Automated Assessment of Cancer Drug Efficacy On Breast Tumor Spheroids in Aggrewell™400 Plates Using Image Cytometry.

Tumor spheroid models have proven useful in the study of cancer cell responses to chemotherapeutic compounds by more closely mimicking the 3-dimensional nature of tumors in situ. Their advantages are often offset, however, by protocols that are long, complicated, and expensive. Efforts continue for the development of high-throughput assays that combine the advantages of 3D models with the convenience and simplicity of traditional 2D monolayer methods. Herein, we describe the development of a breast cancer spheroid image cytometry assay using T47D cells in Aggrewell™400 spheroid plates. Using the Celigo® automated imaging system, we developed a method to image and individually track thousands of spheroids within the Aggrewell™400 microwell plate over time. We demonstrate the use of calcein AM and propidium iodide staining to study the effects of known anti-cancer drugs Doxorubicin, Everolimus, Gemcitabine, Metformin, Paclitaxel and Tamoxifen. We use the image cytometry results to quantify the fluorescence of calcein AM and PI as well as spheroid size in a dose dependent manner for each of the drugs. We observe a dose-dependent reduction in spheroid size and find that it correlates well with the viability obtained from the CellTiter96® endpoint assay. The image cytometry method we demonstrate is a convenient and high-throughput drug-response assay for breast cancer spheroids under 400 µm in diameter, and may lay a foundation for investigating other three-dimensional spheroids, organoids, and tissue samples.

3D host cell and pathogen-based bioassay development for testing anti-tuberculosis (TB) drug response and modeling immunodeficiency.

Tuberculosis (TB) is a global health threat that affects 10 million people worldwide. Human Immunodeficiency Virus (HIV) remains one of the major contributors to the reactivation of asymptomatic latent tuberculosis (LTBI). Over the recent years, there has been a significant focus in developing in-vitro 3D models mimicking early events of Mycobacterium tuberculosis (Mtb) pathogenesis, especially formation of the granuloma. However, these models are low throughput and require extracellular matrix. In this article, we report the generation of a matrix-free 3D model, using THP-1 human monocyte/macrophage cells and mCherry-expressing Mycobacterium bovis BCG (Bacilli Camille Guérin), henceforth referred as 3D spheroids, to study the host cell-bacterial interactions. Using mCherry-intensity-based tracking, we monitored the kinetics of BCG growth in the 3D spheroids. We also demonstrate the application of the 3D spheroids for testing anti-TB compounds such as isoniazid (INH), rifampicin (RIF), as well as a host-directed drug, everolimus (EVR) as single and combinational treatments. We further established a dual infection 3D spheroid model by coinfecting THP-1 macrophages with BCG mCherry and pseudotype HIV. In this HIV-TB co-infection model, we found an increase in BCG mCherry growth within the 3D spheroids infected with HIV pseudotype. The degree of disruption of the granuloma was proportional to the virus titers used for co-infection. In summary, this 3D spheroid assay is an useful tool to screen anti-TB response of potential candidate drugs and can be adopted to model HIV-TB interactions.

In Vitro Miniaturized Tuberculosis Spheroid Model.

Tuberculosis (TB) is a public health concern that impacts 10 million people around the world. Current in vitro models are low throughput and/or lack caseation, which impairs drug effectiveness in humans. Here, we report the generation of THP-1 human monocyte/macrophage spheroids housing mycobacteria (TB spheroids). These TB spheroids have a central core of dead cells co-localized with mycobacteria and are hypoxic. TB spheroids exhibit higher levels of pro-inflammatory factor TNFα and growth factors G-CSF and VEGF when compared to non-infected control. TB spheroids show high levels of lipid deposition, characterized by MALDI mass spectrometry imaging. TB spheroids infected with strains of differential virulence, Mycobacterium tuberculosis (Mtb) HN878 and CDC1551 vary in response to Isoniazid and Rifampicin. Finally, we adapt the spheroid model to form peripheral blood mononuclear cells (PBMCs) and lung fibroblasts (NHLF) 3D co-cultures. These results pave the way for the development of new strategies for disease modeling and therapeutic discovery.

