe a "metastasis-on-a-chip" system allowing real time tracking of fluorescent colon cancer cells migrating from hydrogel-fabricated gut constructs to downstream liver constructs within a circulatory fluidic device system that responds to environmental manipulation and drug treatment. Devices consist of two chambers in which gut and liver constructs are housed independently, but are connected in series via circulating fluid flow. Constructs were biofabricated with a hyaluronic acid-based hydrogel system, capable of a variety of customizations, inside of which representative host tissue cells were suspended and metastatic colon carcinoma tumor foci were created. The host tissue of the constructs expressed normal epithelial markers, which the tumor foci failed to express. Instead, tumor regions lost membrane-bound adhesion markers, and expressed mesenchymal and proliferative markers, suggesting a metastatic phenotype. Metastatic tumor foci grew in size, eventually disseminating from the intestine construct and entering circulation, subsequently reaching in the liver construct, thus mimicking some of the migratory events observed during metastasis. Lastly, we demonstrated the ability to manipulate the system, including chemically modulating the hydrogel system mechanical properties and administering chemotherapeutic agents, and evaluated the effects of these parameters on invasive tumor migration. These results describe the capability of this early stage metastasis-on-a-chip system to model several important characteristics of human metastasis, thereby demonstrating the potential of the platform for making meaningful advances in cancer investigation and drug discovery. Biotechnol. Bioeng. 2016;113: 2020-2032. © 2016 Wiley Periodicals, Inc.

Cell surface assembly of HIV gp41 six-helix bundles for facile, quantitative measurements of hetero-oligomeric interactions.

Helix-helix interactions are fundamental to many biological signals and systems and are found in homo- or heteromultimerization of signaling molecules as well as in the process of virus entry into the host. In HIV, virus-host membrane fusion during infection is mediated by the formation of six-helix bundles (6HBs) from homotrimers of gp41, from which a number of synthetic peptides have been derived as antagonists of virus entry. Using a yeast surface two-hybrid (YS2H) system, a platform designed to detect protein-protein interactions occurring through a secretory pathway, we reconstituted 6HB complexes on the yeast surface, quantitatively measured the equilibrium and kinetic constants of soluble 6HB, and delineated the residues influencing homo-oligomeric and hetero-oligomeric coiled-coil interactions. Hence, we present YS2H as a platform for the facile characterization and design of antagonistic peptides for inhibition of HIV and many other enveloped viruses relying on membrane fusion for infection, as well as cellular signaling events triggered by hetero-oligomeric coiled coils.

T Cells Promote Metastasis by Regulating Extracellular Matrix Remodeling following Chemotherapy.

Metastasis is the main cause of cancer-related mortality. Despite intense efforts to understand the mechanisms underlying the metastatic process, treatment of metastatic cancer is still challenging. Here we describe a chemotherapy-induced, host-mediated mechanism that promotes remodeling of the extracellular matrix (ECM), ultimately facilitating cancer cell seeding and metastasis. Paclitaxel (PTX) chemotherapy enhanced rapid ECM remodeling and mechanostructural changes in the lungs of tumor-free mice, and the protein expression and activity of the ECM remodeling enzyme lysyl oxidase (LOX) increased in response to PTX. A chimeric mouse model harboring genetic LOX depletion revealed chemotherapy-induced ECM remodeling was mediated by CD8+ T cells expressing LOX. Consistently, adoptive transfer of CD8+ T cells, but not CD4+ T cells or B cells, from PTX-treated mice to naïve immunodeprived mice induced pulmonary ECM remodeling. Lastly, in a clinically relevant metastatic breast carcinoma model, LOX inhibition counteracted the metastasis-promoting, ECM-related effects of PTX. This study highlights the role of immune cells in regulating ECM and metastasis following chemotherapy, suggesting that inhibiting chemotherapy-induced ECM remodeling represents a potential therapeutic strategy for metastatic cancer. SIGNIFICANCE: Chemotherapy induces prometastatic pulmonary ECM remodeling by upregulating LOX in T cells, which can be targeted with LOX inhibitors to suppress metastasis.See related commentary by Kolonin and Woodward, p. 197.

