Stem-like Population Research Articles

Optimal control analysis of a leukemia model under imatinib treatment

The paper is devoted to the study of a mathematical model of drug therapy for Chronic Myelogenous Leukemia (CML). The disease dynamics are given by a couple of delay differential equations that describe the interaction between a stem-like population of CML cells and a more mature, differentiated one, without self-renewal properties. A molecular targeted therapy, such as Imatinib, is considered. The objective is to minimize the size of the tumoral cells’ mass while minimizing the amount of drug (thus, minimizing both the adverse effects and the costs). The optimal control is calculated using a discretization scheme. The results are supported by numerical simulations.

Isolation and phenotypic characterization of cancer stem-like side population cells in colon cancer.

Previous studies in cancer biology suggest that chemotherapeutic drug resistance and tumor relapse are driven by cells within a tumor termed 'cancer stem cells'. In the present study, a Hoechst 33342 dye exclusion technique was used to identify cancer stem‑like side population (SP) cells in colon carcinoma, which accounted for 3.4% of the total cell population. Following treatment with verapamil, the population of SP cells was reduced to 0.6%. In addition, the sorted SP cells exhibited marked multidrug resistance and enhanced cell survival rates compared with non‑SP cells. The SP cells were able to generate more tumor spheres and were CD133 positive. Subsequent biochemical analysis revealed that the levels of the adenosine triphosphate‑binding cassette sub‑family G member 2 transporter protein, B‑cell lymphoma anti‑apoptotic factor and autocrine production of interleukin‑4 were significantly enhanced in the colon cancer SP cells, which contributed to drug resistance, protection of the cells from apoptosis and tumor recurrence. Therefore, the findings suggested that treatment failure and colon tumorigenesis is dictated by a small population of SP cells, which indicate a potential target in future therapies.

Cluster of differentiation 44- and octamer-binding transcription factor-4-positive stem-like osteosarcoma cells involved in tumor development.

Osteosarcoma (OS) is an aggressive primary bone cancer that usually affects children and young adolescents. Previous studies have demonstrated the implications of a small sub-population of cancer stem cells on treatment failure and tumor recurrence. The present study analyzed the characteristic features of the stem-like cells within the human OS-55 cell line. It was identified that 2.3% of the OS-55 cells were cancer stem-like side population (SP) cells. Following treatment with verapamil, the population of SP cells was reduced to 0.7%. The sphere formation assay revealed that the OS cells were able to rapidly form tumor spheres (also known as sarcospheres). Immunofluorescence analysis identified that the OS-55 cells expressed the cluster of differentiation 44, octamer-binding transcription factor-3/4A and Nanog stem cell surface markers. The results of the present study suggest that, as with other tumors, OS also contains a sub-population of cancer stem-like cells, which may have important implications in cancer diagnosis and treatment.

Elimination of cancer stem-like cells and potentiation of temozolomide sensitivity by Honokiol in glioblastoma multiforme cells.

Glioblastoma multiforme (GBM) is the most common adult malignant glioma with poor prognosis due to the resistance to radiotherapy and chemotherapy, which might be critically involved in the repopulation of cancer stem cells (CSCs) after treatment. We had investigated the characteristics of cancer stem-like side population (SP) cells sorted from GBM cells, and studied the effect of Honokiol targeting on CSCs. GBM8401 SP cells possessed the stem cell markers, such as nestin, CD133 and Oct4, and the expressions of self-renewal related stemness genes, such as SMO, Notch3 and IHH (Indian Hedgehog). Honokiol inhibited the proliferation of both GBM8401 parental cells and SP cells in a dose-dependent manner, the IC50 were 5.3±0.72 and 11±1.1 μM, respectively. The proportions of SP in GBM8401 cells were diminished by Honokiol from 1.5±0.22% down to 0.3±0.02% and 0.2±0.01% at doses of 2.5 μM and 5 μM, respectively. The SP cells appeared to have higher expression of O 6-methylguanine-DNA methyltransferase (MGMT) and be more resistant to Temozolomide (TMZ). The resistance to TMZ could be only slightly reversed by MGMT inhibitor O 6-benzylguanine (O 6-BG), but markedly further enhanced by Honokiol addition. Such significant enhancement was accompanied with the higher induction of apoptosis, greater down-regulation of Notch3 as well as its downstream Hes1 expressions in SP cells. Our data indicate that Honokiol might have clinical benefits for the GBM patients who are refractory to TMZ treatment.

Isolation, culture and identification of cancer stem-like cells from human thyroid carcinoma tissues

Objective To isolate and culture human thyroid cancer stem-like cells side population(SP) from human thyroid carcinoma specimens and compare the differences in octamer-binding transcription factor 4(Oct-4)and adenosine triphosphate binding cassette transporter G2(ABCG2)expression between SP cells and non-SP cells. Methods SP and non-SP cells were sorted from human thyroid carcinoma cells by fluorescence-activated cell sorting(FACS). The expression of Oct-4 and ABCG2 in SP cells and non-SP cells was detected by Western blotting. Results SP cells with stem cell potency were isolated from human thyroid carcinoma cells. The content of sorted SP cells in experimental group and control group was 1.40% and 0.155% respectively(P< 0.05). The expression of Oct-4 and ABCG2 was significantly higher in SP cells than in non-SP cells. Conclusion SP cells, which have the potency of stem cells, exist in human thyroid carcinoma cells. Key words: Thyroid carcinoma; Side population cells; Cancer stem cells

Parthenolide inhibits cancer stem-like side population of nasopharyngeal carcinoma cells via suppression of the NF-κB/COX-2 pathway.

