Flow cytometry in cancer stem cell analysis and separation

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 cytometry-based sorting principles and future flow cytometric applications for CSC analysis are also discussed.     

Aldehyde dehydrogenase discriminates the CD133 liver cancer stem cell populations

Recent efforts in our study of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) have led to the identification of CD133 as a prominent HCC CSC marker. Findings were based on experiments done on cell lines and xenograft tumors where expression of CD133 was detected at levels as high as 65%. Based on the CSC theory, CSCs are believed to represent only a minority number of the tumor mass. This is indicative that our previously characterized CD133(+) HCC CSC population is still heterogeneous, consisting of perhaps subsets of cells with differing tumorigenic potential. We hypothesized that it is possible to further enrich the CSC population by means of additional differentially expressed markers. Using a two-dimensional PAGE approach, we compared protein profiles between CD133(+) and CD133(-) subpopulations isolated from Huh7 and PLC8024 and identified aldehyde dehydrogenase 1A1 as one of the proteins that are preferentially expressed in the CD133(+) subfraction. Analysis of the expression of several different ALDH isoforms and ALDH enzymatic activity in liver cell lines found ALDH to be positively correlated with CD133 expression. Dual-color flow cytometry analysis found the majority of ALDH(+) to be CD133(+), yet not all CD133(+) HCC cells were ALDH(+). Subsequent studies on purified subpopulations found CD133(+)ALDH(+) cells to be significantly more tumorigenic than their CD133(-)ALDH(+) or CD133(-)ALDH(-) counterparts, both in vitro and in vivo. These data, combined with those from our previous work, reveal the existence of a hierarchical organization in HCC bearing tumorigenic potential in the order of CD133(+)ALDH(+) > CD133(+)ALDH(-) > CD133(-)ALDH(-). ALDH, expressed along CD133, can more specifically characterize the tumorigenic liver CSC population.     

Retinoid signaling regulates breast cancer stem cell differentiation

The cancer stem cell (CSC) hypothesis implicates the development of new therapeutic approaches to target the CSC population. Characterization of the pathways that regulate CSCs activity will facilitate the development of targeted therapies. We recently reported that the enzymatic activity of ALDH1, as measured by the ALDELFUOR assay, can be utilized to isolate normal and malignant breast stem cells in both primary tumors and cell lines. In this study, utilizing a tumorsphere assay, we have demonstrated the role of retinoid signaling in the regulation of breast CSCs self-renewal and differentiation. Utilizing the gene set enrichment analysis (GSEA) algorithm we identified gene sets and pathways associated with retinoid signaling. These pathways regulate breast CSCs biology and their inhibition may provide novel therapeutic approaches to target breast CSCs.     

Aldehyde Dehydrogenase 1 Identifies Cells with Cancer Stem Cell-Like Properties in a Human Renal Cell Carcinoma Cell Line

Cancer stem cells (CSC) or cancer stem cell-like cells (CSC-LCs) have been identified in many malignant tumors. CSCs are proposed to be related with drug resistance, tumor recurrence, and metastasis and are considered as a new target for cancer treatment; however, there are only a few reports on CSCs or CSC-LCs in renal cell carcinoma (RCC). Different approaches have been reported for CSC identification, but there are no universal markers for CSC. We used two different approaches, the traditional side population (SP) approach, and the enzymatic (aldehyde dehydrogenase 1 (ALDH1)) approach to identify CSC-LC population in two RCC cell lines, ACHN and KRC/Y. We found that ACHN and KRC/Y contain 1.4% and 1.7% SP cells, respectively. ACHN SP cells showed a higher sphere forming ability, drug resistance, and a slightly higher tumorigenic ability in NOD/SCID mice than Non-SP (NSP) cells, suggesting that cells with CSC-LC properties are included in ACHN SP cells. KRC/Y SP and NSP cells showed no difference in such properties. ALDH1 activity analysis revealed that ACHN SP cells expressed a higher level of activity than NSP cells (SP vs. NSP: 32.7% vs 14.6%). Analysis of ALDH1-positive ACHN cells revealed that they have a higher sphere forming ability, self-renewal ability, tumorigenicity and express higher mRNA levels of CSC-LC property-related genes (e.g., ABC transporter genes, self-replication genes, anti-apoptosis genes, and so forth) than ALDH1-negative cells. Drug treatment or exposure to hypoxic condition induced a 2- to 3-fold increase in number of ALDH1-positive cells. In conclusion, the results suggest that the ALDH1-positive cell population rather than SP cells show CSC-LC properties in a RCC cell line, ACHN.     

