Date of Award

12-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Neurosciences

Research Advisor

Monica M. Jablonski, Ph.D.

Committee

Vanessa M. Morales-Tirado, M.S., Ph.D. Lawrence M. Pfeffer, Ph.D. Anton J. Reiner, Ph.D. Matthew W. Wilson, M.D.

Keywords

Ocular immunology, Ocular oncology, Ophthalmology, Retinoblastoma, Tumor biology, Tumor microenvironment

Abstract

Retinoblastoma (Rb) is the most common intraocular malignancy in children comprising 4% of all pediatric tumors. Early intervention increases survival rates up to 95% in developed countries; being survival rates associated to socio-economic status. Despite the high survival rates in developed countries, preservation of the eye and vision are continuing challenges in the management of Rb. Vitreous seeds constitute the greatest challenge in treatment of Rb. The unique location of these seeds within the vitreous makes them difficult to treat. Viable seeds showed reduced proliferative capacity and metabolism. making the majority refractory to current chemotherapeutics. This prompted the development of new delivery routes for chemotherapeutics, such as intra-ophthalmic artery, intravitreal, and subconjunctival administration. Still ocular salvage rates have not exceeded 70% in over two decades. Novel, safer therapies are required but there is a fundamental lack of knowledge about the biology of the tumor for the development of targeted therapies; the vitreous, as it surrounds the seeds; and the interactions between the vitreous and the seeds.

Despite the current understanding of how cancer is a multifaceted disease full of complex cellular and protein interactions (commonly referred to as the tumor microenvironment, TME), the immunology of the vitreous microenvironment and the role it plays in the sustainment of seeds in Rb disease is poorly understood. In this study, we begin investigating Rb tumor cells and how they alter the TME by examination of matrix metalloproteinases, a family of enzymes involved in degradation of the extracellular matrix and tissue remodeling heavily implicated in tumor migration and survival. We identified two gelatinases, MMP-2 and MMP-9, to be expressed in Rb cell lines and demonstrated by pharmacological inhibition and genetic knockdown, a role for these gelatinases in Rb cell migration, invasion, and survival. Additionally, we demonstrated how secretion of VEGF, involved in angiogenesis, and TGFβ, involved in metastasis, were altered by MMP-2 and MMP-9 pharmacological inhibition.

As MMPs are pivotal for the tumor and extracellular matrix interactions within the TME leading to tissue invasion, we then transitioned to the vitreous, investigating the vitreous as a TME and how it sustains Rb. Multiple proteins are found within the soluble phase of the vitreous that are associated with ocular pathological processes, including diabetes retinopathy and proliferative vitreoretinopathy. Therefore, we compared the presence of a small cohort of proteins associated to ocular pathologies, to healthy vitreous and to Rb patients’ vitreous samples, identifying high expression of Platelet-Derived Growth Factor Receptor β (PDGFRβ) and its ligand PDGFBB in the Rb samples. Additional studies of ex vivo healthy human vitreous, murine Rb xenografts, and patient-derived Rb xenograft tissues, measured high activity of the PDGF-PDGFRβ signaling pathway in diseased, but not healthy tissue. We focused the next part of this work on the role PDGFRβ plays in vitreous seeds. To investigate this pathway in depth we used established Rb cell lines, namely Y79-the metastatic and aggressive model, and Weri-1 Rb, the non-metastatic model. Our work demonstrated PDGFRβ was overexpressed in Y79 cells, the metastatic model of Rb, in vitro. To provide mechanistic insight, we utilized the tyrosine kinase inhibitor imatinib mesylate (IM), and demonstrated PDGF-PDGFRβ signaling pathway regulates Rb cell proliferation, invasion, and survival. We found increased PDGF-PDGFRβ signaling resulted in higher activity of the p53-inactivator, MDM2, as well as the pro-inflammatory NFβB pathway, both of which are involved in tumor survival. As there is a lack of models for vitreous seeding, we utilized magnetic nanoparticles to generate the first in vitro vitreous seed model and demonstrated similar features between an ex vivo seed and one of our in vitro magnet-generated 3D tumor spheroids, validating our system. Using this novel technology, we recapitulated our in vitro 2D work and demonstrated that inhibition of the PDGF-PDGFRβ signaling pathway results in decreased spheroid size. PDGFRB gene knockdown by siRNA confirmed the results with the pharmacological agent showing these results were PDGF-PDGFRβ signaling-specific.

Next, we tested the effects of IM, as a potential Rb therapy, in retinal endothelial cells h(RECs) as retinal endothelial cell-associated toxicities are one of the challenges with conventional chemotherapies in Rb. We measured the capacity of hREC to proliferate and for tube formation in the presence of the therapy. Our results demonstrated neither proliferation nor tube formation of hRECs changed when exposed to IM. Upon further examination, we demonstrated the absence of PDGFRB mRNA expression in hRECs. Taken together these results illustrate the potential use of anti-PDGFRβ therapy as a targeted therapy in Rb.

The last section of the study investigated the expression and function of members of the ATP-binding cassette (ABC) transporters as well as the multifunctional glycoprotein CD44 in Rb, as one of the properties of vitreous seeds is their resistance to chemotherapy. We discovered heterogeneous expression of multiple ABC transporters in vitro using Y79 Rb cell line and ex vivo using Rb vitreous samples. In addition to expression, the transporters efflux activity was determined. CD44 mRNA, whose protein is considered a promoter of chemoresistance, was highly expressed in naïve vitreous from Rb patients. These results illustrate that chemoresistance is not induced by treatment and is, instead, an innate feature of vitreous seeds. Flow cytometry analyses established (1) a high correlation between active, or phosphorylated PDGFRβ (p-PDGFRβ) and CD44; and (2) inhibition of p-PDGFRβ resulted in decreased CD44, as well.

Collectively, this study demonstrated the Rb TME plays a crucial role in Rb tumor properties. MMP-2 and MMP-9 are highly expressed in Rb and allow degradation of the ECM in tissue, increasing migration and invasion of Rb. Additional work focused on vitreous seeds and identified increasing signaling activity of the PDGF-PDGFRβ signaling pathway in multiple Rb models, including in vitro cell lines, ex vivo Rb patient vitreous samples and tissue, as well as in a murine xenograft system. Next, we identified this pathway as a potential target, as pharmacological and genomic inhibition of the PDGF-PDGFRβ signaling pathway by IM and PDGFRB siRNA resulted in decreased proliferation, invasion, resistance, and survival. These cellular functions appear to be mediated by the downstream targets MDM2 and NFβB. Our initial studies demonstrated a lack of drug-associated toxicity on hRECs with IM. This work is a further step in our quest for targeting vitreous seeding.

ORCID

http://orcid.org/0000-0002-7393-733

DOI

10.21007/etd.cghs.2018.0472

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