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The lymph node paracortex, also known as the T-cell zone, consists of a network of fibroblastic reticular cells (FRCs) that secrete chemokines to induce T-cell and dendritic cell trafficking into the paracortex. To model the lymph node paracortex, we utilized multi-channel microfluidic devices to engineer a 3D lymph node stromal network from human cultured FRCs embedded in a collagen I-fibrin hydrogel. In the hydrogel, the FRCs self-assembled into an interconnected network, secreted the extracellular matrix proteins entactin, collagen IV, and fibronectin, as well as expressed an array of immune cell trafficking chemokines. Although the engineered FRC network did not secrete characteristic CCR7-ligand chemokines (i.e. CCL19 and CCL21), human primary TNF-α matured monocyte-derived dendritic cells, CD45RA+T-cells, and CD45RA-T-cells migrated toward the lymph node stromal network to a greater extent than toward a blank hydrogel. Furthermore, the FRCs co-recruited dendritic cells and antigen-specific T-cells into the lymph node stromal network. This engineered lymph node stromal network may help evaluate how human dendritic cells and T-cells migrate into the lymph node paracortex via CCR7-independent mechanisms.

Mesenchymal stromal cells (MSCs) continue to be proposed for use in clinical trials to treat various diseases due to their therapeutic potential to pleiotropically influence endogenous regenerative processes, such as vasculogenesis. However, the functional heterogeneity of MSCs has hampered their clinical success and poses a significant manufacturing challenge with respect to MSC quality control. Here, we evaluated and qualified a quantitative bioassay based on an enhanced-throughput, microphysiological system to measure the specific paracrine bioactivity of MSCs to stimulate vasculogenesis as a measure of MSC potency. Using this novel bioassay, MSCs derived from multiple donors at different passages were co-cultured with human umbilical vein endothelial cells (HUVECs) and exhibited significant heterogeneity in vasculogenic potency between donors and cell passage. Using our microphysiological system (MPS)-based platform, we demonstrated that variations in MSC vasculogenic bioactivity were maintained when assayed across laboratories and operators. The differences in MSC vasculogenic bioactivity were also correlated with the baseline expression of several genes involved in vasculogenesis (hepatocyte growth factor (HGF), angiopoietin-1 (ANGPT)) or the production of matricellular proteins (fibronectin (FN), insulin-like growth factor-binding protein 7 (IGFBP7)). These findings emphasize the significant functional heterogeneity of MSCs in vasculogenic bioactivity and suggest that changes in baseline gene expression of vasculogenic or matricellular protein genes during manufacturing may affect this bioactivity. The development of a reliable and functionally relevant potency assay for measuring the specific vasculogenic bioactivity of manufactured MSCs will help to reliably assure their quality when used in appropriate clinical trials.

Complex in vitro models (CIVM) offer the potential to improve pharmaceutical clinical drug attrition due to safety and/ or efficacy concerns. For this technology to have an impact, the establishment of robust characterization and qualifi­cation plans constructed around specific contexts of use (COU) is required. This article covers the output from a workshop between the Food and Drug Administration (FDA) and Innovation and Quality Microphysiological Systems (IQ MPS) Affiliate. The intent of the workshop was to understand how CIVM technologies are currently being applied by pharma­ceutical companies during drug development and are being tested at the FDA through various case studies in order to identify hurdles (real or perceived) to the adoption of microphysiological systems (MPS) technologies, and to address evaluation/qualification pathways for these technologies. Output from the workshop includes the alignment on a working definition of MPS, a detailed description of the eleven CIVM case studies presented at the workshop, in-depth analysis, and key take aways from breakout sessions on ADME (absorption, distribution, metabolism, and excretion), pharmacology, and safety that covered topics such as qualification and performance criteria, species differences and concordance, and how industry can overcome barriers to regulatory submission of CIVM data. In conclusion, IQ MPS Affiliate and FDA scientists were able to build a general consensus on the need for animal CIVMs for preclinical species to better determine species concordance. Furthermore, there was acceptance that CIVM technologies for use in ADME, pharmacology and safety assessment will require qualification, which will vary depending on the specific COU.

