Date of Award

January 2020

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Smita Krishnaswamy

Abstract

While several tools have been developed to map axes of variation among individual cells, no analogous approaches exist for identifying axes of variation among multicellular biospecimens profiled at single-cell resolution. Developing such an approach is of great translational relevance and interest, as single-cell expression data are now often collected across numerous experimental conditions (e.g., representing different drug perturbation conditions, CRISPR knockdowns, or patients undergoing clinical trials) that need to be compared. In this work, “Phenotypic Earth Mover's Distance” (PhEMD) is presented as a solution to this problem. PhEMD is a general method for embedding a “manifold of manifolds,” in which each datapoint in the higher-level manifold (of biospecimens) represents a collection of points that span a lower-level manifold (of cells).

PhEMD is applied to a newly-generated, 300-biospecimen mass cytometry drug screen experiment to map small-molecule inhibitors based on their differing effects on breast cancer cells undergoing epithelial–mesenchymal transition (EMT). These experiments highlight EGFR and MEK1/2 inhibitors as strongly halting EMT at an early stage and PI3K/mTOR/Akt inhibitors as enriching for a drug-resistant mesenchymal cell subtype characterized by high expression of phospho-S6. More generally, these experiments reveal that the final mapping of perturbation conditions has low intrinsic dimension and that the network of drugs demonstrates manifold structure, providing insight into how these single-cell experiments should be computational modeled and visualized. In the presented drug-screen experiment, the full spectrum of perturbation effects could be learned by profiling just a small fraction (11%) of drugs. Moreover, PhEMD could be integrated with complementary datasets to infer the phenotypes of biospecimens not directly profiled with single-cell profiling. Together, these findings have major implications for conducting future drug-screen experiments, as they suggest that large-scale drug screens can be conducted by measuring only a small fraction of the drugs using the most expensive high-throughput single-cell technologies—the effects of other drugs may be inferred by mapping and extending the perturbation space.

PhEMD is also applied to patient tumor biopsies to assess intertumoral heterogeneity. Applied to a melanoma dataset and a clear-cell renal cell carcinoma dataset (ccRCC), PhEMD maps tumors similarly to how it maps perturbation conditions as above in order to learn key axes along which tumors vary with respect to their tumor-infiltrating immune cells. In both of these datasets, PhEMD highlights a subset of tumors demonstrating a marked enrichment of exhausted CD8+ T-cells. The wide variability in tumor-infiltrating immune cell abundance and particularly prominent exhausted CD8+ T-cell subpopulation highlights the importance of careful patient stratification when assessing clinical response to T cell-directed immunotherapies.

Altogether, this work highlights PhEMD’s potential to facilitate drug discovery and patient stratification efforts by uncovering the network geometry of a large collection of single-cell biospecimens. Our varied experiments demonstrate that PhEMD is highly scalable, compatible with leading batch effect correction techniques, and generalizable to multiple experimental designs, with clear applicability to modern precision oncology efforts.

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This is an Open Access Thesis.

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