Cancer Stem-like Cells Population Research Articles

Stem Cells and Cancer Stem-Like Cells in Endocrine Tissues

Cancer stem-like cells are a subpopulation of self-renewing cells that are more resistant to chemotherapy and radiation therapy than the other surrounding cancer cells. The cancer stem cell model predicts that only a subset of cancer cells possess the ability to self-renew and produce progenitor cells that can reconstitute and sustain tumor growth. Evidence supporting the existence of cancer stem-like cells in the thyroid, pituitary, and in other endocrine tissues is rapidly accumulating. These cells have been studied using specific biomarkers including: CD133, CD44, Nestin, Nanog, and aldehyde dehydrogenase enzyme. Putative cancer stem-like cells can be studied in vitro using serum-free media supplemented with basic fibroblast growth factor and epidermal growth factor grown in low attachment plates or in extracellular matrix leading to sphere formation in vitro. Cancer stem-like cells can also be separated by fluorescent cell sorting and used for in vitro or in vivo studies. Injection of enriched populations of cancer stem-like cells (also referred to as tumor initiating cells) into immunodeficient mice results in growth of xenografts which express cancer stem-like biomarkers. Human cancer stem-like cells have been identified in thyroid cancer cell lines, in primary thyroid cancers, in normal pituitary, and in pituitary tumors. Other recent studies suggest the existence of stem cells and cancer stem-like cells in endocrine tumors of the gastrointestinal tract, pancreas, lungs, adrenal, parathyroid, and skin. New discoveries in this field may lead to more effective therapies for highly aggressive and lethal endocrine cancers.

CD44 Staining of Cancer Stem-Like Cells Is Influenced by Down-Regulation of CD44 Variant Isoforms and Up-Regulation of the Standard CD44 Isoform in the Population of Cells That Have Undergone Epithelial-to-Mesenchymal Transition

CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumours, and we have previously demonstrated the existence of two different CD44high cancer stem-like cell populations in squamous cell carcinoma, one having undergone epithelial-to-mesenchymal transition and the other maintaining an epithelial phenotype. Alternative splicing of CD44 variant exons generates a great many isoforms, and it is not known which isoforms are expressed on the surface of the two different cancer stem-like cell phenotypes. Here, we demonstrate that cancer stem-like cells with an epithelial phenotype predominantly express isoforms containing the variant exons, whereas the cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition down-regulate these variant isoforms and up-regulate expression of the standard CD44 isoform that contains no variant exons. In addition, we find that enzymatic treatments used to dissociate cells from tissue culture or fresh tumour specimens cause destruction of variant CD44 isoforms at the cell surface whereas expression of the standard CD44 isoform is preserved. This results in enrichment within the CD44high population of cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition and depletion from the CD44high population of cancer stem-like cells that maintain an epithelial phenotype, and therefore greatly effects the characteristics of any cancer stem-like cell population isolated based on expression of CD44. As well as effecting the CD44high population, enzymatic treatment also reduces the percentage of the total epithelial cancer cell population staining CD44-positive, with potential implications for studies that aim to use CD44-positive staining as a prognostic indicator. Analyses of the properties of cancer stem-like cells are largely dependent on the ability to accurately identify and assay these populations. It is therefore critical that consideration be given to use of multiple cancer stem-like cell markers and suitable procedures for cell isolation in order that the correct populations are assayed.

Prostate cancer stem‐like cells/cancer‐initiating cells have an autocrine system of hepatocyte growth factor

Prostate cancer cells include a small population of cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) that have roles in initiation and progression of the cancer. Recently, we isolated prostate CSCs/CICs as aldehyde dehydrogenase 1-highh (ALDH1(high) ) cells using the ALDEFLUOR assay; however, the molecular mechanisms of prostate CSCs/CICs are still elusive. Prostate CSCs/CICs were isolated as ALDH1(high) cells using the ALDEFLUOR assay, and the gene expression profiles were analyzed using a cDNA microarray and RT-PCR. We found that prostate CSCs/CICs expressed higher levels of growth factors including hepatocyte growth factor (HGF). Hepatocyte growth factor protein expression was confirmed by enzyme linked immunosorbent assay and Western blotting. On the other hand, c-MET HGF receptor was expressed in both CSCs/CICs and non-CSCs/CICs at similar levels. Hepatocyte growth factor and the supernatant of myofibloblasts derived from the prostate augmented prostasphere formation in vitro, and prostasphere formation was inhibited by an anti-HGF antibody. Furthermore, c-MET gene knockdown by siRNA inhibited the prostasphere-forming ability in vitro and tumor-initiating ability in vivo. Taken together, the results indicate that HGF secreted by prostate CSCs/CICs and prostate myofibroblasts has a role in the maintenance of prostate CSCs/CICs in an autocrine and paracrine fashion.

TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer

After an initial response to chemotherapy, many patients with triple-negative breast cancer (TNBC) have recurrence of drug-resistant metastatic disease. Studies with TNBC cells suggest that chemotherapy-resistant populations of cancer stem-like cells (CSCs) with self-renewing and tumor-initiating capacities are responsible for these relapses. TGF-β has been shown to increase stem-like properties in human breast cancer cells. We analyzed RNA expression in matched pairs of primary breast cancer biopsies before and after chemotherapy. Biopsies after chemotherapy displayed increased RNA transcripts of genes associated with CSCs and TGF-β signaling. In TNBC cell lines and mouse xenografts, the chemotherapeutic drug paclitaxel increased autocrine TGF-β signaling and IL-8 expression and enriched for CSCs, as indicated by mammosphere formation and CSC markers. The TGF-β type I receptor kinase inhibitor LY2157299, a neutralizing TGF-β type II receptor antibody, and SMAD4 siRNA all blocked paclitaxel-induced IL8 transcription and CSC expansion. Moreover, treatment of TNBC xenografts with LY2157299 prevented reestablishment of tumors after paclitaxel treatment. These data suggest that chemotherapy-induced TGF-β signaling enhances tumor recurrence through IL-8-dependent expansion of CSCs and that TGF-β pathway inhibitors prevent the development of drug-resistant CSCs. These findings support testing a combination of TGF-β inhibitors and anticancer chemotherapy in patients with TNBC.

Cigarette Smoke Promotes Drug Resistance and Expansion of Cancer Stem Cell-Like Side Population

It is well known that many patients continue to smoke cigarettes after being diagnosed with cancer. Although smoking cessation has typically been presumed to possess little therapeutic value for cancer, a growing body of evidence suggests that continued smoking is associated with reduced efficacy of treatment and a higher incidence of recurrence. We therefore investigated the effect of cigarette smoke condensate (CSC) on drug resistance in the lung cancer and head and neck cancer cell lines A549 and UMSCC-10B, respectively. Our results showed that CSC significantly increased the cellular efflux of doxorubicin and mitoxantrone. This was accompanied by membrane localization and increased expression of the multi-drug transporter ABCG2. The induced efflux of doxorubicin was reversed upon addition of the specific ABCG2 inhibitor Fumitremorgin C, confirming the role of ABCG2. Treatment with CSC increased the concentration of phosphorylated Akt, while addition of the PI3K inhibitor LY294002 blocked doxorubicin extrusion, suggesting that Akt activation is required for CSC-induced drug efflux. In addition, CSC was found to promote resistance to doxorubicin as determined by MTS assays. This CSC-induced doxurbicin-resistance was mitigated by mecamylamine, a nicotinic acetylcholine receptor inhibitor, suggesting that nicotine is at least partially responsible for the effect of CSC. Lastly, CSC increased the size of the side population (SP), which has been linked to a cancer stem cell-like phenotype. In summary, CSC promotes chemoresistance via Akt-mediated regulation of ABCG2 activity, and may also increase the proportion of cancer stem-like cells, contributing to tumor resilience. These findings underscore the importance of smoking cessation following a diagnosis of cancer, and elucidate the mechanisms of continued smoking that may be detrimental to treatment.