Combination of dasatinib and curcumin eliminates chemo-resistant colon cancer cells

Metastatic colorectal cancer remains a serious health concern with poor patient survival. Although 5-Fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) is the standard therapy for colorectal cancer, it has met with limited success. Recurrence of the tumor after chemotherapy could partly be explained by the enrichment of the chemo-resistant sub-population of cancer stem cells (CSCs) that possess the ability for self-renewal and differentiation into different lineages in the tumor. Therefore development of therapeutic strategies that target CSCs for successful treatment of this malignancy is warranted. The current investigation was undertaken to examine the effectiveness of the combination therapy of dasatinib (a Src inhibitor) and curcumin (a dietary agent with pleiotropic effect) in inhibiting the growth and other properties of carcinogenesis of chemo-resistant colon cancer cells that are enriched in CSCs sub-population. Remnants of spontaneous adenomas from APC ^{Min +/-} mice treated with dasatinib and/or curcumin were analyzed for several cancer stem cell markers (ALDH, CD44, CD133 and CD166). Human colon cancer cells HCT-116 (p53 wild type; K-ras mutant) and HT-29 (p53 mutant; K-ras wild type) were used to generate FOLFOX resistant (referred to as CR) cells. The effectiveness of the combination therapy in inhibiting growth, invasive potential and stemness was examined in colon cancer CR cells. The residual tumors from APC ^{Min +/-} mice treated with dasatinib and/or curcumin showed 80-90% decrease in the expression of the CSC markers ALDH, CD44, CD133, CD166. The colon cancer CR cells showed a higher expression of CSCs markers, cell invasion potential and ability to form colonospheres, compared to the corresponding parental cells. The combination therapy of dasatinib and curcumin demonstrated synergistic interactions in CR HCT-116 and CR HT-29 cells, as determined by Calcusyn analysis. The combinatorial therapy inhibited cellular growth, invasion and colonosphere formation and also reduced CSC population as evidenced by the decreased expression of CSC specific markers: CD133, CD44, CD166 and ALDH. Our data suggest that the combination therapy of dasatinib and curcumin may be a therapeutic strategy for re-emergence of chemo-resistant colon cancer by targeting CSC sub-population.     

CD166 promotes the cancer stem-like properties of primary epithelial ovarian cancer cells

Cancer stem cells (CSCs) or tumor-initiating cells are thought to play critical roles in tumorigenesis, metastasis, drug resistance, and tumor recurrence. For the diagnosis and targeted therapy of CSCs, the molecular identity of biomarkers or therapeutic targets for CSCs needs to be clarified. In this study, we identified CD166 as a novel marker expressed in the sphere-forming CSC population of A2780 epithelial ovarian cancer cells and primary ovarian cancer cells. The CD166⁺ cells isolated from A2780 cells and primary ovarian cancer cells highly expressed CSC markers, including ALDH1a1, OCT4, and SOX2, and ABC transporters, which are implicated in the drug resistance of CSCs. The CD166⁺ cells exhibited enhanced CSC-like properties, such as increased sphere-forming ability, cell migration and adhesion abilities, resistance to conventional anti-cancer drugs, and high tumorigenic potential in a xenograft mouse model. Knockdown of CD166 expression in the sphere-forming ovarian CSCs abrogated their CSC-like properties. Moreover, silencing of CD166 expression in the sphere-forming CSCs suppressed the phosphorylation of focal adhesion kinase, paxillin, and SRC. These results suggest that CD166 plays a key role in the regulation of CSC-like properties and focal adhesion kinase signaling in ovarian cancer.     