Cancer stem cells play a central role in the pathogenesis of nasopharyngeal carcinoma and contribute to both disease initiation and relapse. In this study, cyclooxygenase-2 (COX-2) was found to regulate cancer stem-like side population cells of nasopharyngeal carcinoma cells and enhance cancer stem-like cells' characteristics such as higher colony formation efficiency and overexpression of stemness-associated genes. The regulatory effect of COX-2 on cancer stem-like characteristics may be mediated by ABCG2. COX-2 overexpression by a gain-of-function experiment increased the proportion of side population cells and their cancer stemness properties. The present study also demonstrated that in contrast to the classical chemotherapy drug 5-fluorouracil, which increased the proportion of side population cells and upregulated the expression of COX-2, parthenolide, a naturally occurring small molecule, preferentially targeted the side population cells of nasopharyngeal carcinoma cells and downregulated COX-2. Moreover, we found that the cancer stem-like cells' phenotype was suppressed by using COX-2 inhibitors NS-398 and CAY10404 or knocking down COX-2 with siRNA and shRNA. These findings suggest that COX-2 inhibition is the mechanism by which parthenolide induces cell death in the cancer stem-like cells of nasopharyngeal carcinoma. In addition, parthenolide exhibited an inhibitory effect on nuclear factor-kappa B (NF-κB) nucler translocation by suppressing both the phosphorylation of IκB kinase complex and IκBα degradation. Taken together, these results suggest that parthenolide may exert its cancer stem cell-targeted chemotherapy through the NF-κB/COX-2 pathway.

Activation of Inflammatory Responses Correlate With Hedgehog Activation and Precede Expansion of Cancer Stem-Like Cells in an Animal Model of Residual Triple Negative Breast Cancer after Neoadjuvant Chemotherapy.

Triple Negative Breast Cancer (TNBC) is characterized as a lack of expression of the hormonal receptors, estrogen and progesterone, and Human epidermal growth factor receptor 2 (HER2) and as such is unresponsive to current targeted therapy. Resistance of breast cancers to treatment is thought to be due to a sub-population of tumor cells called Breast Cancer Stem Cells (BCSCs) and contributes to poor prognosis and increased risk of recurrence. Previously, we have shown that hedgehog activation is induced by chemotherapy and promotes expansion of a stem-like population in breast cancer cell lines. In addition, chemotherapy can induce an inflammatory response and inflammatory factors can lead to activation of Hedgehog (HH) at sites of tissue injury. Therefore, we wanted to investigate how chemotherapy altered hedgehog signaling and correlated with the release of inflammatory cytokines in a mouse model of breast cancer. Patient derived triple negative breast tumor bearing mice were treated with weekly doses of docetaxel. Following treatment, tumor volume decreased reaching a nadir around 15 days after the start of treatment and increased back to pre-treatment size 35-39 days post treatment. Immunohistochemical staining of mice tumors revealed that Sonic hedgehog and nuclear Gli-1 expression transiently increased following docetaxel treatment, reached peak expression at day 8, and subsequently decreased to almost pre-treatment levels following regrowth of the tumor. Similarly, Interleukin 6 (IL-6) and Interleukin 8 (IL-8) expression transiently increased, peaked around day 8, and decreased upon tumor regrowth, however, remained above pre-treatment levels. Expression of the stem cell marker ALDH1A3 proceeded activation of hedgehog signaling and expression of inflammatory cytokines, increasing around day 15 post treatment and continued to be elevated during tumor regrowth. Thus, chemotherapy treatment resulted in activation of the hedgehog pathway and release of inflammatory cytokines leading to long-term expansion of ALDH1A3 positive stem cells, which can contribute to the regrowth of the tumor and promote resistance to treatment.

Hypoxia-induced cell stemness leads to drug resistance and poor prognosis in lung adenocarcinoma.

Since cancer stem cells exhibit embryonic-like self-renewal characteristics and malignant behavior, including drug resistance and metastasis, they may be the origin of tumorigenesis and cancer recurrence. Cancer cell stemness is also highly relevant to cancer in hypoxic environments. In our study, we used cobalt dichloride (CoCl2) to create a hypoxic environment for lung adenocarcinoma A549 cells and the cisplatine-resistant cell line A549/DDP. The cancer stem-like CD166 positive population and the cells' stemness were detected by flowcytometry and quantitative real-time PCR after separation using magnetic antibodies. Drug resistance to cisplatine, docetaxel and pemetrexed was also measured. Finally, a tissue array was used to analyze the relationship between hypoxia-induced stemness and overall survival after radical surgery. Data showed that chemical-induced hypoxia changed cell stemness by enhancing stem cell transcription factors and markers of chemotherapeutic drug resistance. The CD166-positive cancer stem cell-like population showed greater drug resistance than the CD166-negative cells. Tissue array studies also suggested a poorer prognosis for patients whose tissue expressed higher CD166 levels. Our findings indicate that chemical hypoxia may augment cancer cell stemness and drug resistance in CD166-positive stem cells. Therefore, targeting the stem-like cell population, especially CD166-positive cells, may represent a novel therapeutic strategy to treat lung cancer.

