A new bioengineered 3D tumor platform in vitro to replicate tumor-stroma interaction and investigate anti-cancer drug delivery

Nowadays, cancer is still a second leading cause of death after cardiovascular disease in the world. One of the main factors that lead the failure of cancer therapy is related to the fact that little is known about the interaction of cancer cells with microenvironment. Indeed, as hypothesized in the "seed" and "soil" theory by Paget over a century ago, tumor progression is determined not only by tumor cells but also by the surrounding stromal milieu. For these reasons in the last years, increasing attention is focused on the importance of tumor microenvironment in an effort to develop successful strategies in cancer disease treatment. In traditional two-dimensional in vitro models the absence of 3D architecture generates misleading and contradictory results. Hence emerges the need to have an in vitro versatile platform that closely recapitulates pathophysiological features of the native tumor tissue and its surrounding microenvironment. In this PhD thesis a microtissue precursors assembling strategy (µTP), was used and translated to produce 3D tumor engineered models composed by tumor and/or stromal cells. In contrast with the classical spheroid model, the µTP we proposed presents the production of extracellular matrix directly synthesized by stromal cells. First of all, in the chapter 1 a stat of art overview was presented which highlights the importance of tumor microenvironment in cancer research, the existing models for studying tumor development and the nanotechnology contribution in cancer treatment. Then the chapter 2 is focalized on the realization of stromal microtissues fabricated seeding normal or activated fibroblasts on microporous beads, in order to monitor their dynamic evolution in terms of metabolic activity, mechanical properties and ECM composition. In particular it is demonstrated how the microtissue configuration is able to keep phenotypic differences between normal and activated fibroblasts in all the aspects investigated compared to the classical 3D spheroidal model. In the chapter 3 the cross talk between epithelial tumor and the surrounding stroma in a microfluidic device is investigated. Thanks to the combination of 3D microtissues with microfluidic technology, it is possible to detect in real time the modification occurring at cellular and ECM level during the activation period. In the second part of work, the tumor microtissue model is validated as a potential drug-screening platform. In particular, in chapter 4 a commonly drug used in chemotherapy (Doxorubicin) is tested in order to detect the difference in chemoresistance between microtissues and spheroid models, both in monoculture and coculture. Finally, a stimuli-responsive nanoparticles are tested on normal and tumor 3D heterotypic microtissues to demonstrate their significant selectively. At last, the microtissue system may be a useful \emph{in vitro} screening tool for testing innovative approaches of drug delivery, reducing expensive and time-consuming protocol nowadays used in preclinical studies

Structure-Function Studies of Scaffolding Proteins Involved in the Formation of Neuronal Connections: AIDA1 and CASKIN2

Modular proteins serve assembly platforms and often actively regulate cellular signaling events. An intrinsic diversity of interaction modules, typical for scaffolding proteins, facilitates the organization of numerous protein partners into signaling cascades, contributing to the spatial precision, efficiency and fidelity of signal transduction. The role of complex molecular dynamics of postsynaptic density (PSD) proteins in synaptic plasticity is relatively new and yet to be fully understood. AIDA-1 is one of the most abundant members of the PSD protein family. Growing research evidence of multiple protein partnerships suggests that AIDA-1 functions as an essential PSD molecular scaffold, NMDA receptor functional mediator, and a synapse-to-nucleus messenger. The NMR structure of AIDA-1 carboxy-terminal phosphotyrosine binding domain (PTB), presented in this study, provided the structural basis for comparative analysis with the other PTB domain-containing proteins, Fe65 and X11/Mint1, that also participate in amyloid beta precursor protein (APP) processing and amyloid beta peptide (A) secretion. A combination of peptide arrays, mutagenesis and fluorescence based assays was employed to characterize the affinity and specificity of the AIDA-1 PTB domain and APP intracellular domain (AICD) interaction. Another modular protein of these studies is a pre-synaptic scaffolding protein, Caskin2. Presently, its function within the synapse is less clear compared to its more widely studied homolog, Caskin1. However, the structural differences between the two identified by our research suggest the possibility of distinct functional outcomes in the neuron. We demonstrated that Caskin2 Sterile Alpha Motif (SAM) assembles into an oligomeric architecture different from Caskin1, with the minimal repeating unit being a dimer, rather than a monomer. In invertebrates, Caskin has been functionally linked to LAR receptor tyrosine phosphatase functional pathways, implicated in axonogenesis and synaptogenesis. Using a combination of biophysical and biochemical methods, the partnership between Caskin2 and LAR Homo sapiens homologs was confirmed and characterized. These integrated structural and functional studies provide a platform for further elucidation of AIDA-1 and Caskin cellular functions

