Ashley Adile

Introduction to Cancer Stem Cells: Past, Present, and Future

The Cancer Stem Cell (CSC) hypothesis postulates the existence of a small population of cancer cells with intrinsic properties allowing for resistance to conventional radiochemotherapy regiments and increased metastatic potential. Clinically, the aggressive nature of CSCs has been shown to correlate with increased tumor recurrence, metastatic spread, and overall poor patient outcome across multiple cancer subtypes. Traditionally, isolation of CSCs has been achieved through utilization of cell surface markers, while the functional differences between CSCs and remaining tumor cells have been described through proliferation, differentiation, and limiting dilution assays. The generated insights into CSC biology have further highlighted the importance of studying intratumoral heterogeneity through advanced functional assays, including CRISPR-Cas9 screens in the search of novel targeted therapies. In this chapter, we review the discovery and characterization of cancer stem cells populations within several major cancer subtypes, recent developments of novel assays used in studying therapy resistant tumor cells, as well as recent developments in therapies targeted at cancer stem cells.

In Vitro Assays for Screening Small Molecules

Traditionally anti-cancer therapeutics have been designed to target rapidly proliferating cells causing DNA damage and inducing apoptosis. However, with the development of the cancer stem cell (CSC) hypothesis, it has been postulated that a rare, slow dividing tumor cell population is able to escape therapy and contribute to tumor relapse and metastasis. The advances in characterization of CSCs across multiple cancer subtypes have allowed for development of targeted therapies using small molecule inhibitors. In this chapter, we describe two in vitro assays measuring proliferation and secondary sphere formation, which have become gold-standard assays to evaluate the effects of targeted therapies against CSCs. Together these assays constitute a rapid, inexpensive, and highly reproducible pipeline for testing small molecule inhibitors prior to more resource demanding in vivo studies.

Flow Cytometric Analysis of Brain Tumor Stem Cells

As a useful biotechnology, flow cytometry has revolutionized the field of cell analysis through its dynamic system that employs fluidics, optics, and electronics. It was first used to analyze DNA, but is often used to determine biomarker expression, as well as to characterize and sort cells, in accordance with various parameters. A common application of flow cytometry is the identification and isolation of a distinct cancer cell population, known as cancer stem cells (CSCs). Various biomarkers have been used to elucidate this proportion of cells within the brain, termed brain tumor initiating cells (BTICs). Here, we discuss methodology to prepare BTICs for flow cytometric analysis that includes the expression of markers.

BMI1 is a therapeutic target in recurrent medulloblastoma

Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor, representing 20% of newly diagnosed childhood central nervous system malignancies. Although advances in multimodal therapy yielded a 5-year survivorship of 80%, MB still accounts for the leading cause of childhood cancer mortality. In this work, we describe the epigenetic regulator BMI1 as a novel therapeutic target for the treatment of recurrent human Group 3 MB, a childhood brain tumor for which there is virtually no treatment option beyond palliation. Current clinical trials for recurrent MB patients based on genomic profiles of primary, treatment-naive tumors will provide limited clinical benefit since recurrent metastatic MBs are highly genetically divergent from their primary tumor. Using a small molecule inhibitor against BMI1, PTC-028, we were able to demonstrate complete ablation of self-renewal of MB stem cells in vitro. When administered to mice xenografted with patient tumors, we observed significant reduction in tumor burden in both local and metastatic compartments and subsequent increased survival, without neurotoxicity. Strikingly, serial in vivo re-transplantation assays demonstrated a marked reduction in tumor initiation ability of recurrent MB cells upon re-transplantation of PTC-028-treated cells into secondary recipient mouse brains. As Group 3 MB is often metastatic and uniformly fatal at recurrence, with no current or planned trials of targeted therapy, an efficacious targeted agent would be rapidly transitioned to clinical trials.