Flow cytometry in cancer stem cell analysis and separation

Abstract

In recent years, a special type of cancer cell-the cancer stem cell (CSC)-has been identified and characterized for different tumors. CSCs may be responsible for the recurrence of a tumor following a primarily successful therapy and are thought to bear a high metastatic potential. For the development of efficient treatment strategies, the establishment of reliable methods for the identification and effective isolation of CSCs is imperative. Similar to their stem cell counterparts in bone marrow or small intestine, different cluster of differentiation surface antigens have been characterized, thus enabling researchers to identify them within the tumor bulk and to determine their degree of differentiation. In addition, functional properties characteristic of stem cells can be measured. Side population analysis is based on the stem cell-specific activity of certain ATP-binding cassette transporter proteins, which are able to transport fluorescent dyes out of the cells. Furthermore, the stem cell-specific presence of aldehyde dehydrogenase isoform 1 can be used for CSC labeling. However, the flow cytometric analysis of these CSC functional features requires specific technical adjustments. This review focuses on the principles and strategies of the flow cytometric analysis of CSCs and provides an overview of current protocols as well as technical requirements and pitfalls. A special focus is set on side population analysis and analysis of ALDH activity. Flow cytometrybased sorting principles and future flow cytometric applications for CSC analysis are also discussed. ' 2012 International Society for Advancement of Cytometry

Cancer Stem Cells: A Review of Potential Clinical Applications

“…Therefore, numerous in vitro assays have been developed for CSC identification, which include the colony assay, the sphere assay, the ''side population'' (SP) assay by Hoechst labeling, 12 staining for CSC surface antigens (such as ABCG2 and other markers), aldehyde dehydrogenase (ALDH) activity assay, 13 and label-retaining cell assay using PKH (Paul Karl Horan) dyes. These methods are summarized below.…”

“…This phenomenon led to the development of the so-called side population flow cytometry-based isolation technique. 12,14 This technique is being used for isolation of multiple myeloma (MM) stem cells in our laboratory ( Figure 1, A through F). Briefly, MM cells are incubated at 378C, for 90 minutes, in the presence of 2 lg/mL of Hoechst 33342 dye.…”

“…Examples of such markers include CD44, CD24, and CD133. 12,26,27 When sorted by flow cytometry, tumor cells expressing the above molecules were more tumorigenic when transplanted into immunodeficient mice than were all of the other types of cells in the tumor. Examples of CSC markers in different types of human cancers include the following (Table): CD24 and CD44 in breast and pancreatic adenocarcinomas; CD44, CD133, EpCAM, and CD166 in colorectal cancer; CD44 and CD133 in prostate adenocarcinoma; and CD133 and EpCAM-hepatocellular carcinoma, CD34, and CD38 in acute myeloid leukemia.…”

Therapeutic strategies for targeting cancer stem cells

“…[2] Functional CSC properties like intracellular ALDH enzymatic activities and ABC transporter efflux activities of vital DNA dyes such as Hoechst 33342 have been used for CSC isolation. [21,22] Increased aldehyde dehydrogenase isoform 1 (ALDH1) activity has been used to identify and analyze different types of CSCs. Furthermore, CSCs have a distinct efflux mechanism, stemming from their high expression of ABC transporter proteins.…”

“…Flow cytometric SP analysis has been performed with numerous cancer cell lines and the SP has shown enriched CSC activities. [21] A subpopulation of CSCs exhibit intrinsic autofluorescence and were shown to be exclusively linked to a functional CSC phenotype in different epithelial tumors. These autofluorescent cells had CSC characteristics such as high self-renewal, long-term tumorigenic capacity, invasiveness, and chemoresistance.…”