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Deon Doxie, Ph.D.

Department: Cancer Biology, 2018

Faculty Mentor: Jonathan Irish, Ph.D.

Dissertation Title: Quantitative Single Cell Analysis of Cancerous Cells During Therapy

Dissertation Abstract: Single cell tools have great potential to characterize mechanisms of oncogenesis and treatment resistance. To date, single cell quantitative cytometry has been widely applied in the setting of blood cancer. However, few studies use this tool to study solid tumors with the same detail as blood cancers. The work presented in this dissertation applies quantitative single cell biology tools to characterize cancerous cells from research models and primary human tumors. In particular, this work focuses on application of mass cytometry to characterize advanced, metastatic BRAF mutant melanoma. Among patients with metastatic melanoma, >60% have BRAFV600 mutations that induce oncogenic BRAF kinase activity that enhances proliferation of melanoma cells. While much has been learned from relapse melanoma, little is known about the in vivo impact of targeted therapy on persisting cells during therapy. The results of this dissertation generated new methods to obtain high quality viable single cells from human tumors and validated a novel set of mass cytometry reagents focused on identifying and deeply characterizing neural origin cells. These tools were also applied to deeply characterize cell protein expression signatures of melanoma cells from tumor samples obtained before treatment and at the conclusion of neoadjuvant therapy targeting BRAF and MEK proteins. The resulting single cell view of melanoma treatment response revealed initially heterogeneous melanoma were consistently cleared of cells expressing Nestin protein, a hallmark of aggressive melanoma. Mass cytometry analysis of cancerous cells was found to correlate closely with results obtained by traditional histology. An important advantage of mass cytometry was that complex cell phenotypes within the melanoma tumors could be identified and characterized using each of the 32 proteins that were measured simultaneously. As a result, it was also apparent that immune interaction protein, MHC I, was not expressed on melanoma cells after BRAF and MEK targeted therapy. Analysis with computational tools viSNE, FlowSOM, and MEM assisted in revealing and characterizing persisting melanoma cells within tumors. The results of this work revealed new in vivo biology of melanoma cells that contrasted with expectations from laboratory culture models and analysis of tumors at the time of relapse. Thus, the functional qualities of melanoma cells observed in vivo in human tumors demand additional attention and the results of this dissertation provide a roadmap for improving clinical trials and research models, such as patient-derived xenografts and cell lines. The approach here focused on single cell analysis of cancerous cells can now be developed for clinical research studies to identify mechanisms of resistance with single cell precision.