Non-invasive image-based cytometry for high throughput NK cell cytolysis analysis.

Natural Killer (NK) cells are lymphocytes that are the first line of defense against malignantly transformed cells, virally infected cells and other stressed cell types. To study the cytolytic function of NK cells in vitro, a cytotoxicity assay is normally conducted against a target cancerous cell line. Current assay methods are typically performed in mixed 2D cocultures with destructive endpoints and low throughput, thereby limiting the scale, time-resolution, and relevance of the assay to in vivo conditions. Here, we evaluated a novel, non-invasive, quantitative image-based cytometry (qIBC) assay for detection of NK-mediated killing of target cells in 2D and 3D environments in vitro and compared its performance to two common flow cytometry- and fluorescence-based cytotoxicity assays. Similar to the other methods evaluated, the qIBC assay allowed for reproducible detection of target cell killing across a range of effector-to-target ratios with reduced variability. The qIBC assay also allowed for detection of NK cytolysis in 3D spheroids, which enabled scalable measurements of cell cytotoxicity in 3D models. Our findings suggest that quantitative image-based cytometry would be suitable for rapid, high-throughput screening of NK cytolysis in vitro, including in quasi-3D structures that model tissue environments in vivo.

Image-Based Profiling of Patient-Derived Pancreatic Tumor-Stromal Cell Interactions Within a Micropatterned Tumor Model.

Pancreatic cancer is one of the most aggressive cancers with a 5-year patient survival rate of 8.2% and limited availability of therapeutic agents to target metastatic disease. Pancreatic cancer is characterized by a dense stromal cell population with unknown contribution to the progression or suppression of tumor growth. In this study, we describe a microengineered tumor stromal assay of patient-derived pancreatic cancer cells to study the heterotypic interactions of patient pancreatic cancer cells with different types of stromal fibroblasts under basal and drug-treated conditions. The population dynamics of tumor cells in terms of migration and viability were visualized as a functional end point. Coculture with cancer-associated fibroblasts increased the migration of cancer cells when compared to dermal fibroblasts. Finally, we imaged the response of a bromodomain and extraterminal inhibitor on the viability of pancreatic cancer clusters surrounding by stroma in microengineered tumor stromal assay. We visualized a codynamic reduction in both cancer and stromal cells with bromodomain and extraterminal treatment compared to the dimethyl sulfoxide-treated group. This study demonstrates the ability to engineer tumor-stromal assays with patient-derived cells, study the role of diverse types of stromal cells on cancer progression, and precisely visualize a coculture during the screening of therapeutic compounds.

Artificial T Cell Mimetics to Combat Melanoma Tumor Growth.

Despite recent breakthroughs in melanoma treatment with anti-PD-1 immunotherapy, innovative approaches are needed to improve off-target effects. In this study, we report a T cell mimetic microparticle delivery of soluble PD1 aiming at providing a carrier substrate for future combinatorial and targeting efforts. Microparticles of sizes varying from (5 µm to-7 µm) were conjugated with soluble mouse or human PD-1 through nearly irreversible binding between streptavidin and biotin. PD-1 conjugated microparticles (PDMPs) suppressed 3-dimensional tumor growth of human A375 and mouse B16-F10 melanoma cells compared to control microparticles conjugated with the Fc portion of human IgG1 (IgG1MPs). This can be attributed to competitive inhibition by PDMPs on a melanoma cell-intrinsic PD-1/PD-L1 pathway. A single, local administration of mPDMPs in a B16-F10 mouse melanoma model inhibited tumor growth significantly compared to control IgMPs at the same dose. CD45+ immune cells were found to infiltrate tumors treated with mPDMPs as a mechanism for tumor control. These results collectively suggest that PDMPs can target the melanoma cell-intrinsic PD-1/PD-L1 pathway and that these artificial T cell mimetics can be the scaffold for further improvements in anti-tumor immunotherapy.