et al., There is an evolution and increasing need for the utilization of emerging cellular, molecular and in silico technologies and novel approaches for safety assessment of food, drugs, and personal care products. Convergence of these emerging technologies is also enabling rapid advances and approaches that may impact regulatory decisions and approvals. Although the development of emerging technologies may allow rapid advances in regulatory decision making, there is concern that these new technologies have not been thoroughly evaluated to determine if they are ready for regulatory application, singularly or in combinations. The magnitude of these combined technical advances may outpace the ability to assess fit for purpose and to allow routine application of these new methods for regulatory purposes. There is a need to develop strategies to evaluate the new technologies to determine which ones are ready for regulatory use. The opportunity to apply these potentially faster, more accurate, and cost-effective approaches remains an important goal to facilitate their incorporation into regulatory use. However, without a clear strategy to evaluate emerging technologies rapidly and appropriately, the value of these efforts may go unrecognized or may take longer. It is important for the regulatory science field to keep up with the research in these technically advanced areas and to understand the science behind these new approaches. The regulatory field must understand the critical quality attributes of these novel approaches and learn from each other's experience so that workforces can be trained to prepare for emerging global regulatory challenges. Moreover, it is essential that the regulatory community must work with the technology developers to harness collective capabilities towards developing a strategy for evaluation of these new and novel assessment tools.

Mesenchymal stromal cells (MSCs) are increasingly combined with biomaterials to enhance their therapeutic properties, including their immunosuppressive function. However, clinical trials utilizing MSCs with or without biomaterials have shown limited success, potentially due to their functional heterogeneity across different donors and among different subpopulations of cells. Here, we evaluated the immunosuppressive capacity, as measured by the ability to reduce T-cell proliferation and activation, of interferon-gamma (IFN-γ)–licensed MSCs from multiple donors on fibrin and collagen hydrogels, the two most commonly utilized biomaterials in combination with MSCs in clinical trials worldwide according to ClinicalTrials.gov. Variations in the immunosuppressive capacity between IFN-γ–licensed MSC donors on the biomaterials correlated with the magnitude of indoleamine-2,3-dioxygenase activity. Immunosuppressive capacity of the IFN-γ–licensed MSCs depended on the α(V)/α(5) integrins when cultured on fibrin and on the α(2)/β(1) integrins when cultured on collagen. While all tested MSCs were nearly 100% positive for these integrins, sorted MSCs that expressed higher levels of α(V)/α(5) integrins demonstrated greater immunosuppressive capacity with IFN-γ licensing than MSCs that expressed lower levels of these integrins on fibrin. These findings were equivalent for MSCs sorted based on the α(2)/β(1) integrins on collagen. These results demonstrate the importance of integrin engagement to IFN-γ licensed MSC immunosuppressive capacity and that IFN-γ–licensed MSC subpopulations of varying immunosuppressive capacity can be identified by the magnitude of integrin expression specific to each biomaterial.

There are a growing number of globally approved products and clinical trials utilizing autologous and allogeneic therapeutic cells for applications in regenerative medicine and immunotherapies. However, there is a need to develop rapid and cost-effective methods for manufacturing therapeutically effective cells. Furthermore, the resulting manufactured cells may exhibit heterogeneities that result in mixed therapeutic outcomes. Engineering approaches that can provide distinct microenvironmental cues to these cells may be able to enhance the growth and characterization of these cell products. This mini-review describes strategies to potentially enhance the expansion of therapeutic cells with biomaterials and bioreactors, as well as to characterize the cell products with microphysiological systems. These systems can provide distinct cues to maintain the quality attributes of the cells and evaluate their function in physiologically relevant conditions.

Regenerative medicine is a rapidly expanding field given its potential to treat highly complex health conditions and rare diseases. However, the application of the US Food and Drug Administration (FDA) regulatory framework to these innovative products can be challenging given their highly variable and complex nature. Nevertheless, the rapid development of cellular products and their fast-paced innovation requires FDA to develop new tools, standards, and approaches with which to adequately assess their safety, efficacy, quality, and performance. In addition to FDA’s advancement of regulatory science, product developers bear the onus of generating high-quality objective data at key stages throughout the product life cycle that can be effectively and efficiently reviewed. To spur collaboration between FDA and product developers, this review paper provides a discussion about how regulatory science can be leveraged to improve the demonstration of product identity and potency testing for regenerative medicine cellular products.

Improve 19F magnetic resonance imaging uniformity of perfluorocarbon (PFC)-labeled cells by using a secondary inductive resonator tuned to 287 MHz to enhance the induced radio frequency (RF) magnetic field (B1) at 7.05 T. Following Faraday’s induction law, the sign of induced B1 made by the secondary resonator can be changed depending on the tuning of the resonator. A secondary resonator located on the opposite side of the phantom of the 19F surface coil can be shown to enhance or subtract the induced B1 field, depending upon its tuning. The numerical simulation results of rotating transmit B1 magnitude (|B 1 + |) and corresponding experimental 19F images were compared without and with the secondary resonator. With the secondary resonator tuned to 287 MHz, improvements of |B 1 | and 19F image uniformity were demonstrated. The use of the secondary resonator improved our ability to visualize transplanted cell location non-invasively over a period of 6 weeks. The secondary resonator tuned to enhance the induced B1 results in improved image uniformity in a pre-clinical application, enabling cell tracking of PFC-labeled cells with the secondary resonator.

