Side Population Fraction Research Articles

MicroRNA expression profiling identifies miR-328 regulates cancer stem cell-like SP cells in colorectal cancer

Background:Side population (SP) cells and their relationship to stem cell-like properties have been insufficiently studied in colorectal cancer (CRC). MicroRNAs (miRNAs) have attracted much attention but their roles in the maintenance of SP phenotype remain unclear.Methods:The SPs from CRC cell lines and primary cell cultures were analysed for stem cell-like properties. MiRNA microarray analysis identified miR-328 as a potential stemness miRNA of SP phenotype. The level of miR-328 expression in clinical samples and its correlation with SP fraction were determined. Gain-of-function and loss-of-function studies were performed to examine its roles in cancer stem-like SP cells. Furthermore, bioinformatics prediction and experimental validation were used to identify miR-328 target genes.Results:The SP cells sorted from CRC possess cancer stem cell (CSC)-like properties, including self-renewal, differentiation, resistance to chemotherapy, invasive and strong tumour formation ability. MiR-328 expression was significantly reduced in SP cells compared with Non-SP cells (P<0.05). Moreover, miR-328 expression was downregulated in CRC (n=33, P<0.05) and low miR-328 expression tend to correlate with high SP fraction (n=15, r=0.6559, P<0.05, Pearson's correlation). Functional studies indicated that miR-328 expression affects the number of SP cells. In addition, miR-328 overexpression reversed drug resistance and inhibited cell invasion of SP cells. Furthermore, luciferase reporter assay demonstrated that miR-328 directly targets ABCG2 and MMP16 and affects the levels of mRNA and protein expression in SP cells.Conclusion:These findings indicate that CRC contain cancer stem-like SP cells. MiR-328 has an important role in maintaining cancer stem-like SP phenotype that may be a potential target for effective CRC therapy.

Pathobiological Implications of the Expression of EGFR, pAkt, NF-κB and MIC-1 in Prostate Cancer Stem Cells and Their Progenies

The progression of prostate cancers (PCs) to locally invasive, androgen-independent and metastatic disease states is generally associated with treatment resistance and disease relapse. The present study was undertaken to establish the possibility of using a combination of specific oncogenic products, including epidermal growth factor receptor (EGFR), pAkt, nuclear factor-kappaB (NF-κB) and macrophage inhibitory cytokine-1 (MIC-1) as biomarkers and therapeutic targets for optimizing the management of patients with localized PC at earlier disease stages. The immunohistochemical and immunofluorescence data have revealed that the expression levels of EGFR, Ser473-pAkt, NF-κB p65 and MIC-1 proteins were significantly enhanced in the same subset of 76 cases of prostatic adenocarcinoma specimens during the disease progression and these biomarkers were expressed in a small subpopulation of CD133+ PC cells and the bulk tumor mass of CD133− PC cells. Importantly, all of these biomarkers were also overexpressed in 80–100% of 30 PC metastasis bone tissue specimens. Moreover, the results have indicated that the EGF-EGFR signaling pathway can provide critical functions for the self-renewal of side population (SP) cells endowed with stem cell-like features from highly invasive WPE1-NB26 cells. Of therapeutic interest, the targeting of EGFR, pAkt, NF-κB or MIC-1 was also effective at suppressing the basal and EGF-promoted prostasphere formation by SP WPE1-NB26 cells, inducing disintegration of SP cell-derived prostaspheres and decreasing the viability of SP and non-SP WPE1-NB26 cell fractions. Also, the targeting of these oncogenic products induced the caspase-dependent apoptosis in chemoresistant SP WPE1-NB26 cells and enhanced their sensibility to the cytotoxic effects induced by docetaxel. These findings suggest that the combined use of EGFR, pAkt, NF-κB and/or MIC-1 may represent promising strategies for improving the accuracy of current diagnostic and prognostic methods and efficacy of treatments of PC patients in considering the disease heterogeneity, thereby preventing PC progression to metastatic and lethal disease states.

Skeletal Muscle-derived Hematopoietic Stem Cells: Muscular Dystrophy Therapy by Bone Marrow Transplantation

For postnatal growth and regeneration of skeletal muscle, satellite cells, a self-renewing pool of muscle stem cells, give rise to daughter myogenic precursor cells that contribute to the formation of new muscle fibers. In addition to this key myogenic cell class, adult skeletal muscle also contains hematopoietic stem cell and progenitor cell populations which can be purified as a side population (SP) fraction or as a hematopoietic marker CD45-positive cell population. These muscle-derived hematopoietic stem/progenitor cell populations are surprisingly capable of differentiation into hematopoietic cells both after transplantation into irradiated mice and during in vitro colony formation assay. Therefore, these muscle-derived hematopoietic stem/progenitor cells appear to have characteristics similar to classical hematopoietic stem/progenitor cells found in bone marrow. This review outlines recent findings regarding hematopoietic stem/progenitor cell populations residing in adult skeletal muscle and discusses their myogenic potential along with their role in the stem cell niche and related cell therapies for approaching treatment of Duchenne muscular dystrophy.

