Browsing by Author "Farach-Carson, Mary C"

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    A novel paradigm for non-fibrotic regeneration of the cornea: The role of TGF-beta superfamily during embryonic cornea regeneration
    (2015-12-16) Spurlin, James W; Farach-Carson, Mary C; Grande-Allen, Jane; Lwigale, Peter Y; McNew, James A; Stern, Micheal
    Damage to the cornea results in fibrotic scarring, leading to the loss of tissue transparency and reduced visual acuity. In fact, corneal opacity is the world’s third leading cause of blindness. Other than transplantation of the affected tissue, there is no treatment to prevent corneal scarring. For these reasons, there is a need to develop anti-fibrotic therapies to promote corneal regeneration after injury. Embryonic tissue has a remarkable regenerative capacity. However, prior to this study, it was not known if the embryonic cornea possessed the ability to regenerate. I hypothesized wounded embryonic corneas wound exhibit non-fibrotic regeneration, and could be used to elucidate novel mechanisms of cornea regeneration. I developed a multistep microdissection method that allows access to the embryonic cornea and several other tissues undergoing organogenesis. Utilizing this methodology, I found embryonic corneal wounds induce a transient population of scar-forming myofibroblast, and ultimately regenerate scar-free. Immunohistological analysis of wounded embryonic corneas revealed transient change in expression of ECM components, which is restored to normal levels in the healed corneas. Furthermore, I showed that Sema3A mRNA is elevated and innervation of wounded embryonic corneas is inhibited during healing, but regenerated corneas are fully innervated. These findings contribute to the understanding of the events that orchestrate scar-free regeneration of wounded corneas. Since embryonic corneas possess an intrinsic regenerative capacity, the embryonic wound healing model serves as a great tool to study regulatory mechanisms that facilitate non-fibrotic healing. Because scar associated myofibroblasts are inherently transient in the embryonic cornea wound, I sought to determine mechanistic regulation of this cell population during cornea regeneration. I hypothesized the embryonic cornea wound would exhibit unique regulation of myofibroblast inductive growth factor, TGF-beta, during regeneration. Through studying gene expression profiles in the embryonic cornea wound healing model, I determined the spatiotemporal distribution of TGF-beta transcripts and the subsequent activation of the myofibroblast population. Moreover, I identified the expression of candidate TGF-beta antagonists when myofibroblasts are found to exit the regenerating cornea. My data shows BMP3 as a novel antagonist to TGF-beta mediated myofibroblast differentiation in isolated embryonic corneal cells. Interestingly, TGF-beta mediated accumulation of focal adhesion appears to be attenuated by BMP3, implicating the role of cellular adhesion in promoting the myofibroblast phenotype. Collectively, this work demonstrates the utility of the embryonic cornea wound healing model to identify novel mechanisms of scar-free cornea regeneration. Additionally, this novel mechanism of BMP3 antagonism on TGF-beta mediated fibrotic response suggests targeting aspects of cellular adhesion signaling may provide viable therapeutics to mitigate corneal fibrosis.