Multipotent Stromal Cells (MSCs, often called mesenchymal stem cells) have garnered significant attention within the field of regenerative medicine due to their purported ability to differentiate down musculoskeletal lineages. Given the inherent heterogeneity of MSC populations, recent studies have suggested that cell morphology may be indicative of MSC differentiation potential. Toward improving current methods and developing simple yet effective approaches for the morphological evaluation of MSCs, we combined passive pumping microfluidic technology with high dimensional morphological characterization to produce robust tools for standardized high throughput analysis. Using ultraviolet (UV) light as a modality for reproducible polystyrene (PS) substrate modification, we show that MSCs seeded on microfluidic straight channel devices incorporating UV exposed substrates exhibited morphological changes that responded accordingly to the degree of substrate modification. Substrate modification also effected greater morphological changes in MSCs seeded at a lower rather than higher density within microfluidic channels. Despite largely comparable trends in morphology, MSCs seeded in microscale as opposed to traditional macroscale platforms displayed much higher sensitivity to changes in substrate properties. In summary, we adapted and qualified microfluidic cell culture platforms comprising simple straight channel arrays as a viable and robust tool for high-throughput quantitative morphological analysis to study cell-material interactions.

As a result of improved relevance to in vivo physiology, in vitro studies are increasingly performed in diverse, three-dimensional (3D) biomaterials. However, material-cell type pairing effects on cytokine availability remain unclear. We cultured five cell types in agarose, alginate, collagen, Matrigel, or RGD-functionalized polyethylene glycol (PEG) hydrogels. We measured 21 cytokines in the conditioned media, and we identified differences in measured cytokine levels that were cell-type- or material-dependent. We further evaluated our data using principal component analysis. Interestingly, component one identified two classes of biomaterials with characteristic cytokine expression levels. Component two identified cell-type-dependent differences in cytokines related to the wound response. Although elements of soluble cytokine availability are shared despite parameter differences, material and cellular properties variably influenced cytokine levels, underlining the influence of biomaterial-cell type pairings on in vitro assay outcomes. Relationships between material properties, cellular responses, and cytokine availability in 3D in vitro models warrant further investigation.

Personalized cancer therapy focuses on characterizing the relevant phenotypes of the patient, as well as the patient's tumor, to predict the most effective cancer therapy. Historically, these methods have not proven predictive in regards to predicting therapeutic response. Emerging culture platforms are designed to better recapitulate the in vivo environment, thus, there is renewed interest in integrating patient samples into in vitro cancer models to assess therapeutic response. Successful examples of translating in vitro response to clinical relevance are limited due to issues with patient sample acquisition, variability and culture. We will review traditional and emerging in vitro models for personalized medicine, focusing on the technologies, microenvironmental components, and readouts utilized. We will then offer our perspective on how to apply a framework derived from toxicology and ecology towards designing improved personalized in vitro models of cancer. The framework serves as a tool for identifying optimal readouts and culture conditions, thus maximizing the information gained from each patient sample.

The blood-brain barrier (BBB) is notoriously difficult to penetrate for the delivery of drugs and therapeutic compounds due to the unique nature of the tight junctions between endothelial cells. Ultrasound in the presence of microbubbles has been discovered to transiently open the BBB, however, little is known about the mechanism and the effects of excitation parameters at a cellular level. In this study, a static vascular lumen in vitro model, LumeNEXT, was utilized to investigate the excitation parameters in a controlled manner. Fibrin lumen hydrogels were lined with human cerebral microvessel endothelial and astrocyte cells. Cells were exposed to ultrasound with and without the presence of microbubbles. The devices were exposed to ultrasound parameters ranging in frequency from 0.5 to 1.55MHz, duty factor from 0.0001–0.001 and pulse repetition frequency of 1–10 Hz. Acoustic and thermal characterization of the device was performed. Effects of microbubble size and composition on the endothelial permeability were also investigated. The transepithelial electrical resistance (TEER) and cell morphology were monitored post-ultrasound exposure to investigate endothelial permeability duration and extent. Understanding the fundamentals of ultrasound-induced permeability in a controlled manner can lead to further investigations and optimization for in vivo and clinical settings.

