Real-Time Visualization and Manipulation of the Metastatic Trajectory of Breast Cancer Cells
[Technical Report, Final Report]
University of Pittsburgh
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The purpose of this work was to engineer breast cancer cells to irreversibly alter the genome of nearby cells through exosomal transfer of Cre recombinase from the cancer cells to surrounding cells. Our goal was to use this study to activate green fluorescent protein in the host reporter cells in the environment of cancer cells and simultaneously to delete PTEN from those host cells. We hypothesized that PTEN gene deletion in adjacent normal cells would increase the proliferation of low-aggressive or dormant cancer cells. In the first reporting period, we concentrated on optimizing Cre-containing constructs for exosomal packaging. We also initiated breeding and characterization of the mouse model for in vivo experiments. In the second reporting period, we generated in a doxycycline-controlled inducible, established stable D2.OR cell lines with inducible Cre that is functional in mice, and demonstrated robust recombination of the genome of host bystander cells by Cre-exosome producing D2.OR breast cancer cells that were injected subcutaneously. During the current reporting period, we bred floxed PTEN, Zsgreen transgenic mice to homogenousity, and commenced experiments to determine if Cre-exosome expression and resulting host stromal recombination enabled D2.OR cells to escape dormancy. Because D2.OR did not grow out under these conditions, we extended our study, as planned, to other BalbC syngeneic cancer cell lines, 4T1 and D2.A1. These lines were rendered capable of exosomal Cre production and transfer under doxycycline control, and in vivo experiments are in progress.
- Medicine and Medical Research
- Genetic Engineering and Molecular Biology