Proportion Of CD44 Research Articles

418PD - Association Between Cancer Stem Cells and CD133+ Blood Vessels in Glioblastoma Multiforme

ABSTRACT Background Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor in adults. Histologically, GBMs are highly angiogenic. It has been suggested that GBM stem cells might support tumor vascularization. The aim of our study was to evaluate the association between CD133-positive (CD133+) cancer stem cells and blood vessels in GBM tissue. Methods Between Jan 2006 and Dec 2008, 42 patients (30-77 years) received postoperative radiotherapy and chemotherapy. Surgically excised GBM tissues were examined for CD133 expression prior anticancer therapy. After immunohistochemistry, the proportion of CD133+ stem cells (%), the number of CD133+ blood vessels (per microscopic field), and the staining intensity of endothelial CD133 expression (arbitrary score 0-3) were evaluated. Results The expression of CD133 was determined by 2 independent researchers whose results were in good accordance. The proportion of CD133+ GBM stem cells was 33% ± 24% (mean ± SD). GBM tissue consisted of both CD133+ and CD133- blood vessels. However, endothelial staining of CD133 was found in all types of GBM blood vessels, including small capillaries and microvascular proliferations. The number of CD133+ blood vessels per microscopic field was 1.4 ± 1.3 (mean ± SD), and the endothelial CD133 staining intensity 1.0 ± 0.5 (mean ± SD). A significant association was found between the proportion of CD133+ stem cells and the number of CD133+ blood vessels (p = 0.004). Also, a correlation between the number of CD133+ blood vessels and the endothelial CD133 staining intensity was detected (p Conclusion In the present study, a significant association was found between CD133+ GBM stem cells and CD133+ blood vessels. Favourable CD133+ expression has to be taken into account in the strategies under preclinical or clinical development for GBM stem cell targeting. This work was supported by grant ETF8862 and Roche scientific grant. Disclosure J. Jaal: This work was partly supported by Roche scientific grant. M. Kase: This work was partly supported by Roche scientific grant. A. Minajeva: This work was partly supported by Roche scientific grant. K. Niinepuu: This work was partly supported by Roche scientific grant. S. Kase: This work was partly supported by Roche scientific grant. M. Vardja: This work was partly supported by Roche scientific grant. T. Asser: This work was partly supported by Roche scientific grant.

Treatment of breast cancer stem cells with oncolytic herpes simplex virus

Cancer stem cells have recently been isolated from several different solid tumors. In breast cancer, the CD44+CD24−/low population is considered to comprise stem-like cells. The identification of cancer stem cells has provided new targets for the development of therapeutics. Oncolytic herpes simplex viruses (oHSVs) are an effective strategy for killing breast cancer cells and treating breast tumors in preclinical models. Here, we examined the efficacy of the oHSV G47Δ in killing breast cancer stem cells. Human breast cancer cell line SK-BR-3 and human primary breast cancer cells were cultured in suspension under conditions conducive to the growth of stem cells. They generated mammospheres, which had cancer stem cell properties. The proportion of CD44+CD24−/low cells in these mammospheres exceeded 95%, as determined by flow cytometry. The mammospheres were found to be highly tumorigenic when implanted subcutaneously in nude BALB/c mice. G47Δ contains the LacZ gene, and X-gal staining of infected cells in vitro and in vivo showed the replication and spread of the virus. G47Δ was found to be highly cytotoxic to the CD44+CD24−/low population in vitro, even when injected at low multiplicities of infection, and G47Δ treatment in vivo significantly inhibited tumor growth compared with mock treatment. This study demonstrates that oHSV is effective against breast cancer stem cells and could be a beneficial strategy for treating breast cancer patients.

CD44+/CD24- phenotype contributes to malignant relapse following surgical resection and chemotherapy in patients with invasive ductal carcinoma