Multipotent stromal cells (MSCs) are an attractive cell source for bone and cartilage tissue repair strategies. However, the functional heterogeneity of MSCs derived from different donors and manufacturing conditions has limited clinical translation, emphasizing the need for improved methods to assess MSC chondrogenic capacity. We used functionally relevant morphological profiling to dynamically monitor emergent morphological phenotypes of chondrogenically induced MSC aggregates to identify morphological features indicative of MSC chondrogenesis. Toward this goal, we characterized the morphology of chondrogenically stimulated MSC aggregates from eight different human cell-lines at multiple passages and demonstrated that MSC aggregates exhibited unique morphological dynamics that were both cell line- and passage-dependent. This variation in 3D morphology was shown to be informative of long-term MSC chondrogenesis based on multiple quantitative functional assays. We found that the specific morphological features of spheroid area, radius, minimum feret diameter, and minor axis length to be strongly correlated with MSC chondrogenic synthetic activity but not gene expression as early as day 4 in 3D culture. Our high-throughput, nondestructive approach could potentially serve as a tool to identify MSC lines with desired chondrogenic capacity toward improving manufacturing strategies for MSC-based cellular products for cartilage tissue repair.

Heterogeneity in cell function has presented a significant hurdle to the successful clinical translation of many cellular therapies. Current techniques for assessing cell quality and the effects of microenvironmental cues and manufacturing processes on cell behavior often inadequately address heterogeneity due to issues such as population versus single-cell measurements and the therapeutic relevance and throughput/robustness of the assay. Due to the well-established relationship between morphology and cellular function, morphological profiling has become increasingly utilized to better understand functional heterogeneity and its impact on therapeutic development. In this review, we introduce an emerging field we term functionally-relevant morphological profiling with great potential to improve our understanding of cellular heterogeneity through discovering novel quality attributes, optimizing manufacturing, and screening drugs/biomaterials.

The increasing interest in studying cells using more in vivo-like three-dimensional (3D) microenvironments has created a need for advanced 3D screening platforms with enhanced functionalities and increased throughput. 3D screening platforms that better mimic in vivo microenvironments with enhanced throughput would provide more in-depth understanding of the complexity and heterogeneity of microenvironments. The platforms would also better predict the toxicity and efficacy of potential drugs in physiologically relevant conditions. Traditional 3D culture models (e.g., spinner flasks, gyratory rotation devices, non-adhesive surfaces, polymers) were developed to create 3D multicellular structures. However, these traditional systems require large volumes of reagents and cells, and are not compatible with high-throughput screening (HTS) systems. Microscale technology offers the miniaturization of 3D cultures and allows efficient screening of various conditions. This review will discuss the development, most influential works, and current advantages and challenges of microscale culture systems for screening cells in 3D microenvironments.

Microscale 3D in vitro systems have attracted significant interest as tools for cancer research because the microscale systems offer better organization of the cellular microenvironment and enhance throughput of the systems by lowering costs and reducing the amount of reagents and cells. Lumens (i.e., tubular structures) are ubiquitous in vivo being present in blood vessels, mammary ducts, prostate ducts, and the lymphatic system. Lumen structures of varying size and geometry are involved in key normal and disease processes including morphogenesis, angiogenesis, cancer development, and drug delivery. Therefore, there is a need for practical methods that create various lumen structures having different size and geometries to investigate how cells in the lumen structure respond to certain microenvironmental conditions during cancer development and progression. Here, we present a method to create multiple three-dimensional (3D) luminal structures, where parameters, such as size, geometry, and distance, can easily be controlled using simple poly-dimethylsiloxane (PDMS) micro-molds.

The increasing interest in studying the interactions between cells and the extracellular matrix (ECM) has created a need for high throughput low-cost three-dimensional (3D) culture systems. The recent development of tubeless microfluidics via passive pumping provides a high throughput microchannel culture platform compatible with existing high throughput infrastructures (e.g., automated liquid handlers). Here, we build on a previously reported high throughput two-dimensional system to create a robust automated system for 3D culture. Operational controls including temperature and sample handling have been characterized and automated. Human mammary fibroblasts (HMFs) suspended in type I collagen are loaded and cultured in microchannel arrays and used to optimize the system operational parameters. A Peltier cooler maintains the collagen as a liquid at 4 °C during cell seeding, followed by polymerization at 37 °C. Optimization of this platform is discussed (e.g., controlling collagen contraction, increasing cell viability, preventing the removal of microchannel contents), and 3D distribution of HMFs is examined by fluorescent microscopy. Finally, we validate the platform by automating a previously developed 3D breast carcinoma coculture assay. The platform allows more efficient 3D culture experiments and lays the foundation for high throughput studies of cell—ECM interactions.