Epigallocathechin gallate, polyphenol present in green tea, inhibits stem-like characteristics and epithelial-mesenchymal transition in nasopharyngeal cancer cell lines

BackgroundPrevious studies have demonstrated that the consumption of green tea inhibits the growth of various cancers. Most cancers are believed to be initiated from and maintained by a small population of cancer stem-like cells (CSC) or tumor-initiating cells (TIC) that are responsible for tumor relapse and chemotherapeutic resistance. Although epigallocathechin gallate (EGCG), the most abundant catechin in green tea, has been reported to induce growth inhibition and apoptosis in some cancer cells, its effect on CSC is undefined. In this study, we enriched CSC by the sphere formation, and provided an efficient model for further experiments. Using this method, we examined the effects of EGCG regulating the nasopharyngeal carcinoma (NPC) CSC and attempted to elucidate the possible mechanisms.MethodsNPC TW01 and TW06 cell lines were enriched by sphere formation and characterized their phenotypical properties, such as invasion capacity, epithelial-mesenchymal transition (EMT) and gene expression were analyzed by quantitative real-time reverse transcription polymerase chain reaction (q-RT-PCR). EGCG-induced growth inhibition in the parental and sphere-derived cells was determined by MTT and bromodeoxyuridine (BrdU) assay. EGCG-induced apoptosis was analyzed by flow cytometry with Annexin V and PI staining. The effects of EGCG on sphere-derived cell tumorigenicity, migration and invasion were determined by soft agar assay, wound healing, and cell invasion assay. The alternation of protein expression regulated by EGCG on these sphere-derived cells was assessed by immunofluorescence staining and western blot.ResultsNPC sphere-derived cells grown in serum-free non-adherent culture showed increased expression of stem cell markers and EMT markers compared to parental cells grown in conventional culture. Although EGCG induced growth inhibition and apoptosis in the parental cells in a dose-dependent manner, it was not as effective against spheres. However, EGCG potently inhibited sphere formation and can eliminate the stem cell characteristics of NPC and inhibit the epithelial-mesenchymal transition (EMT) signatures.ConclusionsOverall, these findings show that NPC cells with sphere formations possess the properties of CSC. Using this model, we found that EGCG regulated NPC CSC, their self-renewal capacity, and inhibited their invasive characteristics. It supports the pivotal role of EGCG as a dietary compound targeting NPC and may decrease recurrence and metastasis in nasopharyngeal carcinoma cells.

Inhibition of CK2α Down-Regulates Hedgehog/Gli Signaling Leading to a Reduction of a Stem-Like Side Population in Human Lung Cancer Cells

Protein kinase CK2 is frequently elevated in a variety of human cancers. The Hedgehog (Hh) signaling pathway has been implicated in stem cell maintenance, and its aberrant activation has been indicated in several types of cancer, including lung cancer. In this study, we show that CK2 is positively involved in Hh/Gli signaling in lung cancer cell lines A549 and H1299. First, we found a correlation between CK2α and Gli1 mRNA levels in 100 primary lung cancer tissues. Down-regulation of Gli1 expression and transcriptional activity were demonstrated after the silencing of CK2α in lung cancer cells. In addition, CK2α siRNA down-regulated the expression of Hh target genes. Furthermore, two small-molecule CK2α inhibitors led to a dose-dependent inhibition of Gli1 expression and transcriptional activity in lung cancer cells. Reversely, forced over-expression of CK2α resulted in an increase both in Gli1 expression and transcriptional activity in A549 cells. Finally, the inhibition of Hh/Gli by CK2α siRNA led to a reduction of a cancer stem cell-like side population that shows higher ABCG2 expression level. Thus, we report that the inhibition of CK2α down-regulates Hh/Gli signaling and subsequently reduces stem-like side population in human lung cancer cells.

Prostate cancer stem cells: molecular characterization for targeted therapy

Prostate cancer stem cells: molecular characterization for targeted therapy

Abstract 3337: TGF-β from niche fibroblasts increases the stemness cancer stem cell-like side population cells in gastric cancer

