Untangling Intercellular Communication Using Optical Manipulation in 3D Models of Tumor Microenvironment

Abstract

The tumor microenvironment is a tangled web of multiple cell types, extracellular matrix components, and a multitude of cell signaling pathways frequently contribute to poor outcomes, which make cancer the second leading killer in the United States. A better understanding of how these constituents interact will inevitably facilitate development of novel cancer therapeutics and diagnostics. To advance scientific discovery towards this goal, innovative experimental techniques are required. In this dissertation, new research methods for probing cell communication at a single to multi cell level within 3D models of the tumor microenvironment are presented. Optical trapping, composite nanocapsules (i.e., gold-coated liposomes), and 3D cell culture models were the foundation for the development of these research tools. The first aim of this dissertation was to optimize our ability to optically manipulate gold-coated liposomes for the purpose of delivering molecular content to cells. The second aim was to apply optical manipulation of gold-coated liposomes to quantitatively deliver signaling molecules into a single cell to activate communication. The third aim was to develop a 3D model of the tumor microenvironment and demonstrate cell communication within this physiologically accurate architecture. The basis for this work was gold-coated liposomes' strong plasmon resonance with visible to near infrared (NIR) wavelengths of light, which enabled photo-thermal conversion and optical trapping. To identify preferred conditions for optical manipulation of gold-coated liposomes for delivering content into cells, gold-coated liposomes made with different dielectric properties were optically trapped under various laser modulation schemes and thoroughly characterized, enabled by high speed (kHz) imaging. Application of this technique was realized by precise delivery of molecular agents into a single cell (i.e., optical injection). As a demonstration of optical injection, the NIR trapping beam was utilized to propel gold-coated liposomes encapsulating inositol trisphosphate (IP3) into a single cell to initiate calcium (Ca²⁺) signaling. In another method for intracellular delivery, cells were preloaded with similar gold-coated liposomes, internalized by macropinocytosis, and then exposed to on-resonant laser light to trigger on-demand release of IP3 to activate Ca²⁺ signaling. Lastly, a 3D cell culture model of ovarian cancer microenvironment was developed as a platform for interrogating cell signaling. The in vitro model comprised human ovarian cancerous epithelial cells grown upon a collagen and human fibroblast stroma recapitulating architecture of human tissue. Gold-coated liposomes encapsulating signaling molecules, optical manipulation, and a 3D model of ovarian cancer, a trio of versatile experimental tools opens new opportunities for studying the tumor microenvironment.