Orthogonal potency analysis of mesenchymal stromal cell function during ex vivo expansion.

Adult bone marrow mesenchymal stromal cells (MSCs) have cross-functional, intrinsic potency that is of therapeutic interest. Their ability to regenerate bone, fat, and cartilage, modulate the immune system, and nurture the growth and function of other bone marrow hematopoietic stem/progenitor cells have all been evaluated by transplant applications of MSCs. These applications require the isolation and expansion scaled cell production. To investigate biophysical properties of MSCs that can be feasibly utilized as predictors of bioactivity during biomanufacturing, we used a low-density seeding model to drive MSCs into proliferative stress and exhibit the hallmark characteristics of in vitro aging. A low-density seeding method was used to generate MSCs from passages 1-7 to simulate serial expansion of these cells to maximize yield from a single donor. MSCs were subjected to three bioactivity assays in parallel to ascertain whether patterns in MSC age, size, and shape were associated with the outcomes of the potency assays. MSC age was found to be a predictor of adipogenesis, while cell and nuclear shape was strongly associated to hematopoietic-supportive potency. Together, these data evaluate morphological changes associated with cell potency and highlight new strategies for purification or alternatives to assessing MSC quality.

An engineered biomarker system to monitor and modulate immune clearance of cell therapies.

BACKGROUND AIMS: Cell transplants offer a new opportunity to deliver therapies with novel and complex mechanisms of action. Understanding the pharmacology of cell transplants is important to deliver this new therapy effectively. Currently, however, there are limited techniques to easily track cells after intravenous administration due to the dispersion of the graft throughout the entire body. METHODS: We herein developed an engineered cell system that secretes a luciferase enzyme to sensitively detect cell transplants independent of their locale by a simple blood test. We specifically studied a unique feature of cell transplant pharmacology-namely, immune clearance-using mesenchymal stromal cells (MSCs) as a proof-of-concept cell therapy. MSCs are a clinically relevant cell therapy that has been explored in several disease indications due to their innate properties of altering an immune response. RESULTS: Using this engineered reporter, we observed specific sensitivity of cell therapy exposure to the preparation of cells, cytolysis of MSCs in an allogeneic setting and a NK cell-mediated destruction of MSCs in an autologous setting. CONCLUSIONS: Our cellular tracking method has broader implications at large for assessing in vivo kinetics of various other cell therapies.

Three-Dimensional Breast Cancer Models Mimic Hallmarks of Size-Induced Tumor Progression.

Tumor size is strongly correlated with breast cancer metastasis and patient survival. Increased tumor size contributes to hypoxic and metabolic gradients in the solid tumor and to an aggressive tumor phenotype. Thus, it is important to develop three-dimensional (3D) breast tumor models that recapitulate size-induced microenvironmental changes and, consequently, natural tumor progression in real time without the use of artificial culture conditions or gene manipulations. Here, we developed size-controlled multicellular aggregates ("microtumors") of subtype-specific breast cancer cells by using non-adhesive polyethylene glycol dimethacrylate hydrogel microwells of defined sizes (150-600 µm). These 3D microtumor models faithfully represent size-induced microenvironmental changes, such as hypoxic gradients, cellular heterogeneity, and spatial distribution of necrotic/proliferating cells. These microtumors acquire hallmarks of tumor progression in the same cell lines within 6 days. Of note, large microtumors of hormone receptor-positive cells exhibited an aggressive phenotype characterized by collective cell migration and upregulation of mesenchymal markers at mRNA and protein level, which was not observed in small microtumors. Interestingly, triple-negative breast cancer (TNBC) cell lines did not show size-dependent upregulation of mesenchymal markers. In conclusion, size-controlled microtumor models successfully recapitulated clinically observed positive association between tumor size and aggressive phenotype in hormone receptor-positive breast cancer while maintaining clinically proven poor correlation of tumor size with aggressive phenotype in TNBC. Such clinically relevant 3D models generated under controlled experimental conditions can serve as precise preclinical models to study mechanisms involved in breast tumor progression as well as antitumor drug effects as a function of tumor progression. Cancer Res; 76(13); 3732-43. ©2016 AACR.