Abstract Introduction: Cancer Stem Cells (CSCs) are thought to possess tumor initiation and self-renewal and are associated with tumor progression. The niche cells of microenvironment, such as fibroblast might play an important role for the progression of cancer cells. The aim of this study is to examine the effect of cancer-associated fibroblasts on the stemness of CSCs. Material and Methods: A gastric cancer cell line, OCUM-12, and cancer-associated gastric fibroblast, CaF-37, were used. SP cells, known as CSCs rich population, were isolated from OCUM-12 cells by flowcytometry using Hoechest33342, and were named as OCUM-12/SP. The percentage of SP cells was evaluated by flowcytometry, after it was cultured with condition medium from CaF-37 for three days. SP fraction was evaluated by flowcytometry, in the presence or absence of TGF-β receptor inhibitor, FGF receptor inhibitor, and c-Met Receptor inhibitor. In vivo, tumorigenicity of OCUM-12/SP cells were evaluated by subcutaneous inoculation in the presence or abcence of CaF-37. Results: The inoculation of 1.5x105, 5x104, and 1.5x104 OCUM-12/SP cells with 5x105 CaF-37 fibroblasts resulted in tumor formation in all of mice, respectively. However, the inoculation of 1.5x105, 5x104, and 1.5x104 OCUM-12/SP cells alone results in poor tumor formation at 3(60%) of 5 mice, 0(0%) of 5 mice and 0(0%) of 5 mice, respectively. The percentage of SP fraction of OCUM-12/SP was significantly increased in the presence of condition medium from CaF-37. The increase of SP fraction of OCUM-12/SP cells by condition medium from CaF-37 was inhibited by TGF-β receptor inhibitor, but not FGF receptor inhibitor and c-Met inhibitor. Conclusion: TGF-β from the niche, fibroblasts, might sustain the stemness of CSCs and might be associated with the progression of Cancer 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 3337. doi:1538-7445.AM2012-3337

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 323: Targeting B-catenin/CBP interaction in basal like breast cancer and breast cancer stem cells

Abstract Background: Aberrant regulation of the Wnt signaling pathway has been shown to be associated with the development of aggressive types of breast cancer. We hypothesize that increased co-activator interaction of CBP with nuclear β-catenin is associated with epithelial to mesenchymal transition (EMT) in breast cancer with stem-cell (basal) phenotype, disease progression, drug resistance, relapse and metastasis. We further hypothesize that pharmacologic disruption of this complex leads to forced differentiation of cancer stem cells and re-sensitization to conventional therapies. Material/Methods: We have previously developed the small molecule ICG-001 that specifically disrupts the CBP/β-catenin interaction. Several breast cancer cell lines (MDA-MB-231,-468,-435) were treated with ICG-001 and investigated for changes in proliferation, invasive potential and changes in sensitivity to chemotherapy. RNA sequencing and qRT-PCR were used to evaluate gene-expression and alternative splicing events. CoIP and ChIP were used to demonstrate CBP/β-catenin disruption and differential co-activator usage at specific gene promoters. Protein expression levels were assessed by western blot and FACS analysis. Viability assays were performed to determine drug-sensitization. Several stem cell assays were used to study the effect of ICG-001 on cancer stem cell populations. In a human primary breast cancer xenograft model in NSG mice we investigated the in vivo effect of ICG-001. Results: CoIP demonstrated global disruption of the CBP/β-catenin interaction under treatment with ICG-001. Significant down-regulation of EMT related genes (e.g. S100A4, vimentin, CDH2) and up-regulation of epithelial genes (e.g. CD24), in basal-like breast cancer, resulting in a reversal of the stem cell phenotype (CD44+/CD24- to CD24+). ChIP experiments showed re-expression of CD24 being dependent on the usage of the co-activator p300. Re-expression of epithelial splice factors and alternative gene splicing were observed. Cancer stem cell assays revealed that ICG-001 eradicates cancer stem cell-like populations and causes down-regulation of stemness related genes (e.g. nestin, PROCR). An in-vitro invasion assay demonstrated loss of invasive potential. Treatment with ICG-001 resulted in the down-regulation of cell cycle related genes (e.g. Aurora kinases, Cyclins, CDK's) and anti-apoptotic genes (e.g. survivin). Marked inhibition of cancer cell proliferation was observed in vitro. Treatment with ICG-001 re-sensitized hormone receptor-negative cell lines to tamoxifen. In-vivo studies using primary human xenografts in a NSG mouse model suggested similar effects in-vivo. Conclusion: Antagonizing CBP/β-catenin signaling using a small molecule inhibitor should be an attractive strategy for the treatment of drug-resistant aggressive stem-like breast cancer and the eradication of breast 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 323. doi:1538-7445.AM2012-323

Abstract 181: MIR-18b decreases self-renewal of glioblastoma cancer stem-like cells through targeting NOTCH2, NEDD9, and MEKK1

