Abstract LB-306: Multiplex RT-qPCR study on the expression of stem cell markers CD24, CD44, and ALDH1 in circulating tumor cells of breast cancer patients
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Abstract Background: Molecular characterization of Circulating Tumor Cells (CTC) can give important information on their suspected connection to cancer stem cells. In breast cancer, putative cancer stem cells have been identified to have a CD44+CD24-/low and ALDH1+ cell phenotype. Patients and methods: We developed a tetraplex quantitative RT-qPCR assay for CD24, CD44, ALDH1 and HPRT based on the in-silico design of primers and probes with different fluorescence emission spectra. Specificity and sensitivity experiments were performed using the SKBR-3 cancer cell line. We isolated CTC from peripheral blood according to our previously described methodology (Strati et al, BMC cancer 2011) from 40 patients with early breast cancer before the administration of adjuvant chemotherapy, 23 patients with verified metastasis and 18 female healthy volunteers. We evaluated all our data with respect to CD24, CD44, ALDH1 expression of healthy volunteers using the 2βΔΔCt method. Results: In 23 patients with verified metastasis 9.0% were found to have CD44+CD24-/low phenotype and 13% ALDH1high/CD24-/low phenotype. In 40 operable breast cancer patients 8 patients (20.0%) were found to have CD44+CD24-/low phenotype and 12 patients (30.0%) were found to have ALDH1high/CD24-/low phenotype. CK-19, MAGE-A3, HER-2, TWIST1, hTERT α+β+, and mammaglobin gene transcripts were also quantified in the same samples. Discussion: Molecular characterization of CTC has shown that in breast cancer patients CTCs are partly characterized by a putative stem cell phenotype. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome of these patients is known. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-306. doi:1538-7445.AM2012-LB-306Keywords:
CD24
Circulating tumor cell
Multiplex
Identification of cancer stem cells (CSCs) and their behaviors will provide insightful information for the future control of human cancers. This study investigated CD44 and CD24 cell surface markers as breast cancer CSC markers in vitro and in vivo.Flow cytometry with CD44 and CD24 markers was used to sort breast cancer MCF7 cells for scanning electron microscopy (SEM), tumor cell invasion assay, and nude mouse xenograft assay.Flow cytometry assay using CD44 and CD24 markers sorted MCF7 cells into four subsets, i.e., CD44+/CD24-/low, CD44-/CD24+, CD44+/CD24+, and CD44-/CD24-. The SEM data showed that there were many protrusions on the surface of CD44+/CD24-/low cells. CD44+/CD24-/low cells had many microvilli and pseudopodia. The CD44+/CD24-/low cells had a higher migration and invasion abilities than that of the other three subsets of the cells. The in vivo tumor formation assay revealed that CD44+/CD24- cells had the highest tumorigenic capacity compared to the other three subsets.CD44 and CD24 could be useful markers for identification of breast CSCs because CD44+/CD24-/low cells had unique surface ultrastructures and the highest tumorigenicity and invasive abilities.
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Received: September 20, 2010 , Accepted: October 8, 2010 , Published: October 9, 2010 // Tumor initiating or cancer stem cells (CSCs) are suggested to be responsible for tumor initiation and growth. Moreover, therapy resistance and minimal residual disease are thought to result from selective resistance of CSCs. Isolation of CSCs from colon carcinomas can be accomplished by selection of a subpopulation of tumor cells based on expression of one or multiple cell surface markers associated with cancer stemness, like CD133, CD44, CD24, CD29, CD166 and Lgr5. Identification of colon CSCs will lead to a better rational for new therapies that aim to target this fraction specifically. In this review, we analyze known markers used for selection of colon CSCs and their potential function in CSC biology. Moreover, we discuss potential targeting strategies for eradicating CSCs specifically in order to develop more effective therapeutic strategies as well as to address more fundamental questions like the actual role of CSCs in tumor growth.
LGR5
CD24
Targeted Therapy
Stem cell marker
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Tumor initiating or cancer stem cells (CSCs) are suggested to be responsible for tumor initiation and growth. Moreover, therapy resistance and minimal residual disease are thought to result from selective resistance of CSCs. Isolation of CSCs from colon carcinomas can be accomplished by selection of a subpopulation of tumor cells based on expression of one or multiple cell surface markers associated with cancer stemness, like CD133, CD44, CD24, CD29, CD166 and Lgr5. Identification of colon CSCs will lead to a better rational for new therapies that aim to target this fraction specifically. In this review, we analyze known markers used for selection of colon CSCs and their potential function in CSC biology. Moreover, we discuss potential targeting strategies for eradicating CSCs specifically in order to develop more effective therapeutic strategies as well as to address more fundamental questions like the actual role of CSCs in tumor growth.
