Development of Methods to Accurately Quantify Cellular Viability and Multidrug-Resistant Phenotypes within 3D Paper-Based Tumor Models

During cancer progression, cells undergo genetic mutations and epigenetic alterations that, in coordination with influences from the tumor microenvironment, lead to more aggressive cellular phenotypes, which can survive within cytotoxic conditions. Not only can these cancer cells evade antineoplastic chemotherapies, but they adopt a highly invasive mesenchymal phenotype that facilitates their invasion into the surrounding healthy tissue and can lead to metastasis. Multidrug resistance is thought to be the cause of treatment failure for over 90% of cancer patients whose disease has progressed to metastasis. The tumor microenvironment consists of a complex network of vasculature, biotic and abiotic chemical gradients, stroma cells, and extracellular matrix that all play a role in cancer survival and progression. Of particular interest are the consumption-based chemical gradients, specifically oxygen, that form throughout poorly perfused tumors due to cancer cell proliferation outpacing vasculature growth. These gradients lead to the formation of distinct phenotypic regions throughout a tumor mass with well oxygenated proliferative cells adjacent to the vasculature and necrotic cells at the hypoxic tumor core. These cellular phenotypes have different sensitivities to chemotherapies, making tumor models that spatially replicate cellular phenotypes ideal platforms for studies into multidrug resistance. Paper-based cultures (PBCs) are a 3D culture platform that can model tumor cross-sections by stacking multiple layers of cell-laden paper scaffolds together. By limiting the exchange between the PBC stack and medium to a single side, consumption-based abiotic chemical gradients form across the stack. These structures can be easily sectioned into 40 μm thick slices by pealing each scaffold apart. This ability to simply unstack the culture provides a spatial resolution that is ideal for studying how discrete microenvironments throughout the tumor model effect cellular response. A key measurement when studying drug potency and efficacy is cell viability. Determining an accurate assessment of the cellular viability in response to chemotherapeutic dosing is vital not only for drug screens of novel compounds but also in-depth studies of multidrug resistance mechanisms. The majority of viability assays are designed for 2D monolayer cultures and utilizing them with 3D culture platforms requires additional considerations for proper assays selection as 3D cultures have added complexity, which can pose experimental challenges. Indirect viability assay measure cellular phenomena as a marker for viability and provide a bulk readout of the entire sample. In this work, three indirect, end-point viability assays, traditional used with 2D monolayer cultures, were characterized with 3D PBCs. The analytical figures of merit for each assay were determined from calibration curves and the effectiveness of each assay in quantifying small changes in cell number was assessed with dose-response curves. In addition, optical coherence tomography—a biomedical imaging technique that senses near-infrared light backscatter from tissues to identify viable cells—was adapted to PBCs and marks the first instance of in situ longitudinal measurements of a stacked PBC. Indirect viability assays are often sensitive to the cellular microenvironment that influences the cellular phenomena being measured. These environmental influences can alter the assay sensitivity and cause misleading results when chemical gradients are present throughout a sample, as is the case with a PBC tumor model. In this work, we evaluated the influence of the oxygen gradient throughout PBC tumor models on the assay performance of the indirect viability assays and optimized experimental protocols to circumvent this influence. In addition, we adapted flow cytometry, an environmentally independent, direct measurement of cell viability for PBCs. With flow cytometry we were able to determine the distribution of viable cells present throughout PBC tumor models and confirm that multidrug resistance occurs within the hypoxic regions of the tumor model. We are currently using flow cytometry to investigate the possible mechanisms of drug resistance that cells within the hypoxic regions of the tumor model are using to evade drug induced cell death. In summary, my dissertation work has focused on successfully adapting, optimizing, and validating viability assays with PBCs to study the invasive and multidrug-resistant phenotypes that form throughout invasion and tumor model cultures.Doctor of Philosoph

Assessing cell migration in hydrogels: An overview of relevant materials and methods

