Error bars represent SD. a 24-well plate over 23 d in culture. Error bars symbolize SD. (= 5 for each AZ3451 time point). Results showed that the average cell number per acinus increases over time and correlates with that of acinar size (and and and and axis represents days after injection, and axis represents tumor volume (cubic millimeters). Error bars symbolize SEM. ( 0.05) and found a total of 9,227 genes differentially expressed, indicating that changing culture conditions from 2D to 3D exerts AZ3451 a major influence on gene expression in TUM622 cells (Dataset S1). To explore which of the annotated pathways and functional groups of genes are enriched in three sizes, we employed Gene Set Enrichment Analysis (GSEA) and recognized a total of 699 gene sets that are up-regulated in 3D culture [false discovery rate (FDR) 0.25] (Dataset S2). Interestingly, these data include a large number of gene units related to oncogenesis as well as genes and pathways involved in normal lung development, such as canonical Wnt, Notch, and Hedgehog signaling pathways, ES cell genes, and biological processes regulating acinar morphogenesis and polarity, as well as disease prognosis (Dataset S3). To identify the most significant gene units, we applied a more stringent cutoff of FDR 0.05 and a normalized enrichment score of 1.8 and found that the Wnt pathway, ES genes, polarity/acinar morphogenesis, and factors associated with poor prognosis are significantly enriched, while Notch and Hedgehog pathways are not (Fig. 3). Open in a separate windows Fig. 3. Gene units enriched in TUM622 AZ3451 3D vs. 2D culture. ((are up-regulated in 3D compared with 2D culture, while and are largely unchanged (is usually higher than that of or in both 2D and 3D cultures, and the up-regulation of SOX2 in 3D culture is further confirmed at the protein level (by siRNA transfection. Data showed that reducing in TUM622 cells inhibited acinar morphogenesis in TUM622 cells as well as other NSCLC cell lines while having minimal effect on cell viability (expression level in Vector vs. SOX2oe spheroids in 3D culture as quantified by qRT-PCR. Column represents triplicates, and error bars represent SD; 3 10?5. (= 5). Error bars symbolize SD. * 0.05 FLB7527 in Students Vector vs. SOX2oe spheroids. Column represents triplicates, and error bars represent SD. * 0.05. We first generated stable pools of control Vector and SOX2-overexpressing TUM622 cells (termed Vector and SOX2oe cells) by transducing the parental TUM622 collection with lentiviral vectors. In two sizes, SOX2-overexpressing cells adopted a mesenchymal phenotype in comparison with round Vector cells (and locus (Fig. 4and and and and and ?and4and and and and and and 2 10?5. (and and and and value 0.05]. Supplementary Material Supplementary FileClick here to view.(10M, pdf) Supplementary FileClick here to view.(9.7M, xlsx) Supplementary FileClick here to view.(159K, xlsx) Supplementary FileClick here to view.(12K, xlsx) Acknowledgments We thank Jeanine Pignatelli, Matthew Sung, and Jennifer Kahler for critical review of the manuscript; Keith Kobylarz for help with circulation cytometry; Edward Rosfjord, Vladmir Buklan, and Roger Conant for in vivo studies; Magali Guffroy, John Kreeger, and Stephani Bisulco of the Pfizer-Oncology Histopathology and Biomarker group for pathology/histology support; Fred Immermann and Vinicius Bonato for help with statistical analysis; and Maximum Follettie and Veronica Diesl for support with the Affymetrix platform. We also thank the Pfizer Postdoctoral Program and the Oncology R&D group, specifically Karen Widbin, Puja Sapra, and Robert Abraham, for their support.