The transition of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) is a critical step in breast cancer progression. We introduce a simple microfluidic 3D compartmentalized system in which mammary epithelial cells (MCF-DCIS) are co-cultured with human mammary fibroblasts (HMFs), which promotes a transition from DCIS to IDC in vitro. The model enables control of both spatial (distance-dependence) and temporal (transition from larger clusters) aspects within the microenvironment, allowing recapitulation of the in vivo environment in ways not practical with existing experimental models. When HMFs were cultured some distance (0.5-1.5 mm) from the MCF-DCIS cells, we observed an initial morphological change, suggesting soluble factors can begin the transition. However, cell-cell contact with HMFs allowed the MCF-DCIS cells to complete the transition to invasion. Uniquely, the compartmentalized platform enables the analysis of the intrinsic second harmonic generation signal of collagen, providing a label-free quantitative analysis of DCIS-associated collagen remodeling. The arrayed microchannel-based model is compatible with existing infrastructure and, for the first time, provides a cost effective approach to test for inhibitors of pathways involved in DCIS progression to IDC allowing a screening approach to the identification of potential therapeutic targets. Importantly, the model can be easily adapted and generalized to a variety of cell-cell signaling studies.

Microscale 3D in vitro systems have attracted significant interest as tools for cancer research because the microscale systems offer better organization of the cellular microenvironment and enhance throughput of the systems by lowering costs and reducing the amount of reagents and cells. Lumens (i.e., tubular structures) are ubiquitous in vivo being present in blood vessels, mammary ducts, prostate ducts, and the lymphatic system. Lumen structures of varying size and geometry are involved in key normal and disease processes including morphogenesis, angiogenesis, cancer development, and drug delivery. Therefore, there is a need for practical methods that create various lumen structures having different size and geometries to investigate how cells in the lumen structure respond to certain microenvironmental conditions during cancer development and progression. Here, we present a method to create multiple three-dimensional (3D) luminal structures, where parameters, such as size, geometry, and distance, can easily be controlled using simple poly-dimethylsiloxane (PDMS) micro-molds.

In vitro biomimetic modeling of physio-logical structures bridges the gap between 2D in vitro culture and animal models. Lumens (tubular structures) are ubiquitous in vivo, being present in blood vessels, mammary ducts, and the lymphatic system. A method 'LumeNEXT' is presented here that allows the fabrication of 3D embedded lumens where size, structure, distance, and configuration can be controlled using standard poly-dimethylsiloxane micromolding methods.

Botulinum neurotoxin (BoNT) is the most lethal naturally produced neurotoxin. Due to the extreme toxicity, BoNTs are implicated in bioterrorism, while the specific mechanism of action and long-lasting effect was found to be medically applicable in treating various neurological disorders. Therefore, for both public and patient safety, a highly sensitive, physiologic, and specific assay is needed. In this paper, we show a method for achieving a highly sensitive cell-based assay for BoNT/A detection using the motor neuron–like continuous cell line NG108–15. To achieve high sensitivity, we performed a media optimization study evaluating three commercially available neural supplements in combination with retinoic acid, purmorphamine, transforming growth factor β1 (TGFβ1), and ganglioside GT1b. We found nonlinear combinatorial effects on BoNT/A detection sensitivity, achieving an EC(50) of 7.4 U ± 1.5 SD (or ~7.9 pM). The achieved detection sensitivity is comparable to that of assays that used primary and stem cell–derived neurons as well as the mouse lethality assay.