BackgroundInvasive ductal carcinoma is the most common type of breast malignancy, with varying molecular features and resistance to treatment. Although CD44+/CD24- cells are believed to act as breast cancer stem cells and to be linked to poor prognosis in some patients, the association between these cells and tumor recurrence or metastasis in all or some types of invasive ductal carcinoma is unclear.MethodsA total of 147 randomly selected primary and secondary invasive ductal carcinoma samples were assayed for expression of CD44, CD24, ER, PR, and Her2. The association between the proportions of CD44+/CD24- tumor cells and the clinico-pathological features of these patients was evaluated.ResultsCD44+/CD24- tumor cells were detected in 70.1% of the tumors, with a median proportion of 5.8%. The proportion of CD44+/CD24- tumor cells was significantly associated with lymph node involvement (P = 0.026) and PR status (P = 0.038), and was correlated with strong PR status in patients with recurrent or metastatic tumors (P = 0.046) and with basal-like features (p = 0.05). The median disease-free survival (DFS) of patients with and without CD44+/CD24-/low tumor cells were 22.9 ± 2.2 months and 35.9 ± 3.8 months, and the median overall survival (OS) of patients with and without CD44+/CD24-/low tumor cells were 39.3 ± 2.6 months and 54.0 ± 3.5 months, respectively, and with both univariate and multivariate analyses showing that the proportion of CD44+/CD24-/low tumor cells was strongly correlated with DFS and OS.ConclusionThe prevalence of CD44+/CD24- tumor cells varied greatly in invasive ductal carcinomas, with the occurrence of this phenotype high in primary tumors with high PR status and in secondary tumors. Moreover, this phenotype was significantly associated with shorter cumulative DFS and OS. Thus, the CD44+/CD24- phenotype may be an important factor for malignant relapse following surgical resection and chemotherapy in patients with invasive ductal carcinoma.

Effect of heterogeneity of HER2 expression on brain metastases of breast cancer.

635 Background: HER2-overexpressing or triple-negative [ER(-)/PR(-)/HER2(-)] breast cancers are associated with increased risk of brain metastases. The mechanisms leading to metastasis in each subtype are not well known. Methods: We introduced the wild-type HER2 gene into MDA-MB-231-luc-D3H2LN (231-Luc) cells, which are triple-negative breast cancer cells, and established HER2-expressing (63.2%) cells as 231-Luc-HER2 cells. We investigated the tumor formation following orthotopic inoculation and brain metastasis following intracardiac injection into nude mice. Metastasis was detected by bioluminescence imaging and confirmed in H&E staining and immunohistochemistry of vimentin and HER2 expressions. Flow cytometry analysis was used to detect the proportion of CD44+/CD24- cells, a maker for stem-like breast cancer cells. Results: 231-Luc-HER2 cells formed larger tumors in orthotopic xenograft models compared to 231-Luc cells, however, no significant difference was observed in proliferation in vitro. Neither 231-Luc-HER2 nor 231-Luc metastasized in the brain from the breast after orthotopic inoculation. After intracardiac injections of the 231-Luc-HER2 cells, brain metastasis developed (7/13 mice, 53.8%). Immunohistochemical analysis revealed that most metastasized cells expressed HER2, although we had injected a mixture of HER2-positive and HER2-negative cancer cells. Interestingly, administering Lapatinib, a dual EGFR and HER2 tyrosine kinase inhibitor, effectively prevented HER2-positive cells to colonize the brain. However, the HER2-negative 231-Luc-HER2 cells developed into brain metastases. In fact, the 231-Luc cells, which are HER2-negative, also metastasized in the brain (10/16 mice, 62.5%). Flow cytometry analysis of the 231-Luc-HER2 cells showed that HER2-positive cells decreased the population of CD44+/CD24- (HER2+/CD44+/CD24-: 86.8% and HER2-/CD44+/CD24-: 96.3%). Conclusions: The mechanism of brain metastases of HER2-positive breast cancer cells is different from that of HER2-negative breast cancer cells. It is therefore important to consider an additional therapeutic approach when dealing with HER2-negative cells in tumors having the heterogeneity of HER2 expression.

CD44+/CD24− ovarian cancer cells demonstrate cancer stem cell properties and correlate to survival

Cancer cells with the surface marker profile CD44+/CD24- have previously been described to possess cancer stem cell-like properties. This manuscript evaluates those properties in ovarian cancer cell lines. The proportion of CD44+/CD24- cells corresponded to the clinical aggressiveness of each ovarian cancer cell line histologic subtype. CD44+/CD24- cells demonstrated enhanced progressive differentiation as well as showing a 60-fold increase in Matrigel invasion in both SKOV3 and OV90 cell lines (p<0.001 each) compared to other phenotypes. CD44+/CD24- demonstrated significant resistance to all chemotherapy agents used in all cell lines, with a 71-93% increase in resistance compared with baseline. Using a threshold of 25% CD44+/CD24- ovarian cancer cells found in ascites, patients with >25% CD44+/CD24- were significantly more likely to recur (83 vs. 14%, p=0.003) and had shorter median progression-free survival (6 vs. 18months, p=0.01). In conclusion, the CD44+/CD24- phenotype in ovarian cancer cells demonstrate cancer stem cell-like properties of enhanced differentiation, invasion, and resistance to chemotherapy. This CD44+/CD24- phenotype correlates to clinical endpoints with increased risk of recurrence and shorter progression-free survival in patients with ovarian cancer.