Cell-Instructive Graphene-Containing Nanocomposites Induce Multinucleated Myotube Formation.

Myoblast differentiation is a key step in myogenesis and has long been considered to be controlled mainly by biochemical cues such as growth factors. However, the tissue engineering approaches based on biochemical cues demonstrate low reproducibility as a precise spatial control over their bioactivity is challenging. Recently, substrate micro/nano-structure and electro-responsive properties are recognized for their important roles in myoblast differentiation. In this study, we hypothesized that engineering biophysical features such as nano/micro-fibrous structure and conductive properties into a single biomaterial scaffold will instruct the myoblasts to differentiate into multinucleated myotubes even in the absence of differentiation media. We fabricated nanocomposite scaffolds composed of conductive graphene nanosheets and polycaprolactone (PCL), a widely used biocompatible material. The resulting graphene-PCL scaffolds possess excellent conductivity due to graphene nanosheets and great processability, biodegradability and elastic mechanical properties conferred by PCL. Additionally, physicochemical and mechanical properties of nanocomposite scaffolds can be tuned by varying graphene concentration. Further, graphene-PCL nanocomposites and their 8-week degradation products exhibited remarkable cytocompatibility and promoted adhesion and proliferation of C2C12 mouse myoblast cells. Importantly, these nanocomposite scaffolds induced graphene concentration-dependent differentiation of C2C12 cells into multinucleated myotubes even in normal growth media suggesting their cell-instructive potential. Thus, graphene-PCL nanocomposite scaffolds can serve as a strategy to promote skeletal muscle regeneration without biochemical cues.

Suicide Gene-Engineered Stromal Cells Reveal a Dynamic Regulation of Cancer Metastasis.

Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME). The dynamic role of human CAFs in cancer progression has been ill-defined because human CAFs lack a unique marker needed for a cell-specific, promoter-driven knockout model. Here, we developed an engineered human CAF cell line with an inducible suicide gene to enable selective in vivo elimination of human CAFs at different stages of xenograft tumor development, effectively circumventing the challenge of targeting a cell-specific marker. Suicide-engineered CAFs were highly sensitive to apoptosis induction in vitro and in vivo by the addition of a simple small molecule inducer. Selection of timepoints for targeted CAF apoptosis in vivo during the progression of a human breast cancer xenograft model was guided by a bi-phasic host cytokine response that peaked at early timepoints after tumor implantation. Remarkably, we observed that the selective apoptosis of CAFs at these early timepoints did not affect primary tumor growth, but instead increased the presence of tumor-associated macrophages and the metastatic spread of breast cancer cells to the lung and bone. The study revealed a dynamic relationship between CAFs and cancer metastasis that has counter-intuitive ramifications for CAF-targeted therapy.

Carbon-based hierarchical scaffolds for myoblast differentiation: Synergy between nano-functionalization and alignment.