A quantitative proteomics analysis of MCF7 breast cancer stem and progenitor cell populations

Accumulating evidence has demonstrated that breast cancers are initiated and develop from a small population of stem‐like cells termed cancer stem cells (CSCs). These cells are hypothesized to mediate tumor metastasis and contribute to therapeutic resistance. However, the molecular regulatory networks responsible for maintaining CSCs in an undifferentiated state have yet to be elucidated. In this study, we used CSC markers to isolate pure breast CSCs fractions (ALDH+ and CD44+CD24‐ cell populations) and the mature luminal cells (CD49f‐EpCAM+) from the MCF7 cell line. Proteomic analysis was performed on these samples and a total of 3304 proteins were identified. A label‐free quantitative method was applied to analyze differentially expressed proteins. Using the criteria of greater than twofold changes and p value <0.05, 305, 322 and 98 proteins were identified as significantly different in three pairwise comparisons of ALDH+ versus CD44+CD24‐, ALDH+ versus CD49f‐EpCAM+ and CD44+CD24‐ versus CD49f‐EpCAM+, respectively. Pathway analysis of differentially expressed proteins by Ingenuity Pathway Analysis (IPA) revealed potential molecular regulatory networks that may regulate CSCs. Selected differential proteins were validated by Western blot assay and immunohistochemical staining. The use of proteomics analysis may increase our understanding of the underlying molecular mechanisms of breast CSCs. This may be of importance in the future development of anti‐CSC therapeutics.     

Ubiquitin ligase CHIP suppresses cancer stem cell properties in a population of breast cancer cells

Cancer stem cells (CSCs) have several distinctive characteristics, including high metastatic potential, tumor-initiating potential, and properties that resemble normal stem cells such as self-renewal, differentiation, and drug efflux. Because of these characteristics, CSC is regarded to be responsible for cancer progression and patient prognosis. In our previous study, we showed that a ubiquitin E3 ligase carboxyl terminus of Hsc70-interacting protein (CHIP) suppressed breast cancer malignancy. Moreover, a recent clinical study reported that CHIP expression levels were associated with favorable prognostic parameters of patients with breast cancer. Here we show that CHIP suppresses CSC properties in a population of breast cancer cells. CHIP depletion resulted in an increased proportion of CSCs among breast cancers when using several assays to assess CSC properties. From our results, we propose that inhibition of CSC properties may be one of the functions of CHIP as a suppressor of cancer progression.     

Autophagy augments the self-renewal of lung cancer stem cells by the degradation of ubiquitinated p53

It has been postulated that cancer stem cells (CSCs) are involved in all aspects of human cancer, although the mechanisms governing the regulation of CSC self-renewal in the cancer state remain poorly defined. In the literature, both the pro- and anti-oncogenic activities of autophagy have been demonstrated and are context-dependent. Mounting evidence has shown augmentation of CSC stemness by autophagy, yet mechanistic characterization and understanding are lacking. In the present study, by generating stable human lung CSC cell lines with the wild-type TP53 (A549), as well as cell lines in which TP53 was deleted (H1229), we show, for the first time, that autophagy augments the stemness of lung CSCs by degrading ubiquitinated p53. Furthermore, Zeb1 is required for TP53 regulation of CSC self-renewal. Moreover, TCGA data mining and analysis show that Atg5 and Zeb1 are poor prognostic markers of lung cancer. In summary, this study has elucidated a new CSC-based mechanism underlying the oncogenic activity of autophagy and the tumor suppressor activity of p53 in cancer, i.e., CSCs can exploit the autophagy-p53-Zeb1 axis for self-renewal, oncogenesis, and progression.Subject terms: Cancer stem cells, Cancer stem cells     

TRPM7 channels regulate glioma stem cell through STAT3 and Notch signaling pathways