Associations Between Markers of Colorectal Cancer Stem Cells and Adenomas Among Ethnic Groups

Most colorectal tumors develop from adenomatous polyps, which are detected by colonoscopy. African Americans (AAs) have higher incidence of colorectal cancer (CRC) and greater mortality from this disease than Caucasian Americans (CAs). We investigated whether differences in predisposition to CRC and its surrogate (colonic adenomas) between these ethnic groups were related to numbers of cancer stem or stem-like cells (CSCs) in colonocytes. We analyzed colonic effluent from 11 AA and 14 CA patients who underwent scheduled colonoscopy examinations at the John D. Dingell Veterans Affairs Medical Center. We determined proportions of cells that expressed the CSC markers CD44 and CD166 by flow cytometry. The proportion of colonocytes that were CD44(+)CD166(-) in effluent from patients with adenomas was significantly greater than from patients without adenomas (P = 0.01); the proportion of CD44(+)CD166(+) colonocytes was also greater (P = 0.07). Effluent from AAs with adenomas had 60 % more CD44(+)166(-) colonocytes than from CAs with adenomas. Using cutoff values of 8 % for AAs and 3 % for CAs, the proportion of CD44(+)166(-) colonocytes that had positive predictive value for detection of adenomas was 100 % for AAs and CAs, determined by receiver operator characteristic curve analysis. The proportion of CD44(+)166(-) colonocytes in colonic effluent can be used to identify patients with adenoma. AAs with adenomas have a higher proportion of CD44(+)166(-) colonocytes than CA. The increased proportion of CSCs in colonic tissue from AA might be associated with the increased incidence of CRC in this population.

Abstract 138: Deficiency of CD11a Reduces CD8+ T-Cell Activation and Proliferation in Adipose Tissue of Obese Mice

T cells, particularly CD8 + T cells, are major participants in obesity-linked adipose tissue (AT) inflammation. CD11a, a β 2 -integrin, is crucial for T cell adhesion and interactions with antigen-presenting cells. It has been shown that stimulation of CD11a lowered the threshold of T cell activation, yet the role of CD11a in T cell activation in AT remains unknown. We used CD11a -/- mice to test the hypothesis that CD11a deficiency reduces CD8 + T cell activation in AT. Mice fed high-fat diet for 3 months were used as an obesity model, with mice on normal diet as lean controls. CD8 + T cells and related cytokines were examined in AT by flow cytometry or quantitative RT-PCR. Compared to lean wild-type (WT) mice, obese WT mice had increased numbers of activated CD8 + T cells, with higher proportions of CD11a high CD8 + memory T cells, CD44 + CD62L - effector memory CD8 + T cells and CD44 + CD69 + effector CD8 + T cells; and increased mRNA levels of granzyme B and IFN-γ in AT. Compared to obese WT, obese CD11a -/- mice had lower proportions of CD44 + CD62L - effector memory CD8 + T cells and CD44 + CD69 + effector CD8 + T cells, and decreased mRNA levels of granzyme B and IFN-γ (P&lt;0.01), implying decreased activation of CD8 + T cells. IL-2, IL-12 and IL-18 levels were increased in AT of obese WT compared to that of leans, and were lower in AT of obese CD11a -/- mice than that of obese WT. In vitro treatment with IL-2, IL-12 and IL-18 induced proliferation and expression of IFN-γ and CD69 in CD8 + cells isolated from AT of WT mice. Compared to those from WT mice, CD8 + T cells from AT of CD11a -/- mice showed decreased response to cytokine stimulation with fewer cells expressing IFN-γ (7.0±1.1% in CD11a -/- vs. 11.3±1.1% in WT, n=3-5/group, P&lt;0.05). In addition, using Edu labeling techniques, we studied proliferation of CD8 + T cells in AT in vivo. At 3 hours after intraperitoneal injection of Edu, no Edu + T cells appeared in blood but we found CD8 + /Edu + T cells in AT of WT mice, indicating CD8 + T cells proliferated in AT. Compared to obese WT mice, obese CD11a -/- mice had decreased proportion of proliferating CD8 + T cells in AT (1.3±0.4% in CD11a -/- vs. 2.4±0.1% in WT, n=3/group, P&lt;0.01). In conclusion, we demonstrated that CD11a played a crucial role in CD8 + T cell activation and proliferation in AT associated with obesity.

