Section 2.2: Isolation and identification of CSCs (from DOI: 10.1038/s41392-020-0110-5)
From publication: "Targeting cancer stem cell pathways for cancer therapy" published as Signal Transduct Target Ther; 2020 ; 5 8; DOI: https://doi.org/10.1038/s41392-020-0110-5
Section 2.2: Isolation and identification of CSCs
It is known that the proportion of CSCs in tumor tissues is very low and generally accounts for only 0.01-2% of the total tumor mass. In addition, CSCs and normal stem cells also share similar transcription factors and signaling pathways. Therefore, it is more challenging to isolate and identify CSCs. However, an increasing number of techniques and means have emerged.
Table 1 caption: Various biomarkers of cancer stem cells in human cancers
Table 1 data: Cancers Markers Function Breast CD29+,CD49f+,CD90+,CD133+,ALDH+,ESA+/CD44+/CD24,CD44+/CD24- ALDH: An enzyme that plays a role in cell resistanceCD44: A glycoprotein involves in cell migration and self-renewalCD90: A glycoprotein participates in T cell adhesion and signal transductionCD133: A transmembrane glycoprotein that maintains lipid composition in cell membranesCD24: A marker that promotes blood flow in the tumor during metastasisCD49f: A membrane proteins of the integrin family that plays an important role in cell surface adhesion and signaling Prostate EpCAM+,CD117+,alpha2beta1+,ALDH+,CD44+,EZH2+,CXCR4+,E-cadherin+,CD133+ alpha2beta1: A receptor involves in cell adhesion and recognitionE-cadherin: It plays an important role in tumor migration and invasionCXCR4: CXC chemokine receptor works with CD4 protein to support HIV entry into cellsEZH2: A member of the Polycomb family plays an vital role in the central nervous system Brain CD49f+,CD90+,CD44+,CD36+,EGFR+,A2B5+,L1CAM+,CD133+ CD36: The main glycoprotein on the surface of platelet has an important function as an adhesion moleculeEGFR: It binds to epidermal growth factor and promote proliferative migration in tumorsA2B5: A ganglioside marker that identifies subpopulations of nerve cells in the central nervous systemL1CAM: A adhesion molecule that plays an important role in the development of the nervous system include neuronal migration and differentiation Stomach ALDH+, CD44+,CD44V8-10+,CD133+, CD24+,CD54+, CD90+,CD49f+ CD71+,EpCAM+ CD44V8-10: A variant of CD44 with a specific class of CSCsCD54: A class of adhesion molecules express in malignant tumor cells Colorectal CD200+, EpCAM+,CD133+, CD166+,CD206+, CD44+, CD49f+, ALDH+ CD200: A glycoprotein plays an important role in the regulation of immunosuppression and anti-tumor activityCD166: It binds to the T cell differentiation antigen CD6 and involves in cell adhesion and migration processesCD206: A mannose receptor involves in endocytosis, phagocytosis, and immune homeostasisEpCAM: It expresses on most normal epithelial cells and gastrointestinal cancers, and acts as a homotypic calcium-independent cell adhesion molecule Liver CD24+, CD133+,CD13+, CD44+,CD206+, OV-6+,CD90+, EpCAM+ CD13: A receptor for human coronavirus strains, which is the main cause of upper respiratory tract infection and leukemiaOV-6: A marker for rat oval cells and hepatic stem cells AML CD34+,CD38-,CD90+,CD71+,CD19+,CD20+,CD44+,CD10+,CD45RA+,CD123+ CD34: It plays a role in the attachment of stem cells to bone marrow extracellular or stromal cellsCD38: An intracellular Ca2+ mobilization messenger, prognostic markers for patients with chronic lymphocytic leukemiaCD71: A transferrin receptor is important for nerve developmentCD19: A class of signal transduction molecules regulate B lymphocyte differentiationCD20: The protein plays a role in the development and differentiation of B cells into plasma cellsCD10: It inhibits a variety of peptide hormones, include glucagon, encephalin, oxytocin, and bradykininCD45RA: A class of leukocyte activation regulatorsCD123: An interleukin-specific subunit of a heterodimeric cytokine receptor Melanoma CD20+, CD271+,,ALDH+, CD133+ CD271: A nerve growth factor receptor mediates cell survival and cell death in nerve cells Bladder CD44v6+, CD44+,ALDH+ CD44v6: It involves in cell migration, cell adhesion Ovarian CD24+, ALDH+,,CD44+/CD117+,EpCAM+, CD133+ CD117: A class of transmembrane receptors is also known as stem cell factors Pancreas ALDH+, CD133+,CD44+/CD24+/EpCAM+,ABCG2+, CXCR4+, ABCG2: A class of membrane proteins belongs to the ABC transporter superfamily that plays a role in the drug resistance properties of CSCs HNSCC ALDH+, CD44+,,CD166+ Gallbladder CD44+/CD133+ RCC CD133+, ALDH+,,CXCR4+, CD44+,,CD105+ CD105: TGF receptor that involves in TGF-beta signaling plays a role in angiogenesis Lung CD166+, CD90+,,CD87+, ALDH+,,CD44+, CD133+ CD87: A receptor for urokinase plasminogen activator that affects many normal and pathological processes associates with cell surface