Abstract Glioblastoma (GBM) is the most common malignant brain tumor in human with extremely poor prognosis and new treatment for this deadly disease is desperately needed. Cancer Stem-like cells (CSCs) have been prospectively isolated from GBM and shown required for tumor propagation. We and others have demonstrated recently that differentiation of GBM neurospheres decreases GBM cell propagation both in vitro and in vivo (Zhu et al, Cancer Research, 71:6061-72, 2011). In the current study, we found that miR-18b was up-regulated upon differentiation of GBM neurosphere. When we introduced miR-18b into GBM neurospheres by lentivirus, we found that miR-18b decreases clonogenesis and growth of three different GBM neurosphere lines, indicating that GBM CSC population was reduced by miR-18b. Mechanistically, we found that miR-18b down-regulates luciferase activity of 3′-UTR reporters of NOTCH2, NEDD9, and MEKK1. Furthermore, protein expression of NOTCH2, NEDD9, and MEKK1 are also decreased by miR-18b, suggesting that NOTCH2, NEDD9 and MEKK1 are the direct targets of miR-18b in GBM CSCs. In addition, we have demonstrated previously that NOTCH regulates GBM CSC propagation through pAKT (Fan et al, Stem Cells, 28:5-16, 2010). Here, we found that pAKT and pERK1/2 were also down-regulated in miR-10b infected GBM neurosphere lines, indicating that miR-18b reduces GBM CSC propagation through NOTCH2/pAKT pathways. Taken together, we have demonstrated that miR-18b functions as a tumor suppressor and decreases GBM CSC propagation through targeting NOTCH2, NEDD9, and MEKK1, indicating that miR-18b can be a potential therapeutic molecule to target GBM CSCs and improve GBM treatment. 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 181. doi:1538-7445.AM2012-181

Therapeutic implications of an enriched cancer stem-like cell population in a human osteosarcoma cell line

BackgroundOsteosarcoma is a bone-forming tumor of mesenchymal origin that presents a clinical pattern that is consistent with the cancer stem cell model. Cells with stem-like properties (CSCs) have been identified in several tumors and hypothesized as the responsible for the relative resistance to therapy and tumor relapses. In this study, we aimed to identify and characterize CSCs populations in a human osteosarcoma cell line and to explore their role in the responsiveness to conventional therapies.MethodsCSCs were isolated from the human MNNG/HOS cell line using the sphere formation assay and characterized in terms of self-renewal, mesenchymal stem cell properties, expression of pluripotency markers and ABC transporters, metabolic activity and tumorigenicity. Cell's sensitivity to conventional chemotherapeutic agents and to irradiation was analyzed and related with cell cycle-induced alterations and apoptosis.ResultsThe isolated CSCs were found to possess self-renewal and multipotential differentiation capabilities, express markers of pluripotent embryonic stem cells Oct4 and Nanog and the ABC transporters P-glycoprotein and BCRP, exhibit low metabolic activity and induce tumors in athymic mice. Compared with parental MNNG/HOS cells, CSCs were relatively more resistant to both chemotherapy and irradiation. None of the treatments have induced significant cell-cycle alterations and apoptosis in CSCs.ConclusionsMNNG/HOS osteosarcoma cells contain a stem-like cell population relatively resistant to conventional chemotherapeutic agents and irradiation. This resistant phenotype appears to be related with some stem features, namely the high expression of the drug efflux transporters P-glycoprotein and BCRP and their quiescent nature, which may provide a biological basis for resistance to therapy and recurrence commonly observed in osteosarcoma.

Implication of expression of Nanog in prostate cancer cells and their stem cells

Recent studies suggested that the prostate cancer may arise from prostate cancer stem cells that share some same characteristics with normal stem cells. The purpose of this study was to detect the differences of Nanog expression between PC3 prostate cancer cell line and its tumor stem cells, and the relationship was preliminarily examined between Nanog and prostate cancer and its tumor stem cells. By using magnetic active cell sorting (MACS), we isolated a population of CD44(+)/CD133(+) prostate cancer cells that display stem cell characteristics from PC3 cell line. Immunohistochemistry revealed positive expressions of CD44, CD133 and α(2)β(1)-integin in the isolated cells. CCK-8 analysis showed that isolated cells had a strong proliferative ability. The formation of the cell spheres in serum-free medium and holoclones in serum-supplied medium showed that the cells were capable of self-renewing, indicating that the isolated cells were a population of cancer stem-like cells derived from PC3 cell line. Western blotting exhibited that the isolated cells had higher experession of Nanog, an embryonic stem marker, as compared with PC3 cells. Our study showed that Nanog might be helpful in sustaining the self-renewal and the undifferentiation of prostate cancer stem cells, and may serve as a marker for prostate cancer stem cells for isolation and identification.

