Browsing by Author "West, Jennifer L"

Tumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. A major outstanding challenge associated with studying tumor angiogenesis is that existing preclinical models are limited in their recapitulation of in vivo cellular organization in 3D. This disparity highlights the need for better approaches to study the dynamic interplay of relevant cells and signaling molecules as they are organized in the tumor microenvironment. In this thesis, we combined 3D culture of lung adenocarcinoma cells with adjacent 3D microvascular cell culture in 2-layer cell-adhesive, proteolytically-degradable poly(ethylene glycol) (PEG)-based hydrogels to study tumor angiogenesis and the impacts of neovascularization on tumor cell behavior.

In initial studies, 344SQ cells, a highly metastatic, murine lung adenocarcinoma cell line, were characterized alone in 3D in PEG hydrogels. 344SQ cells formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media that significantly increased with exposure to transforming growth factor beta 1 (TGF-β1), a potent tumor progression-promoting factor. Vascular cells alone in hydrogels formed tubule networks with localized activated TGF-β1. To study cancer cell-vascular cell interactions, the engineered 2-layer tumor angiogenesis model with 344SQ and vascular cell layers was employed. Large, invasive 344SQ clusters developed at the interface between the layers, and were not evident further from the interface or in control hydrogels without vascular cells. A modified model with spatially restricted 344SQ and vascular cell layers confirmed that observed 344SQ cluster morphological changes required close proximity to vascular cells. Additionally, TGF-β1 inhibition blocked endothelial cell-driven 344SQ migration.

Two other lung adenocarcinoma cell lines were also explored in the tumor angiogenesis model: primary tumor-derived metastasis-incompetent, murine 393P cells and primary tumor-derived metastasis-capable human A549 cells. These lung cancer cells also formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media. Epithelial morphogenesis varied for the primary tumor-derived cell lines compared to 344SQ cells, with far less epithelial organization present in A549 spheroids. Additionally, 344SQ cells secreted the highest concentration of two of the three angiogenic growth factors assessed. This finding correlated to 344SQ exhibiting the most pronounced morphological response in the tumor angiogenesis model compared to the 393P and A549 cell lines.

Overall, this dissertation demonstrates the development of a novel 3D tumor angiogenesis model that was used to study vascular cell-cancer cell interactions in lung adenocarcinoma cell lines with varying metastatic capacities. Findings in this thesis have helped to elucidate the role of vascular cells in tumor progression and have identified differences in cancer cell behavior in vitro that correlate to metastatic capacity, thus highlighting the usefulness of this model platform for future discovery of novel tumor angiogenesis and tumor progression-promoting targets.

It has been understood for some time that cells are profoundly influenced by their environment. Recently, researchers have made great strides in engineering cell culture platforms that are both physiologically mimetic and reductionist, leading to more biologically relevant cell responses observed within analytically tractable experiments. Moving from stiff tissue culture plastic or glass into hydrogel-based cell culture keeps cells in contact with materials that are stiffness-matched to native tissues, and can reduce or delay de-differentiation into undesirable phenotypes. Incorporating biomolecules like specific adhesion ligands or growth factors into cell culture hydrogels can help drive specific biological outputs, and when coupled with patterning techniques can yield intentional, spatially defined heterogeneity within a cultured cell population. However, biological events or disease states are often are driven by biochemical dynamics, with the time course over which a signal is presented influencing whether cells respond physiologically or pathologically. Unfortunately, dynamic presentations of biomolecules are challenging to replicate within hydrogels due to a lack of ligation mechanisms suitable for dynamically linking relevant biomolecules into the hydrogel matrix in the presence of cells.

In this work, novel protein-based ligation domains were engineered into new strategies for the time-varying presentation of recombinant biomolecules within cell culture hydrogels. SpyCatcher, a protein domain which forms a spontaneous covalent bond with a complementary peptide dubbed “SpyTag”, was used to form a site-specific linkage between a recombinant protein and a synthetic hydrogel. The ligation reaction was shown to proceed under mild conditions appropriate for cell culture, and by adding the cell-adhesive ligand RGDS to the SpyCatcher-tagged protein (forming RGDS-SC), cell spreading within a 3D hydrogel could be switched on by simply adding RGDS-SC topically to cell-laden hydrogels. SnoopCatcher, the chemically orthogonal cousin to SpyCatcher that binds the peptide “SnoopTag”, was then appended with the vascular endothelial growth factor-mimetic peptide QK (KLTWQELYQLKYKGI) to form QK-SnpC, and used in tandem with RGDS-SC to simultaneously control endothelial cell adhesion and mitotic stimulation on synthetic hydrogels containing both Tag peptides.

