TNFα antagonization alters NOS2 dependent nasopharyngeal carcinoma tumor growth.

Tumor necrosis factor (TNFα) is a pro-inflammatory cytokine which mediates via nitric oxide (NO) several carcinogenic processes. Increasing evidences suggest that NO promotes inflammation induced growth of nasopharyngeal carcinoma (NPC). In patients, TNFα synthesis associates with poor survival. To explore the effect of the cytokine on NO production and NOS2 dependent NPC growth, NO2(-) (nitrite) producing cells in patients were analyzed in vitro. We observed that patients' monocytes/macrophages (Mo/Ma) and primary tumor biopsies synthesized significant amounts of NO2(-). Interestingly, tumor explants derived NO2(-) levels were more important in elderly patients in comparison with juveniles. Endogenous TNFα neutralization with an anti-TNFα monoclonal antibody (mAb) successfully inhibited NO2(-) synthesis by blood mononuclear cells and tumor explants. Recombinant TNFα (rTNFα) enhanced NO2(-) synthesis and C666-1 NPC cell proliferation. NOS2 selective inhibition (1400W) and TNFα antagonization with an anti-TNFα mAb potently inhibited rTNFα induced C666-1 proliferation and NO2(-) production. Importantly, primary tumors treated with the anti-TNFα mAb also displayed reduced proliferation index (Ki67). Altogether, our results define monocytes/macrophages and the primary tumor as major sources of circulating NO2(-) in NPC patients and support the idea that antibody dependent inhibition of the TNFα/NOS2 pathway may alter NPC tumor growth.

Combinative in vitro studies and computational model to predict 3D cell migration response to drug insult.

The development of drugs to counter diseases related to cell migration has resulted in a multi-billion dollar endeavor. Unfortunately, few drugs have emerged from this effort highlighting the need for new methods to enhance assays to study, analyze and control cell migration. In response to this complex process, computational models have emerged as potent tools to describe migration providing a high throughput and low cost method. However, most models are unable to predict migration response to drug with direct application to in vitro experiments. In addition to this, no model to date has attempted to describe migration in response to drugs while incorporating simultaneously protein signaling, proteolytic activity, and 3D culture. In this paper, we describe an integrated computational approach, in conjunction with in vitro observations, to serve as a platform to accurately predict migration in 3D matrices incorporating the function of matrix metalloproteinases (MMPs) and their interaction with the Extracellular signal-related kinase (ERK) signaling pathway. Our results provide biological insight into how matrix density, MMP activity, integrin adhesions, and p-ERK expression all affect speed and persistence in 3D. Predictions from the model provide insight toward improving drug combinations to more effectively reduce both speed and persistence during migration and the role of integrin adhesions in motility. In this way our integrated platform provides future potential to streamline and improve throughput toward the testing and development of migration targeting drugs with tangible application to current in vitro assays.

Alteration of cellular behavior and response to PI3K pathway inhibition by culture in 3D collagen gels.

Most investigations into cancer cell drug response are performed with cells cultured on flat (2D) tissue culture plastic. Emerging research has shown that the presence of a three-dimensional (3D) extracellular matrix (ECM) is critical for normal cell behavior including migration, adhesion, signaling, proliferation and apoptosis. In this study we investigate differences between cancer cell signaling in 2D culture and a 3D ECM, employing real-time, live cell tracking to directly observe U2OS human osteosarcoma and MCF7 human breast cancer cells embedded in type 1 collagen gels. The activation of the important PI3K signaling pathway under these different growth conditions is studied, and the response to inhibition of both PI3K and mTOR with PI103 investigated. Cells grown in 3D gels show reduced proliferation and migration as well as reduced PI3K pathway activation when compared to cells grown in 2D. Our results quantitatively demonstrate that a collagen ECM can protect U2OS cells from PI103. Overall, our data suggests that 3D gels may provide a better medium for investigation of anti-cancer drugs than 2D monolayers, therefore allowing better understanding of cellular response and behavior in native like environments.

Quantitative analysis of the effect of cancer invasiveness and collagen concentration on 3D matrix remodeling.

Extracellular matrix (ECM) remodeling is a key component of cell migration and tumor metastasis, and has been associated with cancer progression. Despite the importance of matrix remodeling, systematic and quantitative studies on the process have largely been lacking. Furthermore, it remains unclear if the disrupted tensional homeostasis characteristic of malignancy is due to initially altered ECM and tissue properties, or to the alteration of the tissue by tumor cells. To explore these questions, we studied matrix remodeling by two different prostate cancer cell lines in a three-dimensional collagen system. Over one week, we monitored structural changes in gels of varying collagen content using confocal reflection microscopy and quantitative image analysis, tracking metrics of fibril fraction, pore size, and fiber length and diameter. Gels that were seeded with no cells (control), LNCaP cells, and DU-145 cells were quantitatively compared. Gels with higher collagen content initially had smaller pore sizes and higher fibril fractions, as expected. However, over time, LNCaP- and DU-145-populated matrices showed different structural properties compared both to each other and to the control gels, with LNCaP cells appearing to favor microenvironments with lower collagen fiber fractions and larger pores than DU-145 cells. We posit that the DU-145 cells' preference for denser matrices is due to their higher invasiveness and proteolytic capabilities. Inhibition of matrix proteases resulted in reduced fibril fractions for high concentration gels seeded with either cell type, supporting our hypothesis. Our novel quantitative results probe the dynamics of gel remodeling in three dimensions and suggest that prostate cancer cells remodel their ECM in a synergistic manner that is dependent on both initial matrix properties as well as their invasiveness.