Background Ductal carcinoma in situ (DCIS) is a non-invasive form of breast cancer that is thought to be a precursor to most invasive and metastatic breast cancers. Understanding the mechanisms regulating the invasive transition of DCIS is critical in order to better understand how some types of DCIS become invasive. While significant insights have been gained using traditional in vivo and in vitro models, existing models do not adequately recapitulate key structure and functions of human DCIS well. In addition, existing models are time-consuming and costly, limiting their use in routine screens. Here, we present a microscale DCIS model that recapitulates key structures and functions of human DCIS, while enhancing the throughput capability of the system to simultaneously screen numerous molecules and drugs. Methods Our microscale DCIS model is prepared in two steps. First, viscous finger patterning is used to generate mammary epithelial cell-lined lumens through extracellular matrix hydrogels. Next, DCIS cells are added to fill the mammary ducts to create a DCIS-like structure. For coculture experiments, human mammary fibroblasts (HMF) are added to the two side channels connected to the center channel containing DCIS. To validate the invasive transition of the DCIS model, the invasion of cancer cells and the loss of cell-cell junctions are then examined. A student t-test is conducted for statistical analysis. Results We demonstrate that our DCIS model faithfully recapitulates key structures and functions of human mammary DCIS and can be employed to study the mechanisms involved in the invasive progression of DCIS. First, the formation of cell-cell junctions and cell polarity in the normal mammary duct, and the structure of the DCIS model are characterized. Second, coculture with HMF is shown to induce the invasion of DCIS. Third, multiple endpoint analyses are demonstrated to validate the invasion. Conclusions We have developed and characterized a novel in vitro model of normal and DCIS-inflicted mammary ducts with 3D lumen structures. These models will enable researchers to investigate the role of microenvironmental factors on the invasion of DCIS in more in vivo-like conditions.

Advances in tissue engineering and microtechnology have enabled researchers to more easily generate in vitro tissue models that mimic the tissue geometry and spatial organization found in vivo (e.g., vessel or mammary duct models with tubular structures). However, the widespread adoption of these models for biological studies has been slow, in part due to the lack of direct comparisons between existing 2-dimensional and 3-dimensional cell culture models and new organotypic models that better replicate tissue structure. Using previously developed vessel and mammary duct models with 3-dimensional lumen structures, we have begun to explore this question. In a direct comparison between these next generation organotypic models and more traditional methods, we observed differences in the levels of several secreted growth factors and cytokines. In addition, endothelial vessel geometry profoundly affects the phenotypic behavior of carcinoma cells, suggesting that more traditional in vitro assays may not capture in vivo events. Here, we seek to review and add to the increasing evidence supporting the hypothesis that using cell culture models with more relevant tissue structure influences cell fate and behavior, potentially increasing the relevance of biological findings.—Bischel, L. L., Sung, K. E., Jimenez-Torres, J. A., Mader, B., Keely, P. J., Beebe, D. J. The importance of being a lumen.

Despite advances in medicine and biomedical sciences, cancer still remains a major health issue. Complex interactions between tumors and their microenvironment contribute to tumor initiation and progression and also contribute to the development of drug resistant tumor cell populations. The complexity and heterogeneity of tumors and their microenvironment make it challenging to both study and treat cancer. Traditional animal cancer models and in vitro cancer models are limited in their ability to recapitulate human structures and functions, thus hindering the identification of appropriate drug targets and therapeutic strategies. The development and application of microfluidic 3D cancer models have the potential to overcome some of the limitations inherent to traditional models. This review summarizes the progress in microfluidic 3D cancer models, their benefits, and their broad application to basic cancer biology, drug screening, and drug discovery.

The utilization of 3D, physiologically relevant in vitro cancer models to investigate complex interactions between tumor and stroma has been increasing. Prior work has generally focused on the cancer cells and, the role of fibroblast culture conditions on tumor-stromal cell interactions is still largely unknown. Here, we focus on the stroma by comparing functional behaviors of human mammary fibroblasts (HMFs) cultured in 2D and 3D and their effects on the invasive progression of breast cancer cells (MCF10DCIS.com). We identified increased levels of several paracrine factors from HMFs cultured in 3D conditions that drive the invasive transition. Using a microscale co-culture model with improved compartmentalization and sensitivity, we demonstrated that HMFs cultured in 3D intensify the promotion of the invasive progression through the HGF/c-Met interaction. This study highlights the importance of the 3D stromal microenvironment in the development of multiple cell type in vitro cancer models.

In breast cancer progression, the transition from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) is a life-threatening step. This step is accompanied by a dramatic drop in prognosis. Stromal fibroblasts and epithelial cells are separated by a basement membrane (BM) at the early DCIS stage. However, once the BM is disrupted and stromal invasion of epithelial cells is initiated, direct heterocellular contacts between fibroblasts and epithelial cells often occurs. This suggests that the signaling through the heterocellular contact may be a crucial factor in how the invasive progress continues after it is initiated. Accordingly, the objective of this proposal is to investigate the influence of heterocellular contacts between MCF-DCIS cells and human mammary fibroblasts (HMFs) in breast cancer progression by employing a microfluidic-based compartmentalized 3D co-culture platform enabling both contact-free and contact-associated co-cultures.