The Catcher/Tag systems are advantageous due to their specificity and stability. However, their ligations are irreversible because they form covalent bonds. Incorporating the photocleavable fluorophore PhoCl into the backbone of RGDS-SC (forming PhoCl-SC) allowed for the reversible incorporation of a recombinant protein into synthetic hydrogels. SpyCatcher mediated the spontaneous ligation to SpyTag sites within the gel, and by applying 400 nm light, PhoCl was cleaved, thereby removing the N-terminal RGDS tag from the gel. This reversion was limited to one cycle, and so would not be appropriate for presenting a sequence of several biochemical signals, as would be seen by cells near an area of wound healing for instance. To develop a truly reversible conjugation mechanism, the optogenetic protein LOV2 was engineered into a blue light-mediated non-covalent ligation strategy. The LOVTRAP system, consisting of LOV2 and its binding partner Zdk, was shown to allow synthetic gels containing LOV2 to capture Zdk-tagged proteins in the dark, and then release them upon blue light exposure. Because LOV2 will reset to its dark state via thermal relaxation, this capture and release cycle could be repeated at least 3 times, indicating the reversible association of LOVTRAP was functional in a biomaterial setting.

Protein-based ligation domains are simple to use, as they can be added to recombinant proteins genetically, and are inherently site-specific, alleviating worries that protein activity could be compromised upon conjugation to the gel. Moreover, the Catcher systems and LOVTRAP all bind to their binding partners spontaneously under cell culture conditions, reducing the chance that sensitive cell types would be perturbed by their use. These strategies greatly expand the tool kit for dynamically presenting biomolecules to cells via hydrogel immobilization.

Soft tissues, including neural, adipose, and some vascular tissues, perform processes critical to survival and function such as circulation, cognition, and thermal regulation. Many soft tissue-specific pathologies cause damage to soft tissues that may result in significantly reduced patient health and quality of life. Larger-scale damage to functional soft tissue, as in cases of stroke, traumatic injury, or therapeutic removal such as mastectomy, results in loss of functional tissue on a volume scale that cannot be endogenously regenerated. Adult mammalian brain tissue is particularly limited in its potential to regenerate; thus, cell therapy is a favorable avenue for functional neural tissue regeneration. Transplantation of cells within a biomaterial delivery vehicle improves implanted cell viability and promotes integration of the transplanted cells with the host tissue. Neural tissue cell therapy outcomes may be further improved adding instructional cues to the protective delivery matrix that promote tissue regeneration; however, soft tissue regeneration processes are complex, consist of multiple stages, and in the case of neurogenesis, are not fully understood. Thus, in vitro model systems with tunable control over material properties are needed for modeling soft tissue interactions towards better understanding of their regeneration and design of therapeutic constructs.Current hydrogel models are largely inadequate in achieving soft-tissue mimetic stiffnesses while also providing independent and spatiotemporal control over biochemical cue presentation to cells. Soft tissue culture models made from naturally-derived polymers provide appropriate mechanical properties for soft tissue but sacrifice control over biological signaling. In contrast, synthetic matrix systems offer greater independent control over biochemical signal presentation and mechanical properties; however, they generally do not form stable networks within the mechanical range of soft tissue (E<1 kPa). Further, even the most advanced synthetic tissue culture constructs do not optimally model the dynamic biochemical signaling that occurs in vivo; while molecule-addition and molecule-removal methods do exist separately, current systems are limited in their capability to sequentially add and then remove the same molecule to model transient signaling. Thus, there exists a need for improved soft tissue engineered constructs that accurately recapitulate the stiffness and highly controlled dynamic signaling of natural soft tissue such as neural and fat tissues. Here we present two developments in synthetic hydrogel design for improved accuracy of modeling soft tissue regeneration within a controlled, reductionist environment. We first implemented a novel method of precisely and independently tuning poly(ethylene glycol) (PEG)-based hydrogel stiffness to within the regime of soft tissue by incorporating soluble, allyl-presenting monomers in the hydrogel precursor solution before crosslinking, resulting in allyl-acrylate competition that alters crosslinking mechanics to decrease hydrogel bulk stiffness. PC12 neural cells displayed enhanced neurite outgrowth within this neural stiffness-mimetic environment, both in 2D culture on the surface of PEG-based gels and within a more physiologically relevant degradable PEG-based hydrogel. We then implemented these hydrogels as a platform for investigating the effects of multiple environmental factors on neural stem cell behavior, observing that NSC behavior was influenced by interplay between matrix stiffness, adhesive peptide signaling, and soluble growth factor stimulation. These results indicate that this compliant reductionist hydrogel is an appropriate system for evaluating the influence of single and multiple factors on cell behavior individually and in concert within a controlled environment. This controlled investigation of soft tissue behavior is a promising approach for improved understanding of soft tissue regeneration. We next developed a genetically-encoded method for reversible biochemical signaling within PEG-based hydrogels. We designed and implemented a recombinant protein with a SpyCatcher domain (capable of linking to a SpyTag-functionalized PEG matrix) connected to a cell adhesive RGDS peptide domain by a long-range UV light-cleavable domain known as PhoCl. This protein was shown to bind to SpyTag-functionalized PEG-matrices via isopeptide bonding to present RGDS adhesive ligands to cells, then upon 405 nm light exposure, the PhoCl domain was cleaved to subsequently release the RGDS domain, which diffused out of the matrix. This system was implemented to confer reversible adhesion of cells to the PEG-based hydrogel surface in 2D culture and differential cell spreading over time in 3D culture within cell-degradable PEG-based hydrogels, demonstrating the capability of this system to presenting dynamic signaling events to cells towards modeling soft tissue regeneration processes within in a controlled, ECM-mimetic environment. To address limitations of current in vitro soft tissue culture models, we developed methods for mimicking neural tissue stiffness and presenting reversible biochemical signaling within modularly tunable PEG-based hydrogels. These two synthetic hydrogel design technologies established in this work advance the capabilities for modeling soft tissue behaviors towards designing therapeutic tissue engineered constructs for directing therapeutic regeneration.