plasminogen activation and local degradation of extracellular matrices Malignant mesothelioma CD9+,CD24+,CD26+ CD9: A glycoprotein plays a role in many cellular processes, includes differentiation, adhesion and signal transduction, and plays a key role in cancer cell movement and metastasisCD26: A class of serine exopeptidases is also an intrinsic membrane glycoprotein OSCC CD44+/CD24,-ITGA7+ ITGA7: A integrin plays a role in cell migration, morphogenesis, differentiation, and metastasis and participates in the process of differentiation and migration during myogenesis cSCC CD44+, CD133+ Esophageal ITGA7+, CD44+,ALDH+, CD133+,CD90+ MM CD138-,CD19+,CD27+ CD138: A member of the Syndecan proteoglycan family that involves in cell proliferation, cell migration, and cell-matrix interactionsCD27: A transmembrane glycoprotein involves in the regulation of B cell activation and immunoglobulin synthesis Cervix ABCG2+, CD133+, CD49f+, ALDH+ Nasopharyngeal CD44+, CD133+, ALDH+, CD24+ Laryngeal ALDH+, CD44+, CD133+
Table 1 footer: AML acute myeloid leukemia, HNSCC head and neck squamous cell carcinoma, RCC renal cell carcinoma, OSCC oral squamous cell carcinoma, cSCC cutaneous squamous cell carcinoma, MM multiple myeloma, ALDH aldehyde dehydrogenase, EpCAM epithelial cellular adhesion molecule
CSCs have been identified through different biomarkers in human cancers (Table 1). CSCs can be separated by combining specific biomarkers that are mostly located on the cell surface. The primary separation techniques are fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS). Since Dick JE first screened CSCs from leukemia by using FACS technology, FACS has become the most widely used technique for cell separation. It can perform multibiomarker sorting at one time and has high purity and strong specificity. MACS is a MACS technique. MACS separation is relatively simple, but the technique is cumbersome. Therefore, this method requires high activity of CSCs. These two methods are effective in separating CSCs from large numbers of cells.
Additionally, there are other ways to separate CSCs from tumors. In 1996, Dr. Goodell observed that after adding Hoechst 33342 to a culture of bone marrow cells, a few cells did not accumulate dyes, and he claimed that these few cells were side population (SP) cells. Therefore, SP cells can be separated by fluorescence screening after the outflow of Hoechst 33342. Recently, SP cells have been identified in various normal tissues and tumor cells. SP cells have high homology, self-renewal and multidirectional differentiation potential. Some reports have shown that ABCG2 is highly expressed in SP cells. ABCG2 is highly related to the drug resistance of CSCs and is used as a phenotypic marker for CSCs, including ovarian cancer, AML, breast cancer, lung cancer, nasopharyngeal carcinoma, and hepatocellular carcinoma (HCC). Montanaro et al. explored the optimal concentration of Hoechst 33342 to reduce the toxic effect. The SP sorting method has universal applicability in the separation and identification of CSCs, especially CSCs with unknown cell surface markers, and is an effective method for CSC research.
The colony-forming ability of CSCs is also used for separation and identification. After digestion of the tumor tissues into single cells, low-density cell culture can be conducted in serum-free medium containing epithelial growth factor (EGF) and basic fibroblast growth factor (FGF). Under this condition, a single CSC will form a cell colony or sphere. Taylor et al. successfully isolated CSCs from a variety of neurological tumors by using this colony formation assay. However, the cell purification rate is low, and the CSC specificity is poor in this assay. The in vivo limited dilution assay (LDA) can be used for assessing CSC activity. After low-density transplantation of immune-deficient mice with the limiting dilution method, CSCs can be identified by ELDA software analysis, and this method is affected by cell density and the microenvironment in mice.
Traditional chemotherapeutic drugs mainly affect cancer cells, but CSCs are mostly arrested in the G0 phase and are relatively static, thus evading the killing effect of chemotherapeutic drugs. Hence, the drug-resistant characteristics of CSCs can be used to isolate and identify CSCs. Previous studies have shown that radiotherapy combined with hypoxic culture can also be used to enrich CSCs. In addition, the separation of CSCs can also be accomplished by physical methods. Hepatoma stem cells can be isolated from rat liver cancer tissue by Percoll density gradient centrifugation; a cell fraction with a high nuclear-to-cytoplasmic ratio is obtained. Recently, Rahimi et al. used the miR-302 host gene promoter to overexpress neomycin in cancer cells and selected and collected neomycin-resistant CSCs.
