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Basal cells in a simple secretory epithelium adhere to the extracellular matrix (ECM), providing contextual cues for ordered repopulation of the luminal cell layer. Early high-grade prostatic intraepithelial neoplasia (HG-PIN) tissue has enlarged nuclei and nucleoli, luminal layer expansion and genomic instability. Additional HG-PIN markers include loss of alpha 6 beta 4 integrin or its ligand laminin-332, and budding of tumor clusters into laminin-511-rich stroma. We modeled the invasive budding phenotype by reducing expression of alpha 6 beta 4 integrin in spheroids formed from two normal human stable isogenic prostate epithelial cell lines (RWPE-1 and PrEC 11220). These normal cells continuously spun in culture, forming multicellular spheroids containing an outer laminin-332 layer, basal cells (expressing alpha 6 beta 4 integrin, high-molecular-weight cytokeratin and p63, also known as TP63) and luminal cells that secrete PSA (also known as KLK3). Basal cells were optimally positioned relative to the laminin-332 layer as determined by spindle orientation. beta 4-integrin-defective spheroids contained a discontinuous laminin-332 layer corresponding to regions of abnormal budding. This 3D model can be readily used to study mechanisms that disrupt laminin-332 continuity, for example, defects in the essential adhesion receptor (beta 4 integrin), laminin-332 or abnormal luminal expansion during HG-PIN progression.

Research has shown that autologous progenitor-like cardiac spheroids, when delivered to an infarcted heart, are able to restore mechanical function. These spheroids are made by isolating and expanding autologous cardiac progenitor cells. Though these results are promising, the process for creating cardiac spheroids is inefficient and time consuming, requiring a large amount of cardiac tissue. For every 10,000 cardiac myocytes in the heart there is only one cardiac progenitor cell; requiring a large amount of initial tissue. This clinical limitation could be overcome if cardiac myocytes, which are more abundant than cardiac progenitor cells, could be used to make cardiac spheroids. Research has shown that mesenchymal stem cells when co-cultured with adult cardiac myocytes cause the cardiac myocytes to behave like a progenitor cell. We found that, when co-cultured with mesenchymal stem cells, cardiac mycoytes could be made to form cardiac spheroid bodies. This was done by isolating adult myocytes from rat hearts and co-culturing them with human mesenchymal stem cells. After two weeks, cultures were observed to form spheroid bodies and the number of spheroids formed were compared to a pure myocyte control. Cardiac specific staining confirmed that the spheroids were made from the myocytes. It was also found that the mesenchymal stem cells, when co-cultured in the same well with the myocytes, form significantly more spheroids than myocytes treated with stem cell conditioned media. Further, no other cell type present in the co-cultures are able to create spheroid bodies when co-cultured with mycoytes or stem cells. The ability to create cardiac spheroid like bodies from adult myocytes offers a way to overcome the limitations of the time needed and the large quantity of autologous cardiac tissue required to currently make these types of bodies.

There are a number of pressing issues that the creation of a biomimetic liver culture may be able to solve including catching hepatic mal interactions that are currently missed in preclinical drug screening and offering an alternative solution in the shortage of organs for liver transplant. There are multiple hepatic models that have been created to overcome these issues. However, of the many considerations that need to be taken in the creation of a biomimetic liver model, many fail to capture the functional zone-patterning that is found in vivo. Here is detailed the creation of a hepatic assembloid model that incorporates zone-specific human pluripotent stem cell derived hepatocytes for the recapitulation of zone-patterning in the liver tissues. Use of our lab’s z-wire plate and PGS z-wire scaffold allows for the formation of elongated 3D tissues that resembles the overall morphology of the liver acinus and facilitate the spontaneous development of an aligned vascular-like network. Sustained hepatocyte-specific function in these tissues are promising indicators for application of the hepatic models in drug screening, disease modelling, and regenerative medicine. / Thesis / Master of Applied Science (MASc)

