Isolation and Molecular Characterization of Single Circulating Tumor and Immune Cells to Examine Heterogeneity

Abstract

Cancer patient care is limited by several factors including a lack of methods to detect cancer dissemination and progression, few treatment options, and challenges in detecting tumor heterogeneity. Circulating biomarkers can offer insights into the current state of a patient’s tumor but are underutilized in clinics, as they can be rare, and difficult to detect and analyze. This thesis shows the development and application of new workflows to address some of the current limitations of circulating biomarker isolation technology, and demonstrates the clinical applications of these technologies. First, the sensitivity of an existing technology, the Labyrinth, was improved and validated for the detection of circulating tumor cells (CTCs) in early-stage breast cancer patients. This process utilized low binding materials with antigen-free isolation technology to isolate CTCs from 66.6% of ductal carcinoma in situ (DCIS) patients. We expanded on previous work by performing both single-cell qPCR and targeted bulk sequencing on enriched DCIS CTC samples. This revealed mesenchymal populations of CTCs, suggesting a possible mechanism of early cancer dissemination, and genetic variations previously associated with pathogenic potential and progression. Continuing to further advance this technology and application, a new workflow was developed to take enriched CTC samples from the Labyrinth and further process them to obtain single cells. Single CTCs can be used to explore heterogeneity within the tumor population and identify targetable pathways to improve patient treatment. This was first validated in established cell lines by comparing CNV profiles of cells obtained using our new workflow to profiles of cells obtained using alternative workflows. Additionally, this workflow allowed us to perform novel single cell sequencing of CTC cell lines and freshly isolated cells. The patient sample showed multiple CTC CNVs, including a gain in chromosome 8q, the location of the oncogene Myc. The secretion of extracellular vesicles (EVs) is known to be an important part of cell communication and can help create a tumorigenic environment. However, due to the vesicles’ nanoparticle size, it is difficult to study the secretion profiles on a single cell scale. Therefore, we developed the CellMag-CARWash system to isolate pure single cell populations into single cell droplets using immunomagnetic beads. The system was tested on both cancer cells, MCF7 GFPs, and immune cells, NK92mi, in mixed cell populations and showed over 93% purity in the final cell population. Cell recovery was dependent on cell size, and number of immunomagnetic beads attached to the cell. Cells were placed into media during the droplet generation and cultured for up to 18 hours. After culture, the EV concentration was measured using fluorescent intensity and differences in the amount of EVs secreted per cell were observed. Finally, we developed stimulus dependent degradable hybrid hydrogels for the dual CTC and EV isolation from the same sample. Hydrogels were tested with both cell lines and non-small cell lung cancer patient samples. Concentrations of isolated CTCs and EVs from patient samples using hydrogels were shown to be similar to concentrations obtained using previously established methods. This work demonstrates that hybrid hydrogels can be used for fast multi-biomarker isolation and detection. Overall, in this thesis I developed new workflows and technologies for the isolation of circulating biomarkers, including single CTCs. These advancements will continue to improve the clinical utility of circulating biomarkers and provide insights into the mechanisms of cancer.