&lt;i&gt;In Vitro&lt;/i&gt; Isolation of Intestinal Side Population Cells from Newborn Mice

To isolate side population cells from newborn mice small intestinal mucosa, and to investigate the feasibility of constructing the murine intestinal stem cell population by fluorescence activated cell sorting. Small intestine mucosa organoids of mice were isolated and dissociated into single cells. The side population cells were stained with Hoechst 33342 and propidium iodide, then sorted using fluorescence activated cell sorting. Total RNA and protein were purified from sorted fractions to detect Musashi-1 expressions by RT-PCR and Western-blotting. Single cell suspension from mouse small intestine mucosa contained a viable population of cells, which showed the side population phenotype and were sensitive to verapamil. These cells were enriched for Musashi-1 mRNA and MSI-1 protein expression. Results demonstrated that the side population fraction separated from mice intestinal mucosa is enriched for intestinal stem cells, the murine intestinal stem cell population can be successfully constructed with fluorescence activated cell sorting.

Differentiation Therapy: Targeting Human Renal Cancer Stem Cells with Interleukin 15

Many renal cancer patients experience disease recurrence after immunotherapy or combined treatments due to persistence of cancer stem cells (CSCs). The identification of reliable inducers of CSC differentiation may facilitate the development of efficient strategies for eliminating CSCs. We investigated whether interleukin 15 (IL-15), a regulator of kidney homeostasis, induces the differentiation of CD105-positive (CD105(+)) CSCs from human renal cancers. CD105(+) CSCs were cultured to preserve their stem cell properties and treated with recombinant human IL-15 (rhIL-15) to evaluate their ability to differentiate, to acquire sensitivity to chemotherapeutic drugs, and to form spheroids in vitro and tumors in vivo. Expression of stem cell and epithelial markers were studied by flow cytometry, immunocytochemistry, and immunoblotting. Identification of a CSC side population fraction and its sensitivity to chemotherapy drugs and expression of ATP-binding cassette (ABC) transporters and aldehyde dehydrogenase (ALDH) activities were determined by flow cytometry. Spheroid formation was determined in limiting dilution assay. Xenograft tumors were generated in severe combined immunodeficient mice (n = 12-18 mice per group). All statistical tests were two-sided. CD105(+) CSCs treated with rhIL-15 at 10 pg/mL differentiated into cells expressing epithelial markers. rhIL-15 induced epithelial differentiation of all CD105(+) CSCs subsets and blocked CSC self-renewal (sphere-forming ability) and their tumorigenic properties in severe combined immunodeficient mice. Vinblastine and paclitaxel induced statistically significant higher levels of apoptosis in rhIL-15-differentiated epithelial cells compared with CD105(+) CSCs (mean percentage of apoptotic cells, vinblastine: 33% vs 16.5%, difference = 16.5%, 95% confidence interval = 12.25% to 20.74%, P = .0025; paclitaxel: 35% vs 11.6%, difference = 23.4%, 95% confidence interval = 22.5% to 24.24%, P = .0015). The higher sensitivity of rhIL-15-differentiated epithelial cells to chemotherapeutic drugs was associated with loss of detoxifying mechanisms such as ALDH and ABC transporter activities. IL-15 directs the epithelial differentiation of renal CSCs and meets the criteria for a treatment strategy: CSC pool depletion and generation of differentiated nontumorigenic cells that are sensitive to chemotherapeutic agents.

Abcg2 labels multiple cell types in skeletal muscle and participates in muscle regeneration

Skeletal muscle contains progenitor cells (satellite cells) that maintain and repair muscle. It also contains muscle side population (SP) cells, which express Abcg2 and may participate in muscle regeneration or may represent a source of satellite cell replenishment. In Abcg2-null mice, the SP fraction is lost in skeletal muscle, although the significance of this loss was previously unknown. We show that cells expressing Abcg2 increased upon injury and that muscle regeneration was impaired in Abcg2-null mice, resulting in fewer centrally nucleated myofibers, reduced myofiber size, and fewer satellite cells. Additionally, using genetic lineage tracing, we demonstrate that the progeny of Abcg2-expressing cells contributed to multiple cell types within the muscle interstitium, primarily endothelial cells. After injury, Abcg2 progeny made a minor contribution to regenerated myofibers. Furthermore, Abcg2-labeled cells increased significantly upon injury and appeared to traffic to muscle from peripheral blood. Together, these data suggest an important role for Abcg2 in positively regulating skeletal muscle regeneration.

Side population cells in highly enriched CD34-positive cells from peripheral blood progenitor cells identify an immature subtype of hematopoietic progenitor cells but do not predict time to engraftment in patients treated with high-dose therapy

A Hoechst 33342 dye efflux assay can be used to define a population of immature hematopoietic progenitor cells (HPC) that are called side population (SP) cells. Previously, SP cells examined from bone marrow (BM) and peripheral blood progenitor cells (PBPC) were found to be predominantly CD34 negative. In this study, we show that the level of CD34+ cells within the SP fraction increases from 2% in BM to 15% in mobilized PBPC. Furthermore, SP cells are found in highly enriched CD34+ cells from both BM and PBPC, and these cells define an immature phenotype of HPC. We also observed a higher level of CD133+ cells within the SPCD34+ cell population. Moreover, the frequency of long-term culture-initiating cells (LTC-IC) was markedly increased in SPCD34+ cells. To further investigate whether variations in the level of SP cells in the CD34+ cell fraction influenced short-term engraftment, we studied 20 patients with Hodgkin lymphoma that were autotransplanted with highly enriched CD34+ cells from PB. The percentage of SP cells in the PBCD34+ cell fraction was highly variable, ranging from 0.3 to 22%. No correlation was found between the content of SP cells in the autotransplanted CD34+ cells and time to short-term engraftment. SPCD34+ cells in PBPC define an immature phenotype of HPC with increased numbers of LTC-IC, and they are more frequently found in PBPC than in BM. The number of SP cells does not predict time to engraftment.