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults with median survival time of 14.6 months. A small fraction of cancer stem cells (CSC) initiate and maintain tumors thus driving glioma tumorigenesis and being responsible for resistance to classical chemo- and radio-therapies. It is desirable to identify signaling pathways related to CSC to develop novel therapies to selectively target them. Transient receptor potential cation channel, subfamily M, member 7, also known as TRPM7 is a ubiquitous, Ca^2 + and Mg^2 + permeable ion channels that are special in being both an ion channel and a serine/threonine kinase. In studies of glioma cells silenced for TRPM7, we demonstrated that Notch (Notch1, JAG1, Hey2, and Survivin) and STAT3 pathways are down regulated in glioma cells grown in monolayer. Furthermore, phospho-STAT3, Notch target genes and CSC markers (ALDH1 and CD133) were significantly higher in spheroid glioma CSCs when compared with monolayer cultures. The results further show that tyrosine-phosphorylated STAT3 binds and activates the ALDH1 promoters in glioma cells. We found that TRMP7-induced upregulation of ALDH1 expression is associated with increases in ALDH1 activity and is detectable in stem-like cells when expanded as spheroid CSCs. Finally, TRPM7 promotes proliferation, migration and invasion of glioma cells. These demonstrate that TRPM7 activates JAK2/STAT3 and/or Notch signaling pathways and leads to increased cell proliferation and migration. These findings for the first time demonstrates that TRPM7 (1) activates a previously unrecognized STAT3 → ALDH1 pathway, and (2) promotes the induction of ALDH1 activity in glioma cells.     

Cancer stem cells – the current status of an old concept: literature review and clinical approaches

As regards their morphology and biology, tumours consist of heterogeneous cell populations. The cancer stem cell (CSC) hypothesis assumes that a tumour is hierarchically organized and not all of the cells are equally capable of generating descendants, similarly to normal tissue. The only cells being able to self-renew and produce a heterogeneous tumour cell population are cancer stem cells. CSCs probably derive from normal stem cells, although progenitor cells may be taken into consideration as the source of cancer stem cells. CSCs reside in the niche defined as the microenvironment formed by stromal cells, vasculature and extracellular matrix. The CSC assays include FACS sorting, xenotransplantation to immunodeficient mice (SCID), incubation with Hoechst 33342 dye, cell culture in non-adherent conditions, cell culture with bromodeoxyuridine. CSCs have certain properties that make them resistant to anticancer therapy, which suggests they may be the target for potential therapeutic strategies.     

CD44 and CD24 cannot act as cancer stem cell markers in human lung adenocarcinoma cell line A549

Cancer stem cells (CSCs) are subpopulations of tumor cells that are responsible for tumor initiation, maintenance and metastasis. Recent studies suggested that lung cancer arises from CSCs. In this study, the expression of potential CSC markers in cell line A549 was evaluated. We applied flow cytometry to assess the expression of putative stem cell markers, including aldehyde dehydrogenase 1 (ALDH1), CD24, CD44, CD133 and ABCG2. Cells were then sorted according to the expression of CD44 and CD24 markers by fluorescence-activated cell sorting (FACS) Aria II and characterized using their clonogenic and sphere-forming capacity. A549 cells expressed the CSC markers CD44 and CD24 at 68.16% and 54.46%, respectively. The expression of the putative CSC marker ALDH1 was 4.20%, whereas the expression of ABCG2 and CD133 was 0.93%. Double-positive CD44/133 populations were rare. CD44⁺/24⁺ and CD44⁺/CD24^?/low subpopulations respectively exhibited 64% and 27.92% expression. The colony-forming potentials in the CD44⁺/CD24⁺ and CD44⁺/CD24^?/low subpopulations were 84.37 ± 2.86% and 90 ± 3.06%, respectively, while the parental A549 cells yielded 56.65 ± 2.33% using the colony-formation assay. Both isolated subpopulations formed spheres in serumfree medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). CD44 and CD24 cannot be considered potential markers for isolating lung CSCs in cell line A549, but further investigation using in vivo assays is required.     