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    Complex 3D Culture Models for the Study of Bone Metastatic Prostate Cancer
    (2020-04-24) Sablatura, Lindsey Kay; Farach-Carson, Mary C; Lwigale, Peter Y
    Prostate cancer (PCa) is the most prevalent form of cancer in men. Despite the enormous resource expenditures on research and development, few advances have been made that improve therapeutic outcomes for patients with relapsed or bone metastatic disease. The high attrition rate of potential anti-cancer drugs entering clinical trials indicates the need for a shift in our approach to modeling metastatic tumors in preclinical studies. As evidence mounts of the importance of microenvironmental cues in tumorigenesis, metastasis, treatment response, and recurrence, research has sought to bridge the gap between in vivo biological relevance and in vitro accessibility and throughput. Three-dimensional (3D) culture models allow experimental control over variables not easily manipulatable in vivo using mouse models, including the cell types and extracellular matrix cues present in the tumor microenvironment (TME). These high content models also support real time imaging of cancer cell behavior as they contact with other cells in the TME. Perfusable models can be used further, not only to add and remove nutrients and waste from cultured cells, but also to introduce perturbagens including anti-cancer drugs for testing personalized oncology. With this goal in mind, these studies tested the hypothesis that the use of complex 3D models containing cancer, stroma, and endothelial cells to reconstitute cellular interactions can replicate events that occur in the bone metastatic niche, allowing investigation into new methods of disrupting growth and progression of bone metastatic disease. Work described in chapters 2 and 3 developed a new high-content, perfusable, hydrogel-based system that permitted the tri-culture of diverse PCa patient-derived xenograft cells with microvascular endothelial cells and tumor stromal cells. The applications of this new pre-clinical model for drug screening and the study of cancer biology were demonstrated. Work in chapter 4 constructed a model of the bone marrow TME to examine endothelial cell promotion of PCa death/dormancy in the bone metastatic niche. Together, the work in these chapters establishes a new stepping stone for in vitro models of bone metastatic PCa, providing new tools for drug discovery and cancer research.
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    Engineering Three Dimensional In Vitro Models of Bone Tumors for Drug Testing and Mechanistic Studies
    (2015-04-15) Fong, Li Shan Eliza; Mikos, Antonios G; Farach-Carson, Mary C; Navone, Nora M; Miller, Jordan S; Kasper , Fred K
    Development of anti-cancer therapeutics has been traditionally reliant on two-dimensional (2D) systems and animal models, both of which have major limitations that contribute to the poor clinical translation of preclinical findings. The goal of this thesis work was to develop three-dimensional (3D) in vitro models of bone malignancies for accurate drug testing and mechanistic studies. To this end, I investigated the use of different 3D scaffolds to recreate the distinct in vivo bone niches relevant for these bone cancers in vitro. First, I evaluated the use of electrospun poly(ε-caprolactone) scaffolds to provide 3D architectural cues for the culture of Ewing sarcoma (EWS) cells. 3D-cultured EWS cells were remarkably different from the same cells cultured in 2D, and more similar to those grown in vivo with respect to morphology, growth kinetics, and protein expression. This work underscored the importance of providing a 3D context for tumor growth in vitro. The second part of this thesis investigated the use of 3D hyaluronan (HA) hydrogels to support the culture of bone metastatic prostate cancer (PCa) cells. Due to their high fidelity to the tumor of origin, there is an emerging interest in the use of patient-derived xenograft (PDX) models to overcome the limitations of cancer cell lines. However, existing PDX culture systems are few and limited. Hence, I sought to develop an in vitro PCa PDX model by first establishing a method to enrich for PCa PDX tumor cells, then evaluated the ability of 3D hyaluronan (HA) hydrogels to maintain the viability, morphology, growth and phenotype of the encapsulated tumor cells. This work demonstrated the feasibility of using a 3D scaffold-based approach to culture PDX tumor cells in vitro. Lastly, I incorporated integrin-binding and matrix metalloproteinase-degradable peptides to HA hydrogels to support osteoblast culture with PCa PDX cells in 3D. Through this 3D co-culture system, the in vivo structural organization, phenotype, as well as biochemical crosstalk between PCa and osteoblasts in bone was recapitulated. In this work, I demonstrate for the first time, the feasibility of co-culturing PDX tumor cells with stromal cells in vitro using a tunable 3D system for controlled mechanistic investigations.