Abstract 5191: Periostin signaling in breast cancer stem cells

Abstract Recent evidence suggests that the molecular heterogeneity inherent to breast cancer, which underlies metastasis, resistance to treatment and disease recurrence, can be driven by a distinct subpopulation of tumor cells referred to as cancer stem cells. Breast cancer stem cells are associated with epithelial-mesenchymal transition (EMT) and the basal-like molecular subtype of tumors, exhibit a CD44+/CD24- expression profile and have an enhanced capacity to form mammospheres. However, the extracellular cues and intracellular pathways that cancer stem cells rely on remain unclear. Using a cell line model of breast cancer progression we found a carcinoma line that was enriched for cancer stem cells. Using microarray expression profiling we identified periostin, a secreted extracellular matrix protein, to be overexpressed in this cell line and numerous other basal-like cell lines. Periostin was also overexpressed in populations that were enriched for cancer stem cells through other means such as cell sorting or growth as mammospheres. Furthermore, cancer stem cell-like populations displayed increased surface levels of the alpha-v beta-3 integrin heterodimer, a known receptor for periostin, suggesting that hyperactivation of periostin signaling may be a common feature of the cancer stem cell state. In support of this, cells with high levels of alpha-v beta-3 integrin form significantly more mammospheres and exhibit an enhanced capacity for growth on soft agar, a surrogate marker of tumorigenesis. Conversely, treatment of cells with an alpha-V beta-3 inhibitory antibody decreased mammosphere formation in a dose-dependent manner. Knockdown of periostin expression does not seem to alter the proportion of CD44+/CD24- cells but it does influence their ability to grow as mammospheres and the activity of aldehyde dehydrogenase (ALDH), another marker of cancer stem cells. Finally, as periostin is a TGF-beta inducible gene, we are currently testing the role of periostin in TGF-beta induced EMT and have seen evidence for an attenuation of this process. Ongoing studies are aimed at characterizing the intracellular pathways regulated by periostin and determining the effect of periostin expression on chemotherapeutic resistance and metastasis, two properties which may be enhanced by the presence of cancer stem cells. 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 5191. doi:1538-7445.AM2012-5191

Abstract 3327: Role of microRNA221 in breast cancer stem cell expansion and induction of EMT

Abstract Breast cancer can be grouped by expression profiling into categories that differ in biological and clinical characteristics. Molecular characterization of these subtypes on the basis of stem cell markers has shown that basal and claudin-low tumor bear stem cell like characteristics with a higher population of CD44+/CD24- and CD49f+/EpCAM- cells, which also display characteristics of epithelial mesenchymal tumor transition (EMT). There are several genetic and epigenetic signaling pathways that regulate the stem cell like characteristics of these subtypes. We examined the role of microRNA221 (mir-221) in sub populations of normal breast cells isolated from reduction mammoplasties as well as in cancer cell lines from different molecular subtypes of breast cancer. We found that mir-221 drives cells towards more basal subtype and induces EMT in both normal mammary cells and breast cancer cell lines. On the basis of CD49f and ESA staining in the cells isolated from normal mammary tissue, we found high mir-221 expression in the CD49f+/EpCAM- population. Furthermore, we showed that ALDH+ cells from mammospheres expressed significantly higher mir-221 compared to ALDH- cells. In order to determine the effect of mir-221 on the stem cell population, we overexpressed mir-221 in cells from primary mammospheres and showed that overexpression of mir-221 increases the CD49f+/EpCAM- population with increased expression of mesenchymal biomarkers, an effect also seen in a non-transformed breast cell line MCF10A. Overexpression of mir-221 in luminal breast cancer cell lines MCF-7 and T47D cell lines increased the proportion of CD44+/CD24- cells. Furthermore, overexpression of mir-221 significantly stimulated the tumor growth of MCF7 xenographs in NOD/SCID mice. These studies suggest that mir-221 regulates breast CSCs by promoting EMT. This may play an important role in tumor behavior and treatment resistance. 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 3327. doi:1538-7445.AM2012-3327

Effect of bFGF on the MCF-7 Cell Cycle with CD44+/CD24-: Promoting the G0/G1→G2/S Transition

