CD44 Low Cells Research Articles

Abstract 480: Targeting cancer stem cells through STAT3 inhibition in head and neck cancers

Abstract Objectives: There is increasing evidence that cancer stem cells (CSC) are responsible for tumor recurrence and metastasis in head and neck cancer (HNC). The development of therapeutic strategies that preferentially target CSC may decrease cancer cell self-renewal and enhance progenitor apoptosis, potentially halting tumor growth and metastasis. Signal transducer and activator of transcription (STAT) proteins are important in oncogenic signaling pathways including cell cycle control and regulation of apoptosis. We hypothesized that expression of STAT3 would differ in CSC vs. non-CSC and the novel small molecule inhibitor, FLLL32, which blocks STAT3 function, could be used either alone or in combination with cisplatin, to induce cytotoxicity in both groups. We postulated that CSC would be more sensitive to STAT3 inhibition. Methods: CSC were identified and isolated from oral cavity cancer cell lines on the basis of high expression levels of the cell surface marker CD44. UMSCC-74B and UMSCC-47, were each derived from tongue cancers in male patients; UM-SCC-74B from a tumor that recurred after two rounds of treatment with chemotherapy and radiation and UM-SCC-47 from a previously untreated HPV-positive primary cancer. Quantitative RT-PCR analysis was used to determine relative expression values of STAT3 in CSC and non-CSC. Cell growth inhibition by FLLL32 alone and in combination with cisplatin, was then assessed by MTT assay in each cell line. Results: Both cell lines contain CD44high and CD44low expressing cells. In UMSCC-74B, STAT3 expression was increased 20-fold in CD44high relative to CD44low cells whereas for UMSCC-47, STAT3 expression was 3-fold lower in the CD44high cells. FLLL32 treatment induced growth suppression in UMSCC-74B CD44high and CD44low cells by 36% and 52% (p<0.001), respectively. In UMSCC-47, FLLL32 monotherapy induced growth suppression of CD44high and CD44low cells by 54% and 47% (p<0.001), respectively. Both cell lines and their respective CSC subgroups demonstrated enhanced cisplatin sensitivity when FLLL32 was used in combination. Conclusions: STAT3 expression differs between cell lines and between CSC and non-CSC. The aggressive phenotype of some HNC may be related to high levels of STAT3 expression within their CSC. These cancers may be less susceptible to STAT3 inhibition, alone or with cisplatin. Cancers expressing lower STAT3 expression levels in their CSC population may be more sensitive to STAT3 inhibition. By determining the level of STAT3 expression within CSC, we may be able to identify cancers that are more susceptible to STAT3 inhibition. Ongoing work is aimed at identifying the underlying mechanism for differential STAT3 expression and exploring related changes in key mediators of the STAT3 pathway. This work will underlie the development of novel, CSC-targeted treatment strategies that may be more effective than existing therapies. 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 480. doi:10.1158/1538-7445.AM2011-480

CD44 is associated with proliferation, rather than a specific cancer stem cell population, in cultured canine cancer cells

Background The cancer stem cell hypothesis proposes that tumours are maintained by a population of cancer stem cells (CSC), which must be eradicated to prevent disease relapse after treatment. Cells expressing high levels of CD44 have been identified as candidate CSC in a variety of human tumours. This study sought to investigate CD44 expression and its potential as a CSC marker in canine cancer. Methods CD44 expression in several canine cancer cell lines was determined by flow cytometry. Cells with low and high levels of CD44 expression were examined for differences in growth characteristics, colony forming ability, drug sensitivity and cell cycle profile. Results CD44 High cells demonstrated enhanced growth and colony forming capacity, under both adherent and low-density serum free (“tumoursphere”) conditions. However, no difference in sensitivity to doxorubicin was seen between the two populations. Moreover, whilst most CD44 Low cells were in resting or G 1 growth phase, an increased proportion of CD44 High cells were in G 2M phase of the cell cycle. Upon proliferation in culture, both populations gave rise to progeny with a full spectrum of CD44 expression. Conclusion CD44 expression is associated with proliferation in cultured canine cancer cells, but transient and fluctuating expression may limit its utility as a CSC marker.