Small molecule antibody targeting HLA class I inhibits myeloma cancer stem cells by repressing pluripotency-associated transcription factors.

Cancer stem cells have been proposed to be responsible for tumorigenesis and recurrence in various neoplastic diseases, including multiple myeloma (MM). We have previously reported that MM cells specifically express HLA class I at high levels and that single-chain Fv diabody against this molecule markedly induces MM cell death. Here we investigated the effect of a new diabody (C3B3) on cancer stem cell-like side population (SP) cells. SP fraction of MM cells highly expressed ABCG2 and exhibited resistance to chemotherapeutic agents; however, C3B3 induced cytotoxicity in both SP cells and main population (MP) cells to a similar extent. Moreover, C3B3 suppressed colony formation and tumorigenesis of SP cells in vitro and in vivo. Crosslinking of HLA class I by C3B3 mediated disruption of lipid rafts and actin aggregation, which led to inhibition of gene expression of β-catenin and pluripotency-associated transcription factors such as Sox2, Oct3/4 and Nanog. Conversely, knockdown of Sox2 and Oct3/4 mRNA reduced the proportion of SP cells, suggesting that these factors are essential in maintenance of SP fraction in MM cells. Thus, our findings reveal that immunotherapeutic approach by engineered antibodies can overcome drug resistance, and provide a new basis for development of cancer stem cell-targeted therapy.

P2-01-18: Cancer Stem-Like Cells in Breast Cancer Progression.

Abstract Background: increasing evidences support the presence of cancer stem-like cells (CSLCs) within breast carcinomas. A subset of cells enriched for CSLCs within the tumor can be identified by flow cytometry. It has been supposed that CSLC component is responsible for tumor resistance and metastasis. However, the analysis of such population has never been evaluated during breast cancer progression. Patients and Methods: We prospectively included 98 women with histologically proven breast carcinomas to assess the presence of CSLCs within the primary tumor. According to clinical stage (CS), five patients were CS 0 (palpable DCIS), 16 CS I, 20 CS II and negative lymph nodes (IIA-), 20 CS II and positive lymph nodes (IIA+), 6 IIIa, 17 IIIb, 1IIIc and 2 CS IV. Additionally, ten lymph node metastases from IIA+ patients were analyzed. Invasive ductal carcinoma (IDC) was observed in 79 patients. We excluded from the analysis patients with non ductal histology (n= 11). The percentage of CD44+/CD24- (n= 79), ALDH1+ (ALDEFLUOR®) (n= 29), ABCG2+ (n= 44) and CXCR4+ (n= 46) cells within ESA+ cells was determined by flow cytometry in fresh sampled tumors. The mammosphere assay was studied in 14 samples. The relationship between cytometry analyses and clinical and pathological features was analyzed. Results: The median prevalence of CD44+/CD24- cells within ESA+ population was 1.63% in stage 0, 2.39% in stage I, 0.42% in stage IIA-, 8.15% in stage IIA+, 0.52% in stage III (p=0.005). The CD44+/CD24- cell population was analyzed in 10 lymph node metastasis and, the median prevalence of this population within ESA+ cells was 4.7%. There was no variation of median prevalence of ALDH1+, CXCR4+, ABCG2+ and the median number of spheres among clinical stages (p=0.1, p=0.8, p=0.8, p=0.8, respectively). We didn't observe any association among the expression of, ABCG2+, CXCR4+ with histological grade and the expression of ER and PgR. The median percentage of CD44+/CD24- cells was higher (2.39% vs 0.41%) in ER positive tumors (p=0.003). The median percentage of ESA+/ CXCR4+ cells was higher (42,5% vs 9,7%) in HER2− tumors. There was no correlation between the studied markers and the patients’ age and clinical tumor diameter. The results are summarized in Table 1. Results of association between expression cells surface markers and ALDH1 activity (Aldefluor®) with clinical and pathological features in invasive ductal carcinomas. Discussion: We found a significant variation in CSLC population during breast cancer progression. Our data show a gradual increase in CD44+/CD24- cells within ESA+ population from stage 0 to stage IIA+ and a significant decrease in stage III tumors. This observation supports the hypothesis that a population of CSLCs could be associated with breast cancer progression. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-18.