Engineered microtissue platforms for modeling human pathophysiology and drug metabolism

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2013.Cataloged from PDF version of thesis.Includes bibliographical references (p. 139-160).Over 50% of all drug candidates entering clinical trials are abandoned due to insufficient efficacy or unexpected safety issues despite extensive pre-clinical testing. Liver metabolites that cause toxicity or other side effects cannot always be predicted in animals, in part because of human-specific drug metabolism. Furthermore, while the clinical need for cancer drugs is increasing, anti-tumor activity in animals often leads to a disappointing lack of efficacy in real patients. In vitro models that can better predict human responses to drugs would mitigate the overall costs of development and help bring new therapies to market. In order to improve the predictive power of in vitro tissue models, various features of the microenvironment that modulate cell behavior have been investigated, such as cell-cell interactions, cell-matrix interactions, soluble signals, 3-dimensional (3D) architecture, and mechanical stiffness. Synthetic hydrogels offer a versatile platform within which these cues can be precisely perturbed in a 3D context; however, the throughput of these methods is quite limited. In this thesis, we explore the potential of high-throughput manufacturing and monitoring of populations of miniaturized 3D tissues, termed 'microtissues,' for modeling healthy and diseased tissues in both static and perfused systems. First, we developed a flow-based platform to test tumor proliferation in defined microenvironmental settings with large numbers of replicates (n > 1000). A microfluidic droplet generator was designed to encapsulate tumor cells with stromal cells and extracellular matrix in 100 pm-diameter poly(ethylene glycol) (PEG) microtissues (6000 microtissues/min). Upon screening a small panel of soluble stimuli, TGF-p and the TGF-pR1/2 inhibitor LY2157299 were found to have opposing effects on the proliferation of lung adenocarcinoma cells in microtissues vs. in 2-dimensional culture, affirming a potential role for 3D models in the investigation of cancer therapies. Next, we extend these techniques to the analysis of drug-induced liver injury. Phenotypic maintenance of primary hepatocytes was achieved by controlled pre-aggregation (-50 tm units) with J2-3T3 fibroblasts to establish cell-cell contacts prior to encapsulation into microtissues. Retention of both constitutive and inducible Phase I drug metabolism activity allowed detection of prototypical hepatotoxins through generation of toxic metabolites and emergence of drug-drug interactions, thereby demonstrating the suitability of hepatic microtissues for 3D, high-throughput toxicity screening. Finally, we describe efforts to bridge the gap between multi-organ models and human drug metabolism. Modular human hepatocyte microtissues were entrapped by semi-circular microsieves in a microfluidic perfusion chamber for over 3 weeks. In contrast to immortalized hepatic cell lines, primary hepatocytes stabilized in microtissues exhibited human-specific induction profiles, reflected donor hetereogeneity in CYP2D6 and CYP2C19 enzyme activity levels, and performed xenobiotic detoxification on circulating drugs, establishing the ability to incorporate hepatic functions in 'human-on-a-chip' devices. Collectively, these three applications of cell-laden microtissues demonstrate their versatility and potential impact in both drug development and fundamental studies of the cellular microenvironment.by Cheri Yingjie Li.Ph.D

