Identification of Inhibitors for Tangocytosis Through High-Throughput Screening

Sung, Erica

Undergraduate Honors Thesis

Identification of Inhibitors for Tangocytosis Through High-Throughput Screening Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/5h73px63n

Abstract

Breast cancer is the most common cancer in women, affecting 2.3 million women globally. However, no identified drugs effectively block tumor movement to another site, rendering metastatic breast cancer a chronic and incurable disease. Previously, the Liu lab discovered a process called "tangocytosis," in which an epithelial cell engulfs a tumor cell and transfers the reporter gene from the tumor cell to the epithelial cell. Blocking direct cell-cell interaction prevents subsequent marker gene transfer, which provides a useful tool for studying the cell entrapment process via high-content screening methods. In this thesis, high-content screening using FDA-approved small molecule inhibitors revealed that DNA damage repair drugs can effectively block the gene transfer between the donor and recipient cells.

Following the screening, potent inhibitors of tangocytosis, Clofarabine and Teniposide, were identified and selected for further analysis. It was discovered that these drugs effectively blocked reporter gene transfer and minimized the formation of cell-in-cell structures, suggesting that inhibiting such structures can prevent gene transfer. Additionally, the drugs had polarizing effects on both donor and recipient cells. Pretreating RPE1 cells with the drugs resulted in a decrease in gene transfer upon co-culture while pretreating MDA-MB-231 cells with drugs increased gene transfer upon co-culture. The polarizing effect indicates that gene transfer may be mediated by the upregulation of different proteins in each cell line.

To investigate whether ROCK1, an essential kinase in cell tumor migration, plays a role in the polarization, immunofluorescence staining of ROCK1 was conducted on MDA-MB-231 and RPE1 cells. The results showed that ROCK1 intensities were higher in regions of cell-in-cell structures compared to free cells. In the DMSO control group, ROCK1 intensity was significantly higher in cell-in-cell regions than in absent regions. However, when Clofarabine was added to the co-culture, the difference was no longer significant. The findings suggest that DNA damage repair drugs may reduce gene transfer by reducing the polarization of ROCK1. Due to the high number of hits related to DNA damage repair and the reduction of polarization, the DNA damage repair pathway presents as an essential pathway for successfully blocking gene transfer through the inhibition of cell-in-cell structures.

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