Background: Non-small cell lung cancer (NSCLC) is difficult to treat successfully. This intractability is mainly due to the cancer progressing through invasion, metastasis, chemotherapeutic resistance and relapse. Stemness has been linked to the various steps of cancer progression in a variety of tumors, yet little is known regarding its role in NSCLC. Purpose: In this study, we sought to determine the role of SOX2, a master regulator of pluripotency, in the growth of extracellular matrix (ECM)-detached cells during cancer progression. Methods: We established a three-dimensional (3D) Poly-2-hydroxyethyl methacrylate (poly-HEMA) culture of lung adenocarcinoma (LUAD) A549 cells as an ECM-detached cell growth model and examined the role of stemness genes using siRNA and small molecule inhibitor in comparison to standard two dimensional (2D) culture. Results: In poly-HEMA culture, A549 cells formed substratum-detached spheroids with characteristics of intermediate epithelial to mesenchymal transition (EMT) and exhibited greater expression of SOX2 than did control 2D cells. Knockdown of SOX2 markedly suppressed the growth of A549 cell aggregates in poly-HEMA culture conditions and furthermore increased their sensitivity to the anticancer drug vinblastine with concomitant downregulation of the activity of the anti-apoptotic AKT kinase. Interestingly, a small molecule, RepSox, which replaces SOX2, stimulated A549 cell growth in poly-HEMA 3D culture condition. Conclusion: Our findings strongly indicate that SOX2 contributes to anchorage-independent growth and chemoresistance via its downstream signaling mediator AKT kinase during the disease progression of NSCLC. SOX2 may therefore be an invaluable therapeutic target of NSCLC.

Salivary gland stem cells (SGSCs) are potential cell sources for the treatment of salivary gland diseases. The control of cell survival is an essential factor for applying stem cells to regenerative medicine or stem cell-based research. The purpose of this study was to investigate the effects of the ROCK inhibitor Y-27632 on the survival of SGSCs and its underlying mechanisms. SGSCs were isolated from mouse submandibular glands and cultured in suspension. Treatment with Y-27632 restored the viability of SGSCs that was significantly decreased during isolation and the subsequent culture. Y-27632 upregulated the expression of anti-apoptotic protein BCL-2 in SGSCs and, in the apoptosis assay, significantly reduced apoptotic and necrotic cell populations. Matrigel was used to mimic the extracellular environment of an intact salivary gland. The expression of genes regulating apoptosis and the ROCK signaling pathway was significantly reduced when SGSCs were embedded in Matrigel. SGSCs cultured in Matrigel and treated with Y-27632 showed no difference in the total numbers of spheroids and expression levels of apoptosis-regulating genes. Matrigel-embedded SGSCs treated with Y-27632 increased the number of spheroids with budding structures and the expression of acinar cell-specific marker AQP5. We demonstrate the protective effects of Y-27632 against dissociation-induced apoptosis of SGSCs during their culture in vitro.

Biological systems consist of a variety of distinct cell types that form functional networks. Super-resolution imaging of individual cells is required for better understanding of these complex systems. Direct visualization of 3D subcellular and nano-scale structures in cells is helpful for the interpretation of biological interactions and system-level responses. Here we introduce a modified magnified analysis of proteome (MAP) method for cell super-resolution imaging (Cell-MAP) which preserves cell fluorescence. Cell-MAP expands cells more than four-fold while preserving their overall architecture and three-dimensional proteome organization after hydrogel embedding. In addition, Optimized-Cell-MAP completely preserves fluorescence and successfully allows for the observation of tagged small molecular probes containing peptides and microRNAs. Optimized-Cell-MAP further successfully applies to the study of structural characteristics and the identification of small molecules and organelles in mammalian cells. These results may give rise to many other applications related to the structural and molecular analysis of smaller assembled biological systems.

Tumor cell aggregation is critical for cell survival following the loss of extracellular matrix attachment and dissemination. However, the underlying mechanotransduction of clustering solitary tumor cells is poorly understood, especially in non-small cell lung cancers (NSCLC). Here, we examined whether cell surface protrusions played an important role in facilitating the physical contact between floating cells detached from a substrate. We employed poly-2-hydroxyethyl methacrylate-based 3D culture methods to mimic in vivo tumor cell cluster formation. The suprastructural analysis of human NSCLC A549 cell spheroids showed that finger-like protrusions clung together via the actin cytoskeleton. Time-lapse holotomography demonstrated that the finger-like protrusions of free-floating cells in 3D culture displayed exploratory coalescence. Global gene expression analysis demonstrated that the genes in the organic hydroxyl transport were particularly enriched in the A549 cell spheroids. Particularly, the knockdown of the water channel aquaporin 3 gene (AQP3) impaired multicellular aggregate formation in 3D culture through the rearrangement of the actomyosin cytoskeleton. Moreover, the cells with reduced levels of AQP3 decreased their transmigration. Overall, these data indicate that cell detachment-upregulated AQP3 contributes to cell surface protrusions through actomyosin cytoskeleton remodeling, causing the aggressive aggregation of free-floating cells dependent on the property of the substratum and collective metastasis.