Biofabricated 3D in vitro model of fibrosis-induced abnormal hepatoblast/biliary progenitors' expansion of the developing liver.

Congenital disorders of the biliary tract are the primary reason for pediatric liver failure and ultimately for pediatric liver transplant needs. Not all causes of these disorders are well understood, but it is known that liver fibrosis occurs in many of those afflicted. The goal of this study is to develop a simple yet robust model that recapitulates physico-mechanical and cellular aspects of fibrosis mediated via hepatic stellate cells (HSCs) and their effects on biliary progenitor cells. Liver organoids were fabricated by embedding various HSCs, with distinctive abilities to generate mild to severe fibrotic environments, together with undifferentiated liver progenitor cell line, HepaRG, within a collagen I hydrogel. The fibrotic state of each organoid was characterized by examination of extracellular matrix (ECM) remodeling through quantitative image analysis, rheometry, and qPCR. In tandem, the phenotype of the liver progenitor cell and cluster formation was assessed through histology. Activated HSCs (aHSCs) created a more severe fibrotic state, exemplified by a more highly contracted and rigid ECM, as well higher relative expression of TGF-ß, TIMP-1, LOXL2, and COL1A2 as compared to immortalized HSCs (LX-2). Within the more severe fibrotic environment, generated by the aHSCs, higher Notch signaling was associated with an expansion of CK19+ cells as well as the formation of larger, more densely populated cell biliary like-clusters as compared to mild and non-fibrotic controls. The expansion of CK19+ cells, coupled with a severely fibrotic environment, are phenomena found within patients suffering from a variety of congenital liver disorders of the biliary tract. Thus, the model presented here can be utilized as a novel in vitro testing platform to test drugs and identify new targets that could benefit pediatric patients that suffer from the biliary dysgenesis associated with a multitude of congenital liver diseases.

Cell Viability Assays in Three-Dimensional Hydrogels: A Comparative Study of Accuracy.

Three-dimensional (3D) cell culture systems, such as tumor organoids and multicellular tumor spheroids, have been developed in part as a result of major advances in tissue engineering and biofabrication techniques. 3D cell culture offers great capabilities in drug development, screening, testing, and precision medicine owing to its physiological accuracy. However, since the inception of 3D systems, few methods have been reported to successfully analyze cell viability quantitatively within hydrogel constructs. In this study, we describe and compare commercially available viability assays developed for two-dimensional (2D) applications for use in 3D constructs composed of organic, synthetic, or hybrid hydrogel formulations. We utilized Promega's CellTiter-Glo®, CellTiter-Glo 3D, and CellTiter 96® MTS Assay along with Thermo Fisher's PrestoBlue™ assay to determine if these assays can be used accurately in 3D systems. Compared with direct cell viability commonly used in 2D cell culture, our results show cellular health output inaccuracies among each assay in differing hydrogel formulations. Our results should inform researchers of potential errors when using cell viability measurements in 3D cultures and conclude that microscopic imaging should be used, in combination, for validation. Impact statement Three-dimensional (3D) tissue organoids models are a valuable tool not only for studying drug toxicity but also for understanding human embryonic development, intra-tissue morphogenesis, and mechanisms of disease. In cancer organoids, such 3D models may be used for preclinical chemotherapy screening and for understanding cell death and viability mechanisms under physiologically relevant conditions. Cell viability assays are necessary for assessing the effect of biological reagents on cellular health and have been used on in vitro cell cultures for many years. With the increase of 3D systems in cellular biology research to determine therapeutic efficacy, two-dimensional assays that measure cell viability are being used outside their intended use on 3D constructs. In this study, we assess the accuracy of using various commercially available cell viability assays on different 3D hydrogel constructs to help researchers understand expected variability in their experimentation along microscopic imaging validation.

Manipulating the Tumor Microenvironment in Tumor Organoids Induces Phenotypic Changes and Chemoresistance.

