Novel Microfabricated Systems to Elucidate the Role of Anisotropic Stiffness in the Tumor Microenvironment

Cancer is the second leading cause of death in women and late stage (metastatic) cancers have abysmal survival rates compared to early stage regional cases (27% vs 86%). As a tumor grows, the surrounding extracellular matrix (ECM) is reorganized into a dense, collagen rich matrix. The new matrix of aligned collagen fibers provides unique mechanical cues such as anisotropic stiffness and contact guidance. Matrix turnover also constricts local vasculature and restricts delivery of key nutrients and signaling molecules to malignant cells to outside the tumor creating a chemotactic gradient from outside to inside. In this work, we developed a novel substrate composed of circular and oval cross section PDMS micropillars to study the effect of anisotropic mechanical cues on cell behavior. To prove that cells sense the mechanical properties of their surroundings through force, we downregulated (Y-27632 and blebbistatin) and upregulated (MDA-MB-231 conditioned media) traction force and observed that fibroblasts (NIH-3T3 and adipose derived stem cells (ASC)) feel the mechanical properties of their surroundings by applying force; and downregulating force disrupts mechanosensing. Second, we developed a novel multi-cue microfluidic assay to simulate both biomechanical and biochemical cues of the tumor microenvironment concurrently. NIH-3T3s were used to demonstrate that migration is primarily influenced by TACS mimicking mechanical cues and chemotactic gradients failed to alter migration characteristics significantly. We conclude that mechanical cues dominate chemotactic cues in directed migration

Analysis of barotactic and chemotactic guidance cues on directional decision-making of Dictyostelium discoideum cells in confined environments

Neutrophils and dendritic cells when migrating in confined environments have been shown to actuate a directional choice toward paths of least hydraulic resistance (barotaxis), in some cases overriding chemotactic responses. Here, we investigate whether this barotactic response is conserved in the more primitive model organism Dictyostelium discoideum using a microfluidic chip design. This design allowed us to monitor the behavior of single cells via live imaging when confronted with bifurcating microchannels, presenting different combinations of hydraulic and chemical stimuli. Under the conditions employed we find no evidence in support of a barotactic response; the cells base their directional choices on the chemotactic cues. When the cells are confronted by a microchannel bifurcation, they often split their leading edge and start moving into both channels, before a decision is made to move into one and retract from the other channel. Analysis of this decision-making process has shown that cells in steeper nonhydrolyzable adenosine- 3', 5'- cyclic monophosphorothioate, Sp- isomer (cAMPS) gradients move faster and split more readily. Furthermore, there exists a highly significant strong correlation between the velocity of the pseudopod moving up the cAMPS gradient to the total velocity of the pseudopods moving up and down the gradient over a large range of velocities. This suggests a role for a critical cortical tension gradient in the directional decision-making process

Microfluidics for studying metastatic patterns of lung cancer

The incidence of lung cancer continues to rise worldwide. Because the aggressive metastasis of lung cancer cells is the major drawback of successful therapies, the crucial challenge of modern nanomedicine is to develop diagnostic tools to map the molecular mechanisms of metastasis in lung cancer patients. In recent years, microfluidic platforms have been given much attention as tools for novel point-of-care diagnostic, an important aspect being the reconstruction of the body organs and tissues mimicking the in vivo conditions in one simple microdevice. Herein, we present the first comprehensive overview of the microfluidic systems used as innovative tools in the studies of lung cancer metastasis including single cancer cell analysis, endothelial transmigration, distant niches migration and finally neoangiogenesis. The application of the microfluidic systems to study the intercellular crosstalk between lung cancer cells and surrounding tumor microenvironment and the connection with multiple molecular signals coming from the external cellular matrix are discussed. We also focus on recent breakthrough technologies regarding lab-on-chip devices that serve as tools for detecting circulating lung cancer cells. The superiority of microfluidic systems over traditional in vitro cell-based assays with regard to modern nanosafety studies and new cancer drug design and discovery is also addressed. Finally, the current progress and future challenges regarding printable and paper-based microfluidic devices for personalized nanomedicine are summarized.publishedVersio