To this day, cancer remains the leading cause of mortality worldwide1. A major contributor to cancer progression and metastasis is tumor angiogenesis. The formation of blood vessels around a tumor is facilitated by the complex interplay between cells in the tumor stroma and the surrounding microenvironment. Understanding this interplay and its dynamic interactions is crucial to identify promising targets for cancer therapy. Current methods in cancer research involve the use of two-dimensional (2D) monolayer cell culture. However, cell-cell and cell-ECM interactions that are important in vascularization and the three-dimensional (3D) tumor microenvironment cannot accurately be recapitulated in 2D. To obtain more biologically relevant information, it is essential to mimic the tumor microenvironment in a 3D culture system. To this end, we demonstrate the utility of poly(ethylene glycol) diacrylate (PEGDA) hydrogels modified for cell-mediated degradability and cell-adhesion to explore, in 3D, the effect of various tumor microenvironmental features such as cell-cell and cell-ECM interactions, and dimensionality on tumor vascularization and cancer cell phenotype.

In aim 1, PEG hydrogels were utilized to evaluate the effect of cells in the tumor stroma, specifically cancer associated fibroblasts (CAFs), on endothelial cells (ECs) and tumor vascularization. CAFs comprise a majority of the cells in the tumor stroma and secrete factors that may influence other cells in the vicinity such as ECs to promote the organization and formation of blood vessels. To investigate this theory, CAFs were isolated from tumors and co-cultured with HUVECs in PEG hydrogels. CAFs co-cultured with ECs organized into vessel-like structures as early as 7 days and were different in vessel morphology and density from co-cultures with normal lung fibroblasts. In contrast to normal lung fibroblasts (LF), CAFs and ECs organized into vessel-like networks that were structurally similar to vessels found in tumors. This work provides insight on the complex crosstalk between cells in the tumor stroma and their effect on tumor angiogenesis. Controlling this complex crosstalk can provide means for developing new therapies to treat cancer.

In aim 2, degradable PEG hydrogels were utilized to explore how extracellular matrix derived peptides modulate vessel formation and angiogenesis. Specifically, integrin-binding motifs derived from laminin such as IKVAV, a peptide derived from the α-chain of laminin and YIGSR, a peptide found in a cysteine-rich site of the laminin β chain, were examined along with RGDS. These peptides were conjugated to heterobifunctional PEG chains and covalently incorporated in hydrogels. The EC tubule formation in vitro and angiogenesis in vivo in response to the laminin-derived motifs were evaluated.

Based on these previous aims, in aim 3, PEG hydrogels were optimized to function as a 3D lung adenocarcinoma in vitro model with metastasis-prone lung tumor derived CAFs, HUVECs, and human lung adenocarcinoma derived A549 tumor cells. Similar to the complex tumor microenvironment consisting of interacting malignant and non-malignant cells, the PEG-based 3D lung adenocarcinoma model consists of both tumor and stromal cells that interact together to support vessel formation and tumor cell proliferation thereby more closely mimicking the functional properties of the tumor microenvironment. The utility of the PEG-based 3D lung adenocarcinoma model as a cancer drug screening platform is demonstrated with investigating the effects of doxorubicin, semaxanib, and cilengitide on cell apoptosis and proliferation. The results from drug screening studies using the PEG-based 3D in vitro lung adenocarcinoma model correlate with results reported from drug screening studies conducted in vivo. Thus, the PEG-based 3D in vitro lung adenocarcinoma model may serve as a better tool for identifying promising drug candidates than the conventional 2D monolayer culture method.