Tumors comprised a tightly surrounded tumor microenvironment, made up of non-cellular extracellular matrix (ECM) and stromal cells. Although treatment response is often attributed to tumor heterogeneity, progression and malignancy are profoundly influenced by tumor cell interactions with the surrounding ECM. Here, we used a tumor organoid model, consisting of hepatic stellate cells (HSCs) embedded in collagen type 1 (Col1) and colorectal cancer cell (HCT-116) spheroids, to determine the relationship between the ECM architecture, cancer cell malignancy, and chemoresistance. Exogenous transforming growth factor beta (TGF-ß) used to activate the HSCs increased the remodeling and bundling of Col1 in the ECM around the cancer spheroid. A dense ECM architecture inhibited tumor cell growth, reversed their mesenchymal phenotype, preserved stem cell population, and reduced chemotherapy response. Overall, our results demonstrate that controlled biofabrication and manipulation of the ECM in tumor organoids results enables studying tumor cell-ECM interactions and better understand tumor cell response to chemotherapies.

In Vitro Modeling of the Tumor Microenvironment in Tumor Organoids.

BACKGROUND: The tumor microenvironment (TME) represents the many components occupying the space within and surrounding a tumor, including cells, signaling factors, extracellular matrix, and vasculature. Each component has the potential to assume many forms and functions which in turn contribute to the overall state of the TME, and further contribute to the progression and disposition of the tumor itself. The sum of these components can drive a tumor towards progression, keep a migratory tumor at bay, or even control chemotherapeutic response. The wide potential for interaction that the TME is an integral part of a tumor's ecosystem, and it is imperative to include it when studying and modeling cancer in vitro. Fortunately, the development of tissue engineering and biofabrication technologies and methodologies have allowed widespread inclusion of TME-based factors into in vitro tissue-equivalent models. METHODS: In this review, we compiled contemporary literature sources to provide an overview of the field of TME models, ranging from simple to complex. RESULTS: We have identified important components of the TME, how they can be included in in vitro study, and cover examples across a range of cancer types. CONCLUSION: Our goal with this text is to provide a foundation for prospective research into the TME.

Simulating the human colorectal cancer microenvironment in 3D tumor-stroma co-cultures in vitro and in vivo.

The tumor microenvironment (TME) plays a significant role in cancer progression and thus modeling it will advance our understanding of cancer growth dynamics and response to therapies. Most in vitro models are not exposed to intact body physiology, and at the same time, fail to recapitulate the extensive features of the tumor stroma. Conversely, animal models do not accurately capture the human tumor architecture. We address these deficiencies with biofabricated colorectal cancer (CRC) tissue equivalents, which are built to replicate architectural features of biopsied CRC tissue. Our data shows that tumor-stroma co-cultures consisting of aligned extracellular matrix (ECM) fibers and ordered micro-architecture induced an epithelial phenotype in CRC cells while disordered ECM drove a mesenchymal phenotype, similar to well and poorly differentiated tumors, respectively. Importantly, co-cultures studied in vitro, and upon implantation in mice, revealed similar tumor growth dynamics and retention of architectural features for 28 days. Altogether, these results are the first demonstration of replicating human tumor ECM architecture in ex vivo and in vivo cultures.

Multi-Cell Type Glioblastoma Tumor Spheroids for Evaluating Sub-Population-Specific Drug Response.

Glioblastoma (GBM) is a lethal, incurable form of cancer in the brain. Even with maximally aggressive surgery and chemoradiotherapy, median patient survival is 14.5 months. These tumors infiltrate normal brain tissue, are surgically incurable, and universally recur. GBMs are characterized by genetic, epigenetic, and microenvironmental heterogeneity, and they evolve spontaneously over time and as a result of treatment. However, tracking such heterogeneity in real time in response to drug treatments has been impossible. Here we describe the development of an in vitro GBM tumor organoid model that is comprised of five distinct cellular subpopulations (4 GBM cell lines that represent GBM subpopulations and 1 astrocyte line), each fluorescently labeled with a different color. These multi-cell type GBM organoids are then embedded in a brain-like hyaluronic acid hydrogel for subsequent studies involving drug treatments and tracking of changes in relative numbers of each fluorescently unique subpopulation. This approach allows for the visual assessment of drug influence on individual subpopulations within GBM, and in future work can be expanded to supporting studies using patient tumor biospecimen-derived cells for personalized diagnostics.