Microengineering Aligned Collagen Substrates In Microfluidic Systems

Cells in vivo are surrounded by a fibrous matrix of proteins and macromolecules called the extracellular matrix (ECM), of which type I collagen is the major constituent. During tissue development or cell-matrix interactions, collagen fibers organize into aligned domains with defined degrees of alignment and directionality. Aligned fibers guide stem cell differentiation and influence cell-cell communication and cell motility. In the tumor microenvironment, aligned fibers guide tumor cell invasion and have been linked to poor patient outcomes. Since fiber alignment instructs cell behavior in vivo, there is a need for in vitro models to replicate fiber alignment and thus provide a relevant microenvironment for cells. Microfluidic systems have been established as advanced cell culture platforms to provide precise control over soluble factor concentration, cell patterning, and fluid flow. However, controlling the fiber alignment of a 3D material within them has remained a challenge. This work addresses existing technological challenges to integrate 3D collagen matrices with aligned fibers into microfluidic platforms. To do so, this work i) Demonstrates for the first time that extensional flows can align 3D collagen matrices (250 µm thick) in a microchannel, ii) Develops modular microfluidic platforms with capabilities to directly access and perfuse 3D collagen, and iii) Develops biofabrication capabilities to create interfaces between different ECM materials in 3D, and create tissue barriers using ultrathin nanomembranes. It is anticipated that the novel ECM microengineering capabilities and approach to integrating the engineered matrices into microfluidic devices will provide a path to develop tissue-specific in vitro models with engineered matrices

Microfluidic-based 3d fibroblast migration studies in biomimetic microenvironments

Cell migration in 3D is a fundamental process in many physiological and pathological phenomena. Indeed, migration through interstitial tissue is a multi-step process that turns out from the cell-ECM interaction. It is a dynamic and complex mechanism that depends on the physic-chemical balance between the cell and its surrounding. Early stage of deep dermal wound healing process is a relevant migratory example, in which the fibroblast is the epicenter: the recruitment of the fibroblasts -by chemotaxis of PDGF-BB- to the clotted wound occurs. Likewise, this work focuses on studying the major underlying mechanisms of 3D fibroblast migration and the main microenvironmental cues involved within. To do so, we have confined two physiologically relevant hydrogels, made of collagen and fibrin, within microfluidic platforms. Firstly, an integral comparative study of biophysical and biomechanical properties of both gels is presented. In these results, we have overcome the wide diversity of the existing data and special stress has been done in order to compare the microstructural arrangement, resistance to flow and elasticity. On the other hand, controlled chemical gradients have been generated and characterized within the microfluidic devices. Since biomolecules interact as purely diffusive factors or bound to the matrix proteins, in this work, distribution of PDGF-BB and TGF-ß1 across collagen and fibrin gels has been quantified. Finally, by taking advantage of the biophysico-chemical definition, we have characterized the migratory responses of human fibroblasts within the microsystems in the presence of a chemoattractant (PDGF-BB). Our results demonstrate that the local microarchitecture of the hydrogels determines the migratory properties of human fibroblasts in response to controlled chemotactic and haptotactic gradients, in a myosin II-dependent manner.La migración celular en 3D es fundamental en muchos fenómenos fisiológicos y patológicos. La migración, la cual resulta de la interacción célula-matriz, es un mecanismo dinámico y complejo que depende del equilibrio entre la célula y su entorno físico-químico. Concretamente, la etapa temprana del proceso de cicatrización de heridas profundas es un proceso migratorio ejemplar, en el cual el fibroblasto es el epicentro: se produce el reclutamiento de los fibroblastos -por quimiotaxis de PDGF-BB- del tejido circundante al coágulo. Este trabajo se centra en el estudio de los principales mecanismos subyacentes de la migración de fibroblastos en 3D y las principales señales microambientales involucradas en ella. Para ello, se han empleado modelos in vitro haciendo uso de plataformas microfluídicas para confinar dos hidrogeles fisiológicamente relevantes, compuestos por colágeno y fibrina. En primer lugar, se presenta un estudio comparativo integral de las propiedades biofísicas y biomecánicas de los hidrogeles. En estos resultados, se ha hecho especial hincapié en comparar la conformación microestructural, la resistencia al flujo de fluido y la elasticidad. Por otro lado, se han generado y caracterizado gradientes químicos dentro de los dispositivos. Puesto que las biomoléculas interactúan como factores puramente difusivos o adheridos a las proteínas de la matriz, en este trabajo se ha cuantificado la distribución de PDGF-BB y TGF-β1, en colágeno y fibrina. Finalmente, mediante esta definición físico-química, se ha caracterizado la respuesta migratoria de fibroblastos humanos dentro de los microdispositivos en presencia de un factor químico (PDGF-BB). Los resultados aquí mostrados demuestran que la microarquitectura local de los hidrogeles determina las propiedades migratorias de fibroblastos humanos en respuesta a gradientes quimiotácticos y haptotácticos, de manera dependiente de la miosina II