Multicellular spheroids have emerged as a promising tool for drug delivery, cancer therapy, and tissue engineering. Compared to 2D monolayers, spheroids provide a more realistic representation of the 3D cellular environment, enabling better understanding of the signaling cascades and growth factors involved in vivo. The formation of in vitro spheroids involves the aggregation of several cells that proliferate to grow into larger spheroids. Biophysical cues provide crucial information for the cells to assemble into 3D structures. We used suspended fiber networks to demonstrate a new way to form and spatially pattern spheroids comprised of human pericytes. We show that fiber architecture (aligned vs. crosshatched), diameter (200, 500, and 800 nm), and contractility influence spheroids in their spontaneous formation, growth, and maintenance, and report a dynamic trade of cells between adjacent spheroids through remodeled fiber networks. We found that aligned fiber networks promoted spheroid formation independent of fiber diameter, while large-diameter crosshatched networks abrogated spheroid formation, promoting growth of 2D monolayers. Thus, a mixture of diameters and architectures allowed for spatial patterning of spheroids and monolayers within a single system. We further quantified various dynamic interactions and describe the forces involved during spheroid formation, cell efflux from spheroids, and show the loss and recovery of spheroid forces with pharmacological perturbation of Rho-associated protein kinase (ROCK). Thus, we develop new insights on the dynamics of spheroids using suspended fiber networks of varying diameters and architectures, with the potential to connect matrix biology with developmental, disease, and regenerative biology. / Master of Science / In recent years, studies involving multicellular spherical aggregates or 'spheroids' have gained popularity since they capture the 3D cellular environments seen within the body more realistically when compared to 2D cell culture systems (such as monolayers) traditionally used for biological studies. These spheroids resemble organs and tissues in terms of their structure and function better and are increasingly being studied for an array of applications such as drug delivery, cancer therapy, as implants and in tissue regeneration and tissue engineering. The cellular microenvironment consists of fibrous proteins of varying diameter arranged in various geometric patterns, which can influence the growth and culture of spheroids. Here, we use our Spinneret-Based Tunable Engineered Parameters (STEP) technique to fabricate fibrous networks with precise control over fiber diameter and architecture and study how biophysical cues can influence the formation and culture of spheroids. Using aortic pericytes, we show that fiber architecture (aligned vs. crosshatched) and diameter (200, 500, and 800 nm) can control how pericytes aggregate into either 2D monolayers or 3D spheroids. We study the effect of each of these parameters to show that stiffer, denser fibers are robust networks which the cells refrain from remodeling, and thus lead to monolayers while more compliant and sparser networks are easily remodeled to promote spheroid formation. Thus, we spatially pattern a mixture of 3D spheroids and 2D monolayers in a single system by varying the parameters at different regions. We quantify various interactions such as spheroid formation, spheroid merging, spheroid migration, cell efflux from spheroids and the dynamic contractile forces exerted on the matrix by spheroids during these interactions. We also show that contractility has a major role in spheroid formation and to maintain their structure and look at the changes in the gene expressions associated with contractility during the formation and growth of spheroids. Thus, we develop new knowledge in controlling the growth of pericytes into 2D and 3D structures and show that our fiber networks can be an essential platform for studying spheroids.

