Cooperative adaptation to therapy (CAT) confers resistance in heterogeneous non-small cell lung cancer

Understanding intrinsic and acquired resistance is crucial to overcoming cancer chemotherapy failure. While it is well-established that intratumor, subclonal genetic and phenotypic heterogeneity significantly contribute to resistance, it is not fully understood how tumor sub-clones interact with each other to withstand therapy pressure. Here, we report a previously unrecognized behavior in heterogeneous tumors: cooperative adaptation to therapy (CAT), in which cancer cells induce co-resistant phenotypes in neighboring cancer cells when exposed to cancer therapy. Using a CRISPR/Cas9 toolkit we engineered phenotypically diverse non-small cell lung cancer (NSCLC) cells by conferring mutations in Dicer1, a type III cytoplasmic endoribonuclease involved in small non-coding RNA genesis. We monitored three-dimensional growth dynamics of fluorescently-labeled mutant and/or wild-type cells individually or in co-culture using a substrate-free NanoCulture system under unstimulated or drug pressure conditions. By integrating mathematical modeling with flow cytometry, we characterized the growth patterns of mono- and co-cultures using a mathematical model of intra- and interspecies competition. Leveraging the flow cytometry data, we estimated the model’s parameters to reveal that the combination of WT and mutants in co-cultures allowed for beneficial growth in previously drug sensitive cells despite drug pressure via induction of cell state transitions described by a cooperative game theoretic change in the fitness values. Finally, we used an ex vivo human tumor model that predicts clinical response through drug sensitivity analyses and determined that cellular and morphologic heterogeneity correlates to prognostic failure of multiple clinically-approved and off-label drugs in individual NSCLC patient samples. Together, these findings present a new paradox in drug resistance implicating non-genetic cooperation among tumor cells to thwart drug pressure, suggesting that profiling for druggable targets (i.e. mutations) alone may be insufficient to assign effective therapy.Author summary: Here, we provide mathematical and empirical evidence to support a potentially new paradigm in drug resistance, which we have termed “cooperative adaptation to therapy” (CAT). CAT is defined by a phenomenon wherein drug-sensitive cancer cells with different genetic and phenotypic features within a 3-dimensional heterogeneous tumor induce non-mutational resistance in their neighboring cells under pressure of cancer therapy. To develop this novel conclusion we deployed an interdisciplinary effort including an ex vivo human tumor model, a CRISPR/Cas9 platform with 3-dimensional in vitro experiments, and high throughput flow cytometry. Importantly, we wove these data together using a mathematical model of intra- and interspecies competition to understand how tumor heterogeneity influenced our observations. By estimating the model’s parameters, we determined that the combination of genetic clonal variants in co-cultures allowed for previously drug-sensitive cells to continue to grow despite drug pressure. We were thus able to characterize distinct growth regimens in mono- and co-cultures without and with drug pressure.