Characterizing heterogeneous single-cell dose responses computationally and experimentally using threshold inhibition surfaces and dose-titration assays.

Single cancer cells within a tumor exhibit variable levels of resistance to drugs, ultimately leading to treatment failures. While tumor heterogeneity is recognized as a major obstacle to cancer therapy, standard dose-response measurements for the potency of targeted kinase inhibitors aggregate populations of cells, obscuring intercellular variations in responses. In this work, we develop an analytical and experimental framework to quantify and model dose responses of individual cancer cells to drugs. We first explore the connection between population and single-cell dose responses using a computational model, revealing that multiple heterogeneous populations can yield nearly identical population dose responses. We demonstrate that a single-cell analysis method, which we term a threshold inhibition surface, can differentiate among these populations. To demonstrate the applicability of this method, we develop a dose-titration assay to measure dose responses in single cells. We apply this assay to breast cancer cells responding to phosphatidylinositol-3-kinase inhibition (PI3Ki), using clinically relevant PI3Kis on breast cancer cell lines expressing fluorescent biosensors for kinase activity. We demonstrate that MCF-7 breast cancer cells exhibit heterogeneous dose responses with some cells requiring over ten-fold higher concentrations than the population average to achieve inhibition. Our work reimagines dose-response relationships for cancer drugs in an emerging paradigm of single-cell tumor heterogeneity.