During breast carcinoma progression, the three-dimensional (3D) microenvironment is continuously remodeled, and changes in the composition of the extracellular matrix (ECM) occur. High throughput screening platforms have been used to decipher the complexity of the microenvironment and to identify ECM components responsible for cancer progression. However, traditional screening platforms are typically limited to two-dimensional (2D) cultures, and often exclude the influence of ECM and stromal components. In this work, a system that integrates 3-dimensional cell culture techniques with an automated microfluidic platform was used to create a new ECM screening platform that cultures cells in more physiologically relevant 3D in vitro microenvironments containing stromal cells and different ECM molecules. This new ECM screening platform was used to culture T47D breast carcinoma cells in mono- and co-culture with human mammary fibroblasts (HMF) with seven combinations of three different ECM proteins (collagen, fibronectin, laminin). Differences in the morphology of T47D clusters, and the proliferation of T47D cells were found in ECM compositions rich in fibronectin or laminin. In addition, an MMP enzyme activity inhibition screening showed the capabilities of the platform for small molecule screening. The platform presented in this work enables screening for the effects of matrix and stromal compositions and show promises for providing new insights in the identification of key ECM components involved in breast cancer.

Stromal fibroblasts actively participate in normal mammary gland homeostasis and in breast carcinoma growth and progression by secreting paracrine factors; however, little is known about the identity of paracrine mediators in individual patients. The purpose of this study was to characterize paracrine signaling pathways between breast carcinoma cells and breast carcinoma-associated fibroblasts (CAF) or normal mammary fibroblasts (NF), respectively. CAF and NF were isolated from breast carcinoma tissue samples and adjacent normal mammary gland tissue of 28 patients. The fibroblasts were grown in 3D collagen gel co-culture with T47D human breast carcinoma cells and T47D cell growth was measured. CAF stimulated T47D cell growth to a significantly greater degree than NF. We detected a considerable inter-individual heterogeneity of paracrine interactions but identified FGF2, HB-EGF, heparanase-1 and SDF1 as factors that were consistently responsible for the activity of carcinoma-associated fibroblasts. CAF from low-grade but not high-grade carcinomas required insulin-like growth factor 1 and transforming growth factor beta 1 to stimulate carcinoma growth. Paradoxically, blocking of membrane-type 1 matrix metalloprotease stimulated T47D cell growth in co-culture with NF. The results were largely mirrored by treating the fibroblasts with siRNA oligonucleotides prior to co-culture, implicating the fibroblasts as principal production site for the secreted mediators. In summary, we identify a paracrine signaling network with inter-individual commonalities and differences. These findings have significant implications for the design of stroma-targeted therapies.

Interest in constructing a reliable 3-dimensional (3D) collagen culture platform in microfabricated systems is increasing as researchers strive to investigate reciprocal interaction between extracellular matrix (ECM) and cells under various conditions. However, in comparison to conventional 2-dimensional (2D) cell culture research, relatively little work has been reported about the polymerization of collagen type I matrix in microsystems. We, thus, present a study of 3D collagen polymerization to achieve reproducible 3D cell culture in microfluidic devices. Array-based microchannels are employed to efficiently examine various polymerization conditions, providing more replicates with less sample volume than conventional means. Collagen fibers assembled in microchannels were almost two-times thinner than those in conventional gels prepared under similar conditions, and the fiber thickness difference influenced viability and morphology of embedded human mammary fibroblast (HMF) cells. HMF cells contained more actin stress fibers and showed increased viability in 3D collagen matrix composed of thicker collagen fibers. Relatively low pH of the collagen solution within a physiological pH range (6.5-8.5) and pre-incubation at low temperature (approximately 4 degrees C) before polymerization at 37 degrees C allow sufficient time for molecular assembly, generating thicker collagen fibers and enhancing HMF cell viability. The results provide the basis for improved process control and reproducibility of 3D collagen matrix culture in microchannels, allowing predictable modifications to provide optimum conditions for specific cell types. In addition, the presented method lays the foundation for high throughput 3D cellular screening.