Development of a Colorectal Cancer 3D Micro-tumor Construct Platform From Cell Lines and Patient Tumor Biospecimens for Standard-of-Care and Experimental Drug Screening.

Colorectal cancer is subject to a high rate of mutations, with late stage tumors often containing many mutations. These tumors are difficult to treat, and even with the recently implemented methods of personalized medicine at modern hospitals aiming to narrow treatments, a gap still exists. Proper modeling of these tumors may help to recommend optimal treatments for individual patients, preferably utilizing a model that maintains proper signaling in respect to the derived parent tissue. In this study, we utilized an extracellular matrix-derived hydrogel to create a 3D micro-tumor construct platform capable of both supporting cells for long time durations and for high throughput drug screening. Experiments with cell lines demonstrated long-term viability with maintenance of cell proliferation. Furthermore, studies with several chemotherapeutics utilizing different mechanisms of action displayed differences in efficacy in comparing 3D and 2D cultures. Finally, patient colorectal tumor tissue was acquired and employed to reconstruct micro-tumor constructs, providing a system for the testing of novel chemotherapeutics against tumors in a patient-specific manner. Collectively, the results describe a system capable of high throughput testing while maintaining important characteristics of the parent tissue.

Drug compound screening in single and integrated multi-organoid body-on-a-chip systems.

Current practices in drug development have led to therapeutic compounds being approved for widespread use in humans, only to be later withdrawn due to unanticipated toxicity. These occurrences are largely the result of erroneous data generated by in vivo and in vitro preclinical models that do not accurately recapitulate human physiology. Herein, a human primary cell- and stem cell-derived 3D organoid technology is employed to screen a panel of drugs that were recalled from market by the FDA. The platform is comprised of multiple tissue organoid types that remain viable for at least 28 days, in vitro. For many of these compounds, the 3D organoid system was able to demonstrate toxicity. Furthermore, organoids exposed to non-toxic compounds remained viable at clinically relevant doses. Additional experiments were performed on integrated multi-organoid systems containing liver, cardiac, lung, vascular, testis, colon, and brain. These integrated systems proved to maintain viability and expressed functional biomarkers, long-term. Examples are provided that demonstrate how multi-organoid 'body-on-a-chip' systems may be used to model the interdependent metabolism and downstream effects of drugs across multiple tissues in a single platform. Such 3D in vitro systems represent a more physiologically relevant model for drug screening and will likely reduce the cost and failure rate associated with the approval of new drugs.

Pleural Effusion Aspirate for use in 3D Lung Cancer Modeling and Chemotherapy Screening.

Lung cancer is the leading cause of cancer-related death worldwide yet in vitro disease models have been limited to traditional 2D culture utilizing cancer cell lines. In contrast, recently developed 3D models (organoids) have been adopted by researchers to improve the physiological relevance of laboratory study. We have hypothesized that 3D hydrogel-based models will allow for improved disease replication and characterization over standard 2D culture using cells taken directly from patients. Here, we have leveraged the use of 3D hydrogel-based models to create lung cancer organoids using a unique cell source, pleural effusion aspirate, from multiple lung cancer patients. With these 3D models, we have characterized the cell populations comprising the pleural effusion aspirate and have tracked phenotypic changes that develop during short-term in vitro culture. We found that isolated, patient cells placed directly into organoids created anatomically relevant structures and exhibited lung cancer specific behaviors. On the other hand, cells first grown in plastic dishes and then cultured in 3D did not create similar structures. Further, we have been able to compare chemotherapeutic response of patient cells between 2D and 3D cell culture systems. Our results show that cells in 2D culture were more sensitive to treatment when compared with 3D organoids. Collectively, we have been able to utilize tumor cells from pleural effusion fluid of lung cancer patients to create organoids that display in vivo like anatomy and drug response and thus could serve as more accurate disease models for study of tumor progression and drug development.

Applications of Bioengineered 3D Tissue and Tumor Organoids in Drug Development and Precision Medicine: Current and Future.