Identification of Endothelin‐1 and NR4A2 as CD133‐regulated genes in colon cancer cells

Several in vitro assays have been proposed to identify cancer stem cells (CSCs), including immunophenotyping, sphere assay and side population (SP) assay. CD133 antigen has been proposed as a CSC marker in colon cancer (CC). However, no functional data are available to date and conflicting results have been reported regarding its role as true CSC marker. Here we set out to identify a molecular signature associated with potential CSC. CD133(+) cells isolated from the CaCo-2 CC cell line were analysed by microarray molecular profiling compared to CD133(-) counterparts. Various differentially expressed genes were identified and the most relevant transcripts found to be over-expressed in CD133(+) cells were evaluated by quantitative RT-PCR in the CD133(+) fractions isolated from several CC cell lines. In the attempt to find a correlation between putative CSCs, isolated by means of CD133 immunophenotyping and the SP approach, we demonstrated a significant enrichment of CD133(+) cells within the SP fraction of CC cells, and comparison of the gene expression profiles revealed that Endothelin-1 (END-1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts are highly expressed in both CD133(+) and SP fractions of CC cells. Moreover, depletion of CD133 by siRNA induced a significant attenuation of END-1 and NR4A2 expression levels in CaCo-2 cells, while expression of all three molecules decreased during sodium butyrate-induced differentiation. In conclusion, we have identified a molecular signature associated with potential CSCs and showed for the first time the existence of a functional relationship between CD133, END-1 and NR4A2 expression in colon cancer cells.

Side population cells isolated from human osteosarcoma are enriched with tumor‐initiating cells

It has been proven that "side population (SP)" cells that exclude Hoechst 33342 dye are enriched with cancer stem cells in several tumors. In the present study we aimed to isolate and characterize SP cells from human primary osteosarcoma. Side population cells were detected in osteosarcoma samples. In vitro, SP cells regenerated both SP and non-SP and the clonogenicity of SP cells was higher than that of non-SP cells, just like stem cells. In vivo, SP cells exhibited heightened tumorigenicity and only the SP fraction had the capacity to self- renew both in vitro and in vivo. Furthermore, SP cells exhibited increased multidrug resistance and the RNA expression of ATP-binding cassette protein transporters was increased in the SP group. In addition, "stemness" genes Oct-4 and Nanog were also upregulated in the SP group. However, the expression of other putative stem cell markers (CD44, CD117 and CD133) had no significant difference between SP and non-SP for each individual marker. These findings suggest that SP cells derived from osteosarcoma are enriched with tumorigenic cells with stem-like properties and might be an ideal target for clinical therapy.

Human hepatic cancer stem cells are characterized by common stemness traits and diverse oncogenic pathways

Epigenetic mechanisms play critical roles in stem cell biology by maintaining pluripotency of stem cells and promoting differentiation of more mature derivatives. If similar mechanisms are relevant for the cancer stem cell (CSC) model, then epigenetic modulation might enrich the CSC population, thereby facilitating CSC isolation and rigorous evaluation. To test this hypothesis, primary human cancer cells and liver cancer cell lines were treated with zebularine (ZEB), a potent DNA methyltransferase-1 inhibitor, and putative CSCs were isolated using the side population (SP) approach. The CSC properties of ZEB-treated and untreated subpopulations were tested using standard in vitro and in vivo assays. Whole transcriptome profiling of isolated CSCs was performed to generate CSC signatures. Clinical relevance of the CSC signatures was evaluated in diverse primary human cancers. Epigenetic modulation increased frequency of cells with CSC properties in the SP fraction isolated from human cancer cells as judged by self-renewal, superior tumor-initiating capacity in serial transplantations, and direct cell tracking experiments. Integrative transcriptome analysis revealed common traits enriched for stemness-associated genes, although each individual CSC gene expression signature exhibited activation of different oncogenic pathways (e.g., EGFR, SRC, and MYC). The common CSC signature was associated with malignant progression, which is enriched in poorly differentiated tumors, and was highly predictive of prognosis in liver and other cancers. Epigenetic modulation may provide a tool for prospective isolation and in-depth analysis of CSC. The liver CSC gene signatures are defined by a pernicious interaction of unique oncogene-specific and common stemness traits. These data should facilitate the identifications of therapeutic tools targeting both unique and common features of CSCs.

