Supplementary Figure S1. Characterization of cancer spheroids. Supplementary Figure S2. Characteristics of spheroids based on morphologic classification. Supplementary Figure S3. Validation of spheroid morphology-dependent characteristics. Supplementary Figure S4. Validation of PLS-DA model. Supplementary Figure S5. Network models describing cellular processes associated with spheroid subtypes. Supplementary Figure S6. Drug penetration test in spheroids using bimolecular fluorescence complementation (BiFC). Supplementary Figure S7. Molecular signature of GBM patient-derived round type spheroid. Supplementary Figure S8. Xenograft model to validate JAK-STAT pathway for drug sensitivity.
ARTICLE ABSTRACTTumor permeability is a critical determinant of drug delivery and sensitivity, but systematic methods to identify factors that perform permeability barrier functions in the tumor microenvironment are not yet available. Multicellular tumor spheroids have become tractable in vitro models to study the impact of a three-dimensional (3D) environment on cellular behavior. In this study, we characterized the spheroid-forming potential of cancer cells and correlated the resulting spheroid morphologies with genetic information to identify conserved cellular processes associated with spheroid structure. Spheroids generated from 100 different cancer cell lines were classified into four distinct groups based on morphology. In particular, round and compact spheroids exhibited highly hypoxic inner cores and permeability barriers against anticancer drugs. Through systematic and correlative analysis, we reveal JAK–STAT signaling as one of the signature pathways activated in round spheroids. Accordingly, STAT3 inhibition in spheroids generated from the established cancer cells and primary glioblastoma patient–derived cells altered the rounded morphology and increased drug sensitivity. Furthermore, combined administration of the STAT3 inhibitor and 5-fluorouracil to a mouse xenograft model markedly reduced tumor growth compared with monotherapy. Collectively, our findings demonstrate the ability to integrate 3D culture and genetic profiling to determine the factors underlying the integrity of the permeability barrier in the tumor microenvironment, and may help to identify and exploit novel mechanisms of drug resistance. Cancer Res; 76(5); 1044–54. ©2015 AACR.