PurposeFew cells with stem cell characteristics possess capabilities of self-renewal and differentiation, which leads to high tumorigenesis and resistance to standard chemotherapeutic agents. These cells are mostly quiescent, and arrest occurs at the mitotic G0/G1 phase in mitosis. We explored the effects of basic fibroblast growth factor (bFGF) on the MCF-7 cell cycle with CD44+/CD24-.MethodsCancer-initiating cells were propagated as mammospheres. The CD44+/CD24- subpopulation was sorted by a fluorescence activating cell sorter-Vantage flow cytometer. A cell cycle analysis was performed with different bFGF concentrations.ResultsDifferences in the CD44+/CD24- cell proliferation under different bFGF concentrations were observed (p=0.001). When the bFGF concentration was increased, the proportion of CD44+/CD24- at G0/G1 decreased (p=0.023).ConclusionWe conclude that bFGF may sustain CD44+/CD24- cell proliferation and could promote cell progression through the G0/G1→G2/S phase transition.

Role of CD44(+)/ESA(+) in sorting colonic cancer stem cells

To isolate CD133(+)/CD44(+)/ESA(+) subsets cells from SW480 colon cancer cells, and to observe the tumor formation. CD133(+)/CD44(+)/ESA(+) subsets cells, CD133(-)/CD44(+)/ESA(+) subsets cells and CD133(-)/CD44(-)/ESA(-) subsets cell were sorted by flow cytometry from SW480 colon cancer cells, then three subsets were separately inoculated in five NOD/SCID mice and the growth rates were calculated. The proportion of CD133(-)/CD44(-)/ESA(-), CD133(-)/CD44(+)/ESA(+) and CD133(+)/CD44(+)/ESA(+) subsets cells in SW480 cells were (86.38±10.23)%,(1.26±0.28)% and(0.38±0.07)%. After inoculation, tumor nodules could be formed three days later in CD133(+)/CD44(+)/ESA(+) group, and they could be formed 9 days later in CD133(-)/CD44(+)/ESA(+) group, while they could be formed 15 days later in CD133(-)/CD44(-)/ESA(-) group. Eighteen days later, tumor sizes in three groups were(13.82±5.04) mm(3), (9.25±4.57) mm(3) and (4.76±3.92) mm(3) respectively, and the differences were statistically significant(P<0.05). ESA(+)-CD44(+) is one of the surface markers for colonic cancer stem cells, and CD133(+)-CD44(+)-ESA(+) cells are SW480-like cancer stem cells.

The biological characteristics of glioma stem cells in human glioma cell line SHG44

Gliomas are the most common tumors of the central nervous system (CNS) and a frequent cause of death. The treatment of malignant gliomas is often palliative due to their high recurrence rate. A growing body of evidence suggests that glioma may arise from cancer stem cells (CSC) correlated with neural stem cells (NSC), with the capacity for self-renewal and multipotency. CSCs have been isolated from human gliomas and numerous other solid tumors. It is assumed that a number of established malignant cell lines also contain a rare subpopulation of stem cells. This study was designed to investigate the proportion of CSCs in the human glioma cell line SHG44 and to study the limitations of CD133 immunophenotyping in glioma stem cell research. SHG44 cells were cultured in both serum-containing and serum-free medium. The similar shape in growth curves (in the exponential growth phase) revealed that most cells participated in the population amplification. Time gradient BrdU labeling and monoclonal assay revealed that almost every single cell participated in the division growth (98.82%) and possessed the ability to form clones (96.19%). No significant difference was found in the proportions of CD133+ cells in the serum-containing and serum-free groups (38.25%/37.92%). In addition, no significant difference was noted in the proportions of CD133+ cells among monoclones selected randomly in the serum-containing group. These results suggested that CD133- cells generate CD133+ cells and have the ability to form clones. Thus, we concluded that most SHG44 cells were CSCs and serum-free medium was not necessary for the generation of CSCs. In this line, CD133- cells also possessed clonogenic, self-renewal capacities and were also CSCs.

Expression of Galectin-3 in CD133 + pulmonary adenocarcinoma cells induced CD8 + T cell apoptosis