Stem Cells: Their Role in Breast Cancer Development and Resistance to Treatment

About 20% of the total cells from primary breast tumors could generate palpable tumors in non-obese diabetic severe combined immunodeficient (NOD/SCID) immunocompromised mice. All the tumorigenic cells originate from a normal mammary stem cell. Human mammary stem cells are sensitive to oncogenic mutations and in mouse models they share similarities with breast cancer stem cells (BrCSCs). Tumorigenicity, invasion, progression and metastasization are further BrCSCs properties likely depending on their CD44+/CD24- phenotype. Local invasion and tumor metastasization seem to be facilitated by the epithelial to mesenchymal transition (EMT) program. This program may be reactivated from stable genetic alterations or through exposure of cancer cells to factors present in the surrounding micro-environment, or by an up-regulation of EMT-inducing transcription factors. One main explanation for resistance to treatment by cancer cells is that a rare subpopulation of cells in residual tumors with tumorigenic potential is intrinsically resistant to therapy. Consistent with this hypothesis, in human breast tumors, the subpopulation of tumor-initiating cancer cells with CD44(high)/CD24(low) cell surface-marker profile was found more resistant to cancer therapies (chemo, hormone and radiotherapy) than is the major population of more differentiated breast cancer cells. The reasons for CSC resistance to chemotherapy, hormone therapy and radiotherapy also have been examined and they opened new scenarios for cancer therapy.

The GPI-Anchored Receptor CD24 Increases Cancer Cell Invasion through Enhanced Contractile Forces

The malignancy of a tumor depends on the capability of cancer cells to metastasize. The process of metastasis involves cell invasion through connective tissue and transmigration through endothelial monolayers. The GPI-anchored receptor CD24 expression is increased in several tumor types and is consistently associated with increased metastasis formation in patients. Furthermore, the localization of β1 integrins in lipid rafts depends on CD24. Cell invasion is a fundamental biomechanical process and usually requires cell adhesion to the extracellular matrix (ECM) through mainly β1 heterodimeric integrin receptors. Here, we studied the invasion of human lung A125 cancer cells with different CD24 expression levels in 3D ECMs. We hypothesized that CD24 expression increases the invasiveness of cancer cells by enhanced contractile force transmission or generation.To analyze this, CD24neg cells (CD24-negative) were stably transfected with CD24 and sorted for high and low CD24 expression. The invasiveness of the CD24high and CD24low transfectants were determined in 3D-ECMs. The percentage of invasive cells and their invasion depth was increased in CD24high cells compared to CD24low cells.Knock-down of CD24 and of the β1 integrin subunit in CD24high cells decreased their invasiveness, indicating that the increased invasiveness is CD24 and β1 integrin subunit dependent.Fourier transform traction microscopy revealed that the CD24high cells generated 5-fold higher contractile forces compared to CD24low cells. To test whether contractile forces were functional related to cell invasion. Cell invasiveness was reduced after addition of myosin light chain kinase inhibitor ML-7 as well as Rho kinase inhibitor Y27632 in CD24high cells, but not in CD24neg cells, whereas an increase in prestress in CD24neg cells after addition of LPA increased cell invasiveness. Taken together, these results suggest that CD24 enhances cell invasion through increased transmission or generation of contractile forces.

Abstract P6-15-10: Targeting Delta-Like 4 Ligand (DLL4)/Notch Signaling by a Novel Anti-DLL4 Antibody Inhibits Tumor Growth through Altering Cancer Stem Cell and Epithelial-to-Mesenchymal Transition-Associated Genes in Triple Negative Breast Cancer

Abstract Triple negative breast cancer (TNBC, ER-, PR-Her2-) accounts for 10% of all types of breast cancer and is known to be particularly aggressive and refractory to current therapies. Chemotherapeutic agents, such as taxanes, are currently the only treatment option for this type of breast cancer. However, these treatments often result in local and systemic relapse. While several promising targeted agents have improved the relapse rate in the combination with chemotherapy in patients with pathologic complete response, those who do not achieve pathologic complete response still have a poor prognosis. We previously demonstrated that interfering with Deltalike 4 ligand (DLL4)/Notch signaling by an anti-DLL4 antibody decreased tumor growth and delayed tumor recurrence following paclitaxel treatment through reducing breast cancer stem cell fraction and tumorigenicity (Proc Am Asso Cancer Res. 51:4, 2010). CD44+/CD24low cells have been demonstrated to be highly tumorigenic in breast tumors and are associated with treatment resistance (PNAS U S A, 106: 13820-13825, 2009). To gain insights into the mechanism of action of anti-DLL4-mediated TNBC growth inhibition, we evaluated the effect of anti-DLL4 on breast cancer CD44+/CD24low the expression patterns of a list of genes involved in sternness, differentiation, and key stem cell signaling pathways in TNBC xenograft tumors following paclitaxel with and without antibody treatment in vivo. Our data showed that paclitaxel treatment resulted in significant changes of gene expression patterns in the CD44+/CD24low cells in the residual tumors. We further analyzed these genes and identified several genes related to Notch signaling, stem cell maintenance and epithelial-to-mesenchymal transition that were modulated differentially by treatment with paclitaxel and/or anti-DLL4. In a metastatic TNBC orthotopic tumor model, the anti-DLL4 treatment resulted in tumor growth inhibition at the primary site and distant organs. Taken together, our findings suggest that targeting DLL4 may improve the efficacy of current treatments, delay tumor recurrence, and reduce metastatic growth in TNBC. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-10.

