Deficit of circulating CD19+ CD24hi CD38hi regulatory B cells in severe aplastic anaemia.

Immune aplastic anaemia (AA) is caused by cytotoxic T lymphocytes (CTLs) that destroy haematopoietic stem and progenitor cells. Enhanced type 1 T helper (Th1) responses and reduced regulatory T cells (Tregs) are involved in the immune pathophysiology. CD24hi CD38hi regulatory B cells (Bregs) suppress CTLs and Th1 responses, and induce Tregs via interleukin 10 (IL-10). We investigated circulating B-cell subpopulations, including CD24hi CD38hi Bregs, as well as total B cells, CD4+ T cells, CD8+ T cells and natural killer cells in 104 untreated patients with severe and very severe AA, aged ≥18 years. All patients were treated with standard immunosuppressive therapy (IST) plus eltrombopag. CD24hi CD38hi Bregs were markedly reduced in patients with AA compared to healthy individuals, especially in very severe AA, but residual Bregs remained functional, capable of producing IL-10; total B-cell counts and the other B-cell subpopulations were similar to those of healthy individuals. CD24hi CD38hi Bregs did not correlate with responses to IST, and they recovered to levels present in healthy individuals after therapy. Mature naïve B-cell counts were unexpectedly associated with IST response. Markedly reduced CD24hi CD38hi Bregs, especially in very severe AA, with recovery after IST suggest Breg deficits may contribute to the pathophysiology of immune AA.

[Effectiveness of intraperitoneal chemotherapy for t4 colon cancer]. / Effektivnost' vnutribryushnoi khimioterapii pri rake obodochnoi kishki T4.

OBJECTIVE: To determine the effect of intraperitoneal chemotherapy (IPC) with mitomycin C on expression of intraperitoneal cancer cells markers in patients with T4 colon cancer. MATERIAL AND METHODS: For the period from January 2019 to April 2020, 65 patients with T4 colon cancer were included in prospective comparative study. There were 46 patients in the main group and 19 patients in the control group. In the main group, surgical procedure was followed by IPC with mitomycin C. No IPC was performed in the control group. An effectiveness of IPC was evaluated using CD133, CD24, CD26, CD44, CD184 markers expression in peritoneal lavages. RESULTS: Significant between-group differences were observed for CD133 (p=0.0168), CD24 (p=0.0455) and CD44 (p=0.0012). There was a tendency to decrease in the level of CD184 expression in both groups in the second lavage (p=0.0605). CONCLUSION: IPC in patients with T4 colon cancer can reduce the expression and proliferative potential of free cancer cells.

CD44+/CD24+-Expressing Cervical Cancer Cells and Radioresistant Cervical Cancer Cells Exhibit Cancer Stem Cell Characteristics.

BACKGROUND: Radiation therapy is a mainstay in the treatment of cervical cancer. However, most advanced and metastatic cervical cancers are resistant to radiation therapy because of the presence of cancer stem cells (CSCs). To date, no specific markers were found for cervical CSCs. METHODS: The fraction of CD44+/CD24+ cell subpopulation was detected with flow cytometry (FCM). The clonogenicity and radiosensitivity were detected using colony-formation and radiosensitivity assay. Matrigel-transwell invasion assay was used to compare the invading capacity. We compared the tumor formation capacity using Tumor Xenografts. The expressions of apoptosis related factor, epithelial-mesenchymal transition and stem cell markers were detected with real-time polymerase chain reaction and western blot analysis. RESULTS: This study shows that radiation-resistant cervical cancer cells are rich in CD44+/CD24+-expressing cervical cancer cells. Moreover, these 2 cells exhibit the same CSC characteristics, such as increased expression of Bcl-2, survivin, and stem cell markers being more tumorigenic. These cells also showed phenotypic molecular changes that are consistent with epithelial-mesenchymal transition. CONCLUSION: Our data suggested that CD44+/CD24+-expressing cervical cancer cells may perform an important function in the radioresistance of cervical cancer. The therapy, which focuses on CD44+/CD24+-expressing cervical cancer cells, can increase the radiosensitivity of cervical cancer.