Pleural mesothelioma side populations have a precursor phenotype

DyeCycleViolet was used to set up the side population (SP) functional assay aimed at identifying subpopulations of malignant pleural mesothelioma (MPM) tumor cells with chemoresistance phenotype associated with ABCG2 transporter activity. Self-renewal, chemoresistance and tumorigenicity were tested for SP and non-side population (NSP) cells. Tumors were characterized by mesothelin, calretinin, N-cadherin, D2-40 and Wilms tumor 1 (WT1) immunohistochemistry. Surface expression of mesenchymal stem cell markers CD90, CD73 and CD105 was investigated in SP and NSP cells. We identified SP cells with self-renewal properties and increased chemoresistance in MPM cell lines and tumor-derived primary cell cultures. Compared with the non-SP fraction (NSP), the SP fraction led to the development of tumors including cells with mesothelium precursor phenotype characterized by mesenchymal morphology, being WT1 negative but cytoplasmic D2-40 positive and having a tendency of increased tumorigenicity. The same phenotypic shift was observed in patients with relapsing tumors after chemotherapy. Furthermore, the SP cells were enriched in CD105(-)(/low) expressing cells, which were small sized and had increased tumorigenicity compared with CD105(high) cells. Taken together, our results support the hypothesis that MPM CD105(-)(/low), chemoresistant small sized SP cells may constitute the cellular pool out of which recurrence develops. Further characterization of mechanisms of chemoresistance and self-renewal should lead to targets specific for this subpopulation in MPM patients.

Lenalidomide targets clonogenic side population in multiple myeloma: pathophysiologic and clinical implications

AbstractRecurrence of multiple myeloma (MM) after therapy suggests the presence of tumor-initiating subpopulations. In our study, we performed flow cytometry–based Hoechst 33342 staining to evaluate the existence of a MM population with stem-like features known as side population (SP) cells. SP cells exhibit substantial heterogeneity in MM cell lines and primary MM cells; express CD138 antigen in MM cell lines; display higher mRNA expression and functional activity of ABCG2 transporter; and have a higher proliferation index compared with non-SP cells. We observed evidence for clonogenic potential of SP cells, as well as the ability of SP cells to regenerate original population. Moreover, SP cells revealed higher tumorigenicity compared with non-SP cells. Importantly, lenalidomide decreased the percentage and clonogenicity of SP cells, and also induced phosphorylation changes in Akt, GSK-3α/β, MEK1, c-Jun, p53, and p70S6K in SP cells. Adherence to bone marrow stromal cells (BMSCs) increased the percentage, viability, and proliferation potential of SP cells. Lenalidomide and thalidomide abrogated this stimulatory effect of BMSCs and significantly decreased the percentage of SP cells. Our studies demonstrate a novel mechanism of action for lenalidomide, namely targeting SP fraction, providing the framework for new therapeutic strategies targeting subpopulations of MM cells including presumptive stem cells.

Abstract 494: Stemness characteristics contribute to metastasis and shorter patient survival in nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) has the highest incidence of metastasis among head and neck cancers. The involvement of cancer stem cells (CSCs) in tumorigenesis has been studied in several malignancies, but their presence in NPC has not been extensively evaluated. The range of general characteristics of “stemness” includes increased side-population (SP) fraction, limited functional differentiation or dedifferentiation under the influence of the microenvironment, and expression of putative stem cell markers. We previously isolated a highly metastatic clone (Clone-18) from the NPC cell line CNE-2. Using this clone, we have sought to explore the influence of the stemness characteristics of NPC cells on tumor metastasis. Human tissue samples were collected under informed consent at the Sun Yat-sen University Cancer Center. SP cells were evaluated by flow cytometry. A Transwell assay and a wound-healing assay were used to evaluate the migratory and invasive abilities of the cells. Lung metastasis was evaluated by injecting tumor cells into the tail vein of BALB/c nude mice. Real-time PCR and western blotting were used for a protein kinase C (PKC) pathway study. Tissue microarrays and immunohistochemical staining were used to evaluate the correlation between vimentin and the survival time of patients with nasopharyngeal carcinoma. Clone-18 cells possessed a very high percentage of SP cells (62.1 ± 1.39%) relative to low-metastasis clones (Clone-22, 7.2 ± 1%; Clone-26, 3.9 ± 0.9%) or the parental CNE-2 cells (27.9 ± 2.02%), implying that there were more stem-like cells in the high-metastasis clones. Two stem cell markers, WNT5A and vimentin, were also highly expressed in the high-metastasis clone cells. To confirm the clinical relevance of these findings, human tissue samples were used. The mRNA level for WNT5A was significantly higher in regional metastatic NPC tissues (n = 4) than in primary tumors (n = 20), and it was much higher in liver metastatic NPC (n = 5) than in regional metastatic tissues. In the cohort of 282 NPC patients, a higher vimentin level in the primary tumor, as detected by IHC staining, correlated with shorter overall survival. Suppression of WNT5A by siRNA significantly reduced the migratory and invasive abilities of Clone-18 cells, and this effect was shown to be regulated by activating protein kinase C (PKC). The activated PKC further regulated the expressions of Snail and E-cadherin, which subsequently induced an EMT in NPC cells. In vivo experiments showed that the administration of recombinant WNT5A protein could promote lung metastasis by the low-metastasis Clone-26 cells in the lung metastasis mouse model. In conclusion, the stemness characteristics of NPC cells have been shown to contribute to the migratory, invasive, and metastatic phenotype of this important malignancy, with WNT5A directly regulating these properties and being a critical candidate marker of CSCs. 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 494. doi:10.1158/1538-7445.AM2011-494

Abstract 5049: Cyclopamine modulates ABCG2 activity in glioblastoma side population cells

