The Weisenthal MVV (microvascularity viability) assay was invented by Dr. Weisenthal, based upon a discovery he made involving the presence of endothelial cells in dissociated specimens of human cancer.  The discovery and also the invention which followed the discovery were reported in the Journal of Internal Medicine.  Because the article is copyrighted by the Journal of Internal Medicine, we cannot publish it here.  However, to read the abstract (a summary of the main points of the article) or to order the full text version of the article from the Journal of Internal Medicine, click here.  For a link to a press release which discusses the article and its implications, click here).  The MVV assay allows for reliable identification and characterization of endothelial cells in dissociated specimens of cancer tissue and specifically those comprised of living cancer cells.  The advantage of testing living cancer and endothelial cells is that the effects of different drugs and different combinations of drugs can be assessed directly.  Other model systems, in including those which focus on genetic factors, utilize dead, preserved cells.  These model systems are surrogate methods which show only a theoretical predisposition to drug response based upon a single factor or at best a  small handful of factors.   The preserved cells used in these model systems are never exposed to the actual drugs under consideration.  Other model systems, which also use dead, preserved cells, seek to correlate microvessel counts before and after treatment with anti-angiogenesis drug activity.   To date, there has been little evidence that either of these classes of systems is reliably predictive of patient benefit from angiogenesis inhibiting drugs, from combinations of different angiogenesis-inhibiting drugs, or from combinations of angiogenesis-inhibiting drugs and other types of drugs.

 

In contrast, the MVV assay measures the net effect of the full range of genetic and also mechanical processes which occur within endothelial and cancer cells when these cells actually are exposed to each specific drug or drug combination.

 

An extremely important consideration is that the MVV assay reliably can distinguish anti-tumor drug effects (those that kill the cancer cell by direct insult) from anti-angiogenic drug effects (those which kill endothelial cells and presumptively result in tumor cell killing by indirect means) within the same mixed-cell population.         

 

In order to further assess anti-tumor activity in addition to anti-angiogenic activity, between 2 and 5 additional assay technologies are applied.

 

In the Weisenthal DISC (Differential Staining Cytotoxicity) assay, originated by Dr. Weisenthal while at the NCI, the entire contents of the cell culture are cytocentrifuged onto permanent microscope slides and differentially stained to allow discrimination of normal and neoplastic cells and living and dead cells. The endpoint for cell death is delayed loss of membrane integrity, which has been found to be a surrogate for apoptosis. Advantages of the DISC assay include direct visualization of tumor cells and establishment of a permanent archival record.  The DISC assay was the first of the new-generation functional tumor cell profiling methods to feature the cell death endpoint, upon which nearly all new-generation functional profiling assays subsequently were based.  Interpretation of DISC assay slides is highly labor-intensive but the assay is widely-regarded among experts in the field to be the gold standard owing to the ability examine directly each cell in order to positively discriminate tumor cells from non-tumor cells and to better characterize drug effects upon the entire tumor cell population.  Click here to see a simplified graphic DISC assay methodology flowchart.

 

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay measures mitochondrial metabolism in the entire cell culture. In the assay, yellow tetrazolium salt (MTT) is reduced in metabolically active cells to form purple formazan.  The color can then be quantified by spectrophotometry, enabling an accurate measurement of metabolic activity.

 

The ATP (Adenosine Triphosphate) assay measures cellular ATP content by luminometry, based on the luciferin/luciferase reaction. Cells maintain a critical ATP thresholds whose measurement reflects cell viability, specifically indicating, in functional tumor cell profiling, whether apoptotic cell death has occurred during drug exposure.

 

The redox (resazurin) assay measures total metabolic activity in the entire cell culture, using the Alamar Blue reagent.

 

The caspase 3/7 assay measures the activation of caspases 3 and 7 using luminometry.

 

All of the above assay involve cell death endpoints.

 

 

 

 

 

 

 

 

 

 

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