Bitter melon juice exerts its efficacy against pancreatic cancer via targeting both bulk and cancer stem cells.

Pancreatic cancer (PanC) is one of the deadliest malignancies worldwide and frontline treatment with gemcitabine becomes eventually ineffective due to increasing PanC resistance, suggesting additional approaches are needed to manage PanC. Recently, we have shown the efficacy of bitter melon juice (BMJ) against PanC cells, including those resistant to gemcitabine. As cancer stem cells (CSCs) are actively involved in PanC initiation, progression, relapse and drug-resistance, here we assessed BMJ ability in targeting pancreatic cancer-associated cancer stem cells (PanC-CSCs). We found BMJ efficacy against CD44+ /CD24+ /EpCAMhigh enriched PanC-CSCs in spheroid assays; BMJ also increased the sensitivity of gemcitabine-resistant PanC-CSCs. Exogenous addition of BMJ to PanC-CSC generated spheroids (not pre-exposed to BMJ) also significantly reduced spheroid number and size. Mechanistically, BMJ effects were associated with a decrease in the expression of genes and proteins involved in PanC-CSC renewal and proliferation. Specifically, immunofluorescence staining showed that BMJ decreases protein expression/nuclear localization of CSC-associated transcription factors SOX2, OCT4 and NANOG, and CSC marker CD44. Immunohistochemical analysis of MiaPaCa2 xenografts from BMJ treated animals also showed a significant decrease in the levels of CSC-associated transcription factors. Together, these results show BMJ potential in targeting PanC-CSC pool and associated regulatory pathways, suggesting the need for further investigation of its efficacy against PanC growth and progression including gemcitabine-resistant PanC.

Characterization of primary normal and malignant breast cancer cell and their response to chemotherapy and immunostimulatory agents.

BACKGROUND: The phenomenon of chemotherapy-resistant cancers has necessitated the development of new therapeutics as well as the identification of specific prognostic markers to predict the response to novel drugs. Primary cancer cells provide a model to study the multiplicity of tumourigenic transformation, to investigate alterations of the cellular response to various molecular stimuli, and to test therapeutics for cancer treatment. METHODS: Here, we developed primary cultures of human breast tissue - normal cells (BN1), cancer cells (BC5), and cells from a chemotherapy-treated tumour (BrCCh1) to compare their response to conventional chemotherapeutics and to innate immunity stimulators with that of the immortalized breast cells MCF7, MDA-MB-231, and MCF10A. Expression of the progesterone receptor (PGR), oestrogen receptor (ER) α and ß, human epidermal growth factor receptor (HER) 2 and 3 and aromatase CYP19, as well as expression of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) mRNA in human breast cells were characterized. RESULTS: We revealed that BC5 carcinoma cells were PGRlow/ERbhigh/ERa-/Cyp19+, the BrCCh1 cells that originated from the recurrent tumour were PGR-/ERb+/ERa-/Cyp19+, and normal BN cells were PGR-/ERb+/ERa-/Cyp19high. The treatment of primary culture cells with antitumour therapeutics revealed that BrCCh1 cells were doxorubicine-resistant and sensitive to cisplatin. BC5 cells exhibited low sensitivity to tamoxifen and cisplatin. The innate immunity activators interferon-α and an artificial small nucleolar RNA analogue increased expression of IFIT3 at different levels in primary cells and in the immortalized breast cells MCF7, MDA-MB-231, and MCF10A. The relative level of activation of IFIT3 expression was inversely correlated with the baseline level of IFIT3 mRNA expression in breast cell lines. CONCLUSION: Our data demonstrated that primary cancer cells are a useful model for the development of novel cancer treatments. Our findings suggest that expression of IFIT3 mRNA can be used as a prognostic marker of breast cancer cell sensitivity to immunostimulating therapeutics.

CD24 is a genetic modifier for risk and progression of prostate cancer.

