Single-cell methods for profiling tumor & microenvironment responses to therapeutic challenges

Abstract

Heterogeneity among cells affects function and dysfunction across many complex biological systems. This heterogeneity is particularly important in cancer biology, where variation in the cells composing tumors and their surroundings can affect a patients' response to treatment and subsequent survival. While current methods, such as bulk RNA-Sequencing, are incredibly powerful, they typically measure average phenomena, mischaracterizing the distribution of behaviors within a system. Single-cell technologies - single-cell RNA Sequencing in particular have been foundational in elucidating cellular heterogeneity from first principles, but there are limitations to their application for studying cancer and its response to treatment. Here, we detail efforts to address current needs in profiling treatment responses of tumors and their microenvironments at single-cell resolution.

Specifically, we characterize the underlying cellular diversity of tumor microenvironments, investigate the effect of drug treatment in specific cellular compartments, identify proxies of response in accessible cellular reservoirs, and investigate orthogonal cellular readouts of response. We first apply single-cell RNA Sequencing to study heterogeneity in metastatic melanoma, detailing heterogeneity and potential sources of resistance in cancer cells of profiled patients. Next, we study the effect of drug treatment in leptomeningeal carcinomatosis (LMD), extending previous strategies to utilize pre- and post-treatment patient sampling. We demonstrate the effect of immunotherapy in this microenvironment, and use longitudinal data from specific patients describe the evolution of cancer cell response to treatment. We next expand liquid biopsy profiling to other compartments, specifically circulating tumor cells (CTCs) in blood.

We describe the development of a microfluidic device that captures murine CTCs with minimal sampling. We perform single-CTC RNA-Sequencing to study their response to treatment and relationship to their primary tumors. Finally, we develop a device that simultaneously measures the mass, growth rate and transcriptome of single cells, and use it to investigate the transcriptional activity of cancer cells that continue to grow after therapeutic challenge. Together, this body of work represents contributions towards extending single-cell profiling to understand how cells in naturally occurring and model cancer microenvironments respond to drug treatment.