MICROFLUIDIC MODEL OF VASCULARIZED TUMOR MICROENVIRONMENT: PDMS AND INJECTION-MOLDED PLASTIC 3D CULTURE PLATFORMS
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The “Tumor microenvironment” (TME) has been increasingly recognized as an underlying factor in drug resistance and the recurrence of cancer, both of which are major obstacles to a cure. The TME is a complex, interacting system including the tumor itself, other noncancerous cell types such as immune, stromal and endothelial cells, and the extracellular matrix surrounding these cells. This complexity has largely prevented a comprehensive understanding of TME in conventional in vitro models, which have been too simple to recapitulate the intricate interactions. Therefore, developing in vitro assays that closely replicate the pathophysiology of 3D vascularized TME is critical to understand the formation and development of tumors and unravel the mechanisms by which tumor cells grow, metastasize, and resist against drugs. To this end, this thesis first describes the method to create in vivo like vascularize tumor spheroid Polydimethylsiloxane (PDMS)-based model. We developed vascularized tumor spheroid (VTS) model that reproduces the pathological and morphological characteristics of in vivo vascularized solid TME. We used human hepatocellular carcinoma (HepG2) spheroid to reconstitute VTS. The VTS structure closely recapitulates a vascularized tumor microenvironment where cancer cells can interface with self-assembled vascular endothelial cells within 3D hydrogel. We introduced on-chip tissue clearing technology that enables 3D intact visualization of the tumor-endothelium interaction. We also conducted comparative studies of spheroid morphology, relative tumor suppressor (PTEN and p53) and epithelial-mesenchymal transition (EMT) gene expressions, and microvascular network formation induced by the spheroid. Notably, we observed that cancer-EC hybrid spheroids enhance uniformity of spheroid, tumor aggressiveness, and tumor angiogenesis compared to those with cancer cells only. Moreover, the microvasculature co-cultured with the tumor spheroid showed a higher permeability coefficient compared to control and increased focal intercellular openings (FIOs) of microvascular network located in adjacent to tumor spheroid, demonstrating hallmarks of tumor vasculature; a leaky and fenestrated structure of endothelium. We further visualized and quantitatively demonstrated the effects of the FDA approved cancer drug; Axitinib by monitoring blood vessel area and spheroid tumor size, highlighting the significance of tumor vascularization and revealing the importance of the dose and treatment timing. The 3D VTS model will be a useful platform to better understand how tumor develops and metastasizes in vasculature, screen drugs and their compositions (fine drug cocktails) for heterogeneous cancer treatment, and explore new therapeutic strategies with 3D vascularized models of patient-derived multicellular tumor spheroids. Secondly, this thesis introduces Injection-molded based vascularized tumor microenvironment platform considering standardized application for industrialization. The field of microfluidics-based three-dimensional (3D) cell culture system is rapidly progressing from academic proof-of-concept studies to valid solutions to real-world problems. PDMS-based platform has been widely adopted as in vitro platforms for mimicking tumor microenvironment. However, PDMS has not been welcomed as a standardized commercial application for preclinical screening due to inherent material limitations that make it difficult to scale-up production. Here, we present an injection-molded plastic array 3D spheroid culture platform (Sphero-IMPACT). The platform is made of polystyrene (PS) in a standardized 96-well plate format with a user-friendly interface. This interface describes a simpler design that incorporates a tapered hole in the center of the rail to pattern a large spheroid with 3D extracellular matrix and various cell types. This hole is designed to accommodate standard pipette tip for automated system. The platform that mediate open microfluidics allows implement spontaneous fluid patterning with high repeatability from the end user. To demonstrate versatile use of the platform, we developed 3D perfusable blood vessel network and tumor spheroid assays. In addition, we established a tumor spheroid induced angiogenesis model that can be applicable for drug screening. Sphero-IMPACT has the potential to provide a robust and reproducible in vitro assay related to vascularized cancer research. This easy-to-use, ready-to-use platform can be translated into an enhanced preclinical model that faithfully reflects the complex tumor microenvironment.