The extracellular environment is a key regulator of cell behavior, providing both biochemical factors and mechanical signals to influence the form and function of cells. The process by which cells sense and respond to environmental mechanical signals is often mediated through force-dependent changes in protein structure and function through a poorly understood process known as mechanotransduction. Towards elucidating the molecular processes underlying mechanosensitive regulation, molecular tension sensors (MTSs) have been created to measure forces experienced by specific proteins inside cells. However, an incomplete understanding of the effects of intracellular forces on fluorescent protein (FP) function within the context of MTs limits sensor application and interpretation. To advance our understanding of the molecular events mediating mechanotransduction, it is necessary to improve on existing approaches as well as to develop new technologies for probing mechanical consequences inside cells. In this dissertation we aim to address this limitation by creating a generalizable assay for probing the effects of cell-generated forces on FP function towards improving the use and interpretation of MTSs. Additionally, we describe the development of a new “synthetic” actin crosslinking sensor which leverages FP mechanosensitivity to provide new insights into mechanical processes inside cells.In our initial efforts, we focused on investigating the effects of cell generated forces on FP function within vinculin-based MTSs. We chose vinculin as our model system as vinculin is a well-studied mechanosensitive protein, known to play a critical role in force transmission inside cells. Additionally, the vinculin tension sensor (VinTS) has been extensively characterized, validated, and utilized in a broad array of applications. Leveraging the relationship between FRET measurements and fluorophore stoichiometry with vinculin MTSs, we developed a generalizable assay for evaluating changes in ensemble MTS measurements in terms of fluorophore contributions. Furthermore, we validated this new method on an extensive MTS data set containing over 2000 cells expressing vinculin sensors. Our analysis revealed that FP stoichiometry within VinTS was modulated significantly within individual focal adhesions (FAs) in an actomyosin-dependent manner, and that both load magnitude and load duration likely play a role. Additionally, we found that this force-mediated loss of FP function, or “mechanical quenching,” is a reversible process, consistent with nonequilibrium transitions in protein structure. To investigate FP mechanosensitivity further, we developed an engineered FRET-based actin crosslinking (ABD) sensor to serve as an improved experimental platform, within which FPs would be subjected to higher loads in a manner free of endogenous biochemical regulation. Within this new system, higher tensile loading and FP mechanical quenching was observed at dynamic actin networks. Furthermore, we found that FP mechanical quenching within these sensors was mediated by non-muscle myosin II (NMII) activity and appears to be reversible. In addition, we found that FPs exhibit different sensitivities to intracellular mechanical loads. To probe the molecular origins of ABD sensors loading within cells, we manipulated the organization and dynamics of actin structures by tuning substrate stiffness within engineered in vitro culture systems. Using this approach, we found that ABD sensors reported increased loads and FP mechanical quenching at dynamic actin networks in response to softer substrates. By coupling FRET-based MTSs with the tunability of in vitro culture models, we demonstrated the application of ABD sensors to probe changes in tensile loading in response to environmental mechanical cues. In summary, this dissertation describes the development of novel tools for studying the effects of intracellular forces on FP function within the context of FRET-based tension sensors. Using these tools, we found that FPs, like mechanosensitive signaling proteins, can undergo nonequilibrium transitions in response to cell-generated forces. Based on these observations, we propose that FP mechanical quenching within MTSs could potentially serve as an entirely new way to visualize and probe mechanical consequences within force-sensitive proteins. By exploiting the mechanosensitivity of FPs as a mechanical consequence, new insights into molecular force-sensitive processes inside cells may be obtained.

X-ray computed tomography (CT) is one of the most useful diagnostic tools for clinicians, with widespread availability, fast scan times, and low cost. CT imaging can reveal a patient’s anatomy in exquisite detail and is extremely useful in the diagnosis of a wide variety of diseases. However, CT is currently limited to anatomical imaging due to the lack of appropriate contrast agents and imaging protocols that would allow for molecular imaging, so clinicians must instead rely on other modalities which are more expensive and less readily available. Dual energy CT, a relatively new technique in which two x-ray energies are used for a single scan, can provide valuable information about tissue material composition. This information can potentially be used for molecular imaging if it is coupled with appropriately-designed contrast agents.

This work aims to extend the use of CT into the molecular imaging realm by developing and testing nanoparticle contrast agents for use with dual energy CT. Several studies were carried out, each of which focused on a different application of using nanoparticle contrast agents together with dual energy CT for molecular imaging.

A commercial blood pool iodine contrast agent for pre-clinical CT (Exia-160) has been shown to accumulate in the myocardium and continue to enhance the myocardium after the contrast agent has been cleared from the bloodstream. It was hypothesized that this agent would not accumulate in infarcted myocardium, which would allow for specific identification of myocardial infarction by CT. Mice were injected with the contrast agent following myocardial infarction, and dual energy CT was used to identify the iodine within the myocardium and separate the iodine from the calcium in the neighboring ribs. Regions of myocardial infarction showed no enhancement on CT, while the healthy myocardium was highly enhanced. Size and position of myocardial infarction determined by dual energy CT were compared with the standard molecular imaging technique for measuring myocardial viability (SPECT). It was found that dual energy CT using this nanoparticle contrast agent reliably agreed with the gold standard molecular imaging method.

Molecular imaging for the improved detection and characterization of lung tumors was also explored through two different studies. The first study used both gold nanoparticles and iodine-containing liposomes together with dual energy CT in order to measure tumor vascular functional parameters, including tumor fractional blood volume and vascular permeability. These dual energy CT measurements were confirmed with ex vivo tissue analysis to demonstrate the validity and accuracy of the in vivo dual energy CT method. The second study used antibody-targeted gold nanoparticles to image EGFR-positive tumors. Two different types of antibodies were tested: a clinically used humanized anti-EGFR antibody, and a small llama-derived single domain anti-EGFR antibody. The single domain antibody showed improved blood half-life and reduced immune clearance compared to the full-sized antibody when attached to gold nanoparticles, but the higher affinity of the full-sized antibody led to much higher overall tumor accumulation. This antibody significantly increased the accumulation of gold nanoparticles in tumors expressing high levels of EGFR. Together, these two studies showed that dual energy CT and nanoparticle contrast agents can be used to measure a wide variety of tumor functional parameters, including tumor vascular density, vascular permeability, and receptor expression. All these parameters can be combined with the anatomical CT imaging to better characterize lung tumors and differentiate between benign and malignant lesions.