LGR5
CD24
Stem cell marker
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[Purpose] Breast cancer cells with a CD44+/CD24-phenotype have been suggested to have tumor-initiating properties with stem cell-like. It is not yet known whether CD44/CD24 phenotype up-regulate Twist gene and increase micrometasis risk. [Methods] The MCF7 breast cancer cell line was cultured by mammosphere and implanted into node mice. Double-staining immunohistochemistry was used to quantify CD44 and CD24 expression in cytospin and tissue samples from animal model and 92 cases with breast tumor. Twist gene expression were examined by RT-PCR. [Results] The cells marked with CD44+/CD24-existed in cell line MCF-7. In animal model,the samples from metastasis tissues had more CD44+/CD24-cells and stronger expression for Twist. In human tumor samples,20.7% contained cells with CD44 +/CD24-phenotype. The CD44+/CD24-phenotype was no relationship with estrogen and progesterone receptors as well as HER2(r =0.452) . In the samples from human metastasis tissues,Twist was over-expression. [Conclusions] MCF-7 breast cancer cell line and human cancer tissue exist CD44+/CD24-cells. Breast cancer stem cells involve distant metastasis through up-regulating the Twist expression in animal model.
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CD44+/CD24 -/low and ALDH1+ are both markers of breast cancer stem cell (BCSC), we compared the biological characteristics of CD44+/CD24 -/low and ALDH1+ BCSCs in the four molecular subtypes of breast cancer.Four fresh blocks of breast cancer tissue were obtained from patients with Luminal A, Luminal B, human epidermal receptor-2 (HER2)-overexpression and triple-negative breast cancer, respectively. Flow cytometry was used to sort CD44+/CD24 -/low and ALDH1+ BCSCs. The mammosphere (MS) formation experiment and NOD/SCID mouse xenograft experiment were performed to compare the self-renewal and tumorigenic abilities of CD44+/CD24 -/low with those of ALDH1+ BCSCs.The proportions of CD44+/CD24 -/low BCSC and ALDH1+ BCSC in Luminal A, Luminal B, HER2 overexpression and triple-negative subtypes were 12.1% and 8.7%, 2.7% and 5.7%, 0.8% and 8.7%, 0.7% and 4.5%, respectively. The MS formation experiment demonstrated that ALDH1+ BCSC formed significantly more MSs than CD44+/CD24 -/low BCSC in the four molecular subtypes (P<0.001). The NOD/SCID mouse xenograft experiment demonstrated that the sizes of grafted tumors formed by ALDH1+ BCSC were larger than those formed by CD44+/CD24 -/low BCSC in the four molecular subtypes (P<0.05).In the four molecular subtypes of breast cancer, ALDH1+ BCSC demonstrated better self-renewal and tumorigenic abilities than CD44+/CD24 -/low BCSC.
CD24
Triple-negative breast cancer
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Identification and isolation of breast cancer stem cells (CSCs) based on CD44/CD24 expression and/or enzymatic activity of aldehyde dehydrogenase 1 (ALDH1). However, the differences among the CD44+/CD24‑/low cells, ALDH1+ cells and the overlap between the sub‑populations have not been frequently investigated. Thus, it is imperative to improve the understanding of breast CSC with different stem markers. CD44+/CD24‑/low, ALDH1+ and ALDH1+CD44+/CD24‑/low cell populations were isolated from fresh breast cancer tissues and analyzed by flow cytometry and immunofluorescence. Mammosphere formation, cell proliferation assay and Transwell experiments, were used to analyze self‑renewal, proliferation and invasion, respectively, for each sub‑population. Finally, in vivo experimentation in mice was performed to evaluate the tumorigenic abilities of the sub‑populations. The sub‑populations of CD44+/CD24‑/low, ALDH1+ and ALDH1+CD44+/CD24‑/low in human breast cancer cells, represented the 7.2, 4.6 and 1.5% of the total tumor cell population, respectively. ALDH1+CD44+/CD24‑/low cells had the strongest ability of self‑renewal, invasion, proliferation and tumorigenicity compared with the other sub‑populations (P<0.05). In conclusion, different phenotypes of CD44+/CD24‑/low, ALDH1+ and ALDH1+CD44+/CD24‑/low were isolated and demonstrated that breast CSCs are heterogeneous, and they exhibit distinct biological characteristics. As ALDH1+CD44+/CD24‑/low cells demonstrated the strongest stem‑like properties, it may be a useful specific stem cell marker. The utilization of more reliable biomarkers to distinguish the breast CSC pool will be important for the development of specific target therapies for breast cancer.