CD24 plays an oncogenic role in the onset and progression of various human cancers, including prostate cancer. In the present study, we identified two linkage disequilibrium blocks with four recombination hotspot motifs in human CD24 locus. To elucidate whether genetic variants of CD24 are associated with susceptibility to prostate cancer and its disease status, we conducted a case-control association study with two P170 C/T and P-534 A/C polymorphisms of CD24 in 590 patients with prostate cancer and 590 healthy controls. A significant increased risk of prostate cancer was found in men with the P170T/T genotype over the P170C/C genotype (odd ratio = 1.74, 95% confidence interval = 1.16-2.63, P = 0.008), and in men with the P-534C/C genotype over the P-534A/A genotype (odd ratio = 1.47, 95% CI = 1.18-2.26, P = 0.003). Cochran-Armitage trend analysis showed that the P170T allele was significantly correlated with an increased risk of prostate cancer progression (P = 0.029, trend between genotypes and stages) and this observation was also validated in an independent sample cohort. Next, we found that tumors with P170T or P-534C alleles had more twofold increased protein expressions of CD24 as compared to those with P170C or P-534A alleles, respectively. Likewise, tumors with a combination of P170T/T and P-534C/C genotypes were associated with a high mRNA level of CD24. Our data suggest a significant association of CD24 genetic variants with prostate cancer onset and progression, which provides new insight into molecular genetics of prostate cancer; however, these findings need to be validated in multiple independent cohorts. © 2016 Wiley Periodicals, Inc.

Identification and genetic manipulation of human and mouse oesophageal stem cells.

OBJECTIVE: Human oesophageal stem cell research is hampered by the lack of an optimal assay system to study self-renewal and differentiation. We aimed to identify and characterise human and mouse oesophageal stem/progenitor cells by establishing 3-dimensional organotypic sphere culture systems for both species. DESIGN: Primary oesophageal epithelial cells were freshly isolated and fluorescence-activated cell sorting (FACS)-sorted from human and mouse oesophagus and 3-dimensional organotypic sphere culture systems were developed. The self-renewing potential and differentiation status of novel subpopulations were assessed by sphere-forming ability, cell cycle analysis, immunostaining, qPCR and RNA-Seq. RESULTS: Primary human and mouse oesophageal epithelial cells clonally formed esophagospheres consisting of stratified squamous epithelium. Sphere-forming cells could self-renew and form esophagospheres for over 43 passages in vitro and generated stratified squamous epithelium when transplanted under the kidney capsule of immunodeficient mice. Sphere-forming cells were 10-15-fold enriched among human CD49f(hi)CD24(low) cells and murine CD49f(+)CD24(low)CD71(low) cells compared with the most differentiated cells. Genetic elimination of p63 in mouse and human oesophageal cells dramatically decreased esophagosphere formation and basal gene expression while increasing suprabasal gene expression. CONCLUSIONS: We developed clonogenic and organotypic culture systems for the quantitative analyses of human and mouse oesophageal stem/progenitor cells and identified novel cell surface marker combinations that enrich for these cells. Using this system, we demonstrate that elimination of p63 inhibits self-renewal of human oesophageal stem/progenitor cells. We anticipate that these esophagosphere culture systems will facilitate studies of oesophageal stem cell biology and may prove useful for ex vivo expansion of human oesophageal stem cells.

Forced extinction of CD24 stem-like breast cancer marker alone promotes radiation resistance through the control of oxidative stress.

Along with CD44, CD24 is a key marker of breast cancer stem cells (CSCs), frequently defined by CD24(-)/CD44(+) labeling. Among all phenotypes classically attributed to breast CD24(-)/CD44(+) cancer cells, radiation resistance has been extensively described and seen as being implicated in radiotherapy failure. Our previous data indicated that CD24(-) cells constitute a radiation-resistant subpopulation transitory selected by high doses of ionizing radiation. However, little is known about the biological role of CD24 in breast cancers, and no function has been assigned to CD24 in radiation response. Here, CD24 expression was induced in CD24(-) cells or knocked-down in CD24(+) cells. We show that forced extinction of CD24 expression is associated with decreased proliferation rate, lower levels of reactive oxygen species (ROS) and decreased genomic instability. On the opposite when CD24 is artificially expressed in CD24(-) cells, proliferation rates in vitro and in vivo, ROS levels and genomic instability are enhanced. Moreover, we observe that loss of CD24 expression leads to radiation resistance, by preventing radiation-induced cell death and promoting generation of progeny in relation to lower G2/M blockade and a smaller proportion of polyploid cells. Finally, control of ROS levels appears to be the key event in the CD24-mediated radiation response. For the first time, CD24 is proposed as a direct actor in radiation response of breast cancer cells, independently of CD44 expression. These findings could have interesting applications in evaluating the intrinsic radiation response of primary tumors.