Pattern Formation and Organization of Epithelial Tissues

Developmental biology is a study of how elaborate patterns, shapes, and functions emerge as an organism grows and develops its body plan. From the physics point of view this is very much a self-organization process. The genetic blueprint contained in the DNA does not explicitly encode shapes and patterns an animal ought to make as it develops from an embryo. Instead, the DNA encodes various proteins which, among other roles, specify how different cells function and interact with each other. Epithelial tissues, from which many organs are sculpted, serve as experimentally- and analytically-tractable systems to study patterning mechanisms in animal development. Despite extensive studies in the past decade, the mechanisms that shape epithelial tissues into functioning organs remain incompletely understood. This thesis summarizes various studies we have done on epithelial organization and patterning, both in abstract theory and in close contact with experiments. A novel mechanism to establish cellular left-right asymmetry based on planar polarity instabilities is discussed. Tissue chirality is often assumed to originate from handedness of biological molecules. Here we propose an alternative where it results from spontaneous symmetry breaking of planar polarity mechanisms. We show that planar cell polarity (PCP), a class of well-studied mechanisms that allows epithelia to spontaneously break rotational symmetry, is also generically capable of spontaneously breaking reflection symmetry. Our results provide a clear interpretation of many mutant phenotypes, especially those that result in incomplete inversion. To bridge theory and experiments, we develop quantitative methods to analyze fluorescence microscopy images. Included in this thesis are algorithms to selectively project intensities from a surface in z-stack images, analysis of cells forming short chain fragments, analysis of thick fluorescent bands using steerable ridge detector, and analysis of cell recoil in laser ablation experiments. These techniques, though developed in the context of zebrafish retina mosaic, are general and can be adapted to other systems. Finally we explore correlated noise in morphogenesis of fly pupa notum. Here we report unexpected correlation of noise in cell movements between left and right halves of developing notum, suggesting that feedback or other mechanisms might be present to counteract stochastic noise and maintain left-right symmetry.PHDPhysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/138800/1/hjeremy_1.pd

Cell adhesion and cell mechanics during zebrafish development

During vertebrate development, gastrulation leads to the formation of three distinct germlayers. In zebrafish a central process is the delamination and the ingression of single cells from a common ancestor tissue - that will lead to the formation of the germlayers. Several molecules have been identified to regulate this process but the precise cellular mechanisms are poorly understood. Differential adhesiveness, a concept first introduced by Steinberg over 40 years ago, has been proposed to represent a key phenomena by which single hypoblast cells separate from the epiblast to form the mesendoderm at later stages. In this work it is shown that differential adhesion among the germlayer progenitor cells alone cannot predict germlayer formation. It is a combination of several mechanical properties such as cell cortex tension, cell adhesion and membrane mechanical properties that influence the migratory behavior of the constituent cells

Biomaterials‐Based Approaches to Tumor Spheroid and Organoid Modeling

Evolving understanding of structural and biological complexity of tumors has stimulated development of physiologically relevant tumor models for cancer research and drug discovery. A major motivation for developing new tumor models is to recreate the 3D environment of tumors and context‐mediated functional regulation of cancer cells. Such models overcome many limitations of standard monolayer cancer cell cultures. Under defined culture conditions, cancer cells self‐assemble into 3D constructs known as spheroids. Additionally, cancer cells may recapitulate steps in embryonic development to self‐organize into 3D cultures known as organoids. Importantly, spheroids and organoids reproduce morphology and biologic properties of tumors, providing valuable new tools for research, drug discovery, and precision medicine in cancer. This Progress Report discusses uses of both natural and synthetic biomaterials to culture cancer cells as spheroids or organoids, specifically highlighting studies that demonstrate how these models recapitulate key properties of native tumors. The report concludes with the perspectives on the utility of these models and areas of need for future developments to more closely mimic pathologic events in tumors.State‐of‐the‐art approaches using natural, synthetic, and composite biomaterials for 3D tumor modeling are presented in this Progress Report. Furthermore, it is discussed how these models uniquely reproduce key properties of native tumors to facilitate basic and applied cancer research and cancer drug discovery efforts.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142941/1/adhm201700980.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142941/2/adhm201700980-sup-0001-S1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142941/3/adhm201700980_am.pd

Contributions of cluster shape and intercellular adhesion to epithelial discohesion and emergent dynamics in collective migration