Yes / We report the first application of a microfluidic device to observe chemotactic migration in multicellular spheroids. A microfluidic device was designed comprising a central microchamber and two lateral channels through which reagents can be introduced. Multicellular spheroids were embedded in collagen and introduced to the microchamber. A gradient of fetal bovine serum (FBS) was established across the central chamber by addition of growth media containing serum into one of the lateral channels. We observe that spheroids of oral squamous carcinoma cells OSC–19 invade collectively in the direction of the gradient of FBS. This invasion is more directional and aggressive than that observed for individual cells in the same experimental setup. In contrast to spheroids of OSC–19, U87-MG multicellular spheroids migrate as individual cells. A study of the exposure of spheroids to the chemoattractant shows that the rate of diffusion into the spheroid is slow and thus, the chemoattractant wave engulfs the spheroid before diffusing through it. / This work has been supported by National Research Program of Spain (DPI2011-28262-c04-01) and by the project "MICROANGIOTHECAN" (CIBERBBN, IMIBIC and SEOM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Three dimensional multicell tumor spheroids (MCTS) provide an experimental model where the influence of microenvironmental conditions on protein expression can be determined. Sequential trypsin digestion of HT29 colon carcinoma MCTS enabled segregation into four populations comprising proliferating cells from the surface (SL), an intermediate region (IR), nonproliferating hypoxic cells from the perinecrotic region (PN), and a necrotic core (NC). Total protein was extracted from each population and subjected to iTRAQ-based quantitative proteomics analysis. From a total of 887 proteins identified, 209 were observed to be up-regulated and 114 were down-regulated in the PN and NC regions relative to the SL. Among the up-regulated proteins, components of glycolysis, TCA cycle, lipid metabolism, and steroid biosynthesis increased progressively toward the PN and NC regions. Western blotting, immunohistochemistry, and enzyme assays confirmed that significant changes in the expression of proteins involved in cellular metabolism occur in the nonproliferating fraction of cells within the viable rim. The presence of full length, functional proteins within the NC was unexpected, and further analysis demonstrated that this region contains cells that are undergoing autophagy. This study has identified possible targets that may be suitable for therapeutic intervention, and further studies to validate these are required.

Multicellular tumour spheroids (TS) are an in vitro model of avascular tumours, and have been widely used to investigate tumour growth, metabolism and hypoxia. The geometry of the TS lends itself to mathematical representation, and theoretical models of TS growth and the development of hypoxia are abundant. With some notable exceptions however, these models have been developed independently of the biological data collection process and are overwhelmingly based upon data from multiple sources. Thus, whilst mathematical modeling has the potential to help explain and guide biological experiments, without reliable data it is unlikely to live up to this expectation. In this thesis, a combination of experimental and theoretical approaches was used to characterize the relationship between proliferation, hypoxia and metabolism during the growth of TS derived from the DLD-1 human colon adenocarcinoma cell line. Experimental data were collected over the entire period of TS growth, generating a high volume of predominantly imaging data. To facilitate the extraction of quantitative information from this, a suite of image analysis software, which is readily applicable to other data sets, was developed. During growth, the DLD-1 TS maintained a macroscopic spherical geometry but at the microscale level the TS boundary was increasingly irregular, with TS disintegrating rapidly after 20 days. Immunofluorescence (IF) studies showed that hypoxia developed soon after TS initiation, followed by the characteristic onset of necrosis. Reduced proliferation was found to be concomitant with the development of hypoxia, although some cells retained proliferative capacity even under severely hypoxic conditions. Towards the end of culture, TS were primarily comprised of severely hypoxic and necrotic cells, a probable cause of disintegration. Mathematical simulation of oxygen gradients in TS using literature-based values for the maximal rate of oxygen consumption was used to estimate the partial oxygen pressure (pO₂) at which the IF marker of hypoxia was bound. Assuming a spatially-invariant rate of oxygen consumption, the model predicted that the onset of hypoxic binding occurs at pO₂ levels similar to those reported in the literature, however the onset of necrosis was overestimated. Mathematical simulations predicted that oxygen consumption decreases as TSs increase in size, supporting previous observations. The Warburg Effect, where glucose metabolism is favoured even under aerobic conditions, is a hallmark of tumours. Although development of the glycolytic phenotype during TS growth was observed in the form of an elevated activity of the lactate dehydrogenase V (LDHV) enzyme, the activity and expression of other glycolytic enzymes, such as hexokinase II (HKII), was unaltered. Whilst the spatial distribution of HKII was unrestricted throughout the TS's viable fraction, LDHV expression was elevated in regions of hypoxia, suggesting constant adaptation of tumour cells to their microenvironment. In addition to the above findings, the data generated have been collected and analysed in the context of the requirements of theoretical modelling at each step; thus, they can be used to parameterise and inform more sophisticated models of tumour metabolism.

