The CSC population possesses several characteristics that can be useful for cancer therapy development, primarily focusing on the elimination of these cells.
Usually, a distinctive profile of surface and functional markers characterizes the CSC population, and their identification and purification usually begins with the description of such markers[3,29]. Moreover, there is an increasing interest in identifying the role of each marker in CSCs, as well as targeting CSC-specific pathways, which could increase the radio- and chemo-sensitivity of CSCs.
To date, several CSC markers from distinct tumor types have been proposed and validated through different experimental models (Table 1 and Figure 1). Some of these markers are discussed below.
Nowadays, there are CSC markers that are widely used to identify several tumor types. Such markers have been reported in CSC-enrichment culture models from cell lines or primary cultures derived from patient samples and serial xenotransplantation of putative CSCs in mouse models, which must be able to recapitulate the original heterogeneous populations and be directly validated in human tumor samples. It is important to note that the use of a single marker to define a CSC population is not recommended. For this purpose, a phenotypic profile that combines various markers should be established, as well as carrying out self-renewal assays (Figure 1)[2,25].
CD133, also known as prominin-1, is a transmembrane cell surface glycoprotein traditionally used as a hematopoietic stem cell marker that is effective for detection of non-stem cells from various tumor and tissue samples. The Dirks laboratory used the CSC marker CD133 for brain CSC identification. The purified CD133+ population from primary human brain tumors samples showed higher proliferation and self-renewal capacity in neurosphere formation assays than CD133- cells. Moreover, the inoculation of only a few CD133+ cells was sufficient to produce a tumor, which was then successfully transplanted. In 2013, the Pelicci laboratory reported that CD133 was found in an interconvertible state in glioblastoma patient-derived neurospheres and that the use of short hairpin RNA (shRNA) against CD133 diminished their self-renewal and tumorigenicity potential. Interestingly, some studies have proposed that CD133 could maintain CSC properties through the Wnt/β-catenin signaling pathway.
CD133 has also been tested in colorectal cancer cell lines and tumor tissue samples[39,40] through the use of various techniques, including flow cytometry and serial xenotransplantation in mice. Additionally, CD133+ CSCs have been reported in many other solid cancer models, including endometrial cancer, lung cancer, small cell lung cancer, laryngeal cancer[45,46], liver cancer, colorectal cancer, and gastric cancer.
CD133 has been found in samples that represent higher stage tumors and are predictors of poor prognosis. For this reason, CD133 is considered a promising therapeutic target. This year, a phase I trial for testing the efficacy of CD133-directed CAR-T cells showed that CD133+ cells were successfully eliminated after CART-133 infusion.
CD44 is a multifunctional glycoprotein involved in cell adhesion, signaling, proliferation, migration, hematopoiesis, and lymphocyte activation. It functions as a receptor for hyaluronan and other extracellular matrix components. CD44 is widely used as a CSC marker, especially for tumors of epithelial origin, and it is used alone or in combination with CD24 for the identification of breast CSCs. CD24 is a small surface protein that is found in many tumor types. However, reports from cancer cell lines show that there is a substantial variation in CD24 expression even among the same tumor types.
Though CD24- cells are commonly associated with CSC phenotypes, there are some cases in which CD24+ has been found to be a marker for cell populations with CSC features. For example, in nasopharyngeal carcinoma (NPC) cell lines and in HPV-16 SiHa cervical cancer cells, isolated CD44+CD24+ cells were radioresistant and more tumorigenic than those negative for the same markers. The same CD44+CD24+ phenotype was used to identify gastric CSCs.
A known classic publication demonstrated that only a small population isolated from breast tumors, defined as CD44+CD24-/low, has the capacity to sustain tumor growth in NOD/SCID mice and generate heterogeneous cell populations as the original breast tumor. Later, in human prostate cancer samples, CSCs characterized through immunofluorescence with the CD44+/β2β1hi/CD133+ phenotype were identified and characterized. The next year, CD44+ prostate cancer cell populations were obtained. Also, CD44 and CD133 expression was evaluated in gastric adenocarcinoma tumors by immunohistochemistry, and it was found that both markers could be correlated with clinical and pathological parameters.