As a physical system, a cell interacts with its environment through physical and chemical processes. The cell can change these interactions through modification of its environment or its own composition. This dissertation presents the overarching hypothesis that both biochemical regulation of intercellular adhesion and physical interaction between cells are required to account for the emergence of cluster migration and collective dynamics observed in epithelial cells. Collective migration is defined as the displacement of a group of cells with transient or permanent cell-cell contacts. One mode, cluster migration, plays an important role during embryonic development and in cancer metastasis. Despite its importance, collective migration is a slow process and hard to visualize, and therefore it has not been thoroughly studied in three dimensions (3D). Based on known information about cluster migration from 2D studies of epithelial sheets and 3D single cell migration, this dissertation presents theoretical and experimental techniques to assess the independent contribution of physical and biochemical factors to 3D cluster migration. It first develops two computational models that explore the interaction between cells and the ECM and epithelial discohesion. These discrete mechanistic models reveal the need to account for intracellular regulation of adherens junctions in space and time within a cluster. Consequently, a differential algebraic model is developed that accounts for cross-reactivity of three pathways in a regulatory biochemical network: Wnt/β-catenin signaling, protein N-glycosylation, and E-cadherin adhesion. The model is tested by matching predictions to Wnt/β-catenin inhibition in MDCK cells. The model is then incorporated into a self-propelled particle (SPP) model, creating the first SPP model for study of adhesive mammalian cellular systems. MDCK cell clusters with fluorescent nuclei are grown, seeded, and tracked in 3D collagen gels using confocal microscopy. They provide data on individual cell dynamics within clusters. Borrowed from the field of complex systems, normalized velocity is used to quantify the order of both in vitro and simulated clusters. An analysis of sensitivity of cluster dynamics on factors describing physical and biochemical processes provides new quantitative insights into mechanisms underlying collective cell migration and explains temporal and spatial heterogeneity of cluster behavior

Development of a three-dimensional organotypic skin model for in vitro study of skin disease state

The skin functions as a protective physical barrier against the outside and has a primary role to protect body from external influences such as pathogenic microorganisms and mechanical injuries. Moreover in the skin, the interaction of keratinocytes, fibroblasts and melanocytes is tightly controlled by various factors and cascades. Under normal conditions, there is a balance between cell types via cell–cell contact and the extracellular matrix (ECM). The ECM provides structural scaffolding for cells, as well as contextual information. The disruption of these equilibria can result in an uncontrolled stroma degeneration (as involved in scarring) or uncontrolled proliferation of epithelial cells (as in malignant melanoma). Wound healing is a highly organized series of processes resulting in tissue integrity and function of the damaged tissue; this process needs a complex microenvironement to be studied. Carcinogenesis is defined as complex, adaptive process, controlled by intricate communications between the host and the tissue microenvironment. Thus, the microenvironment and the stroma play an important role in both wound healing process and tumour development. In this perspective, in the present PhD thesis a tissue engineering bottom-up approach was used to fabricate a 3D dermis tissue. This model was composed by a cell-synthesized and responsive extracellular matrix that resembles the in vivo dermis, and it was used as living platform to study in vitro skin alteration and diseases. The first model of skin alteration dealt with the wound healing process, by exploiting its self-repairing capability. Interestingly, the relationship between cell migration, differentiation marker and ECM production and remodeling during repair process followed the same in vivo timing. Moreover, the presence of a responsive dermis allowed possibility to evaluate granulation tissue and to study and understand processes involved in scarring. Indeed, due to the endogenous nature of the stroma, the model proposed could represent a valuable tool to in vitro study tissue status at both cellular and extracellular level after a physical damage. At least, once demonstrated dermis responsivity, we investigated epidermal counterpart. In this perspective, we fabricated a 3D human skin equivalent (3D-HSE) model with the same endogenous stroma as dermis component to study cell-ECM – with native basement membrane (BM) – and cell–cell communications, in the presence of an aggressive form of skin cancer: melanoma. As a fact, carcinogenesis can disrupt these forms of communication, thus altering cell biology of human skin. Consequently, we investigated the role of skin cells and BM components on melanoma biology and invasive ability in reconstructed human skin equivalent. We first made-up and characterized a human skin model that resembled the architecture of skin in situ, than we carried out an analogous procedure for the equivalent engineered tumor model. On the basis of our results, we can assert that there is communication between skin cells and melanoma cells and the outcome is dictated by the nature of the melanoma cells. Thus, the bioengineered 3D melanoma skin model may become a valuable tool to investigate the underlying mechanics of melanoma infiltration. The proposed study does not recapitulate yet melanoma metastasis process as a whole; however, the present engineered 3D tissue represents a reliable model for investigating the phenotype and behavior of melanoma cells derived from primary sites. Indeed, the 3D melanoma skin model is suitable to study the biological properties of radial growth phase invasion. This study represents a preliminary model for investigating all aspects of melanoma metastasis and it has great potential for improving our understanding of the interactive biology between melanoma cells and their immediate surroundings and evaluating melanoma cells influence on epidermis structure and differentiation. In conclusion, the present 3D engineered skin model represents a valid platform to study scar formation and a valuable tool for studying healthy and disease skin or for screening test in vitro. Moreover, this platform could provide an in vivo skin substitute in clinical applications