The distribution of CD44+/CD24- cancer stem cells in breast cancer and its relationship with prognostic factors.

PURPOSE: The purpose of this study was to investigate the correlation between the percentages of CD44+/CD24- cancer stem cells (CSCs) and the clinicopathological and prognostic factors in breast cancer patients. METHODS: Twenty three women who underwent surgery for breast cancer were enrolled in this study. The mean age of the patients was 46.65 years and 52% had early-stage disease. Tumor tissues obtained during surgery were digested enzymatically. CD44+/CD24- cell phenotype was identified by using surface marker antibodies and percentages were determined by surface marker expression of the cells. RESULTS: Sixty five percent of the tumors were positive for estrogen (ER)/ progesterone receptors (PR) and 38% of the tumors were positive for HER-2. All of the patients with hormone receptor positive tumors had ER positive tumors, while only 11 patients had PR positive breast cancer. CD44+/CD24- cells were present in all tumor tissues. The mean proportion of the CD44+/CD24- cells was 1.43±1.6. The mean percentages of CD18+ cells and MUC1+ were 27.9±26.5% and 6.07±11.34%, respectively. The percentage of CD18+ cells was significantly higher in PR positive tumors (p=0.042). There was no significant correlation between the percentage of CD44+/CD24- cells and clinicopathological features. CONCLUSION: This study showed that CD44+/CD24- cells were present in all tumor tissues. The percentage of CD44+/CD24- cells was higher in early-stage disease, yet without statistical significance. No correlation was found between prognostic factors and the percentage of the CD44+/CD24- cells.

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.

Rapid phenotypic fingerprinting of cell products by robust measurement of ubiquitous surface markers.

Flow cytometry is one of the most versatile and powerful approach for the quantitative analysis of cell suspensions. With widespread applications in basic and clinical research, its commonest use is in the detection of cell populations labelled against markers specific for a particular phenotype. In this study, we aimed to expand the potential of flow cytometry by describing a method based on robust automated quantification of ubiquitous cell surface markers. We demonstrate that automatic fluorescence standardization combined with whole cell population analysis yields highly reproducible results and can alleviate many of the difficulties associated with conventional analyses. This new generic approach is very flexible for quantifying the uniqueness of entire cell populations regardless of their composition. It provides quantitative phenotypic fingerprints rapidly, does not incorporate subjective factors, is more amenable to standardization, and is easily transferable across a wide diversity of applications, such as quality control for cell manufacture and authentication of cell products.

Siglec-10 is associated with survival and natural killer cell dysfunction in hepatocellular carcinoma.

BACKGROUND: The interaction between Siglec-10 and its ligand, CD24, selectively represses tissue damage-caused immune responses. However, the nature of Siglec-10 and CD24 in human hepatocellular carcinoma (HCC) is still poorly defined. Hereon, the expression, function, and regulation of CD24 and Siglec-10 in HCC were investigated in the present study. METHODS: Flow cytometry was performed to examine the expression of Siglec-10 in HCC tissues and adjacent non-tumor tissues of HCC patients. To further determine whether Siglec-10 expression is associated with the clinical characteristics and survival, conventional immunohistochemistry was performed in 96 HCC patients. Additionally, the role of Siglec-10 in the regulation of natural killer (NK) cell dysfunction was evaluated. Finally, CD24 expression in HCC was also assessed. RESULTS: Siglec-10 was expressed most on NK cells in HCC (40.7 ± 4.5%). Compared with surrounding non-tumor tissues, tumor tissues had higher Siglec-10 expression (31.0 ± 1.7% versus 40.7 ± 4.5%, n = 10, P < 0.05), and the expression was negatively associated with patient survival. Siglec-10(+)CD56(+) NK cells exhibited reduced effector function, as shown by decreased granules and cytokine expressions compared with Siglec-10(-)CD56(+) NK cells. Moreover, the number of CD24(+)CD45(-) cells in HCC tissues was higher than that in adjacent non-tumor tissues (9.4 ± 0.9% versus 3.1 ± 0.9%, n = 15, P < 0.05). CONCLUSIONS: These findings suggest that Siglec-10 is associated with decreased survival and impaired NK cell function in human HCC. This process may function via the CD24-Siglec-10 interaction, which may represent a therapeutic target in HCC patients.

