Engineered microsystems and their application in the culture and characterization of three-dimensional (3D) breast tumor models
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Microsystems are a broad category of engineered technologies in the micro and nano scale that have a diverse range of applications. They are emerging as a powerful tool in the field of biomedical research, drug discovery, as well as clinical diagnostics and prognostics, especially with regards to cancer. One of the major challenges in precision and personalized medicine in cancer lies in the technical difficulties of ex-vivo cell culture and propagation of the limited number of primary cells derived from patients. Therefore, our aims are to 1. Develop a biologically relevant platform for culturing cancer cells and characterize how it influences the cell growth and phenotype compared to conventional 2-dimensional(2D) cell culturing techniques, 2. Isolate secondary metabolites from endophytic fungi and screen them on the platform for potential anticancer properties in a preliminary drug discovery pipeline, 3. Design and develop biosensors for quantifying cell responses in real-time within these systems. Several biomaterial scaffolds with microscale architectures have been utilized for engineering the tumor extracellular matrix, but very few studies have thoroughly characterized the phenotypic changes in their cell models, which is critical for translational applications of biomaterial systems. The overall objective of these studies is to engineer a biomimetic platform for the culture of breast cancer cells in vitro and to quantify and profile their phenotypic changes. In order to do this, we first evaluated a blank-slate matrix consisting of thiolated collagen, hyaluronic acid and heparin, cross-linked chemically via Michael addition reaction using diacrylate functionalized poly (ethylene glycol). The hydrogel network was used with triple-negative breast cancer cells and showed significant changes in characteristics, with cells self-assembling to form a 3D spheroid morphology, with higher viability, and exhibiting significantly lower cell death upon chemotherapy treatment, as well as had a decrease in proliferation. Furthemore, the transcriptomic changes quantified using RNA-Seq and Next-Gen Sequencing showed the dramatic changes in some of the commonly targeted pathways in cancer therapy. Furthermore, we were able to show the importance of our biomimetic platform in the process of drug discovery using fungal endophytes and their secondary metabolites as the source for potential anticancer molecules. Additionally, we developed gold nanoparticle and antibody-based (ICAM1 and CD11b) sensors to quantify cell responses spatiotemporally on our platform. We were able to show quenching of the green fluorescent fluorophores due to the Förster Resonance Energy Transfer mechanism between the fluorophore and the gold nanometal surface. We also observed antigen-dependent recovery of fluorescence and inhibition of energy transfer upon the antibody binding to the cell-surface receptors. Future efforts are directed towards incorporating the hydrogel system with antigen-dependent sensors in a conceptually-designed microfluidic platform to spatiotemporally quantify the expression of surface proteins in various cells of the tumor stroma. This includes the migration,infiltration, and polarization of specific immune cells. This approach will provide further insight into the heterogeneity of cells at the single-cell resolution in defined spaces within the 3D microfluidic platform.
General Audience Abstract
Microsystems are a broad category of engineered technologies in the micro and nano scale that have a diverse range of applications. They are emerging as a powerful tool in the field of biomedical research, drug discovery, as well as clinical diagnostics and prognostics, especially with regards to cancer. However, a major challenge in being able to offer personalized medicine to cancer patients comes from the difficulty of growing cells from the patient's tumor biopsy in a laboratory for further screening and analysis. There are also limited resources available for real-time expression of proteins on cell-surfaces, that could be potential biomarkers and targets for treatment. Various natural and synthetic polymers are biocompatible and have been used widely in engineering the tumor extracellular matrix. However, the effect of hydrogels derived from these polymers on the specific tumor cells are not always well characterized. Our studies explore the influence of a biohybrid hydrogel on breast cancer cells and our results show that the microscale architecture of the hydrogel platform works as a suitable scaffold for recapitulating the 3-dimensional(3D) breast tumor microenvironment, and can also be employed in the drug discovery process. Additionally, we developed a nano-scale biosensor to enable the quantification of specific cell-surface proteins in real-time. Ongoing and future efforts are focused on designing and fabricating a microfluidic device with precise control over the design of space and special chambers for cell culture. These will be used for studying interactions of various cells in the tumor microenvironment that influence cancer progression. Integrating these micro-scale systems, including sensors will allow researchers to quantify cell behavior in response to the variable factors they are exposed to, as well as provide insight to answer fundamental questions about cancer biology that are limited by the conventional 2D cell culture systems.
- Doctoral Dissertations