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SummaryTargeted therapeutics have advanced cancer treatment, but single agent activity remains limited by de novo and acquired resistance. Combining targeted drugs is broadly seen as a way to improve treatment outcome, motivating the ongoing search for efficacious combinations. To identify synergistic targeted therapy combinations and study the impact of tumor heterogeneity on combination outcome, we systematically tested over 5,000 two drug combinations at multiple doses across a collection of 81 non-small cancer cell lines. Both known and novel synergistic combinations were identified. Strikingly, very few combinations yield synergy across the majority of cell line models. Importantly, synergism mainly arises due to sensitization of single agent resistant models, rather than further sensitize already sensitive cell lines, frequently via dual targeting of a single or two highly interconnected pathways. This drug combinations resource, the largest of its kind should help delineate new synergistic regimens by facilitating the understanding of drug synergism in cancer.

Summary Two-dimensional (2D) culture of tumor cells fails to recapitulate some important aspects of cellular organization seen in in vivo experiments. In addition, cell cultures traditionally use non-physiological concentration of nutrients. Here, we describe a protocol for a facile three-dimensional (3D) culture format for cancer cells. This 3D platform helps overcome the 2D culture limitations. In addition, it allows for longitudinal modeling of responses to cancer therapeutics. For complete details on the use and execution of this protocol, please refer to Lhuissier et al. (2017), Lehmann et al. (2016), Liu et al. (2016), and Duval et al. (2011)., Graphical Abstract, Highlights • A detailed protocol on hydrogel-based 3D culture of patient-derived tumor cell lines • No binding sites for cells in hydrogel polymers allowing for pure interaction of cells • Longitudinal 3D proliferation assays and drug-response assessments • Quick and easy recovery of 3D-cultured cells for downstream experiments, Two-dimensional (2D) culture of tumor cells fails to recapitulate some important aspects of cellular organization seen in in vivo experiments. In addition, cell cultures traditionally use non-physiological concentration of nutrients. Here, we describe a protocol for a facile three-dimensional (3D) culture format for cancer cells. This 3D platform helps overcome the 2D culture limitations. In addition, it allows for longitudinal modeling of responses to cancer therapeutics.

Cancer-associated fibroblasts (CAFs) are highly heterogeneous. With the lack of a comprehensive understanding of CAFs' functional distinctions, it remains unclear how cancer treatments could be personalized based on CAFs in a patient's tumor. We have established a living biobank of CAFs derived from biopsies of patients' non-small lung cancer (NSCLC) that encompasses a broad molecular spectrum of CAFs in clinical NSCLC. By functionally interrogating CAF heterogeneity using the same therapeutics received by patients, we identify three functional subtypes: (1) robustly protective of cancers and highly expressing HGF and FGF7; (2) moderately protective of cancers and highly expressing FGF7; and (3) those providing minimal protection. These functional differences among CAFs are governed by their intrinsic TGF-β signaling, which suppresses HGF and FGF7 expression. This CAF functional classification correlates with patients' clinical response to targeted therapies and also associates with the tumor immune microenvironment, therefore providing an avenue to guide personalized treatment.