Objective To investigate the expression and function of Galectin-3 in CD133 + pulmonary adenocarcinoma cells.Methods CD133 + cells were separated by magnetic activated cell sorting (MACS) from resected pulmonary adenocarcinoma specimens of 10 patiens.The proportion of CD133 +cells was measured by flow cytometry (FCM).The expression of Galectin-3 in CD133 + or CD133- cells was quantitated by fluorescent quantitation real time-polymerase chain reaction (fqRT-PCR) and Western blotting.We also investigated whether the supemants of CD133 + cells could mediate apoptosis of CD8 + T cells in vitro.Results 90％ cells separated by MACS were positive for CD133,which was proved by FCM.The expression of Galectin-3 was 1.24 folds and 1.5 folds higher in CD133 + cells than that in CD133 - cells detected by fqRT-PCR and Western blotting.The supernants of CD133 + cells more effectively induced apoptosis of CD8+ T cells with apoptosis rate being (27.1 ± 2.6) ％ than those of CD133 - cells with apoptosis rate being ( 10.1 ±2.2)％,which could be down-regulated by lactose and anti-CD133 monoclonal antibody.Conclusion Galectin-3 was highly expressed in CD133 + pulmonary adenocarcinoma cells,exerted strong biological activity,and could obviously mediate CD8 + T cell apoptosis in vitro. Key words: Galectin-3 ; CD133 ; Pulmonary adenocarcinoma; Apoptosis

CD44+/CD24−/low cancer stem/progenitor cells are more abundant in triple-negative invasive breast carcinoma phenotype and are associated with poor outcome

Women classified as having triple-negative tumors have a poor prognosis. The importance of CD44(+)/CD24(-/low) (stem/progenitor cell-phenotype) in breast cancer patients has also been appreciated. However, correlation between triple negativity and CD44(+)/CD24(-/low) with tumor recurrence remains elusive. In the present study, we evaluated tumor specimens of 50 breast cancer patients with known hormone receptor status for whom we had follow-up information and outcome data available, and performed immunohistochemistry analysis to determine CD44 and CD24 expression. Gene expression arrays were also independently performed on 52 breast cancer specimens with banked frozen tissue. Lastly, we used FVBN202 transgenic mouse model of breast carcinoma and determined the hormone receptor status, the proportion of CD44(+)/CD24(-/low) breast cancer stem-like cells, and the behavior of the tumor. We determined that patients with triple-negative tumors had significantly higher incidence of recurrence or distant metastasis associated with increased frequency of breast cancer stem cell phenotypes compared with those with non-triple-negative tumors. Preclinical studies in FVBN202 transgenic mice confirmed these findings by showing that relapsed tumors were triple negative and had significantly higher frequency of breast cancer stem cells compared with their related primary tumors. Unlike non-triple-negative primary tumors, relapsed triple-negative tumors were tumorigenic at low doses when inoculated into FVBN202 transgenic mice. These findings suggest that CD44(+)/CD24(-/low) breast cancer stem-like cells play an important role in the clinical behavior of triple-negative breast cancer and that development of therapeutic targets directed to breast cancer stem-like cells may lead to reduction in the aggressiveness of triple-negative breast cancers.

Expression and function of Galectin-1 in CD133+ human pulmonary adenocarcinoma cells

Objective To investigate the expression and function of Galectin-1 in CD133+ pulmonary adenocarcinoma cells. Methods CD133 + cells were separated by magnetic activated cell sorting (MACS) from excised pulmonary adenocarcinoma speciments of 9 patiens. The proportion of CD133 + cells was measured by flow cytometry (FCM). The expression of Galectin-1 in CD133 + or CD133 - cells was quantitated by fluorescent quantitation real-time polymerase chain reaction (fqRT-PCR), Western blotting and enzyme linked immunosorbent assay (ELISA) respectively. CD133 + cells were transfected with small interfering RNA (siRNA) of Galectin-1 to study the effect of Galectin-1 inhibition on cancer cells growth and clonality. Tumorigenesis in nude mice was also performed in vivo. Results 92.6% cells separated by MACS were positive for CD133, which was proved by FCM. The expression of Galectin-1 in CD133 + cells was 1. 748 folds and 1. 135 folds higher than that in CD133 - celts detected by fqRT-PCR and Western blot respectively. Downregulation of Galectin-1 ex vivo also resulted in (36. 75 ±: 1.35 ) % decrease of proliferation rate of cancer cells. Conclusion Galectin-1 which can be efficiently inhibited by Galectin-1 siRNA was significantly highly expressed in CD133 + cells and associated with the proliferation and clonality of CD133 + cells. Key words: Galectin; CD133; Pulmonary adenocarcinoma

CD44+/CD24− cells and lymph node metastasis in stage I and II invasive ductal carcinoma of the breast