Cell migration is essential in numerous living processes, including embryonic development, wound healing, immune responses, and cancer metastasis. From individual cells to collectively migrating epithelial sheets, the locomotion of cells is tightly regulated by multiple structural, chemical, and biological factors. However, the high complexity of this process limits the understanding of the influence of each factor. Recent advances in materials science, tissue engineering, and microtechnology have expanded the toolbox and allowed the development of biomimetic in vitro assays to investigate the mechanisms of cell migration. Particularly, three-dimensional (3D) hydrogels have demonstrated a superior ability to mimic the extracellular environment. They are therefore well suited to studying cell migration in a physiologically relevant and more straightforward manner than in vivo approaches. A myriad of synthetic and naturally derived hydrogels with heterogeneous characteristics and functional properties have been reported. The extensive portfolio of available hydrogels with different mechanical and biological properties can trigger distinct biological responses in cells affecting their locomotion dynamics in 3D. Herein, we describe the most relevant hydrogels and their associated physico-chemical characteristics typically employed to study cell migration, including established cell migration assays and tracking methods. We aim to give the reader insight into existing literature and practical details necessary for performing cell migration studies in 3D environments.publishedVersio

DEVELOPMENT OF PAPER-BASED BREAST CANCER MODELS TO PROBE THE RELATIONSHIP BETWEEN THE TUMOR MICROENVIRONMENT AND THE ESTROGEN RECEPTOR ALPHA SIGNALING PATHWAY

Approximately 320,000 new cases of breast cancer were diagnosed in the United States in 2018, resulting in 41,000 deaths. Nearly 70% of these cancers express estrogen receptor alpha (ER) and rely on estrogenic molecules for tumor growth and progression. To treat these cancers, endocrine therapies that target the ER signaling pathway have been developed, yet 50% of recurrent ER (+) breast cancers develop resistance to these therapies. Recent work has shown that the tumor microenvironment plays an important role in modulating the ER signaling pathway, leading to the progression of these cancers and the development of endocrine resistance. A better understanding of how components of the tumor microenvironment regulate the ER signaling pathway is key to better treating ER (+) breast tumors. In vitro platforms afford researchers excellent experimental control, enabling studies that examine the effects of the tumor microenvironment on ER (+) breast cancer. Traditionally, two-dimensional (2D) monolayer cultures have been used. However, these cultures cannot adequately recapitulate the complexity of the tumor microenvironment, where cells extend in three dimensions and are suspended in an extracellular matrix (ECM). Three-dimensional (3D) culture formats restore key features of the tumor microenvironment including increased cell-cell and cell-ECM interactions, along with gradients of oxygen, nutrients, and signaling molecules, that better recapitulate the in vivo environment. Paper-based cultures are an emerging, modular 3D culture platform that, while simple to prepare, allow for excellent experimental control over the cell density and type, ECM composition and stiffness, and the formation of gradients across the culture. Paper-based cultures are prepared by wax-patterning thin paper scaffolds to outline discrete cell culture zones. Cell-laden hydrogels are embedded in the paper scaffolds, which increase the structural integrity of the hydrogel, and allow for easy manipulation and analysis of the culture. In this work, I developed a series of paper-based breast cancer models that enabled the study of how environmental exposures, tissue dimensionality, and hypoxia impact ER (+) breast cancer. In Chapter 2, I developed a paper-based screening platform capable of quantitatively assessing ER modulators in 96 chemically isolated 3D cultures. I measured the potency and efficacy of ER modulators in a 3D culture format with a luciferase-based reporter assay and quantified the invasion and proliferation of two ER (+) cell lines in the presence of estradiol. The results demonstrate the potential of this platform to support increasingly complex and physiologically relevant tissue-like structures for environmental chemical risk assessment. In Chapter 3, I investigated the role of hypoxia on the ER signaling pathway in a breast cancer cell line. In vitro 2D cultures suggest that hypoxia may lead to endocrine resistance by loss in ER activity that is driven by protein depletion. However, clinical samples show a positive correlation between hypoxia markers and ER expression. I compared the impact of hypoxia on ER protein expression and transcriptional activity, finding that hypoxia differentially regulates ER protein levels in a culture format-dependent manner. I investigated possible causes for this divergence in response observed between the two culture formats. The results from this study revealed that ER protein levels in hypoxia are not an accurate indicator of ER transcriptional activity in a 3D culture format, and highlight the importance of considering tissue dimensionality for in vitro studies of ER (+) breast cancer. In the effort to build more representative in vitro models, in Chapter 4 I constructed a paper-based breast tumor model that develops diffusion-generated gradients of oxygen throughout the model; these gradients are representative of hypoxia in vivo. I further studied the impact of hypoxia and estrogen and compared the results across three in vitro culture formats: 2D monolayer cultures, 3D single-zone cultures, and 3D tumor stacks. In each culture format, I compared the impact of hypoxia and estrogen on the expression of ER and HIF-1, ER transcriptional activation, cell viability, and gene expression. The results from this study indicate a clear divergence in response of ER (+) breast cancer cell lines to both estrogen and hypoxia and further highlight the importance of considering culture format for in vitro studies. In summary, my dissertation work focused on the development of physiologically-relevant paper-based breast cancer models that enabled studies of the impact of tumor microenvironment, namely environmental exposures and hypoxia, on ER (+) breast cancer. I developed a paper-based screening platform for ER modulators in monocultures of ER (+) cell lines, with the potential to support increasingly physiologically relevant tissue structures. I also developed a breast cancer tumor model, characterized the effects of hypoxia on the ER signaling pathway, and compared the responses from the 3D culture format to those observed in 2D cultures. Future work will continue the development of increasingly complex and physiologically relevant tissue and tumor paper-based structures, which permit the simple analysis of discrete cell populations from distinct chemical environments. These models will enable the identification of key microenvironmental regulators (i.e., environmental chemicals, oxygen, paracrine signaling) of the ER signaling pathway and the evaluation of cancer therapies in tumor models of varying, controllable microenvironmental conditions.Doctor of Philosoph