The use of dual energy CT for measuring tumor vascular permeability changes after gold nanoparticle-augmented radiation therapy was also demonstrated. Liposomal iodine was injected into mice following radiation therapy in order to measure vascular permeability. Dual energy CT was used to differentiate the signal of the liposomal iodine from the CT signal of the gold nanoparticles already in the tumor. Tumor permeability was measured with CT using multiple combinations of gold nanoparticles and radiation doses to find the optimal conditions for enhancing the effect of radiation therapy on the vasculature. These conditions were then used to increase the delivery of a liposomal chemotherapy agent to tumors. Tumors treated with the gold-augmented radiation therapy and liposomal drug showed significant growth delay compared to the other groups, confirming the predictions made in the dual energy CT imaging.

Finally, a protease-responsive contrast agent was developed for use with dual energy CT imaging. Clusters of gold nanoparticles cross-linked together by protease-sensitive peptides were injected into mice along with liposomal iodine. In the presence of tumor proteases, the clusters degraded and the concentration of gold within the tumor decreased. Clusters without the protease-sensitive peptide did not degrade and did not leave the tumors. The ratio of iodine to gold in each tumor was measured, and it was found that the ratio was significantly higher in mice injected with the degradable gold clusters compared to mice injected with non-degradable control clusters. This demonstrated the ability to use multiple contrast agents with dual energy CT for enzyme-specific ratiometric molecular imaging.

This work confirms that dual energy CT can be used along with multiple nanoparticle contrast agents for molecular imaging applications. With continued contrast agent development and further application of dual energy CT, these methods can potentially be applied clinically to improve the power of CT imaging and improve diagnosis of a wide variety of pathologies.

Mortality and morbidity rates caused by acute liver failure (ALF), acute-on-chronic liver failure (ACLF) and chronic liver disease continue to rise because of drug induced failure or viral hepatitis, currently with 2,000 cases annually in the United States. Liver transplantation is the only intervention that has shown the most promising patient outcomes, but this approach has major shortcomings like shortage of donor livers, lifelong immunosuppression and a risk of organ rejection after transplantation. Additionally, with rapid liver deterioration and subsequent multi-organ failure characterized by ALF and ACLF conditions, there are high mortality rates as patients await a liver transplant or wait for their livers to regenerate. As such, bioartificial liver support devices provide an alternative to improve patient survival through either offloading liver functions to allow for liver regeneration or by allowing the patient time to receive a liver transplant. These bioartificial liver support devices are designed to perform essential liver functions through incorporation of an active cellular component that performs the liver functions and their success is therefore heavily reliant on the performance of the incorporated cell lines. Because of this, limited sources of these characteristic cell lines with hepatic function is a great challenge being faced in the research and development of the devices.

Adipose-derived stem cells (ADSCs) are a great candidate as a stem cell source for differentiation of hepatocyte-like cells because they can be easily obtained in large quantities with little donor site morbidity or discomfort and have been successfully differentiated into multiple cell lineages. In this study, we investigate the possibility of differentiating human ADSCs into functional hepatocyte-like cells. Furthermore, we investigated the ability to differentiate ADSCs into hepatocyte-like cells in both 2D and 3D environments. We found that induced ADSCs can produce high levels of some hepatocyte functions, like albumin secretion. However, other functions, like urea secretion and cytochrome P450 metabolic activity, while present, are not yet at sufficient levels to be comparable to primary hepatocytes.

Macrophages, key cells of the immune system, are often present at active sites of angiogenesis. It has been found that macrophages can play a critical role in supporting blood vessel development as the removal of these cells results in impaired vessel development. Because of the supportive role macrophages can play in vessel formation, macrophages can be considered as a novel cell source to support vessel development

Within the field of tissue engineering, one major limitation towards the development of large scale tissues is the need for vascularization of the tissues to support oxygen and nutrient demands. The need for vascularization combined with the roles of macrophages in vessel development introduce a unique opportunity to utilize cells of the immune system (in this case, macrophages) to support vessel development within tissue engineered constructs. In this thesis, we identify the roles of macrophages in vessel development utilizing a cell-adhesive and proteolytically-degradable poly(ethylene glycol) (PEG)-based hydrogel.

In our initial studies, we introduced the notion that macrophages enhance vessel formation of endothelial cells when both cells are simultaneously encapsulated into the PEG-based hydrogel. We next assessed the macrophage response to the presence of endothelial cells in our PEG-based hydrogel. Macrophages became more spread depending on the density of endothelial cells they were encapsulated with. We found that a 1:1 ratio of endothelial cells to macrophages resulted in the most spread population of macrophages within the PEG-based hydrogels. Macrophages also closely associated with endothelial cells in a proximity dependent manner; macrophages closest to endothelial cells were more spread than macrophages further away from the endothelial cells. We next classified the types of associations seen between the macrophages and the endothelial cells: macrophages closely associating with endothelial cells and macrophages bridging neighboring endothelial cells. The close association seen between the macrophages and endothelial cells mimics the close contact seen between endothelial cells and support cells. The bridging association seen mimics the cell-chaperoning behavior of macrophages during in vivo vessel formation. It has been seen that macrophages can physically connect two endothelial tip cells, thus acting as the cell-chaperone. The bridging association seen in this work complements the cell-chaperone behavior seen in vivo.