While several scaffolds have been proposed for skeletal muscle regeneration, multiscale hierarchical scaffolds with the complexity of extracellular matrix (ECM) haven't been engineered successfully. By precise control over nano- and microscale features, comprehensive understanding of the effect of multiple factors on skeletal muscle regeneration can be derived. In this study, we engineered carbon-based scaffolds with hierarchical nano- and microscale architecture with controlled physico-chemical properties. More specifically, we built multiscale hierarchy by growing carbon nanotube (CNT) carpets on two types of scaffolds, namely, interconnected microporous carbon foams and aligned carbon fiber mats. Nanostructured CNT carpets offered fine control over nano-roughness and wettability facilitating myoblast adhesion, growth and differentiation into myocytes. However, microporous foam architecture failed to promote their fusion into multinucleated myotubes. On the other hand, aligned fibrous architecture stimulated formation of multinucleated myotubes. Most importantly, nanostructured CNT carpets interfaced with microscale aligned fibrous architecture significantly enhanced myocyte fusion into multinucleated mature myotubes highlighting synergy between nanoscale surface features and micro-/macroscale aligned fibrous architecture in the process of myogenesis. STATEMENT OF SIGNIFICANCE: Due to limited regenerative potential of skeletal muscle, strategies stimulating regeneration of functional muscles are important. These strategies are aimed at promoting differentiation of progenitor cells (myoblasts) into multinucleated myotubes, a key initial step in functional muscle regeneration. Recent tissue engineering approaches utilize various scaffolds ranging from decellularized matrices to aligned biomaterial scaffolds. Although, majority of them have focused on nano- or microscale organization, a systematic approach to build the multiscale hierarchy into these scaffolds is lacking. Here, we engineered multiscale hierarchy into carbon-based materials and demonstrated that the nanoscale features govern the differentiation of individual myoblasts into myocytes whereas microscale alignment cues orchestrate fusion of multiple myocytes into multinucleated myotubes underlining the importance of multiscale hierarchy in enhancing coordinated tissue regeneration.

Production of Uniform 3D Microtumors in Hydrogel Microwell Arrays for Measurement of Viability, Morphology, and Signaling Pathway Activation.

Despite significant investments in cancer research and drug discovery/development, the rate of new cancer drug approval is ≤5% and most cases of metastatic cancer remain incurable. Ninety-five percent of new cancer drugs fail in clinical development because of a lack of therapeutic efficacy and/or unacceptable toxicity. One of the major factors responsible for the low success rate of anticancer drug development is the failure of preclinical models to adequately recapitulate the complexity and heterogeneity of human cancer. For throughput and capacity reasons, high-throughput screening growth inhibition assays almost exclusively use two-dimensional (2D) monolayers of tumor cell lines cultured on tissue culture-treated plastic/glass surfaces in serum-containing medium. However, these 2D tumor cell line cultures fail to recapitulate the three-dimensional (3D) context of cells in solid tumors even though the tumor microenvironment has been shown to have a profound effect on anticancer drug responses. Tumor spheroids remain the best characterized and most widely used 3D models; however, spheroid sizes tend to be nonuniform, making them unsuitable for high-throughput drug testing. To circumvent this challenge, we have developed defined size microwell arrays using nonadhesive hydrogels that are applicable to a wide variety of cancer cell lines to fabricate size-controlled 3D microtumors. We demonstrate that the hydrogel microwell array platform can be applied successfully to generate hundreds of uniform microtumors within 3-6 days from many cervical and breast, as well as head and neck squamous cell carcinoma (HNSCC) cells. Moreover, controlling size of the microwells in the hydrogel array allows precise control over the size of the microtumors. Finally, we demonstrate the application of this platform technology to probe activation as well as inhibition of epidermal growth factor receptor (EGFR) signaling in 3D HNSCC microtumors in response to EGF and cetuximab treatments, respectively. We believe that the ability to generate large numbers of HNSCC microtumors of uniform size and 3D morphology using hydrogel arrays will provide more physiological in vitro 3D tumor models to investigate how tumor size influences signaling pathway activation and cancer drug efficacy.

Nanoclay-enriched poly(ɛ-caprolactone) electrospun scaffolds for osteogenic differentiation of human mesenchymal stem cells.