Over the past decade, advances in biomedical and tissue engineering technologies, such as cell culture techniques, biomaterials, and biofabrication, have driven increasingly widespread use of three-dimensional (3D) cell culture platforms and, subsequently, the use of organoids in a variety of research endeavors. Given the 3D nature of these organoid systems, and the frequent inclusion of extracellular matrix components, these constructs typically have more physiologically accurate cell-cell and cell-matrix interactions than traditional 2D cell cultures. As a result, 3D organoids can serve as better model systems than their 2D counterparts. Moreover, as organoids can be biofabricated from highly functional human cells, they have certain advantages over animal models, being human in nature and more easily manipulated in the laboratory. In this review, we describe such organoid technologies and their deployment in drug development and precision medicine efforts. Organoid technologies are rapidly being developed for these applications and now represent a wide variety of tissue types and diseases. Evidence is emerging that organoids are poised for widespread adoption, not only in academia but also in the pharmaceutical industry and in clinical diagnostic applications, positioning them as indispensable tools in medicine.

Optimization of collagen type I-hyaluronan hybrid bioink for 3D bioprinted liver microenvironments.

Current 3D printing of tissue is restricted by the use of biomaterials that do not recapitulate the native properties of the extracellular matrix (ECM). These restrictions have thus far prevented optimization of composition and structure of the in vivo tissue microenvironment. The artificial nature of currently used biomaterials affects cellular phenotype and function of the bioprinted tissues, and results in inaccurate modeling of disease and drug metabolism significantly. Collagen type I is the major structural component in the ECM, and is widely used as a 3D hydrogel, but is less applicable for 3D bioprinting due to low viscosity and slow polymerization. We have hypothesized that a combination of hyaluronic acid with collagen I yields a bioink with the properties required for extrusion bioprinting, while supporting native cell-matrix interactions and preservation of the native microenvironment properties. To test this hypothesis, we tested the viscoelastic properties of three bioink formulations -2:1, 3:1, and 4:1 collagen type I to hyaluronic acid, and examined cellular behavior in order to determine an optimal formulation that allows for bioprinting while supporting biological activity. We then employed this formulation to bioprint 3D liver tissue constructs containing primary human hepatocytes and liver stellate cells and tested the effects of acetaminophen, a common liver toxicant. Our results have shown that the combination of methacrylated collagen type I and thiolated hyaluronic acid yield a simple, printable bioink that allows for modulation that was directly related to stromal cell elongation. Further, the bioink adequately allowed for implementation as a support hydrogel for hepatocytes which were able to remain viable over two weeks and responded to drug treatment appropriately.

Environmental Toxin Screening Using Human-Derived 3D Bioengineered Liver and Cardiac Organoids.

INTRODUCTION: Environmental toxins, such as lead and other heavy metals, pesticides, and other compounds, represent a significant health concern within the USA and around the world. Even in the twenty-first century, a plethora of cities and towns in the U.S. have suffered from exposures to lead in drinking water or other heavy metals in food or the earth, while there is a high possibility of further places to suffer such exposures in the near future. METHODS: We employed bioengineered 3D human liver and cardiac organoids to screen a panel of environmental toxins (lead, mercury, thallium, and glyphosate), and charted the response of the organoids to these compounds. Liver and cardiac organoids were exposed to lead (10 µM-10 mM), mercury (200 nM-200 µM), thallium (10 nM-10 µM), or glyphosate (25 µM-25 mM) for a duration of 48 h. The impacts of toxin exposure were then assessed by LIVE/DEAD viability and cytotoxicity staining, measuring ATP activity and determining IC50 values, and determining changes in cardiac organoid beating activity. RESULTS: As expected, all of the toxins induced toxicity in the organoids. Both ATP and LIVE/DEAD assays showed toxicity in both liver and cardiac organoids. In particular, thallium was the most toxic, with IC50 values of 13.5 and 1.35 µM in liver and cardiac organoids, respectively. Conversely, glyphosate was the least toxic of the four compounds, with IC50 values of 10.53 and 10.85 mM in liver and cardiac organoids, respectively. Additionally, toxins had a negative influence on cardiac organoid beating activity as well. Thallium resulting in the most significant decreases in beating rate, followed by mercury, then glyphosate, and finally, lead. These results suggest that the 3D organoids have significant utility to be deployed in additional toxicity screening applications, and future development of treatments to mitigate exposures. CONCLUSION: 3D organoids have significant utility to be deployed in additional toxicity screening applications, such as future development of treatments to mitigate exposures, drug screening, and environmental toxin detection.