Combined inhibitory effect of formestane and herceptin on a subpopulation of CD44+/CD24low breast cancer cells

In breast cancer, stromal cells surrounding cancer epithelial cells can influence phenotype by producing paracrine factors. Among many mediators of epithelial-stromal interactions, aromatase activity is perhaps one of the best studied. Clinical data suggest that estrogen-independent signaling leads to increased proliferation even during therapy with aromatase inhibitors (AIs). Molecular mechanism of crosstalk between the estrogen receptor (ER) and the epidermal growth factor receptor (HER) family have been implicated in resistance to endocrine therapy, but this interaction is unclear. The ability of aromatase to induce estradiol biosynthesis provides a molecular rationale to combine agents that target aromatase activity and the HER pathway. We targeted stromal-epithelial interactions using formestane, which exerts antiaromatase activity, combined with the monoclonal anti-HER2 antibody herceptin, in a subpopulation of CD44+/CD24low cells sorted from epithelial-mesenchymal co-cultures of breast cancer tissues. The growth inhibition was respectively 16% (P < 0.01) in the response to herceptin, 25% to formestane (P < 0.01), and 50% (P < 0.001) with the combination of the two drugs, suggesting that herceptin cooperates with formestane-induced inhibition of aromatase and this effect could be mediated through HER family receptors. In cells which expressed ERalpha, formestane/herceptin combination suppressed the mRNA expression of aromatase and HER2 and decreased cyclin D1 expression. These results show that combination therapies involving AIs and anti-HER2 can be efficacious for the treatment of cancer in experimental models and suggest that subtyping breast tumors gives useful information about response to treatment.

Abstract 3316: CD44High head and neck cancer cells demonstrate increased cell growth and chemotherapeutic resistance

Abstract Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is a tumor made up of a heterogeneous population of cancer cells. Differences in individual cell phenotype may extrapolate to clinical behavior at the tumor level. CD44 is a cell surface marker highly expressed in putative cancer stem cells. Here, we demonstrate that fluorescence activated cell sorting (FACS) for the CD44 cell surface marker isolates a population of HNSCC cells with increased growth rate, colony forming ability, and chemoresistance. Methods: FACS was used to isolate CD44High and CD44Low cells from the HNSCC cell line SCC1. CD44High, CD44Low and unsorted cells were compared with respect to cell growth using the MTT assay for 8 days. They were also seeded into agar and grown for 6 weeks to determine colony forming potential. Sorted and unsorted cells were also compared for their sensitivity to cisplatin (20 µM). Live cells were measured using the MTT assay and compared to untreated cells at the same time point in order to calculate relative growth rates. Additionally, SCC1 cells were analyzed using FACS with and without cisplatin to determine changes in the population of CD44High cells. Primary tumor specimens and normal controls were analyzed for various cancer stem cell markers via immunohistochemistry. Results: CD44High SCC1 cells grew the fastest in culture with CD44Low cells growing significantly slower. Unsorted cells had a similar growth rate to CD44High cells, which is not surprising given that CD44High cells made up roughly 60% of the total population. CD44High cells in soft agar developed significantly more colonies with a larger average colony size after 6 weeks in culture compared to unsorted and CD44Low cells. CD44High SCC1 cells also were less sensitive to cisplatin with no difference in cell growth between treated and untreated cells. Using FACS to analyze unsorted cells after treatment with cisplatin, we observed enrichment in the CD44High cell population. Control non-cancer specimens demonstrated expression of p16 with low levels of BMI and CD44 on immunohistochemical analysis. Poorly differentiated HNSCC tumors demonstrated absence of p16, high BMI, and high CD44. Conclusion: The cell surface marker CD44 is expressed in a subset of HNSCC cells that have higher growth rates, tumorogenicity, and resistance to chemotherapy. These HNSCC stem cells may be the reason for resistance and recurrence. Understanding the mechanism behind their specific phenotype will be valuable in advancing treatments for HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3316.