Understanding Mechanisms of Metastasis of Aggressive Breast Cancers via Microfluidic Means

The spread of cancer from its site of origin to other organs is called metastasis, and it is this stage of the disease that is responsible for over 90% of cancer deaths. Tumors are comprised of a heterogeneous population and not every cell in a primary tumor has the intrinsic capability to metastasize. Understanding what gives certain metastatically enabled cells this potential will ultimately provide insight into how to target and prevent metastases. In order to form a metastasis, a cancer cell must: move, invade through often stiff supporting tissue, enter the vasculature via small intercellular spaces, survive the hydrodynamic forces of circulation, squeeze through vessel endothelium once again, and finally proliferate. Imbued with the knowledge of this metastatic journey of a cancer cell, it is understandable how very physical and mechanical in nature the process is. Therefore, to study the steps of metastasis effectively requires the ability to precisely control physical attributes of a cell’s surroundings. The engineering field of microfluidics affords this opportunity and in this work I advanced our present knowledge of the metastatic process by using microfluidic techniques in four fundament studies of critical steps required for metastases. In one study, cancer cells are challenged with a geometrically confining migration space which mimics the constraints of a lymphatic capillary and the early necessary intravasation metastatic step. After migration, motile and non-motile cells are recaptured and analyzed for genetic differences which allow for intravasation. In another study, the effects of secreted factors from normal immune cells in the tumor microenvironment are tested for their stimulation of cancer cell migration – the first required step of metastasis – in the most aggressive form of breast cancer that is considered metastatic at its inception. A third study leveraged the adhesive properties of cancer cells as a novel paradigm for circulating tumor cell capture and analysis independent of dynamic cell surface markers. Lastly, specifically designed microfluidic assays were used to determine a multiparametric cellular phenotype of the most aggressive subpopulation of cancer cells’ biomechanical properties, which may confer the capability to effectively traverse the inefficient steps of metastasis.PHDCellular & Molec Biology PhDUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/143962/1/allensg_1.pd

Bioelectronic microfluidic wound healing: a platform for investigating direct current stimulation of injured cell collectives

Upon cutaneous injury, the human body naturally forms an electric field (EF) that acts as a guidance cue for relevant cellular and tissue repair and reorganization. However, the direct current (DC) flow imparted by this EF can be impacted by a variety of diseases. This work delves into the impact of DC stimulation on both healthy and diabetic in vitro wound healing models of human keratinocytes, the most prevalent cell type of the skin. The culmination of non-metal electrode materials and prudent microfluidic design allowed us to create a compact bioelectronic platform to study the effects of different sustained (12 hours galvanostatic DC) EF configurations on wound closure dynamics. Specifically, we compared if electrotactically closing a wound\u27s gap from one wound edge (i.e., uni-directional EF) is as effective as compared to alternatingly polarizing both the wound\u27s edges (i.e., pseudo-converging EF) as both of these spatial stimulation strategies are fundamental to the eventual translational electrode design and strategy. We found that uni-directional electric guidance cues were superior in group keratinocyte healing dynamics by enhancing the wound closure rate nearly three-fold for both healthy and diabetic-like keratinocyte collectives, compared to their non-stimulated respective controls. The motility-inhibited and diabetic-like keratinocytes regained wound closure rates with uni-directional electrical stimulation (increase from 1.0 to 2.8% h−1) comparable to their healthy non-stimulated keratinocyte counterparts (3.5% h−1). Our results bring hope that electrical stimulation delivered in a controlled manner can be a viable pathway to accelerate wound repair, and also by providing a baseline for other researchers trying to find an optimal electrode blueprint for in vivo DC stimulation