Abstract PL05-04: Epigenetic marks as biomarkers and their mechanisms of maintenance as targets in ovarian cancer.

Abstract PL05-04: Epigenetic marks as biomarkers and their mechanisms of maintenance as targets in ovarian cancer.

Liposarcoma cells with aldefluor and CD133 activity have a cancer stem cell potential

Aldehyde dehydrogenase (ALDH) has recently been shown to be a marker of cancer stem-like cells (CSCs) across tumour types. The primary goals of this study were to investigate whether ALDH is expressed in liposarcomas, and whether CSCs can be identified in the ALDHhigh subpopulation. We have demonstrated that ALDH is indeed expressed in 10 out of 10 liposarcoma patient samples. Using a liposarcoma xenograft model, we have identified a small population of cells with an inducible stem cell potential, expressing both ALDH and CD133 following culturing in stem cell medium. This potential CSC population, which makes up for 0, 1-1, 7% of the cells, displayed increased self-renewing abilities and increased tumourigenicity, giving tumours in vivo from as few as 100 injected cells.

Endosialin: A novel malignant cell therapeutic target for neuroblastoma

Endosialin emerged recently as a potential therapeutic target for sarcoma. Since some sarcoma subtypes, such as Ewing's sarcoma, show characteristics of neuroendocrine differentiation, we wondered whether cancers with neuro-endocrine properties and/or neuroectodermal origin, such as neuroblastoma, small cell lung cancer and melanoma, may express endosialin. Endosialin protein expression was surveyed in neuroblastoma, small cell lung cancer and melanoma in human clinical specimens by immunohistochemistry (IHC) and in human cell lines by flow cytometry. Side population cells were examined to determine whether cancer stem cells can express endosialin. Endosialin-expressing neuroblastoma cell lines were implanted in immunodeficient mice and allowed to grow. The xenograft tumors were resected and tested for endosialin expression by IHC. In human clinical specimens, vascular endosialin staining was observed in neuroblastoma, small cell lung cancer and melanoma. Malignant cell staining was strongest in neuroblastoma, weak in melanoma and rare in small cell lung cancer. In human cell lines, endosialin was detected in neuroblastoma cell lines, including cancer stem cell-like side population (SP) cells, but was absent in melanoma and was both rare and weak in small cell lung cancer. Human neuroblastoma xenograft tumors were found to be positive for endosialin. Our work suggests that endosialin may be a suitable therapeutic target for neuroblastoma.

Correction: Chemopreventive Effect of PSP Through Targeting of Prostate Cancer Stem Cell-Like Population

There are multiple errors in the legends of Figure 1 and Figure 2. The correct legends are: Figure 1. PSP down-regulates prostate CSC markers in PC-3 cells. (A) Western blotting of prostate CSC markers CD44 and CD133 in PC-3 cells after PSP treatment. Note that PSP significantly down-regulates both stem cell markers in a dose- and time-dependent manner. (B) Viability of PC-3 cells after treatment with 5, 25, 125, 250, and 500 μg/ml of PSP for 48 or 72 hr was measured with MTT assay. Results are presented as mean ± s.d. (C) Flow cytometry analysis of PC-3 cells after treatment with 250 μg/ml of PSP for 72 hr. Note that no significant difference in cell cycle distribution was observed. (D and E) Western blotting results for stem cell maintenance proteins and apoptotic markers in PC-3 cells after PSP treatment. Note that no changes in Bax and Bcl-2 or cleavage of PARP were detected. Figure 2. Effects of PSP on CSC properties. (A) Spheroid formation assay was performed with PC-3 and Du145 cells. Two hundred of cells were seeded onto polyHEMA pre-coated plates and treated with either 500 mg/mL of PSP or vehicle for 14 days. The number of prostaspheres formed was counted, and the result was presented as the mean ± s.d. Note that PSP treatment efficiently suppresses the spheroid formation ability of PC-3 cells. Image of the prostaspheres was captured under microscope. Note that no prostaspheres can be found in cells treated with 500 mg/mL of PSP. (B) PSP inhibited the formation of secondary prostaspheres. Primary prostaspheres were dissociated and re-seeded into polyHEMA pre-coated plate. PSP was added 24 hr after the plating. Note that prostasphere formation was inhibited by more than 70% and 90% in the presence of 250 mg/mL and 500 mg/mL of PSP respectively . *P<0.001, **P<0.0001, t test.