Abstract 3363: Identification and characterization of stem-like cells in human esophageal epithelial and adenocarcinoma cells

Abstract The aim of this study was to identify and characterize stem-like cells in human normal esophageal epithelial and adenocarcinoma cell lines. The immortalized esophageal epithelial cell line, Het-1A, and the primary esophageal adenocarcinoma (EADC) cell line, OE33, were first studied by flow cytometry and immunofluorescence using a panel of antibodies to putative stem cell biomarkers which included CD34, CD44, CD44/CD24, CD117/integrin α6, Musashi I, EpiCam, and Oct 4. Fluorescence activated cell sorting (FACS) was then used to isolate 15% of cells with the highest CD44 staining (CD44high) or reactive oxygen species (ROS) (ROShigh) and 15% of cells with the lowest (including negative) CD44 staining (CD44low) or ROS (ROSlow). Each cell population was further characterized using sphere-forming assays, soft agar clonogenic assays, flow cytometry for ROS, and synchrotron mid-infrared (IR) spectromicroscopy. CD44 positivity was found in 90% of Het-1A and 75% of OE33 cells overall. For both Het-1A and OE33 cell lines, spheroid formation (mean±SD/5000 cells) was significantly higher in cells expressing high levels of CD44 (Het-1A:138.33±2.08 vs. 97.67±1.53, p&amp;lt;0.01; OE33: 150.00±31.19 vs. 100.67±7.02, p=0.056) and low levels of ROS (Het-1A: 149.33±20.43 vs. 92.00±8.89, p=0.011; OE33: 199.67±31.63 vs. 119.33±14.47, p=0.016). Colony formation (mean±SD/1000 cells) was also found to be significantly higher in CD44high (Het-1A: 15.33±4.51 vs. 8.00±1.00, p=0.051; OE33: 92.67±11.02 vs. 18.00±1.73, p&amp;lt;0.01) and ROSlow (Het-1A: 45.00±1.00 vs. 31.67±3.79, p&amp;lt;0.01; OE33: 33.00±5.29 vs. 14.67±5.03, p=0.012) cell populations. Whereas OE33 CD44high cells were found to express consistently lower levels of ROS compared with OE33 CD44low cells (p&amp;lt;0.01), no differences in ROS expression were found in Het-1A cells sorted by CD44 expression. Spectromicroscopic analysis revealed only small non-significant differences between cell populations sorted by CD44 and ROS expression. We conclude that the higher rates of spheroid formation and clonogenicity of Het-1A and OE33 cells expressing high levels of CD44 (CD44high) and low levels of ROS (ROSlow) reflect an enrichment of stem-like cell populations in esophageal cell lines. However, the significant differences in ROS levels between CD44high OE33 cells and CD44high Het-1A cells suggests unique and biologically distinct pathways in stem-like cells derived from normal and malignant esophageal epithelia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3363.

Ten color flow cytometry reveals CD126 as a marker for mature SP4 and SP8 thymocytes (42.10)

Abstract We have developed a 10 color flow cytometric staining protocol for the simultaneous detection and characterization of 21 thymocyte subpopulations. These subpopulations include 17 previously described subsets and 4 new subsets. Importantly, this methodology uses only commercially available monoclonal antibodies and therefore could be easily employed by any investigator with access to a standard 4 laser flow cytometer. Using this novel staining scheme we have analyzed mouse thymocytes at discreet stages of development for expression of multiple cytokine receptors. As previously described, we found IL-7Ralpha to be present on some early subsets, upregulated briefly after beta-selection, downregulated on ISP and DP cells, and then re-expressed after positive selection. IL-4Ralpha was also expressed within the early DN1 and DN2 subsets. However, its expression was downregulated prior to beta-selection (in the DN3a subset) and was re-expressed in the DP cells prior to positive selection. IL-6Ralpha (CD126) showed a dramatically different pattern of expression. It was low or negative on all thymocyte subsets with the exception of the CD24low SP4 and SP8 subsets. The CD24low cells represent the more mature SP populations and therefore CD126 expression appears to be a good marker for the detection of mature thymocytes.

CD24 staining of mouse mammary gland cells defines luminal epithelial, myoepithelial/basal and non-epithelial cells

