Results: 2

1.
Figure 1

Figure 1. High-throughput approaches to target breast CSC. From: Targeting Breast Cancer Stem Cells.

A) Breast CSC can be isolated by sorting using the Aldefluor assay or CD44+CD24- populations (red box) or via culturing as mammospheres. Within these populations, the CSC frequency is enriched with some percentage of non-tumorigenic bulk cells remaining. B) To screen the CSC population, cells are plated in 384-well plates in conditions that maintain tumor-initiation capacity, such as in serum-free suspension (mammosphere) or in EGF- and FGF-containing media on laminin-coated plates (Pollard et al., 2009). Small-molecule compound collections are added to plates at one compound/well, typically at high nanomolar or low micromolar concentrations. Additionally, the whole genome or selected gene sets (kinases, phosphatases, ‘druggable’) can be targeted using either siRNA or retroviral or lentiviral shRNA libraries with usually 4-5 constructs per gene. shRNA libraries can provide long-term knockdown whereas siRNA knockdown is more transient. Cellular endpoints can include growth inhibition (Gupta et al., 2009), flow cytometry (Krutzik and Nolan, 2006), sphere formation (Diamandis et al., 2007), or cell migration (Wurdak et al., 2010).

Sean P. McDermott, et al. Mol Oncol. ;4(5):404-419.
2.
Figure 2

Figure 2. Validation of potential “hits” as anti-CSC agents. From: Targeting Breast Cancer Stem Cells.

A) Traditionally, “hits” are identified using a Z- or B-score for each compound/RNA and at least 3 standard deviations (green line) from the mean (blue line). Hits (red dots) are “cherry-picked” for validation studies (right) at additional doses to show specificity for CSC (blue circles) and not normal stem cells (red triangles). B) To confirm validated “hits” target CSCs, activity must be validated using primary tumor xenografts in immunocompromised mice. In the advanced setting (top), cells enriched for breast CSC are injected into mice, allowed to grow to a palpable size and then mice are treated with candidate drugs or siRNA/shRNA. A CSC-specific agent (blue triangles) would have minimal effect in this assay since tumor growth is driven primarily by progenitors and not CSC. However, an agent that targets CSC and the bulk tumor cells (red squares) would show dramatic tumor reduction. This effect could be achieved using a CSC-specific agent and a chemotherapeutic agent targeting the bulk population. In an early (i.e. adjuvant) setting (bottom), treatment is initiated soon after injection of CSC-enriched cells. A selective anti-CSC agent (blue triangles) would be predicted to have a much greater effect when administered in this setting.

Sean P. McDermott, et al. Mol Oncol. ;4(5):404-419.

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