The presence of tumor-initiating cells (CD44(+)/CD24(-)) in solid tumors has been reported as a possible cause of cancer metastasis and treatment failure. Nevertheless, little is know about the presence of CD44(+)/CD24(-) cells within the primary tumor and metastasis. The proportion of CD44(+)/CD24(-) cells was analyzed in 40 samples and in 10 lymph node metastases using flow cytometry phenotyping. Anti-human CD326 (EpCam; FITC), anti-human CD227 (MUC-1; FITC), anti-human CD44 (APC), and anti-human CD24 (PE), anti-ABCG2 (PE), and anti-CXCR4 (PeCy7) were used for phenotype analysis. The mean patient age was 60.5 years (range, 33-87 years); mean primary tumor size (pT) was 1.8 cm (0.5-3.5 cm). The Wilcoxon or Kruskal-Wallis test was used for univariate analyses. Logistic regression was used for multivariate analysis. The median percentage of CD44(+)/CD24(-) cells within primary invasive ductal carcinomas (IDC) was 2.7% (range, 0.2-71.2). In lymph node metastases, we observed a mean of 6.1% (range, 0.07-53.7). The percentage of CD44(+)/CD24(-) cells in IDCs was not associated with age, pT, tumor grade and HER2. We observed a significantly enrichment of CD44(+)/CD24(-) and ABCG2(+) cells in ESA(+) cell population in patients with positive lymph nodes (P = 0.02 and P = 0.04, respectively). Our data suggest that metastatic dissemination is associated with an increase in tumor-initiating cells in stage I and II breast cancer.

Effect of fibroblasts on breast cancer cell mammosphere formation and regulation of stem cell-related gene expression

The purpose of this study was to investigate the regulatory effects of breast cancer fibroblasts (BCFs) vs. normal mammary fibroblasts (NMFs) on mammosphere formation and stem cell-related gene expression in breast cancer cells. Breast cancer cells (MCF-7) were cultured in suspension to generate primary and secondary mammospheres. The proportion of CD44+/CD24low/- cells was assessed by flow cytometry (FCM), and Wnt1, Notch1, β-catenin, CXCR4, SOX2 and ALDH3A1 gene expression was detected by quantitative real-time PCR. The fibroblasts from either breast cancer tissue or normal mammary tissue were purified from tissue specimens and co-cultured with breast cancer cells. The mammosphere formation efficacy was approximately 180/10,000 MCF-7 cells. FCM analysis showed that, compared to the 2.1% positive expression in the MCF-7 monolayer culture cells, the expression of CD44+/CD24low/- in MCF-7 mammosphere cells was significantly elevated to 10.4% (P<0.01). The proportion of the CD44+/CD24low/- subpopulation of the cells in mammospheres was nearly 5-fold higher than that of general MCF-7 cells. Compared with MCF-7 monolayer culture cells, mammosphere cells showed significantly (P<0.01) enhanced expression of Wnt1 [fold-change (FC), 2.25], Notch1 (FC, 2.45), β-catenin (FC, 1.72), CXCR4 (FC, 4.68), SOX2 (FC, 4.25) and ALDH3A1 (FC, 5.38). When BCFs were co-cultured with MCF-7 cells under mammosphere culture conditions, the length of time of mammosphere formation decreased, the volume of the mammo-spheres increased and the mammosphere-forming efficiency (MFE) was higher than that of NMFs and the control group. Both the BCF and NMF groups showed enhanced gene expression for the following genes: Wnt1 (FC, 3.18 and 1.27, respectively), β-catenin (FC, 1.75 and 1.22, respectively), Notch1 (FC, 2.09 and 1.31, respectively), CXCR4 (FC, 2.77 and 1.33, respectively), SOX2 (FC, 2.77 and 1.80, respectively) and ALDH3A1 (FC, 5.23 and 1.85, respectively). Cancer fibroblast cells can promote the MFE and up-regulate stem cell-related gene expression in breast cancer cells.

Abstract 3331: An increase in cancer stem cell population after primary systemic therapy is a poor prognostic factor in breast cancer

