A CRISPR/Cas9-based Lineage Tracing System to Track Metastatic Progression

Agarwal Picture

When cancer cells spread, they detach from the primary tumor, invade, and disseminate to new tissue environments to colonize distant sites in the body. This process, known as the metastatic cascade, is the cause of death for nearly all patients with solid tumors. Therefore, understanding the drivers of metastasis and selective pressures that exist during this process can lead to therapies to prolong the lives of cancer patients. This summer, I used my Dean’s Summer Research Fellowship to continue my research in this area. The overall goal of my project is to use CRISPR/Cas9-based lineage tracing to better understand the selective pressures that occur during invasion, a key step in metastasis. The application of CRISPR/Cas9 technology to lineage trace single cells (McKenna, 2016), allows us to track the evolutionary dynamics of cells pre- and post-invasion and define the fitness bottlenecks that occur during invasion.

To mimic the first process of invasion that occurs in vivo, I used Corning BioCoat Matrigel Invasion Chambers and three barcoded human sarcoma cell lines of varying invasive capabilities (HOS, 143B, HT1080). A population of HOS cells with edited barcodes underwent five successive rounds of selection through the invasion assays. Cells that invaded the chamber membrane were dissociated and propagated to be reapplied to the selective invasion assay. Three replicates from the HOS cell line underwent this repeated selection with the use of control migration chambers as negative control. Another set of chambers was used to quantitate invasion by staining and enumerating the cells that invaded the basement membrane by fluorescent imaging. This process was used to compare the number of invasive cells to the number of migratory cells in the control condition. An average of 60% of the original population succeeded in invasion during the first passage though the assay. This percent invasion appeared to increase through the successive passages to a final average percent invasion of 97%. This suggests that this in vitro model of invasion could have applied a selective pressure to the cells that resulted in an enrichment of invasive phenotype in the population through successive exposure to the assay.

To investigate whether this apparent change in phenotype correlates with a change in the frequencies of CRISPR/Cas9-based barcodes in the population, invasive cells were propagated and samples were collected for next generation sequencing after each passage through the invasion assay. This sequencing data will be used to determine how the pattern of barcodes in invasive cells after each passage compares to the original heterogeneity of barcodes in the initial population.

This summer, I also planned another experiment that would investigate the change in population structure after a selective event using doxorubicin, a common chemotherapy drug. To prepare for this future experiment, I constructed dose response curves to determine the percent of cells that are viable after exposure to different concentrations of doxorubicin. This experiment was conducted with all three cell lines – HOS, 143B, and HT1080. This data will be used in future experiments to test how the pattern of barcodes in cancer cell populations change in response to different drug treatment plans that vary in both dose concentration and treatment timing.