|Hyuna Kim, 2nd Year
Program: Molecular and Cellular Biology
Advisor: Shelly Peyton, PhD.
Education: Korea University, BS, Biotechnology/Medical Science, 2016
A Study of Relationship between Tumor Microenvironment and Drug Efficacy
Before in vivo test of drug efficacy, newly developed drugs are tested in vitro, mostly using a two-dimensional (2D) cell culture environment. Although 2D in vitro screening is convenient and high-throughput, it fails to mimic a tumor microenvironment, resulting in false-positives or drug inefficiencies; drug resistance are often observed when the drug is tested in vivo, even if it showed high efficiency in vitro. Therefore, studying a tumor microenvironment, as one of key factors that alter drug efficacy in vivo, is of critical importance because the microenvironment is the one lacked in a 2D cell culture system. My overarching goal is to find out the relationship between tumor microenvironment and drug efficacy. In my study, I mainly use synthetic three-dimensional (3D) polyethylene glycol (PEG) hydrogel with different stiffness and the hydrogels incorporated with different binding or degradable peptides.
1) Optimize RNA extraction from synthetic PEG-Maleimide hydrogels: In order to study distinct gene expressions in different tumor microenvironment, a proper extraction of RNA is critical. While a 3D cell culture system brings great advantages of mimicking tumor microenvironment, the PEG hydrogel structure itself makes the RNA extraction process difficult. Thus, in this aim, I have tested several different extraction methods to maximize the RNA yields from 3D PEG hydrogels. Prior to the cell lysis process, PEG gels need to be broken down into smaller pieces. I compared two different gel destruction methods: homogenizer vs. flash-freezing. When using a homogenizer, I was able to get more than twice of RNAs than using a flash-freezing and thawing method. I also compared two different RNA isolation methods: using magnetic beads vs. RNA binding column. When extracting RNAs from cells cultured on tissue culture polystyrene, two methods showed no or little differences. On the other hand, when extracting RNAs from cells cultured in 3D PEG hydrogels, the column-based method showed much greater RNA yields. When using magnetic beads, the destructed gel pieces interrupted RNA-to-beads binding and resulted in low RNA yields as well as high impurities. To sum up, it is the most efficient RNA extraction method to use a homogenizer for gel destruction and use a column-based method for RNA isolation when extracting RNAs from 3D PEG hydrogels.
2) Compare cell growth and marker expression at a single cell level: I have monitored cancer cell proliferation (HCC1143, triple negative breast cancer) under different extracellular matrix (ECM) environment and under different growth factor stimulation. I tracked individual cell proliferation for 50 hours and generated cell lineage trees. With the analysis of cell lineage trees, I was able to compare cell proliferation at the single cell level as well as to compare overall proliferation tendency with various conditions. I was also able to compare different marker expressions with immunofluorescence staining; Vimentin, Cytokeratin (CK) 5, CK8, and CK14. Although the cell line was identical, they showed distinct marker expressions under different culture conditions. In this study, I will investigate if cells treated with drugs would change their cancer subtypes and/or change their specific marker expressions. Furthermore, I will test if the changed subtype or marker expression would revert to the initial state when removing drugs. I will also compare the cell growth after adding and removing drugs, at a single cell level with the cell lineage tree analysis.
3) Examine the relationship between biophysical properties of ECM and drug efficacy: Taking advantage of the analyzing methods I described above, I aim to study the role of ECM on drug responses. I hypothesize that matrix stiffening is one of the factors that makes cancer cells become resistant to chemotherapeutics. The rationale for this is that tumor microenvironment gets more stiffened as disease develops. To test the hypothesis, I will generate diverse 3D hydrogels: varying the Young’s Modulus that can represent tissue elasticity or stiffness and varying the concentration of Arg-Gly-Asp (RGD) ligands that can correspond with integrin binding. I have been preparing gels with different stiffness by changing the arm length of PEG-Maleimide or changing the weight percentage of the polymer, and I have been testing the stiffness of the gels using a micro-indentation method. With regards to cell lines, I will use four different cell lines: MDA-MB-453, MDA-MB-468, Hs578T, and BT20. These cell lines are triple-negative subtypes of breast cancer, and they overexpress epidermal growth factor receptor except for MDA-MB-453; I will use MDA-MB-453 as a control of normally expressed EGFR. Since I will use EGFR targeting drugs to test drug efficacy, those cell lines will fit to my research. This way, I will be able to find out a link between the physical properties of ECM and ECM-derived drug resistance.