Graduation Date

Summer 8-14-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Genetics, Cell Biology & Anatomy

First Advisor

Shantaram S. Joshi, Ph.D.

Second Advisor

Graham J. Sharp, Ph.D.

Third Advisor

Timothy McGuire, Pharm D.

Fourth Advisor

Hamid Band M.D., Ph.D.

Abstract

Intercellular communication between tumor cells and stroma within the hypoxic microenvironment promote aggressiveness and poor patient prognoses in ways that remain unclear. Here we show that hypoxic Ewing’s sarcoma (EWS) cells release exosomes that promote sphere formation, a stem-like phenotype, in EWS cells by enhancing survival and a cancer-associated phenotype in fibroblasts (CAFs). Given that hypoxia enriches for stem-like cells, we exposed EWS cells to hypoxia and hypoxic EWS-derived exosomes and assessed the stem cell phenotype. Hypoxia and hypoxic EWS-derived exosomewere found to enhance TIC formation in vitro and in vivo by reprogramming EWS cells. Analysis of the hypoxic exosomal miRNA cargo identified a HIF-1α regulated miRNA, miR-210, as a potential mediator of sphere formation in cells exposed to hypoxic exosomes. Knockdown of HIF-1α in hypoxic EWS cells led to decreased exosomal miR-210 levels and reduced the capacity of hypoxic exosomes to form spheres. Inhibition of miR-210 in hypoxic spheres attenuated sphere formation and overexpression of miR-210 in normoxic spheres significantly enhanced the number of EWS spheres. Our results indicate that hypoxic exosomal miR-210 targets the proapoptotic protein CASP8AP2 in recipient cells. Moreover, the suppression of CASP8AP2 led to a reduction in apoptotic cells and increased sphere formation.

Exosomes co-cultured with fibroblasts in a soft agar assay and 2D culture assessed induction of the CAF phenotype. Fibroblasts preconditioned with exosomes were injected into NSG mice to determine the effects of exosomes on tumorigenicity. Exosomes increased expression of CAF markers and tumorigenicity in fibroblasts. Network analysis of hypoxic-derived exosomes revealed enrichment of miRNA targeting the Akt pathway. Our observations suggest that hypoxic-derived exosomes enhance an aggressive phenotype in EWS and fibroblasts by increasing Akt signaling.

Together, the findings in this study suggest that hypoxic exosomes promote stemness in EWS cells by delivering enriched miR-210 that is capable of down-regulating apoptotic pathways, resulting in the survival of cells with increased sphere formation. Future studies will further investigate the effect of exosomal miRNA on target genes and the role these interactions play in driving aggressiveness in hypoxic EWS cells. Future work will investigate the role of exosomal miRNA on the Akt pathway in target cells.

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Available for download on Sunday, August 07, 2022

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