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theses

Given its extremely poor prognosis, there is a pressing need for improved understanding of the biology of glioblastoma multiforme (GBM), including the roles of tumor subpopulations, such as macrophages, that contribute to tumor growth and therapeutic resistance. DNA alkylating agents, such as temozolomide and carmustine, are the current gold standard drug treatment for GBM, yet resistance occurs and is attributable to a subset of cancer stem cells (CSCs). The mechanisms by which the CSC state is produced and promotes drug resistance are not well understood, but molecular pathways are beginning to emerge. We investigated the interplay between CSCs and macrophages via co-culture. The results showed that macrophages communicate with and influence CSC functions via a paracrine loop. Specifically, the levels of an M2 macrophage-specific secreted cytokine, TGF-β1, were elevated in the presence of CSCs, but whether the cells were plated as contacting or non-contacting co-cultures did not affect the results. Furthermore, co-culturing with CSCs resulted in enhanced expression of M2 markers in macrophages that were previously polarized to the M1 phenotype. A major player in the cross talk is the inflammatory transcription factor, STAT3, produced by CSCs, which recruits and subsequently modulates macrophages to become immunosuppressive, maintaining CSC stemness. Another intriguing target is the transcription factor, HIF-2α, the expression of which is markedly increased in hypoxic CSCs. Here, we utilized short interfering RNA (siRNA) to silence CSC targets. For the delivery of siRNA cargo, cationic amphiphilic macromolecule (CAM) – lipid complexes were co-developed. We discovered that addition of certain levels of lipid induces pH-dependent instabilities that promote intracellular delivery and release from endosomes, leading to efficient gene silencing. We utilized these CAM-lipid complexes or Lipofectamine for silencing experiments. It was observed in both glioblastoma patient and cell line-derived CSCs that neurosphere formation, proliferation rate, chemoresistance, migration towards macrophage conditioned media, and matrix degradation were elevated compared to bulk tumor. Addition of STAT3 siRNA potentiated modestly the effects of carmustine on CSCs. HIF-2α siRNA had a significant effect on hypoxic CSC functions and induced their differentiation. Thus, disrupting CSC stemness or their cross talk with macrophages may be viable avenues for next-generation cancer therapeutics.

ETD_5988

Ph.D.

Includes bibliographical references

by Leora Molly Nusblat

doi:10.7282/T3CF9PCM

ETD doctoral

The author owns the copyright to this work.