Abstract The Sonic Hedgehog pathway (HH) has been implicated in the maintenance of stem cells in adult tissues. Abnormal HH activation is also associated with initiation of neoplasia by cancer stem cells. Cyclopamine has been used as an inhibitor of the HH, with potential effectiveness in several xenograft models, including gliomas. Side Population (SP) cells are stem cells with expression of ABCG2, a multidrug ABC transporter, able to extrude dyes as Hoechst 33342 (Ho342). SP cells, then, present low fluorescence after staining with Ho342. ABC transporters have been identified as drug-resistance proteins. HH regulates the expression of ABC transporters, and promotes multi-drug resistance by increasing drug efflux by the ABC transporters. Different research groups have shown that SP cells can be depleted following cyclopamine treatment, an observation that would support that SP cells are HH pathway-dependent. We hypothesized that cyclopamine might interact with ABCG2 and limit the number of SP cells. Therefore, we analyzed the effect of cyclopamine and temozolomide on the MXRA cell line (KB cells transfected with the full-length ABCG2 cDNA), and on five astrocytoma cell lines, both in the parental cell lines and in their SP cells. We first evaluated the modulatory effect of cyclopamine on ABCG2 function by measuring the intracellular accumulation of two substrates of ABCG2, Ho342 and mitoxantrone. Secondly, we analyzed the effect of these drugs in the phenotype of SP cells (the cells were incubated with only Ho342, or a combination of Ho342 and cyclopamine, or a combination of Ho342, cyclopamine and temozolomide; afterwards we analyzed the number of SP cells). We saw that cyclopamine significantly decreased the fraction of SP cells but not through a depletion of the SP compartment, but as a consequence of the molecular mechanisms involved in the affinity of cyclopamine and Ho342 for ABCG2, as cyclopamine modulated the ABCG2 transporter by increasing Ho342 and mitoxantrone uptake. Temozolomide had no effect on the SP cells. Therefore, cyclopamine, although decreases the SP fraction of cancer stem cells in glioblastoma cell lines, does not deplete the SP compartment, but modulates ABCG2 activity. 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 5049. doi:10.1158/1538-7445.AM2011-5049

Abstract 5200: Effect of cancer-associated fibroblast on the progression of gastric cancer stem cells

Abstract Introduction: Cancer stem cells (CSCs) are thought to possess the ability to initiate tumor growth and sustain self-renewal, which explains the tumorigenicity and the maintenance of bulk. There is an increasing evidence of the importance of the host microenvironment in conditioning the CSCs status, such as the stemness and the tumor initiation. Recently, cancer-associated fibroblasts in the cancer microenvironment have been suggested to be associated with the tumor progression. Then, the aim of this study is to clarify the relation between CSCs and cancer-associated fibroblasts. Material and Methods: A gastric cancer cell line, OCUM-12, and cancer-associated gastric fibroblasts, CaF-37, were used. Side population (SP) cells, known as CSCs rich population, were isolated from OCUM-12 cells by flowcytometry using Hoechst 33342, and were named as OCUM-12/SP. OCUM-12/SP cells or OCUM-12 cells were cultured with or without CaF-37 fibroblasts. The percentage of SP cells was evaluated by flowcytometry, and the growth of cancer cells was examined by cell number count. In vivo tumorigenicity of OCUM-12/SP cells was evaluated by subcutaneous inoculation in the presence or absence of CaF-37 fibroblasts. Results: The percentage of SP fraction of OCUM-12/SP cells was increased in the presence of CaF-37 fibroblasts. In contrast, CaF-37 fibroblasts significantly decreased the proliferation of OCUM-12/SP cells, while that of OCUM-12 cells was significantly increased by CaF-37 cells. The inoculation of 3×105, 1×105, and 3×104 OCUM-12/SP cells with 1×106 CaF-37 fibroblasts resulted in tumor formation in 5 (100%) of 5 mice, 4 (80%) of mice, 5 (100%) of 5 mice, respectively. However, the inoculation of 3×105, 1×105, and 3×104 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.Conclusion: Cancer-associated fibroblast might sustain the CSCs phenotype of stemness, and might increase the tumor initiation. 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 5200. doi:10.1158/1538-7445.AM2011-5200

Abstract 3411: Analysis of gene profile and identification of molecular targets in endometrial cancer stem cells