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    Interplay of Perlecan and MMP-7/Matrilysin Regulates Metastatic Prostate Cancer Cell Behavior: Basic and Clinical Implications
    (2015-04-23) Grindel, Brian John; Farach-Carson, Mary C; Lwigale, Peter; Segatori, Laura
    Perlecan/HSPG2 is a large extracellular heparan sulfate proteoglycan concentrated at tissue borders and separating epithelium and stroma. Along with its proteolytic consumers, the matrix metalloproteinases (MMPs), perlecan helps orchestrate development and homeostasis in nearly all studied multicellular organisms. However, both molecule classes can be coopted by prostate cancer (PCa) to advance the disease to its most deadly metastatic form. This work aimed to understand that relationship both at the basic and clinical level. Perlecan with its HS chains and tight domain structure is generally resistant to proteolysis, but a PCa cell must produce an associated enzyme to cleave the border proteoglycan in order to metastasize. This work was the first to identify an active protease produced by PCa cells that can completely digest intact perlecan. Following in silico proteolytic analysis, matrilysin/MMP-7, was identified as a likely candidate for in vitro assays. MMP-7, unlike other enzymes tested, cleaved perlecan when presented in multiple contexts. Perlecan and a subdomain, domain IV-3 (Dm IV-3), but not other subdomains (Dm I, IV-1 and IV-2), induced a striking clustering phenotype. MMP-7 incubation completely reversed this effect to favor cell dispersion and adhesion. Proteomic signaling arrays point towards global Src kinase activation as a major influence of perlecan DmIV-3 effects. To determine if this perlecan/MMP-7 relationship exists in PCa subjects, I performed a tissue microarray, along with β2-microglobulin (β2M), a GF that binds perlecan and induces MMP secretion. Besides increased levels of the two proteins within the patients (cancer/normal), MMP-7 and perlecan levels statistically correlated in multiple grades and localized at tissue interfaces. Additionally, I developed a new assay probing the perlecan fragment signature in the same PCa subjects’ serum. Perlecan fragments were largely increased in PCa and some of the fragments were associated with MMP-7 expression in the subjects. Overall, this work demonstrates a unique interplay between perlecan and its efficient proteolyzer, MMP-7, a relationship that is relevant from the cell and tissue to the clinic and which is likely to contribute to PCa progression to metastatic lethal disease.
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    Targeted Diagnostic Imaging and Image Post-processing of Colorectal Cancer using Hyperpolarized MRI
    (2022-04-22) McCowan, Caitlin; Kemere, Caleb; Farach-Carson, Mary C; Bhattacharya, Pratip
    Colorectal cancer (CRC) is the third leading cause of cancer related deaths. While there are current methods for screening, such as colonoscopy, a number of patients remain undiagnosed until metastasis has occurred, resulting in limited treatment with unfavorable prognosis. Colonoscopy is the current standard of care, but it carries the risk of intestinal perforation and has difficulty detecting small, flat lesions. Patients diagnosed with late-stage cancer have a 5-year survival rate of 14.7%, accounting for approximately 22% of colorectal cancer patients. As early detection is key for favorable patient outcome, an improved screening method is paramount for patients unable to undergo invasive procedures. A less invasive, more quantitative method of diagnosis can be achieved using hyperpolarized magnetic resonance imaging (MRI) with a biomarker targeted imaging agent. MRI is an ideal candidate as it does not utilize ionizing radiation and offers deep tissue imaging. However, low sensitivity and low specificity have hindered its application. Hyperpolarization is a technique that can increase the sensitivity of MRI by over 10,000-fold, through dynamic nuclear polarization. Hyperpolarized MRI is compatible with several nuclear isotopes, including biocompatible elements like silicon and carbon. Additionally, by targeting mucin 1 (MUC1), a transmembrane protein overly expressed by CRC cells on their surfaces, with an imaging agent, increased specificity can be achieved. Here I describe our investigation using two new MRI-based methods to image CRC. In the first, targeted ²⁹Si microparticles were used for in vivo diagnostic imaging of CRC in a humanized MUC1-expressing mouse model. Additionally, I describe a post-processing algorithm I developed that reduces false signal, background noise, and artifacts in hyperpolarized MR images from these mice, and allows for cross-study comparison. In the second method, I developed a MUC1-targeted urease imaging construct that can be detected with hyperpolarized ¹³C-based MRI through a catabolic reaction that converts ¹³C-urea to carbon dioxide and ammonia. The targeting agent uses protein L to bind the targeting antibody and thus presents the opportunity to be used to detect a wide range of cancer-associated targets for which protein L binding antibodies are available. Together, these studies advance the field by providing new methods for non-invasive targeted imaging of CRC and potentially other cancer types.