Although CD44 is widely reported as a CSC marker, it is very important to note that it is a ubiquitously expressed molecule derived from a gene with 18 exons. When all variable exons are spliced out, the standard form (CD44s) is expressed, and when alternative splicing occurs, variant forms (CD44v) are expressed. In spite of this, there are only a few reports in which CD44 isoforms are considered when evaluating CSCs. In 2005, Mackenzie and his group demonstrated the existence of two CSC populations, both expressing CD44high (and CD44+), derived from head and neck cutaneous squamous cell carcinoma. One was associated with EMT properties and the other one possessed an epithelial phenotype. They demonstrated that the CD44high cells that undergo EMT preferably expressed the CD44s isoform; while the epithelial CD44high cells expressed the CD44v isoform. Using RNAseq, another group later confirmed these results. The CD44v6 isoform was identified as the predominant isoform in a prostate cancer epithelial cell line.
A very important contribution from the Mackenzie laboratory is that they demonstrated that the use of enzymes (for example, trypsin or collagenase) for cell extraction from tissues caused destruction of cell surface CD44v isoforms, leaving only the CD44s isoform. Moreover, CD44-specific antibodies are not able to distinguish between all isoforms. Specifically, in breast cancer, CD44v was found to be associated with better prognosis while CD44s was related to poor prognosis. As a consequence, CD44 is the most frequently found CSC marker[64,65]. Other examples are found in colorectal cancer, in which CD44 was found together with CD133[66,67], head and neck squamous cell carcinoma[68,69], ovarian CSCs, and gastric cancer using the specific isoform CD44v8-10.
CD49f or integrin α6, is a transmembrane glycoprotein that functions as the receptor for the extracellular matrix protein laminin[72,73]. CD49f is widely distributed in stem cells and in the brain; because of its role in tumor cell proliferation, survival, self-renewal and tumor growth, it has been proposed that it could be used as a CSC marker.
In sphere colony forming cell culture using prostate cancer cells, CD49f was shown to be a better marker than CD133 and CD44. In gastric cancer, CD49high cells displayed CSC characteristics, including resistance to doxorubicin, 5-fluorouracil and doxifluridine.This has also been reported in breast and colon cancer.
Besides the examples mentioned above, there are other surface markers that have been proposed as CSC markers, such as CXCR4 and LGR5, among others.
Another strategy for CSC identification and purification is the use of functional or intracellular markers (Figure 1), which are considered to be more stable than surface markers. The principal functional CSC marker is aldehyde dehydrogenase or ALDH, part of an enzyme superfamily encoded by 19 genes that metabolize endogenous and exogenous aldehydes. It is present in practically all organisms, and its levels and isozymes vary depending on tissue and organ.
For ALDH identification, specific ALDH antibodies are available; nonetheless, we suggest that the most appropriate way for ALDH identification is the measurement of its activity using the commercial ALDH fluorescent substrate ALDEFLUOR® kit assay by Stem Cells Technologies, Inc. (Vancouver, BC, Canada). Cells that display high ALDH activity, (named ALDHhigh or ALDH+ or ALDHbr), can be identified and isolated using flow cytometry. Several works have shown that high ALDH activity is often associated with CSCs derived from solid tumor types. These cells are generally characterized by a higher proliferation potential, colony-forming capacity, self-renewal, in vivo tumorigenic capacity, metastasis, and drug resistance. For instance, ALDHhigh CSCs have been identified in colon cancer[81,82], lung cancer, cervical cancer[14,84,85], breast cancer, pancreatic cancer[87,88], and melanoma[89,90], to mention some examples.
As for surface markers, ALDH is often reported in combination with other cell markers to increase the accuracy of CSC validation. In some cases, high ALDH activity is found together with high expression of markers like CD133. Some cases have been identified in ovarian cancer[91,92], invasive ductal breast carcinoma tumors, and lung cancer. The combination ALDH+/CD44+ has been evaluated in various tumors such as breast cancer and lung cancer.