Scaffold dimensionality and confinement determine single cell morphology and migration

This thesis describes a highly interdisciplinary approach to discern the differing impact of scaffold dimensionality and physical space restrictions on the behavior of single cells. Rolled-up nanotechnology is employed to fabricate three-dimensional (3D) SiO/SiO2 microtube geometries of varied diameter, that after a biofunctionalization step are shown to support the growth of U2OS and six different types of stem cells. Cell confinement quantifiable through the given microtube diameter is tolerated by U2OS cells through a remarkable elongation of the cell body and nucleus down to a certain threshold, while the integrity of the DNA is maintained. This confinement for NSPCs also leads to the approaching of the in vivo morphology, underlining the space-restrictive property of live tissue. The dimensionality of the cell culture scaffold however is identified as the major determiner of NSPC migration characteristics and leads to a morphologically distinct mesenchymal to amoeboid migration mode transition. The 3D microtube migration is characterized by exclusively filopodia protrusion formation, a higher dependence on actin polymerization and adopts aspects of in vivo-reported saltatory movement. The reported findings contribute to the determination of biomaterial scaffold design principles and advance our current understanding of how physical properties of the extracellular environment affect cell migration characteristics

BIOENGINEERED SCAFFOLDS TO INDUCE ALIGNMENT AND PROMOTE AXON REGENERATION FOLLOWING SPINAL CORD INJURY

Scaffolds delivered to injured spinal cords to stimulate axon connectivity often act as a bridge to stimulate regeneration at the injured area, but current approaches lack the permissiveness, topology and mechanics to mimic host tissue properties. This dissertation focuses on bioengineering scaffolds through the means of altering topology in injectables and tuning mechanics in 3D-printed constructs as potential therapies for spinal cord injury repair. A self-assembling peptide scaffold, RADA-16I, is used due to its established permissiveness to axon growth and ability to support vascularization. Immunohistochemistry assays verify that vascularized peptide scaffolds promote axon infiltration, attenuate inflammation and reduce astrogliosis. Furthermore, magnetically-responsive (MR) RADA-16I injections are patterned along the rostral-caudal direction in both in-vitro and in-vivo conditions. ELISA and histochemical assays validate the efficacy of MR hydrogels to promote and align axon infiltration at the site of injury. In addition to injectable scaffolds, this thesis uses digital light processing (DLP) to mimic the mechanical heterogeneity of the spinal cord caused by white and gray matter, and demonstrate that doing so improves axon infiltration into the scaffold compared to controls exhibiting homogeneous mechanical properties. Taken together, this work contributes to advancing the field of tissue engineering and regenerative medicine by demonstrating the potential of bioengineered scaffolds to repair the damaged spinal cord