Abstract Background; Cancer stem cells (CSCs) have been known to be the major source of tumor propagation and might be an attractive therapeutic target. Our previous study showed that CD133+ Ishikawa and MFE 280 endometrial cancer cells have the ability of tumorigenicity, self-renewal and differentiation capacity in vitro and in vivo, while CD133- cells did not. Furthermore, CD133+ cells showed more resistance to cisplatin- or paclitaxel-induced cytotoxicity than CD133- cells. These results indicate that CD133 is the CSC maker in endometrial cancer cells. In the present study, we sought to analyze gene profile and identify molecular targets in endometrial cancer stem cells. Methods; We examined microarray analysis to investigate the molecules essential for the CSCs characteristics by 3D-Gene chip (TORAY). Results; Microarray analysis of total 25,000 genes showed that about 500 genes were highly expressed in CD133+ cells, compared to CD133- cells. The ALDH1 gene was up-regulated in CD133+ cells, in accordance with the previous reported expression of ALDH1 on breast and colorectal CSCs. We also found out the elevated expression of ABC transporter genes, ABCG2, ABCG3, ABCG4, ABCG8, ABCC8 and ABCC9. It is well known that cells expressing those genes are resistant to chemotherapeutic agents and associated with side population (SP). Fluorescence activated cell sorter analysis demonstrated that the ratio of SP fraction in CD133+ Ishikawa cells is slightly higher than in CD133- Ishikawa cells (0.8% vs 0.6%). Isolated SP/CD133+ cells ells showed more extensive proliferation and tumorigenicity, compared with non-SP/CD133+, SP/CD133- and non-SP/CD133- cells. Finally, we focused on high expression of MMP14 (MT1-MMP) gene in CD133+ cells. Invasion assay revealed that CD133+ Ishikawa and MFE 280 cells have more invasive property than CD133- Ishikawa and MFE 280 cells. The inhibition of MT1-MMP gene by siRNA significantly reduced the invasive ability of CD133+ cells. These results suggest that MT1-MMP is one of the critical molecules that generate biological features of CSC and may be a potential therapeutic target in endometrial cancer. Conclusions; We demonstrated that SP/CD133+ cells might be more defined CSCs in endometrial cancer and identified important molecules essential for characterizing endometrial CSCs in which MT1-MMP plays critical roles in generating biological features of CSCs in this tumor type. These findings shed light on establishment of molecular target therapy in endometrial cancer. 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 3411. doi:10.1158/1538-7445.AM2011-3411

Combination of CD34/CD38 Immunophenotypes and Side Population (SP) Reveals the Putative Leukemia Stem Cell/Leukemia Initiating Cell In Acute Myeloid Leukemia.

AbstractAbstract 1582Although the originally defined CD34+CD38- leukemia stem cell (LSC)/leukemia initiating cell (LIC) in acute myeloid leukemia (AML) still serves as a lead in studies on the characterization of LSC/LIC, in more immuno-compromised mouse models, other stem cell immunophenotypes, i.e. CD34-CD38- and CD34-CD38+ turn out to have LSC/LIC activity (Taussig et al, Blood 2010; 115: 1976). Two functional LSC phenotypes offer the ability not only to restrict the LSC/LIC compartment to a lower frequency compartment, but also to include such CD34- negative immunophenotypes. These concern high activity of aldehyde dehydrogenase (ALDH) or high efflux of Hoechst 44432 (resulting in side population, SP). The present study deals with the possible overlap between CD34/CD38 and SP defined stem cell compartments. Based on our previous results (van der Pol, Haematologica 2003; 88: 983), we defined truly CD34 negative AML as AML with a very small CD34+ population only (usually≤1%), with particular scatter properties, and proven to be of normal origin. Using expression of cell surface markers present on LSC/LIC, but absent on HSC (van Rhenen et al, Leukemia 2007; 21: 1700; Blood 2007; 110: 2659), the LSC/LIC compartment in this type of leukemia (5 cases studied) was shown to be present in the CD34-CD38+ compartment, while the HSC was found in the CD34+CD38- compartment. When determining aberrant cell surface markers within the SP, the only malignant cells present in the SP population were CD34-CD38+. To assess the primitive character of the putative LSC/LIC sub-populations, cell sorting experiments were performed in which all aberrant marker positive SP cells along with marker positive non-SP cells (n=2) and all aberrant marker negative SP cells and marker negative non-SP cells (n=2), were assessed in the liquid culture stem cell assay. SP marker positive cells had 280- and 725-fold more stem cell activity compared to non-SP cells (14,500 versus 20 and 8,400 versus 30, respectively), while for marker negative cells only the SP fraction contained stem cell activity (8,000 and 8,200 colonies per million input cells). Even though the frequency of SP cells is far below that of non-SP cells, the absolute number of colonies was higher in SP than in non-SP cells. In CD34 positive leukemia, in contrast to CD34 negative AML, the SP fraction was largely filled with cells with all combinations of CD34 and CD38, which all fulfill the criteria (marker positivity) for malignancy. Liquid culture assay results were: for marker positive cells, colonies were formed only in the SP compartment in 2/3 cases studied (6,500 and 4,400 colonies per million input cells). In the remaining case colony formation was 195-fold higher in SP versus non-SP (4,100 versus 21 colonies per million input cells). For marker negative cells, in all three cases only the SP fraction produced colonies (5,300, 1,4500 and 90 per million input cells). Malignant character of marker positive cells was confirmed using FISH analysis, immediately after cell sorting prior to cell culturing, and at the end of the liquid culture on plucked colonies. These results show that both in CD34 negative and CD34 positive leukemia the SP fraction contains most primitive cells and has much higher clonogenic potency compared to non-SP cells. In CD34 negative AML, SP HSC are CD34+CD38- and SP LSC/LIC are CD34-CD38+. In CD34 positive AML, SP HSC are CD34+CD38- and SP LSC/LIC may be CD34+CD38-, but also CD34+CD38+, CD34-CD38+ and CD34-CD38-. The absence of CD34 negative engraftment, seen in earlier studies, likely results from relatively high immune competence of the NOD/SCID mice in those studies. The results suggest that CD34 negative leukemia may contain more immune sensitive LSC/LIC. Above that, CD34 negative AML is characterized by lower multidrug resistance (abstract Schuurhuis, Kelder et al, subm for this meeting). From these characteristics favorable outcome for the particular class of patients, defined as truly CD34 negative, can be predicted. This was indeed the case in our large study (394 patients): median overall survival not reached (>41 months) for CD34 negative patients (n=85) and 19 months for CD34 positive patients (n=309) (p=0.006). Our results warrant to consider CD34 negative patients as a completely different entity of AML patients, in terms of stem cell characteristics, which may evoke different therapeutic strategies compared to CD34 positive patients. Disclosures:No relevant conflicts of interest to declare.