Aldehyde Dehydrogenase 1 (ALDH1) Is a Potential Marker for Cancer Stem Cells in Embryonal Rhabdomyosarcoma

Cancer stem cells (CSCs) are defined as a small population of cancer cells with the properties of high self-renewal, differentiation, and tumor-initiating functions. Recent studies have demonstrated that aldehyde dehydrogenase 1 (ALDH1) is a marker for CSCs in adult cancers. Although CSCs have been identified in some different types of pediatric solid tumors, there have been no studies regarding the efficacy of ALDH1 as a marker for CSCs. Therefore, in order to elucidate whether ALDH1 can be used as a marker for CSCs of pediatric sarcoma, we examined the characteristics of a population of cells with a high ALDH1 activity (ALDH1^high cells) in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children. We used the human embryonal RMS (eRMS) cell lines RD and KYM-1, and sorted the cells into two subpopulations of ALDH1^high cells and cells with a low ALDH1 activity (ALDH1^low cells). Consequently, we found that the ALDH1^high cells comprised 3.9% and 8.2% of the total cell population, respectively, and showed a higher capacity for self-renewal and tumor formation than the ALDH1^low cells. With regard to chemoresistance, the survival rate of the ALDH1^high cells was found to be higher than that of the ALDH1^low cells following treatment with chemotherapeutic agents for RMS. Furthermore, the ALDH1^high cells exhibited a higher degree of pluripotency and gene expression of Sox2, which is one of the stem cell markers. Taken together, the ALDH1^high cells possessed characteristics of CSCs, including colony formation, chemoresistance, differentiation and tumor initiation abilities. These results suggest that ALDH1 is a potentially useful marker of CSCs in eRMS.     

Targeting HIF1α eliminates cancer stem cells in hematological malignancies

Molecular targeting of cancer stem cells (CSCs) has therapeutic potential for efficient treatment of cancer, although relatively few specific targets have been identified so far. Hypoxia-inducible factor (HIF) was recently shown to regulate the tumorigenic capacity of glioma stem cells under hypoxic conditions. Surprisingly, we found that, under normoxia, HIF1α signaling was selectively activated in the stem cells of mouse lymphoma and human acute myeloid leukemia (AML). HIF1a shRNA and HIF inhibitors abrogated the colony-forming unit (cfu) activity of mouse lymphoma and human AML CSCs. Importantly, the HIF-inhibitor echinomycin efficiently eradicated mouse lymphoma and serially transplantable human AML in xenogeneic models by preferential elimination of CSCs. Hif1α maintains mouse lymphoma CSCs by repressing a negative feedback loop in the Notch pathway. Taken together, our results demonstrate an essential function of Hif1α-Notch interaction in maintaining CSCs and provide an effective approach to target CSCs for therapy of hematological malignancies.     

Blocking HIF1α activity eliminates hematological cancer stem cells

Selective targeting of cancer stem cells (CSCs) has the potential to prevent cancer relapse. Wang et?al. (2011) report that hypoxia-inducible factor 1α (HIF1α) represses Notch signaling to maintain CSC subsets from lymphoma, and that blocking HIF1α activity eliminates lymphoma and human acute myeloid leukemia (AML) CSCs.     

Reversing the Intractable Nature of Pancreatic Cancer by Selectively Targeting ALDH-High,Therapy-Resistant Cancer Cells

Human pancreatic ductal adenocarcinoma (PDAC) is a cancer with a dismal prognosis. The efficacy of PDAC anticancer therapies is often short-lived; however, there is little information on how this disease entity so frequently gains resistance to treatment. We adopted the concept of cancer stem cells (CSCs) to explain the mechanism of resistance and evaluated the efficacy of a candidate anticancer drug to target these therapy-resistant CSCs. We identified a subpopulation of cells in PDAC with CSC features that were enriched for aldehyde dehydrogenase (ALDH), a marker expressed in certain stem/progenitor cells. These cells were also highly resistant to, and were further enriched by, treatment with gemcitabine. Similarly, surgical specimens from PDAC patients showed that those who had undergone preoperative chemo-radiation therapy more frequently displayed cancers with ALDH strongly positive subpopulations compared with untreated patients. Importantly, these ALDH-high cancer cells were sensitive to disulfiram, an ALDH inhibitor, when tested in vitro. Furthermore, in vivo xenograft studies showed that the effect of disulfiram was additive to that of low-dose gemcitabine when applied in combination. In conclusion, human PDAC-derived cells that express high levels of ALDH show CSC features and have a key role in the development of resistance to anticancer therapies. Disulfiram can be used to suppress this therapy-resistant subpopulation.