This work also explored the roles of macrophage phenotypes in governing the role of macrophages in vessel formation. Macrophages are highly plastic cells that alter their function based on environmental cues. There are two main paradigms of macrophage phenotypes: M1, pro-inflammatory macrophages, and M2, pro-tissue healing macrophages. This work explored the roles of macrophage phenotypes to vessel formation in the PEG-based hydrogels. M0 and M2 macrophages were found to support vessel development when encapsulated with endothelial cells. M1 macrophages significantly retarded vessel formation when encapsulated with endothelial cells. The endothelial cell and M2 macrophage co-culture secreted VEGF while the M1 macrophages retarded endothelial cell proliferation.

Due to the diverging effects of macrophage phenotype on vessel formation, we developed of PEG-based hydrogels capable of presenting a stimulating microenvironment to macrophages and endothelial cells. We found that a M2 stimulating hydrogel enhanced vessel formation when endothelial cells and macrophages were encapsulated in the hydrogel.

Overall, this dissertation demonstrates the role of macrophages in supporting vessel formation in PEG-based hydrogels. Findings in this thesis have helped to elucidate the diverging roles of macrophage phenotypes in supporting vascularization of PEG-based hydrogels. Moreover, this work has created PEG-based materials can be manipulated macrophage phenotype. This work highlights the usefulness of macrophages in vessel development and the usefulness of a macrophage-directing platform to enhance vascularization of tissue engineered constructs.

The aortic valve regulates the unidirectional flow of oxygenated blood from the left ventricle to the systemic circulation. When severe congenital defects occur in aortic valves, valve replacement is inevitable in children. However, current options including mechanical valves and bioprosthetic valves, lack the ability to grow and remodel, which necessitates multiple valve replacements as children grow. Tissue engineering provides a possible avenue to generate a living valve substitute that can grow and remodel via combining cells, scaffolds and environmental cues. The cells used in this work were valvular interstitial cells (VICs), the predominant cell population in the valves, and responsible for extracellular matrix (ECM) synthesis in the valve tissue. VICs are highly heterogeneous and dynamic in phenotype, with the majority assuming a quiescent, fibroblast phenotype in healthy adult valves1,2. During valve injury or disease conditions, VICs may undergo myofibroblast activation or osteogenic differentiation3,4. Myofibroblast activation is characterized by the expression of smooth muscle -actin (SMA), and may cause valve fibrosis3,4; osteogenic differentiation is characterized by the upregulation of alkaline phosphatase (ALP), followed by tissue calcification, which is the leading cause of valve disease in the elderly (>60 years of age) and the failure of bioprosthetic valves5. However, the most common method of in vitro VIC culture on two-dimensional (2D) stiff substrates leads to myofibroblast activation of VICs. For better physiological relevance and future application in valve substitutes, there is a need to understand and regulate VIC behaviors within three-dimensional (3D) scaffolds that are more reminiscent to their native environments. This dissertation describes the development of a poly(ethylene glycol) (PEG)-based hydrogel platform to support VIC growth in 3D, and the exploration of free and immobilized bioactive cues to dictate VIC phenotype and behaviors toward the development of a living valve substitute.

Otherwise bioinert, PEG hydrogels were functionalized with cell-adhesive ligands RGDS and proteolytically degradable sequences (GGGPQGIWGQGK). The functionalized hydrogels supported VIC growth, proliferation and ECM remodeling (secretion of matrix metalloproteinase-2 and deposition of collagens) in 3D during the culture period of 4 weeks. The soft hydrogels with compressive moduli of ~4.3 kPa quickly reverted VICs from myofibroblast activation to a quiescent phenotype upon encapsulation, evidenced by the loss of αSMA expression. The functionalized PEG hydrogels are preferable to 2D stiff substrates for preservation of the native phenotype of VICs and resistance to calcification.

In an effort to potentially promote deposition of ECM components by encapsulated VICs, ascorbic acid (AA), which is a cofactor in the post-translational modification of collagen molecules6 and has been reported to increase collagen section by several other cell types7–9, was added to the culture media of cell-laden hydrogels. AA treatment promoted VIC-mediated ECM remodeling without negatively influencing their quiescent phenotype in hydrogels. AA also enhanced VIC spreading and proliferation while inhibiting apoptosis.

ECM-mimicking adhesive peptides with specific affinity to different receptors were immobilized on PEG hydrogels in order to regulate VIC adhesion, phenotype and ECM production. Expression of adhesion receptors by VICs was assessed via flow cytometry and used to guide the choice of peptides studied. The peptide RGDS with affinity to multiple integrin receptors, and specific receptor-targeting peptides DGEA (integrin 21), YIGSR (67 kDa laminin/elastin receptor; 67LR), and VAPG (67LR) were chosen based on the receptor expression profiles as well as the potential outcomes of each receptor binding. DGEA, YIGSR, and VAPG alone were insufficient to induce stable VIC adhesion. As a result, these peptides were studied in combination with 1 mM RGDS. For VICs cultured on 2D hydrogel surfaces, YIGSR and VAPG down-regulated the expression of αSMA (myofibroblast activation marker) whereas DGEA promoted VIC adhesion and VIC-mediated ECM deposition while inhibiting the activity of ALP (osteogenic differentiation marker). Further, YIGSR and DGEA in combination promoted ECM deposition while inhibiting both myofibroblastic and osteogenic differentiation. However, VICs behaved differently when cultured within 3D hydrogels, with VICs assuming a quiescent, fibroblastic phenotype without any calcification under all peptide conditions tested. DGEA promoted ECM deposition by VICs within hydrogels without causing VIC activation.