Cancer Tissue Engineering: development of new 3D models and technologies to support cancer research

The activity of research of this thesis focuses on the relevance that appropriate models reproducing the in vivo tumor microenvironment are essential for improving cancer biology knowledge and for testing new anticancer compounds. Animal models are proven not to be entirely compatible with the human system, and the success rates between animal and human studies are still unsatisfactory. On the other hand, 2D cell cultures fail to reproduce some aspects of tumor system. These limitations have a significant weight especially during the screening of novel antitumor drugs, as it was demonstrated that cells are less sensitive to treatments when in contact with their microenvironment. To obtain the same tumor cell inhibition levels observed in vivo, the culture environment has to reflect the 3D natural environment. Natural or synthetic hydrogels reported successful outcomes in mimicking ECM environment. During this PhD, I developed different gel-based scaffolds to be use as substrates for the culture of breast cancer cells. In detail, I developed different gels for low and highly aggressive cancer cell lines (i.e. MCF-7 and MDA-MB-231), obtaining significant results as regards the reproduction of key features normally present into the in vivo environment. Considering the importance of the metastasis process in breast cancer evolution, I then focused on a new set-up for the observation of cancer cell motility and invasion. In particular, I combined a bioreactor-based bioengineering approach with single cell analysis of Circulating Tumor Cells (CTCs). This part of work was carried out at the Dipartiment of Biomedicine of the University of Basel (CH) that, among its equipment, has a cell celector machine for single cell analysis. At the end of this work, I provided a proof-of-concept that the approach can work, as well as evidence that the cells can be extracted from the device and used for molecular analysis

Microfabricated platforms for epithelial cultures and cell-based assays

Epithelial cells, the major target of adult cancers have served as in vitro culture models for cancer related research for many years. Despite the increased use of these models, their potential as a cell-based screening tool for therapeutics has been hampered by the lack of existing miniaturized platforms that can: (1) facilitate single cell analysis (2) support cell cultivation in a physiological context, and (3) enable parallelization for highthroughput screening. Recent advancements in microfabrication technology has led to the development of miniaturized systems capable of handling very small (microliter – nanoliter) volumes of fluid, biomaterials, and bioparticle solutions.The central goal of this thesis was to develop miniaturized technologies that transcend the limitations of conventional macroscopic two-dimensional culture systems, by approaching the native environment of cells in vivo through technologies that allow direct screening of cultivated cells with drugs and targeted therapeutics in an appropriate context. To this end, we have developed miniaturized cell culture platforms towards three distinct applications: breast cancer research, liver biology/pathology, and studies of hepatitis B viral replication. Pertaining to breast cancer research, we have developed new techniques to create micropatterns of the most popular biomatrix for 3D epithelial cell culture (Matrigel). Moreover, we demonstrated that the micropatterned matrix can support the 3D cultivation of normal and breast cancer cell lines with comparable phenotypes to standard 3D culture techniques. In addition, by combining this micropatterned Matrigel with microfluidic techniques, we were able to develop a new platform to study individual cancer cell migration and invasion.Unlike previous demonstrations of micro-scale 3D systems that rely on just coating surfaces with thin layers of matrices, we took an approach that fully recapitulates standard 3D culture assays, where cells are totally embedded or overlayed with ECM to mimic the 3D microenvironment necessary to establish tissue-specific functions as well as mechanical and structural signals. Our development of a microfabricated platform for studying breast cancer cells can not only recapitulate normal epithelial structure and function but can also allow for more detailed understanding of tissue dysfunction in disease states and provides a more realistic milieu for modeling therapeutic intervention.We have also developed a novel “mini-liver” culture system using a microfluidic platform that simulates the basic functional unit of the liver (the hepatic sinusoid). The microfluidic platform is composed of a layered co-culture of hepatocytes and sinusoidal endothelial cells (the two major cell types in the liver) in a microchannel that mimics the liver sinusoid. Unlike previous liver models that rely on the culture of hepatocytes alone or random co-culture of hepatocytes with other cell types, we have developed a more realistic model that recreates important aspects of the liver including the architecture, fluidic environment, and more importantly the cellular composition. Moreover, we demonstrated the utility of our microchannel-based platform for meaningful biological experiments; we developed a microfluidic based in vitro assay for studying hepatitis B viral replication in hepatocytes. This platform will find many useful applications infundamental liver biology research, viral-mediated liver cancer studies, as well as potential use as a novel pharmaceutical platform for epithelial-based screening of genes and targeted cancer therapeutics.Ph.D., Mechanical Engineering and Mechanics -- Drexel University, 200