Enterobacter Strains Might Promote Colon Cancer.

Many studies have been performed to determine the interaction between bacterial species and cancer. However, there has been no attempts to demonstrate a possible relationship between Enterobacter spp. and colon cancer so far. Therefore, in the present study, it is aimed to investigate the effects of Enterobacter strains on colon cancer. Bacterial proteins were isolated from 11 Enterobacter spp., one Morganella morganii, and one Escherichia coli strains, and applied onto NCM460 (Incell) and CRL1790 (ATCC) cell lines. Cell viability and proliferation were determined in MTS assay. Flow Cytometry was used to detect CD24 level and apoptosis. Real-Time PCR studies were performed to determine NFKB and Bcl2 expression. Graphpad Software was used for statistical analysis. The results showed that proteins, isolated from the Enterobacter spp., have significantly increased cell viability and proliferation, while decreasing the apoptosis of the cell lines tested. The data in the present study indicated that Enterobacter strains might promote colon cancer. Moreover, Enterobacter spp. could be a clinically important factor for colon cancer initiation and progression. Studies can be extended on animal models in order to develop new strategies for treatment.

Gene expression characteristics of breast cancer stem cells.

PURPOSE: Breast cancer stem cells have been found to be responsible for tumorigenic potential and resistance to therapy. This study aimed at comparing gene expression profiles in breast cancer, based on the differences of stem cells in their biological characteristics. METHODS: Four breast cancer cell lines with different molecular and biological characteristics were used to analyze 84 breast cancer-related gene expressions. These were the ductal human epithelial breast cancer cell line T47D (HTB-133) with metastatic origin, the invasive ductal human breast carcinoma cell line MDA-MB-231 (HTB-26), the ductal human epithelial breast cancer cell line BT-474 (HTB-20) and the human metastatic breast adenocarcinoma cell line MCF-7 (HTB-22). RESULTS: There were significant differences between the breast cancer cells and the stem cells, particularly in angiogenesis, migration, proliferation and the expression of the DNA repair genes. CONCLUSION: These data indicated the absence of a general cancer stem cell in breast cancer. Our study supports the use of the term "breast cancer initiating cells" instead of breast cancer stem cells. All of these genetic differences should be taken into account in the planning of final therapeutic approach.

Determination of 35 cell surface antigen levels in malignant pleural effusions identifies CD24 as a marker of disseminated tumor cells.

Many targets have been identified in solid tumors for antibody therapy but it is less clear what surface antigens may be most commonly expressed on disseminated tumor cells. Using malignant pleural effusions as a source of disseminated tumor cells, we compared a panel of 35 antigens for their cancer specificity, antigen abundance and functional significance. These antigens have been previously implicated in cancer metastasis and fall into four categories: (i) cancer stem cell, (ii) epithelial-mesenchymal transition, (iii) metastatic signature of in vivo selection and (iv) tyrosine kinase receptors. We determined the antigen density of all 35 antigens on the cell surface by flow cytometry, which ranges from 3 × 10(3) -7 × 10(6) copies per cell. Comparison between the malignant and benign pleural effusions enabled us to determine the antigens specific for cancer. We further chose six antigens and examined the correlation between their expression levels and tumor formation in immunocompromised mice. We concluded that CD24 is one of the few antigens that could simultaneously meet all three criteria of an ideal target. It was specifically and abundantly expressed in malignant pleural effusions; CD24(high) tumor cells formed tumors in mice at a faster rate than CD24(low) tumor cells, and shRNA-mediated knockdown of CD24 in HT29 cells confirmed a functional requirement for CD24 in the colonization of the lung. Concomitant consideration of antigen abundance, specificity and functional importance can help identify potentially useful markers for disseminated tumor cells.