CD24
Stem cell marker
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The CD44+/CD24– phenotype identifies cancer stem cell (CSC) properties in canine mammary carcinoma (MC); however, the histopathological features associated with this phenotype remain to be elucidated. Here, we determined whether the CD44+/CD24– phenotype was associated with hormonal receptor (HR; estrogen receptor [ER] and/or progesterone receptor [PR]) status and/or triple (ER, PR, and human epithelial growth factor receptor 2)–negative (TN) subtype; conventional histological evaluation was also performed. We found that, as single markers, both CD44+ and CD24+ were associated with less aggressive histological types, low grade, and a non-TN subtype; both markers were associated with HR positivity. On the other hand, a CD44+/CD24– phenotype was associated with higher grade of carcinoma. Therefore, our results suggest that immunohistochemical phenotyping for CD44/CD24 is useful for the evaluation of tumor behavior as well as CSC-like properties in canine MCs.
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Progesterone receptor
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e13084 Background: There are currently limited effective therapeutic options for metastatic breast cancer, and none target the metastatic process. MiR-10b is a major driver of breast cancer cell invasion and migration. Our lab has demonstrated its importance in metastatic cell viability, positioning miR-10b as a target for metastatic breast cancer. Our previous studies demonstrated that delivery of a novel therapeutic consisting of antisense anti-miR-10b oligomers conjugated to magnetic nanoparticles (termed MN-anti-miR10b) prevented metastases in vivo. These studies led us to hypothesize that MN-anti-miR10b affects the viability of a stem cell-like population of cells within primary tumors that otherwise would have formed metastases. Here, we investigate the relationship between miR-10b and breast cancer cell stemness and the effects of miR-10b inhibition by MN-anti-miR10b on breast cancer cell stemness. Methods: MDA-MB-231 breast cancer cells were sorted based on surface markers into stem-like (CD44+/CD24-) and non-stem-like (CD44-/CD24-) populations, and their miR-10b expression levels were quantitated. Then, unsorted MDA-MB-231 cells were treated with MN-anti-miR10b to determine the effects of miR-10b inhibition on the expression of EPCAM, a marker for cancer cell stemness. Lastly, MCF-7 breast cancer cells were cultured in mammosphere medium (which selects for stem-like cells and induces spheroid formation) and treated with MN-anti-miR10b to establish the phenotypic effect of miR-10b inhibition on cancer cell stemness. Results: We found that stem-like MDA-MB-231 cells displayed >2-fold miR-10b expression compared to non-stem-like cells. Accordingly, alongside a 70% reduction in miR-10b expression, treatment of MDA-MB-231 cells with MN-anti-miR10b reduced EPCAM expression by 65%. Treatment of MCF-7 spheroids with MN-anti-miR10b resulted in significantly greater cell dissociation from the spheroid compared to controls. Conclusions: These results support the hypothesis that inhibition of miR-10b with MN-anti-miR10b inhibits breast cancer cell stemness. This provides an explanation for the therapeutic efficacy of MN-anti-miR10b observed in mouse breast cancer metastasis models. Future studies will further investigate this relationship and shed light on how miR-10b can be optimally targeted to treat metastatic breast cancer in humans.
CD24
Viability assay
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Hepatocellular carcinoma (HCC) is a type of liver cancer, in which CD44 isoforms have been proposed as markers to identify cancer stem cells (CSCs). However, it is unclear what characteristics are associated with CSCs that exclusively express CD44 isoforms. The objective of the present study was to determine the expression of CD44 isoforms and their properties in CSCs. Analysis of transcriptomic data from HCC patient samples identified CD44v8-10 as a potential marker in HCC. In SNU-423 cells, CD44 expression was detected in over 99% of cells, and two CD44 isoforms, namely, CD44std and CD44v9, were identified in this cell line. CD44 subpopulations, including both CD44v9+ (CD44v9) and CD44v9- (CD44std) cells, were obtained by purification using a magnetic cell separation kit for human CD44v9+ cancer stem cells. CD44v9 cells showed greater potential for colony and spheroid formation, whereas CD44std cells demonstrated significant migration and invasion capabilities. These findings suggested that CD44std and CD44v9 may be used to identify features in CSC populations and provide insights into their roles in HCC.
Stem cell marker
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CD24
Stem cell marker
Identification
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