Abstract Introduction: The cancer stem cell (CSC) hypothesis has important clinical implications for cancer therapeutics because of the proposed role of CSCs in chemoresistance. The aim of this study was to investigate changes in the CSC populations before and after primary systemic therapy (PST) and their prognostic role in human breast cancer. Methods: Paired samples (before and after PST) of breast cancer tissue were obtained from clinical stage II or III patients (n=92) undergoing PST and subsequent breast resection. The proportions of putative CSCs with CD44+/CD24- or ALDH1+ phenotypes were determined by immunohistochemistry. Results: A higher proportion of CD44+/CD24- tumor cells and ALDH1 positivity in pre-chemotherapy biopsy was correlated with higher histologic grade, estrogen receptor negativity, high Ki-67 proliferation index and basal-like subtype of breast cancer. ALDH1 positivity in pre-chemotherapy biopsy was also associated with a higher rate of pathologic complete response (pCR) following PST. In comparisons of putative CSC populations before and after PST, the proportions of CD44+/CD24- and ALDH1+ tumor cells were significantly increased after PST. The cases with increased CD44+/CD24- tumor cell populations after PST showed high Ki-67 proliferation index in post-chemotherapy specimens and those with increased ALDH1+ tumor cell population after PST were associated with estrogen receptor negativity and p53 overexpression. Furthermore, cases showing such an increase had significantly shorter disease-free survival time than those with no change or a reduced number of CSCs. Conclusions: The present study provides the clinical evidence that the putative CSCs in breast cancer are chemoresistant and are associated with tumor progression, emphasizing the need for targeting of CSCs in the breast cancer therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3331. doi:10.1158/1538-7445.AM2011-3331

Primary breast cancer patients with high risk clinicopathologic features have high percentages of bone marrow epithelial cells with ALDH activity and CD44+CD24lo cancer stem cell phenotype

Cancer stem cells (CSCs) are purported to be epithelial tumour cells expressing CD44(+)CD24(lo) that exhibit aldehyde dehydrogenase activity (Aldefluor(+)). We hypothesised that if CSCs are responsible for tumour dissemination, disseminated cells in the bone marrow (BM) would be positive for putative breast CSC markers. Therefore, we assessed the presence of Aldefluor(+) epithelial (CD326(+)CD45(dim)) cells for the presence of the CD44(+)CD24(lo) phenotype in BM of patients with primary breast cancer (PBC). BM aspirates were collected at the time of surgery from 66 patients with PBC. Thirty patients received neoadjuvant chemotherapy (NACT) prior to aspiration. BM was analysed for Aldefluor(+) epithelial cells with or without CD44(+)CD24(lo) expression by flow cytometry. BM aspirates from three healthy donors (HD) were subjected to identical processing and analyses and served as controls. Patients with triple-receptor-negative (TN) tumours had a significantly higher median percentage of CD44(+)CD24(lo) CSC within Aldefluor(+) epithelial cell population than patients with other immunohistochemical subtypes (P=0.018). Patients with TN tumours or with pN2 or higher pathologic nodal status were more likely to have a proportion of CD44(+)CD24(lo) CSC within Aldefluor(+) epithelial cell population above the highest level of HD. Furthermore, patients who received NACT were more likely to have percentages of Aldefluor(+) epithelial cells than the highest level of HD (P=0.004). The percentage of CD44(+)CD24(lo) CSC in the BM is higher in PBC patients with high risk tumour features. The selection or enrichment of Aldefluor(+) epithelial cells by NACT may represent an opportunity to target these cells with novel therapies.

CD44+/CD24− Cells Are Transit Progenitors and Do Not Determine the Molecular Subtypes and Clinical Parameters in Breast Carcinomas

CD44+/CD24− cells have been associated with breast cancer stem/progenitor cell features. However, the status of this phenotype cells in normal, benign and malignant breast tissues has not been studied, and the clinical correlation of this subpopulation in breast cancer is not fully understood. The present study sought to identify these cells in a series of normal, benign, and malignant breast tissues and explore their correlation to the molecular subtypes of breast carcinoma and conventional pathological features. Double-staining immunohistochemistry (DIHC) of CD44 and CD24 was performed on 30 normal breast tissues, 30 breast fibroadenomas (FA), 60 breast invasive ductal carcinomas (IDC), and 3 breast cancer cell lines (MCF-7, MDA-MB-468, and MDA-MB-231). In the normal breast tissues and FAs, three phenotypes were observed including CD44+/CD24+, CD44+/CD24−, and CD44−/CD24− cells. In the IDCs, CD44−/CD24+ cells were detected, in addition to the three aforementioned phenotypes. The strong positive rate (+++, incidence >60%) of CD44+/CD24− was significantly increased from normal breast tissue, FAs to IDCs (0.0%→6.7%→21.7%). However, the CD44+/CD24− cells didn’t correlate with ages of patients, lymph node metastasis, tumor size, molecular subtypes, and the expression of ER, PR, HER-2, PS2, Bcl-2, nm23. The proportion of CD44+/CD24− cells in MCF-7, MDA-MB-468, and MDA-MB-231 was about 1, 5, and 80%, respectively. The results indicate that the CD44+/CD24− cells are transit progenitors and have no association with the molecular subtypes and clinicopathological parameters in the IDCs.