Resistance of cancer cells to CTL-mediated immunotherapy. Resistance to targeted anti-cancer therapeutics. Benjamin Bonavida, Salem Chouaib (Eds). Springer International Publishing Switzerland (2015)
CSCs are hypothesized to derive from normal stem cells through an aberrant step of differentiation or after a reprogrammed leading to a less differentiated status . In light of this, it is possible to identify CSCs by using stemness characteristic markers, such as transcription factors acting during early embryogenesis, or genes involved in pluripotency maintenance. Similarly, cancer stem/progenitor cells can be recognized by following specific proteins that intervene in early organogenesis, from the three different germ layers.
In association with Oct4, Sox2 forms a trimeric complex involved in embryonic development. These markers are transcription factors that perform their function by binding to DNA and activating some important genes, such as YES1, FGF4, UTF, and ZFP206. Nanog is a transcription factor induced by Oct4 involved in stem cell self-renewal and pluripotency and hence, preventing differentiation. CSC identification can be obtained by following genes belonging to stem cell pathways, such as Wnt, Hedgehog, and Notch (classified also by EMT-inducing signaling pathways) (www.uniprot.org).
In proceeding with differentiation, embryonic stem cells undergo a phenotype change in their tissue destination. To analyze the differentiation towards each lineage, it is possible to use ectodermal (i.e., Notch, Nestin, p63), mesodermal (i.e., BMP4, Nodal, CD34, Cryptic), and endodermal (i.e., a-fetoprotein, beta-catenin, CXCR4, SOX17) markers. In relation to tissue differentiation and development, these marker classes belong to all cells with the same tissue derivation. For this reason, they are commonly used and constitute a simple screen panel for CSC characterization. Being that most markers are intracellular, they cannot be used for FACS sorting or beads separation. Hence, the challenge of many research groups is the identification of membrane markers that can be stable and specific to a definite pathology.
The cells with the capacity to efflux Hoechst 33342 vital dye, that were first identified in mouse bone marrow, are referred to as “side population” (SP) because they are composed of unstained cells in the left lower quadrant of a FACS profile . SP has been used to isolate malignant cells since their ability to efflux dyes correlates to multidrug resistance mediated by the ABC transporters over-expression . Moreover, these cell subsets are highly enriched for the capacity to initiate tumor formation upon serial transplantation and express stem-like genes .
The use of Hoechst dye to isolate stem-like cells has met with criticisms. In fact, this is a dynamic process, based on dye efflux, in which variables in staining times, dye and cellular concentrations can affect the SP phenotype. Since the DNA binding induced by Hoechst staining promotes a toxic effect in living cells, the SP cells, isolated through this method, may be a population able to resist the lethal effects rather than stem-like cells. Furthermore, flow cytometry gating strategies, used to define SP cells, cannot be associated with gating strategies involved in staining using other markers .
A similar method of characterization of CSCs is the analysis of the cell subset expressing an high Aldehyde Dehydrogenase (ALDH) activity, which is involved in early cellular differentiation, detoxification, and drug resistance, through the oxidation of intracellular aldehydes . ALDH belongs to the oxidoreductase enzyme family and is highly expressed in stem and progenitor cells, thus it is used as a functional marker for CSC isolation from solid tumors (i.e., breast, lung, ovarian, prostate, head-neck, and thyroid cancer), as well as in multiple myelomas and acute leukemia . Using the ALDEFLUOR™ assay, it is possible to isolate cancer stem and progenitor cells through cell sorting with a positive selection, without compromising their vitality.
In solid tumors, several cell surface markers are used to isolate cell subsets enriched with CSCs, such as CD44 [46–49], CD24 [57, 50], EpCAM [46, 51], THY1 (also called CD90) , and CD133 [51, 53–57].
CD133, also known as Prominin-1, is a pentaspan transmembrane glycoprotein originally identified as a marker for human CD34+ hematopoietic stem and progenitor cells by Miraglia et al. . It was recognized as an important marker in the identification and isolation of cell subsets with “stemness” properties in many tumor tissues, such as brain , kidney , prostate , hepatic , and colon [53, 57]. Nonetheless, the usage of CD133 as an identification and isolation marker in colon CSCs is controversial because its expression pattern is not completely elucidated. In line with this, CD133+ and CD133cell fractions have been reported to display similar “stemness” and differentiation potential, including the ability to generate tumors similar to the parental ones . Kamper and colleagues explained the contradictions found in the literature by studying possible regulation mechanisms of epitope expression. CD133 is expressed in both CSCs and differentiated tumor cells. Whereas the CD133 mRNA and protein expression remained unchanged, differentiation led to down-regulation of the AC133 epitope, correlating with differential glycosylation and reduced antibody detection .
The CD133 polarized localization suggests its role in regulating proliferation but its functions remains still unclear. Recent studies highlight that CD133 could be involved in tumor angiogenesis since CD133+ glioma cells have shown to produce vascular endothelial growth factors . In the intestine, CD133 has been proposed as a stem cell marker susceptible to neoplastic transformation, being prone to activate Wnt signaling . Therefore, it is important to note that CSC identification and isolation requires the use of more than one specific marker.
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