Development of New Treatment Strategies Targeting Clonogenic and Tumorigenic Myeloma Side Population.

AbstractAbstract 1581 Introduction:Recurrence of multiple myeloma (MM) after establishment of complete remission indicates the presence of a small fraction of resistant tumor cells which persists after treatment and expresses stem cell-like characteristics.In our previous studies, we confirmed the existence of a MM population with “stem-like” features, known as side population (SP) cells. SP cells exhibit substantial heterogeneity not only in MM cell lines, but also in primary samples. Importantly, SP cells manifest significant clonogenic potential in vitro compared to the main population (MP). In our current study, we examined whether the clonogenicity of SP cells in vitro is associated with tumorigenic potential of SP cells in vivo. Methods and Results:To evaluate tumorigenicity of SP cells, several dilutions of sorted SP and MP cells were injected into immunocompromised mice. In CB17/SCID mice, sorted SP cells (range 105 - 106 cells/mouse) from OPM1 and KMS11 cell lines induced significantly greater tumor growth compared to MP cells (90-70% less tumor mass than SP tumors). At lower innocula of cells (10 × 103 cells/mouse), SP cells resulted in tumors, whereas no tumors were produced by MP cells. In NOD/SCID mice, lower numbers of SP cells (1 × 103 cells/mouse) produced tumor. These data strongly support the tumorigenic potential of SP sub-population and provide the rationale for targeting these cells in novel therapeutic strategies in MM.Indeed, our previous studies show that lenalidomide significantly decreased the percentage and clonogenicity of SP cells. We therefore expanded these studies to next examine whether other novel agents target SP cells. Novel agents pomalidomide (1 μM; decreased by 97%) and histone deacetylase (HDAC) inhibitor vorinostat (0.4 μM; decreased by 86%) significantly affected SP cells. Natural plant compound sulforaphane (2 μM; decreased by 86%), but not isothiocyanate PEITC, also decreased percentage of SP cells.We similarly evaluated the anti-SP effect of compounds known or proposed to target crucial signaling pathways in hematopoietic/cancer stem cells including cantharidin (terpenoid inhibitor of PPA2) and its demethylated analog norcantharin, deguelin (PI3K/Akt inhibitor), plumbagin (quinonoid substrate of ABCG2) and ionophore salinomycin. All these compounds significantly decreased or completely eliminated SP cells at low nontoxic doses in flow cytometry-based Hoechst 33342 staining assays. However, clonogenicity of SP cells treated by salinomycin was only slightly affected compared to control SP cells by clonogenic assay in vitro.In addition, Hedgehog pathway inhibitor cyclopamine (10 μM; decreased by 82%), as well as its analog tomatidine (10 μM; decreased by 64%), significantly decreased the percentage of SP cells, while sonic hedgehog agonist purmorphamine (0.8 μM; decreased by 17%) only modestly decreased the percentage of SP cells. To study these substantial inhibitory effects of tested compounds, the clonogenic assays were performed in vitro. Finally, the effect of drugs and compounds against SP cells was also evaluated in co-culture with bone marrow stromal cells, which confer growth and survival in MM. Importantly, the SP fraction was decreased by these agents, whereas no effect of hedgehog agonist and plumbagin was detected. Conclusions:Our studies confirmed the tumorigenic potential of SP cells isolated from established MM cell lines and identify the impact of targeted therapies against these cells, providing the rationale for further studies to evaluate the clinical potential of therapeutic targeting of SP cells in MM. Disclosures:Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

Preclinical Studies of Salinomycin In Multiple Myeloma (MM) Models: Targeting of Side Population (SP) Cells In the Context of Tumor – Microenvironment Interactions.