Breast cancer progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) is a critical step in breast cancer. This invasive transition of DCIS is defined by stromal invasion and is a life-threatening step accompanied by a dramatic drop in prognosis. In addition, the invasive transition of DCIS is largely driven by stromal alteration; thus it is critical to understand how DCIS alters the surrounding microenvironment, thus causing the cancer cells to become invasive. This study investigates the role of stromal fibroblasts in the vicinity of DCIS during the invasive transition using an innovative multidisciplinary approach that combines 3D cancer biology, microfluidics, and high-resolution imaging. We recently developed an efficient 3D microfluidic system that supports the transition from DCIS to IDC. The in vitro system employs microchannels with two inputs and one output enabling MCF10-DCIS.com cells (MCF-DCIS) and human mammary fibroblasts (HMF) to be loaded in two adjacent (side-by-side) compartments. This platform allows investigations of effects of spatial organization on the transition by independently analyzing their morphology and the modifications to the surrounding collagen architecture. Importantly, the compartmentalized platform enables monitoring of both MCF-DCIS and HMF independently including quantitative measures of the collagen architecture associated with each cell type. We observed that the HMF near MCF-DCIS became more protrusive versus HMF relatively far from MCF-DCIS. We have also begun to identify how the HMF become activated and protrusive when co-cultured with MCF-DCIS and to understand the biological function and impact of protrusive HMF during DCIS progression to IDC. We verified that the signaling based on Cathepsin D produced from MCF-DCIS and low-density lipoprotein receptor-related protein-1 (LRP1) from HMF is involved in regulation of the protrusive activity of HMF. Additionally, knocking down LRP1 in HMF inhibited the invasive transition of MCF-DCIS. This study demonstrates one possible route through which MCF-DCIS activate pre-existing fibroblasts and subsequently, leads to the modification of the ECM and the progression to IDC. Citation Format: Kyung Eun Sung, Carolyn Pehlke, Erwin Berthier, Kevin W. Eliceiri, Andreas Friedl, David J. Beebe. A microscale 3-D in vitro model of the breast cancer progression from DCIS to IDC: Deciphering the role of the stromal fibroblasts. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B17.

Introduction: Oncolytic virotherapy can have a potentially high impact in the treatment of non-small cell lung cancer (NSCLC), as there are documented preclinical activities and target specificity. Oncolytic viruses have unique advantages over small molecular drugs, such as their ability to replicate, spread, and carry anti-tumoral therapeutic genes; however, their challenges include concerns over systemic toxicity, requiring intratumoral administration and restricting its applicability. This highlights the clinical significance of tumor microenvironment in oncolytic virotherapy. Although studies have shown significant stromal effects on carcinogenesis, the effects of the tumor microenvironment on viral susceptibility have been largely unstudied. Microscale 3D co-culture assays were recently developed and validated by our laboratory for studies on tumor invasion, and we have used them here to manipulate, control, and examine cell-cell interactions. Here we developed an in vitro microfluidic model to examine tumor-stromal interaction on oncolytic virotherapy. Methods: To determine the effect of tumor microenvironment on oncolytic virotherapy, we examined VSV infection in 3D microfluidic cultures of representative NSCLC cell lines (A549 lung adenocarcinoma, H226 squamous cell carcinoma) in vitro, either as a monoculture, a co-culture in combination with normal lung fibroblasts WI38, or a monoculture in WI38-conditioned media. The infection of VSV-GFP1, a recombinant virus that expresses green fluorescent protein as a byproduct of infection, in lung cancer cells was investigated using a novel microfluidic-based method. As GFP is only produced during viral replication, GFP fluorescence serves as a surrogate for viral infection. Time-lapse fluorescence microscopy of a single VSV-GFP plaque on a lung cancer cell monolayer in 3D microfluidic cultures was conducted over a 30-hour period post infection. Viral production was quantified using conventional plaque assays. Statistical analysis was based on the two-sample Student's t-test at a two-tailed level of 0.05. Results: Up to 37% infection in 3D microfluidic monocultures of A549 lung adenocarcinoma cells compared to 12% infection in co-cultures with WI38 fibroblasts was observed over 30 hours post infection. The rate of VSV infection in A549 decreased significantly in the presence of WI38 lung fibroblasts (2% vs. 0.6% cells/hour; P=0.01). Up to 12% infection in 3D microfluidic monocultures of A549 in WI38-conditioned media was observed over 30 hours post infection; the rate of its VSV infection was equivalent to that in co-cultures with WI38 fibroblasts (0.6% cells/hour). VSV production decreased by 100 folds in A549 monocultures in WI38-conditioned media compared to that in conventional media. Conclusions: VSV spread and production in A549 lung adenocarcinoma cells decreased significantly in the presence of WI38 lung fibroblasts or WI38-conditioned media. As WI38-conditioned media have the same inhibitory effects on viral infection as do WI38 fibroblasts, secreted soluble factors appear to be more important than direct cell-cell contact in causing this phenomenon. Future studies on stroma-targeted therapy may alleviate the inhibitory effects of tumor microenvironment on virotherapy and increase the bioavailability of oncolytic viruses to the tumors systemically. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C236.