Musculoskeletal tissue engineering aims at repairing and regenerating damaged tissues using biological tissue substitutes. One approach to achieve this aim is to develop osteoconductive scaffolds that facilitate the formation of functional bone tissue. We have fabricated nanoclay-enriched electrospun poly(ɛ-caprolactone) (PCL) scaffolds for osteogenic differentiation of human mesenchymal stem cells (hMSCs). A range of electrospun scaffolds is fabricated by varying the nanoclay concentrations within the PCL scaffolds. The addition of nanoclay decreases fiber diameter and increases surface roughness of electrospun fibers. The enrichment of PCL scaffold with nanoclay promotes in vitro biomineralization when subjected to simulated body fluid (SBF), indicating bioactive characteristics of the hybrid scaffolds. The degradation rate of PCL increases due to the addition of nanoclay. In addition, a significant increase in crystallization temperature of PCL is also observed due to enhanced surface interactions between PCL and nanoclay. The effect of nanoclay on the mechanical properties of electrospun fibers is also evaluated. The feasibility of using nanoclay-enriched PCL scaffolds for tissue engineering applications is investigated in vitro using hMSCs. The nanoclay-enriched electrospun PCL scaffolds support hMSCs adhesion and proliferation. The addition of nanoclay significantly enhances osteogenic differentiation of hMSCs on the electrospun scaffolds as evident by an increase in alkaline phosphates activity of hMSCs and higher deposition of mineralized extracellular matrix compared to PCL scaffolds. Given its unique bioactive characteristics, nanoclay-enriched PCL fibrous scaffold may be used for musculoskeletal tissue engineering.

Highly elastomeric poly(glycerol sebacate)-co-poly(ethylene glycol) amphiphilic block copolymers.

Poly(glycerol sebacate) (PGS), a tough elastomer, has been proposed for tissue engineering applications due to its desired mechanical properties, biocompatibility and controlled degradation. Despite interesting physical and chemical properties, PGS shows limited water uptake capacity (∼2%), thus constraining its utility for soft tissue engineering. Therefore, a modification of PGS that would mimic the water uptake and water retention characteristics of natural extracellular matrix is beneficial for enhancing its utility for biomedical applications. Here, we report the synthesis and characterization of highly elastomeric poly(glycerol sebacate)-co-polyethylene glycol (PGS-co-PEG) block copolymers with controlled water uptake characteristics. By tailoring the water uptake property, it is possible to engineer scaffolds with customized degradation and mechanical properties. The addition of PEG results in almost 15-fold increase in water uptake capacity of PGS, and improves its mechanical stability under dynamic loading conditions. PGS-co-PEG polymers show elastomeric properties and can be subjected to serve deformation such as bending and stretching. The Young's modulus of PGS-co-PEG can be tuned from 13 kPa to 2.2 MPa by altering the amount of PEG within the copolymer network. Compared to PGS, more than six-fold increase in elongation was observed upon PEG incorporation. In addition, the rate of degradation increases with an increase in PEG concentration, indicating that degradation rate of PGS can be regulated. PGS-co-PEG polymers also support cell proliferation, and thus can be used for a range of tissue engineering applications.

Hyperbranched polyester hydrogels with controlled drug release and cell adhesion properties.

Hyperbranched polyesters (HPE) have a high efficiency to encapsulate bioactive agents, including drugs, genes, and proteins, due to their globe-like nanostructure. However, the use of these highly branched polymeric systems for tissue engineering applications has not been broadly investigated. Here, we report synthesis and characterization of photocrosslinkable HPE hydrogels with sustained drug release characteristics for cellular therapies. These HPE can encapsulate hydrophobic drug molecules within the HPE cavities due to the presence of a hydrophobic inner structure that is otherwise difficult to achieve in conventional hydrogels. The functionalization of HPE with photocrosslinkable acrylate moieties renders the formation of hydrogels with a highly porous interconnected structure and mechanically tough network. The compressive modulus of HPE hydrogels was tunable by changing the crosslinking density. The feasibility of using these HPE networks for cellular therapies was investigated by evaluating cell adhesion, spreading, and proliferation on hydrogel surface. Highly crosslinked and mechanically stiff HPE hydrogels have higher cell adhesion, spreading, and proliferation compared to soft and complaint HPE hydrogels. Overall, we showed that hydrogels made from HPE could be used for biomedical applications that require spatial control of cell adhesion and controlled release of hydrophobic clues.