Illuminating the lateral organization of cell-surface CD24 and CD44 through plasmon coupling between Au nanoparticle immunolabels.

CD44 and CD24 are important cell surface glycoproteins whose relative expression levels are used to identify so-called cancer stem cells (CSCs). While current diagnostic applications of CD44 and CD24 focus primarily on their expression levels, we demonstrate here that noble metal nanoparticle (NP) immunolabeling in combination with plasmon coupling microscopy (PCM) can reveal more subtle differences, such as the spatial organization of these surface species on subdiffraction limit length scales. We quantified both expression and spatial clustering of CD44 and CD24 on MCF7 and SKBR3 breast cancer cells through analysis of the labeling intensity and the electromagnetic coupling of the NP labels, respectively. The labeling intensity was well correlated with the receptor expression, but the inspection of the labeled cell surface in the optical microscope revealed that the NP immunolabels were not homogeneously distributed. Consistent with a heterogeneous spatial distribution of the targeted CD24 and CD44 in the plasma membrane, a significant fraction of the NPs were organized into clusters, which were easily detectable in the optical microscope as discrete spots with colors ranging from green to orange. To further quantify the spatial organization of the targeted proteins, we characterized individual NP clusters through spatially resolved elastic scattering spectroscopy. The statistical analysis of the single cluster spectra revealed a higher clustering affinity for CD24 than for CD44 in the investigated cancer models. This preferential clustering was removed upon lipid raft disruption through cholesterol sequestration. Overall, these observations confirm a preferential enrichment of CD24 in lipid rafts and a more random distribution of CD44 in the plasma membrane as cause for the observed differences in protein clustering.

Genetic and epigenetic analysis of putative breast cancer stem cell models.

BACKGROUND: Cancer stem cell model hypothesizes existence of a small proportion of tumor cells capable of sustaining tumor formation, self-renewal and differentiation. In breast cancer, these cells were found to be associated with CD44⁺CD24-low and ALDH⁺ phenotype. Our study was performed to evaluate the suitability of current approaches for breast cancer stem cell analyses to evaluate heterogeneity of breast cancer cells through their extensive genetic and epigenetic characterization. METHODS: Breast cancer cell lines MCF7 and SUM159 were cultured in adherent conditions and as mammospheres. Flow cytometry sorting for CD44, CD24 and ALDH was performed. Sorted and unsorted populations, mammospheres and adherent cell cultures were subjected to DNA profiling by array CGH and methylation profiling by Epitect Methyl qPCR array. Methylation status of selected genes was further evaluated by pyrosequencing. Functional impact of methylation was evaluated by mRNA analysis for selected genes. RESULTS: Array CGH did not reveal any genomic differences. In contrast, putative breast cancer stem cells showed altered methylation levels of several genes compared to parental tumor cells. CONCLUSIONS: Our results underpin the hypothesis that epigenetic mechanisms seem to play a major role in the regulation of CSCs. However, it is also clear that more efficient methods for CSC enrichment are needed. This work underscores requirement of additional approaches to reveal heterogeneity within breast cancer.

Profiling of normal and malignant breast tissue show CD44high/CD24low phenotype as a predominant stem/progenitor marker when used in combination with Ep-CAM/CD49f markers.