A tough act to follow: collagen hydrogel modifications to improve mechanical and growth factor loading capabilities

[EN] Collagen hydrogels are among the most well-studied platforms for drug delivery and in situ tissue engineering, thanks to their low cost, low immunogenicity, versatility, biocompatibility, and similarity to the natural extracellular matrix (ECM). Despite collagen being largely responsible for the tensile properties of native connective tissues, collagen hydrogels have relatively low mechanical properties in the absence of covalent cross-linking. This is particularly problematic when attempting to regenerate stiffer and stronger native tissues such as bone. Furthermore, in contrast to hydrogels based on ECM proteins such as fibronectin, collagen hydrogels do not have any growth factor (GF)-specific binding sites and often cannot sequester physiological (small) amounts of the protein. GF binding and in situ presentation are properties that can aid significantly in the tissue regeneration process by dictating cell fate without causing adverse effects such as malignant tumorigenic tissue growth. To alleviate these issues, researchers have developed several strategies to increase the mechanical properties of collagen hydrogels using physical or chemical modifications. This can expand the applicability of collagen hydrogels to tissues subject to a continuous load. GF delivery has also been explored, mathematically and experimentally, through the development of direct loading, chemical cross-linking, electrostatic interaction, and other carrier systems. This comprehensive article explores the ways in which these parameters, mechanical properties and GF delivery, have been optimized in collagen hydrogel systems and examines their in vitro or in vivo biological effect. This article can, therefore, be a useful tool to streamline future studies in the field, by pointing researchers into the appropriate direction according to their collagen hydrogel design requirements.This work was supported by Medical Research Scotland, EPSRC (through a programme grant EP/P001114/1) and a programme of research funded by the Sir Bobby Charlton Foundation. M.S.S. acknowledges support from a grant from the UK Regenerative Medicine Platform 'Acellular/Smart Materials - 3D Architecture' (MR/R015651/1). The graphical abstract was created using BioRender.com.Sarrigiannidis, S.; Rey, JM.; Dobre, O..; González-García, C.; Dalby, M.; Salmerón Sánchez, M. (2021). A tough act to follow: collagen hydrogel modifications to improve mechanical and growth factor loading capabilities. Materials Today Bio. 10(1):1-22. https://doi.org/10.1016/j.mtbio.2021.10009812210

Engineering microscale topographies to control the cell-substrate interface

a b s t r a c t Cells in their in vivo microenvironment constantly encounter and respond to a multitude of signals. While the role of biochemical signals has long been appreciated, the importance of biophysical signals has only recently been investigated. Biophysical cues are presented in different forms including topography and mechanical stiffness imparted by the extracellular matrix and adjoining cells. Microfabrication technologies have allowed for the generation of biomaterials with microscale topographies to study the effect of biophysical cues on cellular function at the cellesubstrate interface. Topographies of different geometries and with varying microscale dimensions have been used to better understand cell adhesion, migration, and differentiation at the cellular and sub-cellular scales. Furthermore, quantification of cellgenerated forces has been illustrated with micropillar topographies to shed light on the process of mechanotransduction. In this review, we highlight recent advances made in these areas and how they have been utilized for neural, cardiac, and musculoskeletal tissue engineering application