Mesenchymal stem cells support growth and organization of host-liver colorectal-tumor organoids and possibly resistance to chemotherapy.

Despite having yielded extensive breakthroughs in cancer research, traditional 2D cell cultures have limitations in studying cancer progression and metastasis and screening therapeutic candidates. 3D systems can allow cells to grow, migrate, and interact with each other and the surrounding matrix, resulting in more realistic constructs. Furthermore, interactions between host tissue and developing tumors influence the susceptibility of tumors to drug treatments. Host-liver colorectal-tumor spheroids composed of primary human hepatocytes, mesenchymal stem cells (MSC) and colon carcinoma HCT116 cells were created in simulated microgravity rotating wall vessel (RWV) bioreactors. The cells were seeded on hyaluronic acid-based microcarriers, loaded with liver-specific growth factors and ECM components. Only in the presence of MSC, large tumor foci rapidly formed inside the spheroids and increased in size steadily over time, while not greatly impacting albumin secretion from hepatocytes. The presence of MSC appeared to drive self-organization and formation of a stroma-like tissue surrounding the tumor foci and hepatocytes. Exposure to a commonly used chemotherapeutic 5-FU showed a dose-dependent cytotoxicity. However, if tumor organoids were allowed to mature in the RWV, they were less sensitive to the drug treatment. These data demonstrate the potential utility of liver tumor organoids for cancer progression and drug response modeling.

Bioengineered Submucosal Organoids for In Vitro Modeling of Colorectal Cancer.

The physical nature of the tumor microenvironment significantly impacts tumor growth, invasion, and response to drugs. Most in vitro tumor models are designed to study the effects of extracellular matrix (ECM) stiffness on tumor cells, while not addressing the effects of ECM's specific topography. In this study, we bioengineered submucosal organoids, using primary smooth muscle cells embedded in collagen I hydrogel, which produce aligned and parallel fiber topography similar to those found in vivo. The fiber organization in the submucosal organoids induced an epithelial phenotype in spheroids of colorectal carcinoma cells (HCT-116), which were embedded within the organoids. Conversely, unorganized fibers drove a mesenchymal phenotype in the tumor cells. HCT-116 cells in organoids with aligned fibers showed no WNT signaling activation, and conversely, WNT signaling activation was observed in organoids with disrupted fibers. Consequently, HCT-116 cells in the aligned condition exhibited decreased cellular proliferation and reduced sensitivity to 5-fluorouracil chemotherapeutic treatment compared to cells in the unorganized construct. Collectively, the results establish a unique colorectal tumor organoid model to study the effects of stromal topography on cancer cell phenotype, proliferation, and ultimately, chemotherapeutic susceptibility. In the future, such organoids can utilize patient-derived cells for precision medicine applications.

Optical Tracking and Digital Quantification of Beating Behavior in Bioengineered Human Cardiac Organoids.

Organoid and organ-on-a-chip technologies are rapidly advancing towards deployment for drug and toxicology screening applications. Liver and cardiac toxicities account for the majority of drug candidate failures in human trials. Liver toxicity generally produces liver cell death, while cardiac toxicity causes adverse changes in heart beat kinetics. In traditional 2D cultures, beating kinetics can be measured by electrode arrays, but in some 3D constructs, quantifying beating kinetics can be more challenging. For example, real time measurements of calcium flux or contractile forces are possible, yet rather complex. In this communication article, we demonstrate a simple sensing system based on software code that optically analyzes video capture files of beating cardiac organoids, translates these files in representations of moving pixels, and quantifies pixel movement activity over time to generate beat kinetic plots. We demonstrate this system using bioengineered cardiac organoids under baseline and drug conditions. This technology offers a non-invasive, low-cost, and incredibly simple method for tracking and quantifying beating behavior in cardiac organoids and organ-on-a-chip systems for drug and toxicology screening.