BACKGROUND: Accumulating evidence supports cancer to initiate and develop from a small population of stem-like cells termed as cancer stem cells (CSC). The exact phenotype of CSC and their counterparts in normal mammary gland is not well characterized. In this study our aim was to evaluate the phenotype and function of stem/progenitor cells in normal mammary epithelial cell populations and their malignant counterparts. METHODS: Freshly isolated cells from both normal and malignant human breasts were sorted using 13 widely used stem/progenitor cell markers individually or in combination by multi-parametric (up to 9 colors) cell sorting. The sorted populations were functionally evaluated by their ability to form colonies and mammospheres, in vitro. RESULTS: We have compared, for the first time, the stem/progenitor markers of normal and malignant breasts side-by-side. Amongst all markers tested, we found CD44high/CD24low cell surface marker combination to be the most efficient at selecting normal epithelial progenitors. Further fractionation of CD44high/CD24low positive cells showed that this phenotype selects for luminal progenitors within Ep-CAMhigh/CD49f + cells, and enriches for basal progenitors within Ep-CAM-/low/CD49f + cells. On the other hand, primary breast cancer samples, which were mainly luminal Ep-CAMhigh, had CD44high/CD24low cells among both CD49fneg and CD49f + cancer cell fractions. However, functionally, CSC were predominantly CD49f + proposing the use of CD44high/CD24low in combination with Ep-CAM/CD49f cell surface markers to further enrich for CSC. CONCLUSION: Our study clearly demonstrates that both normal and malignant breast cells with the CD44high/CD24low phenotype have the highest stem/progenitor cell ability when used in combination with Ep-CAM/CD49f reference markers. We believe that this extensive characterization study will help in understanding breast cancer carcinogenesis, heterogeneity and drug resistance.

Putative cancer stem cell marker expression in oral epithelial dysplasia and squamous cell carcinoma.

Multifactorial conditions underlie progression of potentially malignant oral lesions (PMOL) to oral squamous cell carcinoma (OSCC) and there is currently need for better prediction of malignant transformation. The hypothesised existence of cancer stem cells in dysplastic oral tissues provides the potential for more informed assessment of PMOL progression. Semi-quantitative immunohistochemical assessment of four putative cancer stem cell markers (CD24, CD44, CD271 and ALDH1) was conducted with a training cohort of 107 patient biopsies to establish clinically applicable score threshold values that were subsequently applied to a blind diagnosis in an independent validation cohort of 278 biopsies. Stain intensity scores for ALDH1, CD24 and CD44, but not CD271 were greater for OSCC than normal tissues. The intensity of ALDH1 and CD24 immunostaining correlated with increased oral epithelial disease severity, and CD24 was effective in distinguishing OSCC from non-malignant tissues, correctly diagnosing 71% of OSCC cases in the validation cohort. Importantly, CD24 immunostaining was effective in diagnosing the presence of dysplasia, correctly discriminating 69% of dysplasia tissues from normal tissues, although no distinction between mild and severe grades of dysplasia was achieved. The results highlight CD24 immunostain intensity as an effective marker of oral dysplasia and OSCC. In conclusion, CD24 immunostain intensity scoring may serve as a helpful technique to assist with the histological recognition of dysplasia in oral biopsies, but not for distinguishing between grades of dysplasia.

The immunohistochemical expression of CD24 and CD171 adhesion molecules in borderline ovarian tumors.

CD24 and CD171 are cell adhesion proteins, which have been shown to be overexpressed in several carcinomas and to be associated with a poor clinical outcome. Our aim was to determine the expression of these two adhesion molecules in ovarian borderline neoplasms. We investigated 50 ovarian borderline tumors (serous, mucinous and endometrioid) as well as 29 benign cystadenomas and 25 carcinomas, which were used as controls. Paraffin sections were stained immunohistochemically for CD24 and CD171, and their expression was recorded in a semi-quantitative manner. In normal epithelium and benign ovarian cystadenomas both the CD24 and CD171 expression was negative to low, while their expression was significantly increased in borderline and malignant ovarian tumors. High-grade carcinomas, and carcinomas with metastases to the omentum presented considerably higher CD24 expression than low-grade carcinomas, and carcinomas without metastases. In addition, a few borderline and many malignant tumors presented cytoplasmic CD24 immunoreactivity, whereas all benign and most borderline tumors showed apical localization of this molecule. In conclusion, borderline tumors and carcinomas of the ovary present increased expression of CD24 and CD171 in relation to their benign counterparts, as is the case in malignant tumors of other organs. Change of staining pattern of CD24 (apical to cytoplasmic) apparently relates to a more aggressive phenotype.