Leveraging Microtechnology to Study Multicellular Microvascular Systems and Macromolecular Interaction

Biological systems are large-scale, complex systems comprised of many hierarchical subsystems interacting physico-chemically in a dynamic and coordinated fashion. The complex interactions of subsystems (in micro-scale) lead to the formation of emergent properties (in macro-scale); these are properties that are not visible if individual subsystems are studied. The inherent high-throughput characteristics of microfabrication technology (microtechnology) along with its ability to manipulate biological species at the micro-scale makes it an ideal tool to elucidate the mechanisms leading to the formation of emergent properties at the macro-scale. In this dissertation, by combining microtechnologies with advanced computational algorithms, we demonstrate system-level analysis of biological systems in development and disease. The abundance of high quality molecular and genetic data along with the drastic increase in computational power resulted in considerable progress in genomics, epigenomics and proteomics, but not for the so-called cellomics as we define it here: high-throughput study of single-cell phenotype and heterotypic cell-cell interaction via micromanipulation and bioinformatics analysis. Lack of high-throughput robust experimental tools is the major roadblock to cellomics. Using microtechnologies, in the context of developmental biology we studied vascular tissue morphogenesis (vasculogenesis). Formation of microvessels is of critical significance in development and for vascularizing newly engineered tissues in regenerative medicine. First, we sought to map the heterogeneous morphodynamic behavior of individual clonal cells in the process of capillary-like structure (CLS) formation (Chapter 2 and 3). Then we looked into deciphering the role of extracellular matrix (ECM) mediated mechanical signals in deriving the process of CLS formation (Chapter 4). In the second half of this thesis, we demonstrated the capabilities of microtechnologies and advanced computational algorithms in tackling the challenging problems in disease: global health diagnostics and cancer drug screening. First, we studied the performance of microfluidic-based diagnostic as a large-scale complex system under real-world constraints (Chapter 5). Then, we present the development of two microfluidic-based platforms to study the heterotypic interaction of cells in both a biomimetic in vitro and a realistic in vivo setting. We developed an implantable construct carrying a densely-packed heterogeneous panel of tumor cells. This platform could ultimately be used to test anti-cancer drug efficacy against a large number of genotypes in an in vivo setting (Appendices A and B). Together, these methods provide a powerful suite of tools for high-throughput analysis of biological species at the micro-scale and could potentially unlock the mysteries behind the emergent properties observed at the macro-scale

Mechanical Studies of the Third Dimension in Cancer: From 2D to 3D Model

From the development of self-aggregating, scaffold-free multicellular spheroids to the inclusion of scaffold systems, 3D models have progressively increased in complexity to better mimic native tissues. The inclusion of a third dimension in cancer models allows researchers to zoom out from a significant but limited cancer cell research approach to a wider investigation of the tumor microenvironment. This model can include multiple cell types and many elements from the extracellular matrix (ECM), which provides mechanical support for the tissue, mediates cell-microenvironment interactions, and plays a key role in cancer cell invasion. Both biochemical and biophysical signals from the extracellular space strongly influence cell fate, the epigenetic landscape, and gene expression. Specifically, a detailed mechanistic understanding of tumor cell-ECM interactions, especially during cancer invasion, is lacking. In this review, we focus on the latest achievements in the study of ECM biomechanics and mechanosensing in cancer on 3D scaffold-based and scaffold-free models, focusing on each platform's level of complexity, up-to-date mechanical tests performed, limitations, and potential for further improvements