funded investigators

Benjamin G. Neel, M.D., Ph.D. Garry P. Nolan, Ph.D.
Ontario Cancer Institute Stanford University

Characterization of Ovarian Tumor Initiating Cells by Mass Cytometry

 Serous ovarian cancer (SOC) is the leading cause of morbidity/mortality from gynecologic malignancy. Current therapies significantly increase survival, yet the vast majority of SOC patients (70 percent to 90 percent) recur within five years and die of their disease. The cancer stem cell (CSC) hypothesis holds that only some tumor cells have the potential to initiate and maintain the tumor. Moreover, CSC might be resistant to many therapeutic agents that are effective against bulk tumor cells, which could contribute to treatment failure.

Drs. Neel and Nolan have established a large collection of primary human SOC samples and developed a robust, quantitative assay for SOC tumor-initiating cells (TIC). Using this assay, they found that TIC from primary SOC can be identified based on their expression of the surface marker CD133. However, they also observed heterogeneity and instability of the TIC phenotype with disease progression. Microarray analysis of our TIC-enriched fractions has identified specific cell signaling pathways that might be important to CSC.

Although these results indicate that CD133 cannot be used to unambiguously identify CSC in all SOC cases, there might still be a marker "above" CD133 in the tumor hierarchy that has such properties. For example, a marker/marker combination that is functionally required for maintenance of CSC or that is obligately expressed on cells with CSC properties would be expected to identify TIC throughout disease progression. Candidate approaches to marker identification are laborious, costly and require access to large amounts of tissue.

A new technique, inductively-coupled plasma mass spectrometry (mass cytometry), which combines atomic mass spectrometry and flow cytometry, provides a novel platform for examining the expression of a large number of markers simultaneously. They hypothesize that using multiparametric mass cytometry to concomitantly assess many markers/marker combinations will provide a rapid and possibly clinically applicable way to identify TIC in SOC. To this end, a large panel of antibodies to surface antigens that we have shown to label small fractions of SOC cells will be converted to CyTOF reagents. They will characterize the expression of these antigens, along with intracellular proteins associated with "stemness", in 25 primary SOC samples.

Using novel software and hardware developed by their collaborators in Dr. Nolan's lab, they will ask if these samples exhibit a hierarchical organization. Markers/marker combinations that co-stain with self-renewal genes/miRNAs will be tested for their ability to enrich for TIC in their xenograft assay. TIC-enriched populations will be characterized using expression microarrays to identify differences in their gene expression compared with the bulk/non-tumor initiating cells. These differences will be validated using CyTOF analysis of primary SOC cells, as well as conventional and newly derived serum-free/primary cell lines. Their ability to identify and characterize TIC in this disease may facilitate development of new and more effective therapeutic strategies for ovarian cancer, thereby benefitting patients with this devastating disease.