The results of this research provide a clearer understanding of VIC biology and pathology under biomimetic conditions and lay the groundwork for constructing living valve substitutes using the tissue engineering approach. The hydrogel platform developed in this work may also be applied to study the initiation and progression of valvular diseases.

Cancer is currently the second leading cause of death in the United States. Although many treatment options exist, some of the most common, including radiotherapy and chemotherapy, are restricted by dose-limiting toxicities. In addition, the largest hurdle for translating novel biological therapies such as siRNA into the clinic is lack of an efficient delivery mechanism to get the therapeutic into malignant cells. This work aims to improve this situation by engineering a minimally invasive controlled release system that specifically delivers therapeutics to the site of malignant tissue. This platform consists of two novel material components: a thermally responsive poly[N-isopropylacrylamide-co-acrylamide] (NIPAAm-co-AAm) hydrogel and gold-silica nanoshells. Therapeutic molecules are encapsulated within a poly(NIPAAm-co-AAm) hydrogel carrier, leading to increased serum stability, circulation time, and decreased exposure to off-site tissues. Additionally, gold-silica nanoshells embedded within this hydrogel will be used to optically trigger therapeutic release from the carrier. This hydrogel-nanoshell composite material was designed to be swollen under physiologic conditions (37 oC), and expel large amounts of water and absorbed molecules at higher temperatures (40-45 oC). This phase transition can be optically triggered by embedded gold-silica nanoshells, which rapidly transfer near-infrared (NIR) light energy into heat due to the surface plasmon resonance phenomena. NIR light can deeply penetrate biological tissue with little attenuation or damage to tissue, and upon exposure to such light a rapid temperature increase, hydrogel collapse, and drug expulsion will occur. Ultimately, these drug-loaded hydrogel-nanoshell composite particles would be injected intravenously, passively accumulate in tumor tissue due to the enhanced permeability and retention (EPR) effect, and then can be externally triggered to release their therapeutic payload by exposure to an external NIR laser. This dissertation describes the synthesis, characterization, and validation of such a controlled therapeutic delivery platform.

Initial validation of poly(NIPAAm-co-AAm)-gold nanoshell composites to act as a material in site-specific cancer therapeutic delivery was accomplished using bulk hydrogel-nanoparticle composite disks. The composite material underwent a phase transition from a hydrated to a collapsed state following exposure to NIR light, indicating the ability of the NIR absorption by the nanoshells to sufficiently drive this transition. The composite material was loaded with either doxorubicin or a DNA duplex (a model nucleic acid therapeutic), two cancer therapeutics with differing physical and chemical properties. Release of both therapeutics was dramatically enhanced by NIR light exposure, causing 2-5 fold increase in drug release. Drug delivery profiles were influenced by both the molecular size of the drug as well as its chemical properties.

Towards translation of this material into in vivo applications, the hydrogel-nanoshell composite material was synthesized as injectable-sized particles. Such particles retained the same thermal properties as the bulk material, collapsing in size from ~330 nm to ~270 nm upon NIR exposure. Furthermore, these particles were loaded with the chemotherapeutic doxorubicin and NIR exposure triggered a burst release of the drug payload over only 3 min. In vitro, this platform provided increased delivery of doxorubicin to colon carcinoma cells compared to free-drug controls, indicating the irradiated nanoshells may increase cell membrane permeability and increase cellular uptake of the drug. This phenomena was further explored to enhance cellular uptake of siRNA, a large anionic therapeutic which cannot diffuse into cells easily.

This work advances the development of an injectable, optically-triggered delivery platform. With continued optimization and in vivo validation, this approach may offer an novel treatment option for cancer management.