Alterations in energy metabolism due to factors including cellular stress from the hostile tumour micro-environment are a emerging cancer hallmark. Distinct hypoxic and quiescent cell populations develop, which are resistant to chemotherapy due to lack of proliferation, drug inactivity in the altered redox status of the cell and enhanced drug biotransformation. The present study characterises the metabolic strategies employed by these distinct populations of cancer cells. The in vitro 3-dimensional tumour spheroid model, which reflects tumour architecture and behaviour, cultured under different micro-environmental conditions was utilized in this study. Metabolic enzyme activity and expression, overall metabolic flux rates for nutrients, metabolomics profiles of specific pathways and tissue status were assessed. Metabolic adaptations consistent with the Warburg effect were observed in fully oxygenated, proliferative tumour spheroids, with glucose being metabolised to produce lactate. Additionally, metabolomics investigations determined glucose was metabolised by the pentose phosphate pathway, demonstrated by high enrichment of glucose-derived carbon in 6-phophogluconate. The extraction of 39.7 ± 7.6 μ moles (mg protein) ^-1 glutamine from the medium over 24 hours was observed in these spheroids, consistent with glutaminolysis pathway activity. A 2-fold higher rate of glycolytic flux (measured by production of 3h2O from 5-³H-glucose) was measured in hypoxic tumour spheroids, despite reduced levels of glycolytic enzymes being determined. Surprisingly, although lower rates of glycolysis (2.6-fold) were measured in quiescent spheroids, increased glycolytic enzyme activities (HK 1.9 fold, PK 2 fold and LDH 1.8 fold), glucose (1.9 fold over 24 hours) and glutamine uptake (5.5 fold over 12 hours) as well as lactate production (1.8 fold) were measured, relative to their proliferating counterparts. This study demonstrates that metabolic strategies employed by tumour spheroids differ upon exposure to distinct micro-environmental stresses, additionally identifying hexokinase as a potential therapeutic target for the inhibition of glycolysis under all micro-environmental stress conditions analysed.

Head and neck squamous cell carcinoma (HNSCC) is a type of cancer that begins in the epithelial cells that line the mucosal surfaces of the head and neck, including the oral cavity, pharynx, larynx, paranasal sinuses, nasal cavity, and salivary glands. Head and neck cancer is the sixth most common type of cancer with a 5-year survival rate of 60% for all cases. Over the past few years, a subset of cells with stem-like properties, called cancer stem cells, are believed to have tumor-initiation capabilities and are responsible for maintaining on-going tumor growth. Previous data from our lab suggested that cells grown in suspension, called spheroids, may have stem cell like properties. We employed a model system where a primary HNSCC cell line, HN4, was used to set up spheroids. We found that expression of EPS8 and its downstream targets, FOXM1 and CXCL5, was increased in HN4 spheroids. In addition, we measured the expression of Nanog, as it is a transcription factor involved in the self-renewal of human embryonic stem cells. We also used a metastatic HNSCC cell line, HN12, to see how it compared to spheroids. We wanted to investigate the hypothesis that activation of Src potentiates EPS8 function to deregulate downstream signaling pathways. We used a small molecule tyrosine kinase inhibitor, Dasatinib, on HN4 spheroids and HN12 cells. We found that when Src is inhibited, EPS8 expression is decreased in HN4 spheroids and it also interferes with spheroid formation. The results of the current study were also able to show that the proliferation capability of HN12 cells is greatly diminished when treated with Dasatinib, due to G1 arrest in the cell cycle. When we measured for FOXM1, which is a cell cycle regulator, we found the levels were reduced in Dasatinib treated cells, preventing the cells from completing mitosis. With all of the data taken together, it suggests that Src does in fact play a role in regulating the downstream signaling pathways of EPS8, and its inhibition leads to the loss of cell proliferation. Additional studies need to be performed to discover whether Src inhibition will stop the proliferation of cancer stem cells, which are believed to be more resistant to cytotoxic therapies.