Dissecting multi drug resistance in head and neck cancer cells using multicellular tumor spheroids.

One of the hallmarks of cancers is their ability to develop resistance against therapeutic agents. Therefore, developing effective in vitro strategies to identify drug resistance remains of paramount importance for successful treatment. One of the ways cancer cells achieve drug resistance is through the expression of efflux pumps that actively pump drugs out of the cells. To date, several studies have investigated the potential of using 3-dimensional (3D) multicellular tumor spheroids (MCSs) to assess drug resistance; however, a unified system that uses MCSs to differentiate between multi drug resistance (MDR) and non-MDR cells does not yet exist. In the present report we describe MCSs obtained from post-diagnosed, pre-treated patient-derived (PTPD) cell lines from head and neck squamous cancer cells (HNSCC) that often develop resistance to therapy. We employed an integrated approach combining response to clinical drugs and screening cytotoxicity, monitoring real-time drug uptake, and assessing transporter activity using flow cytometry in the presence and absence of their respective specific inhibitors. The report shows a comparative response to MDR, drug efflux capability and reactive oxygen species (ROS) activity to assess the resistance profile of PTPD MCSs and two-dimensional (2D) monolayer cultures of the same set of cell lines. We show that MCSs provide a robust and reliable in vitro model to evaluate clinical relevance. Our proposed strategy can also be clinically applicable for profiling drug resistance in cancers with unknown resistance profiles, which consequently can indicate benefit from downstream therapy.

Cancer/testis antigen expression in human mesenchymal stem cells: down-regulation of SSX impairs cell migration and matrix metalloproteinase 2 expression.

Several families of genes by and large located on the X chromosome encode proteins of unspecified function. Commonly known as cancer/testis (CT) antigens, they are considered, under normal conditions, only to be expressed in cells of the germ line and placenta. CT genes are also often expressed in cancer cells, hence their classification. Here we report that their expression in normal cells is wider spread and can be observed in cells with the potential for self-renewal and pleuripotency, namely, stem cells. Several CT genes and their products, CT antigens, including SSX, NY-ESO-1, and N-RAGE, were expressed in undifferentiated mesenchymal stem cells (MSCs) and down-regulated after osteocyte and adipocyte differentiation. To elucidate the possible overlapping function played by these genes in cancer and stem cells, a comparative analysis of the localization of their proteins was made. In addition, localization relative to other MSC markers was examined. This revealed that SSX localizes in the cytoplasm and overlap occurs in regions where matrix metalloproteinase 2 (MMP2) and vimentin accumulate. Nevertheless, it was found that no protein interactions between these molecules occur. Further investigation revealed that the migration of a melanoma cell line (DFW), which expresses SSX, MMP2, and vimentin, decreases when SSX is down-regulated. This decrease in cell migration was paralleled by a reduction in MMP2 levels. Analogous to this, SSX expression is down-regulated in MSCs after differentiation; concomitantly a reduction in MMP2 levels occurs. In addition, E-cadherin expression increases, mimicking a mesenchymal epithelial transition. These results afford SSX a functional role in normal stem cell migration and suggest a potentially similar function in cancer cell metastases.

Phenotype-based drug screening in primary ovarian carcinoma cultures identifies intracellular iron depletion as a promising strategy for cancer treatment.

Primary cultures of patient tumor cells (PCPTC) have been used for prediction of diagnosis-specific activity and individual patient response to anticancer drugs, but have not been utilized as a model for identification of novel drugs in high throughput screening. In the present study, ovarian carcinoma cells from three patients were tested in response to a library of 3000 chemically diverse compounds. Eight hits were retrieved after counter screening using normal epithelial cells, and one of the two structurally related hit compounds was selected for further preclinical evaluation. This compound, designated VLX 50, demonstrated a broad spectrum of activity when tested in a panel of PCPTCs representing different forms of leukemia and solid tumors and displayed a high tumor to normal cell activity. VLX 50 induced delayed cell death with some features of classical apoptosis. Significant in vivo activity was confirmed on primary cultures of human ovarian carcinoma cells in mice using the hollow fiber model. Mechanistic exploration was performed using gene expression analysis of drug exposed tumor cells to generate a drug-specific signature. This query signature was analyzed using the Gene Set Enrichment Analysis and the Connectivity Map database. Strong connections to hypoxia inducible factor 1 and iron chelators were retrieved. The mechanistic hypothesis of intracellular iron depletion leading to hypoxia signaling was confirmed by a series of experiments. The results indicate the feasibility of using PCPTC for cancer drug screening and that intracellular iron depletion could be a potentially important strategy for cancer therapy.