IntroductionBreast cancer is thought to arise in mammary epithelial stem cells. There is, therefore, a large amount of interest in identifying these cells. The breast is a complex tissue consisting of two epithelial layers (an outer myoepithelial/basal layer and an inner luminal epithelial layer) as well as a large non-epithelial component (fibroblasts, endothelial cells, lymphocytes, adipocytes, neurons and myocytes). The definitive identification of a mammary epithelial stem cell population is critically dependent on its purity. To date, this has been hampered by the lack of suitable markers to separate out the two epithelial layers, and to remove contaminating non-epithelial cells.MethodsMouse mammary glands were dissociated and stained with CD24. Cells were sorted into separate populations based on CD24 expression and assessed for luminal epithelial and myoepithelial/basal markers by direct fluorescent microscopy and real time PCR. The stem/progenitor potential of these cell populations was assessed in vivo by cleared mammary fat pad transplantation.ResultsThree populations of CD24 expressing cells were identified: CD24Negative, CD24Low and CD24High. Staining of these cells with cytokeratin markers revealed that these populations correspond to non-epithelial, myoepithelial/basal and luminal epithelial cells, respectively. Cell identities were confirmed by quantitative PCR. Cleared mammary fat pad transplantation of these cell populations revealed that extensive mammary fat pad repopulation capacity segregates with the CD24Low cells, whilst CD24High cells have limited repopulation capacity.ConclusionDifferential staining of mammary epithelial cells for CD24 can be used to simultaneously isolate pure populations of non-epithelial, myoepithelial/basal and luminal epithelial cells. Furthermore, mammary fat pad repopulation capacity is enriched in the CD24Low population. As separation is achieved using a single marker, it will be possible to incorporate additional markers to further subdivide these populations. This will considerably facilitate the further analysis of mammary epithelial subpopulations, whilst ensuring high purity, which is key for understanding mammary epithelial stem cells in normal tissue biology and carcinogenesis.

Distinct methylation of the interferon gamma (IFN-gamma) and interleukin 3 (IL-3) genes in newly activated primary CD8+ T lymphocytes: regional IFN-gamma promoter demethylation and mRNA expression are heritable in CD44(high)CD8+ T cells.

Differential genomic DNA methylation has the potential to influence the development of T cell cytokine production profiles. Therefore, we have conducted a clonal analysis of interferon (IFN)-gamma and interleukin (IL)-3 gene methylation and messenger (m)RNA expression in primary CD8+ T cells during the early stages of activation, growth, and cytokine expression. Despite similar distributions and densities of CpG methylation sites, the IFN-gamma and IL-3 promoters exhibited differential demethylation in the same T cell clone, and heterogeneity between clones. Methylation patterns and mRNA levels were correlated for both genes, but demethylation of the IFN-gamma promoter was widespread across >300 basepairs in clones expressing high levels of IFN-gamma mRNA, whereas demethylation of the IL-3 promoter was confined to specific CpG sites in the same clones. Conversely, the majority of clones expressing low or undetectable levels of IFN-gamma mRNA exhibited symmetrical methylation of four to six of the IFN-gamma promoter CpG sites. Genomic DNA methylation also has the potential to influence the maintenance or stability of T cell cytokine production profiles. Therefore, we also tested the heritability of IFN-gamma gene methylation and mRNA expression in families of clones derived from resting CD44(low)CD8+ T cells or from previously activated CD44(high)CD8+ T cells. The patterns of IFN-gamma gene demethylation and mRNA expression were faithfully inherited in all clones derived from CD44(high) cells, but variable in clones derived from CD44(low) cells. Overall, these findings suggest that differential genomic DNA methylation, including differences among cytokine genes, among individual T cells, and among T cells with different activation histories, is an important feature of cytokine gene expression in primary T cells.

CD8+ Memory T Cells (CD44high, Ly-6C+) Are More Sensitive than Naive Cells (CD44low, Ly-6C−) to TCR/CD8 Signaling in Response to Antigen

AbstractMemory CD8+ T cells from mice previously primed with alloantigen (alloAg) can respond in vitro to IL-2 and purified class I alloAg presented on microspheres, while no response can be detected using cells from naive mice. Similar results have been obtained using cells from OT-1 mice expressing a transgenic TCR that is specific for OVA257–264 (SIINFEKL) peptide bound to H-2Kb. A population of resting memory cells (defined on the basis of low forward scatter and CD44high, Ly-6C+, CD25−, CD69− surface phenotype) that is present in the OT-1 mice exhibits a substantially higher sensitivity to Ag-stimulation than do naive cells (CD44low, Ly-6C−) expressing the same TCR. CD44high cells respond vigorously to H-2Kb immobilized on microspheres and pulsed with peptide, while CD44low cells respond weakly and only at high class I density and peptide concentration. The Ag-presenting surface only has ligands for TCR and CD8 (class I and peptide), thus ruling out the possibility that differences are due to ligand binding by other adhesion or costimulatory receptors that are expressed at high levels on the memory cells. Experiments using anti-TCR mAb as the stimulus and coimmobilized non-Ag class I as a ligand for CD8 suggest that the difference between naive and memory cells may be at the level of stimulation through the TCR. Thus, in addition to expressing increased levels of adhesion receptors that may enhance responses to Ag on APCs, memory CD8+ T cells appear to be intrinsically more sensitive than naive cells to stimulation through the TCR/CD8 complex.