AbstractAbstract 1574Cancer cells with stem cell-like features are a topic of intense research because their resistance to existing drugs is considered a culprit for relapses, even in patients with complete remission defined by clinical, biochemical and imaging parameters or by sensitive molecular techniques. Salinomycin, an antibacterial and coccidiodostatic ionophore, is reported (Cell 2009;138(4):645-59) to be >100-fold more potent against breast cancer cells with stem cell-like phenotype after mesenchymal transdifferentiation due to stable transfection with shRNA against CDH1 than against the parental cells. We evaluated whether salinomycin could also exhibit a similar activity against stem cell-like cells in multiple myeloma (MM). To establish a comparative reference for such potential activity, we first tested salinomycin (0-10 uM for up to 72hrs) against a panel of 15 MM cell lines and observed IC50 values <1 uM in 10/15 cell lines tested, including >80% reduction of tumor cell viability in 6/15 cell lines tested at 0.5 uM, i.e. levels lower than the IC50 values for in vitro activity of salinomycin against breast cancer cells with (HMLE-shCDH1, IC50 ∼1 uM) or without (HMLE-shControl, IC50 >>10 uM) stem cell-like features. CD138+ purified primary tumor cells from 3 MM patients responded to salinomycin with IC50 values (105, 332 and 750 nM, respectively) in the same range as MM cell lines. In vitro combinations with bortezomib, doxorubicin, melphalan, and dexamethasone showed overall no antagonism, while evidence of additive or even synergistic effect could be identified in certain dose ranges. Because MM cell lines and primary tumor cells responded concordantly to salinomycin and with higher sensitivity than breast cancer stem cell-like cells, we hypothesized that MM cells may in general be more responsive to salinomycin than other tumors. Since tumor-stromal interactions can increase the expression of transcriptional signatures of “stemness” in MM cells, we embarked on characterizing the anti-MM properties of salinomycin using compartment-specific bioluminescence imaging (CSBLI) assays. These showed that co-culture with stromal cells did not confer resistance to salinomycin in 5 MM cell lines (MM.1S, OCI-My5, KMS-11, KMS-18, NCI-H929) and in fact enhanced its activity against 4 of them. Side population (SP) cells, defined by their ability to efflux Hoechst stain, represent a stem cell-like population which was identified in MM cell lines and could represent the functional equivalent of the mesenchymally transdifferentiated breast cancer stem cell-like cells. We observed that salinomycin reduces the SP fraction of MM cell lines at doses >20 times lower than those required for in vitro effect against the bulk <<main population>> of the respective cell lines. Interestingly, the anti-SP effect of salinomycin was more pronounced in the presence of stroma, similarly to the CSBLI studies on the entire MM cell population and consistent with our prior observation that tumor-stroma interaction enhances transcriptional signatures of ≪stemness≫ in the tumor compartment. However, when we tested the in vivo anti-MM activity of salinomycin in an orthotopic model of i.v. injected Luc+ MM cells, no anti-MM activity (in terms of tumor burden decrease or overall survival prolongation) was observed at the maximum tolerated dose (1 mg/kg i.p. daily, which is consistent with most studies reported thus far in the literature). Ex vivo treatment of KMS-11 cells with salinomycin doses (100 nM for 72 hrs) selectively targeting SP cells was followed by s.c. injection of these cells or vehicle-treated controls in sublethallly irradiated SCID/NOD mice, but no statistically significant improvement in tumor burden or overall survival was observed. Our in vitro results indicate that salinomycin exhibits intriguing in vitro anti-MM activity, not only against SP cells but also against the bulk ≪main≫ MM cell population, even in the presence of stromal support. In contrast, the in vivo activity of salinomycin is compromised by side effects in the orthotopic model of MM lesions, while short term ex vivo exposure of tumor cells is conceivably insufficient to eradicate clonogenic cells and lead to appreciable delay in tumor growth in vivo. Our studies point to intriguing features as well as notable challenges that have to overcome before salinomycin or other more selective agents of this class can be safely tested in clinical trials in MM. Disclosures:McMillin:Axios Biosciences: Equity Ownership. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

Prolonged Drug Selection of Breast Cancer Cells and Enrichment of Cancer Stem Cell Characteristics

Cancer stem cells are presumed to have virtually unlimited proliferative and self-renewal abilities and to be highly resistant to chemotherapy, a feature that is associated with overexpression of ATP-binding cassette transporters. We investigated whether prolonged continuous selection of cells for drug resistance enriches cultures for cancer stem-like cells. Cancer stem cells were defined as CD44+/CD24⁻ cells that could self-renew (ie, generate cells with the tumorigenic CD44+/CD24⁻ phenotype), differentiate, invade, and form tumors in vivo. We used doxorubicin-selected MCF-7/ADR cells, weakly tumorigenic parental MCF-7 cells, and MCF-7/MDR, an MCF-7 subline with forced expression of ABCB1 protein. Cells were examined for cell surface markers and side-population fractions by microarray and flow cytometry, with in vitro invasion assays, and for ability to form mammospheres. Xenograft tumors were generated in mice to examine tumorigenicity (n = 52). The mRNA expression of multidrug resistance genes was examined in putative cancer stem cells and pathway analysis of statistically significantly differentially expressed genes was performed. All statistical tests were two-sided. Pathway analysis showed that MCF-7/ADR cells express mRNAs from ABCB1 and other genes also found in breast cancer stem cells (eg, CD44, TGFB1, and SNAI1). MCF-7/ADR cells were highly invasive, formed mammospheres, and were tumorigenic in mice. In contrast to parental MCF-7 cells, more than 30% of MCF-7/ADR cells had a CD44+/CD24⁻ phenotype, could self-renew, and differentiate (ie, produce CD44+/CD24⁻ and CD44+/CD24+ cells) and overexpressed various multidrug resistance-linked genes (including ABCB1, CCNE1, and MMP9). MCF-7/ADR cells were statistically significantly more invasive in Matrigel than parental MCF-7 cells (MCF-7 cells = 0.82 cell per field and MCF-7/ADR = 7.51 cells per field, difference = 6.69 cells per field, 95% confidence interval = 4.82 to 8.55 cells per field, P < .001). No enrichment in the CD44+/CD24⁻ or CD133+ population was detected in MCF-7/MDR. The cell population with cancer stem cell characteristics increased after prolonged continuous selection for doxorubicin resistance.