Tumor growth, survival, and metastasis depend upon interactions with the matrix and cells which compose the tumor microenvironment (TME). Tumor cell interactions with transformed extracellular matrix (ECM) and immune cells, such as tumor associated neutrophils (TANs) have been shown to affect tumor progression both clinically and in animal models. Unfortunately, while the complexity of the TME is difficult to recapitulate in standard cell culture, it is also difficult to analyze and to influence in vivo. Researchers have sought to circumvent these challenges by developing 3D models in naturally derived matrices like collagen and Matrigel, but these scaffolds often sacrifice biochemical tunability and fail to meet the stiffness regimes often seen in malignant ECM. Thus, there is a need for highly controlled 3D culture systems which mimic the biophysical properties and cell-cell interactions of the TME to better investigate how these interactions direct cancer development and progression. To meet this goal, researchers have employed synthetic hydrogel systems for 3D in vitro cell culture. Our lab has previously engineered a synthetic scaffold to serve as an ECM by incorporating a matrix metalloproteinase cleavable peptide into a biocompatible poly(ethylene glycol) (PEG) backbone and grafting in an integrin-binding peptide. This system allows independent tuning of adhesivity and matrix stiffness and supports tumor cell growth and spheroid formation. Here, we present two applications of this system to model cell-matrix interactions in the TME. First, we orthogonally tuned the adhesion ligand concentration and stiffness of our PEG-based hydrogels to investigate the individual and interactive impact of these matrix properties in a physiologically relevant regime. We assessed the tumor progression of a fibrosarcoma cell line (derived from mesenchymal cells) and a triple-negative breast carcinoma cell line (TNBC, derived from epithelial cells) cultured in and on these hydrogels. We observed that the cell proliferation, invasion, and focal complex formation in the fibrosarcoma cells responded to changes in matrix stiffness, while the same behaviors in the TNBC cell line occurred in response to changes in matrix adhesivity. to discern the differential behavior of these broad classes of tumors. We found no interactive effect between the two matrix properties within the conditions tested. These results helped to reiterate the importance of independently tunable systems in assessing cell response to specific matrix properties and established our system’s ability to discern differential tumorigenic behavior across diseases. Next, we incorporated neutrophil extracellular traps (NETs) in our system to study their impact on tumorigenesis in TNBC cells in a highly controlled environment. We observed that NETs helped to increase cell survival, proliferation, and pro-metastatic morphological phenotype. We also showed that the presence of NETs influenced the secretion of IL-8, a pro-NETosis factor, and that conditioned media from cells cultured in these gels influenced NETosis in an IL-8 dependent manner. The results observed in this system correlate with murine models and clinical studies in the literature and help to provide additional insight of the individual factors at play in the NET-mediated promotion of TNBC progression and metastasis. To expand upon current models of cell-matrix interactions within the TME, we applied an existing model to assess the differences in behavior between a fibrosarcoma and TNBC cell line and developed a new model for assessing NETs as a matrix biomolecule in TME models. These two reductionist models help to advance our understanding of the roles specific aspects of the TME play in tumor progression, ultimately helping to drive better rational design of TME-targeted therapies to improve clinical outcomes of cancer patients.

Liver tissue engineering has made tremendous progress over the last decades, but continues to be limited by dedifferentiation of hepatocytes and insufficient vascularization of engineered constructs.1 Functional hepatocytes are the parenchymal liver cells and they are responsible for many of the over 500 essential liver functions. Thus, they are necessary for advancement toward effective liver disease treatments, predictive models for drug screening, and whole-organ tissue engineering.2

Liver-specific functions of hepatocytes are known to be regulated by complex microenvironmental cues that are not fully understood and are difficult to recapitulate in vitro. These signals are known to include homotypic interactions with other hepatocytes, heterotypic interactions with non-parenchymal cells, biochemical cues from surrounding extracellular matrix, and signals and sustenance from the underlying vasculature.3 The objective of this work was to investigate material properties and culture conditions that are conducive to the long-term culture of functional primary hepatocytes in order to design a platform capable of supporting them. We found that coculturing hepatocytes in adhesive and degradable hydrogels with capillary forming endothelial cells (EC) and supporting pericytes formed vascularized liver tissue-like constructs. Over time, we found that hepatocytes and vascular cells in these constructs formed close synergistic associations. While hepatocytes enhanced vascularization, microvascular networks supported at least 3 major hepatocyte functions for at least 2 months.

Then we translated our platform into a bioartificial liver for acute liver failure therapy. Bioartificial livers, are extracorporeal life-saving treatments similar to dialysis for patients with acute liver failure who are awaiting a transplant. These devices often to employ hepatocytes in cartridges filled with semi-porous hollow fibers to perfuse patient blood and temporarily off-load the patient’s liver functions, reducing blood toxicity and preserving the function of other organs in the body while the liver repairs itself. However, they are limited in that the hepatocytes used are either short-lived hepatocytes or hepatocyte derivatives that have lost essential functions during their transformation. They are also enormously large, costly, challenging to maintain, and difficult to use. Since our platform is prevascularized and capable of supporting hepatocyte function, we examined its potential to alleviate some of these issues.

Not only were ECs and pericytes able to form robust vascular networks in the context of a perfused hollow fiber-based cartridge, but they were able to anastomose to the hollow fibers and enhance mass transport of nutrients into the surrounding hydrogel and hepatocytes. Thus, we developed a module that effectively bridges macro-to-micro scale mass transport using hollow fibers as conduits for efficient, artery scale fluid transport and cell-assembled microvasculature to perfuse the engineered tissue and facilitate exchange of nutrients and metabolic waste with functional cells, interstitial fluids, and the bulk fluid from the hollow fiber. Upon the addition of hepatocytes in the bioreactor platform, we observed maintenance of liver synthetic and metabolic functions for at least 1 month, the length of time it takes a severely damaged liver to regenerate without further complications. The findings in this work serve as a proof-of-concept for future studies that further optimize the bioreactor further and examine its potential toward viable liver disease treatments.