Smad7 maintains epithelial phenotype of ovarian cancer stem-like cells and supports tumor colonization by mesenchymal-epithelial transition.

Epithelial ovarian carcinoma (EOC) is a lethal gynecological malignancy. Epithelial-mesenchymal transition (EMT) has an important role in the tumorigenesis and progression of EOC. During the process of EMT, the transforming growth factor-β (TGF-β)-Smad signaling pathway has been indicated to regulate cell motility and tumor development. Among numerous EMT-associated transcripts, Smad7 is considered to be an inhibitor, however its involvement together with TGF-β1 in the progression of ovarian cancer remains to be elucidated. The present study demonstrated that Smad7 was overexpressed in SK-OV-3 and stem-like side populations of EOC cells, both of which grow in an epithelial pattern. The transformation of cells from an epithelial to a mesenchymal phenotype was stimulated by TGF-β1 with a corresponding increase in Smad7 expression in SK-OV-3 cells. These results indicate that Smad7 is a regulator in the maintenance of the epithelial phenotype in EOC cells, and may serve as an inhibitory element which targets TGF-β-stimulated EMT. Furthermore, inhibition of Smad7 resulted in cellular mesenchymal transformation, with an increased expression of N-cadherin and a decreased expression of E-cadherin. The invasiveness and migratory capabilities of Smad7 small hairpin RNA transduced EOC cells was also reduced. The findings of the present study have identified Smad7 as a fundamental factor in the maintenance of epithelial growth of EOC cells. Reversal of EMT results in a mesenchymal-epithelial transition, which is necessary for EOC cell colonization at metastatic sites.

Evaluation of stem-like side population cells in a recurrent nasopharyngeal carcinoma cell line.

BackgroundSide population (SP) assay identifies cells with dye/drug extrusion ability, a characteristic of stem cells. Here, we determined if SP cells exist in a verified cell line originating from recurrent nasopharyngeal carcinoma (NPC) and a xenograft established from recurrent metastatic NPC. These cells were evaluated for stem-like properties via functional assays as well as for tumourigenicity.MethodsWe used Hoechst 33342 to identify the SP from non-SP (NSP) cells in HK1 NPC cell line and xeno-284 NPC xenograft. The cells were assayed for in vitro characteristics of cancer stem cells (CSC), gene expression and tumourigenicity ability. Student’s t test was used to test for significance.ResultsFive to ten percent and less than 0.5% of HK1 and xeno-284 NPC cells, respectively, were SP cells. Fumitremorgin C (FTC), as opposed to verapamil, was effective in causing the cells to retain Hoechst 33342 dye. HK1 SP cells formed more holoclones, had more aldehyde dehydrogenase (ALDH) activity, divided asymmetrically and contained slow-proliferating cells. ABCG2, SOX2, TERT, MYC, Hedgehog, Notch, TGFβ and Wnt signalling pathway genes were significantly upregulated in the SP cells. However, despite these differences in vitro, both HK1 SP and NSP cells had an overall similar tumourigenic potential in vivo.ConclusionsHK1 SP cells were ABCG2-specific as confirmed by FTC inhibition and gene expression data. Despite data from in vitro and gene expression experiments suggesting stem-like features, there was no significant difference in tumourigenic potential between SP and NSP cells. We conclude that SP assay alone is not sufficient to identify CSCs in HK1 cells. Our work also suggests the presence of a stem-cell like population among NPC cells which do not display increased tumourigenicity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-014-0101-0) contains supplementary material, which is available to authorized users.

Abstract A70: Chemotherapy-resistant subpopulations in a tumor-initiating cell model of human osteosarcoma

Abstract Introduction: Tumor-initiating cells (TICs) are a subpopulation of therapy resistant cells in osteosarcoma. To date, TICs have been experimentally defined based on the chosen isolation technique. Common to all isolation methods, TICs demonstrate a high degree of the multi-drug resistance phenotype, are more invasive, metastasize readily, and have increased capacity for tumorigenesis. Given this phenotype TICs represent a viable therapeutic target. Cultivating cells in low-adherence, serum-deprived conditions results in spherical colony formation. Certain growth factors present in serum drive epithelial-to-mesenchymal transition, and typically confound TIC models of carcinoma. The effect of serum on mesenchymal-based sarcoma TIC models is less clear. Recent literature suggests that merely the presence of low-adherence conditions can enrich in TICs. We have shown that osteosarcoma cells cultured in low-adherence conditions in the presence of 10% fetal bovine serum (FBS) form spheres, exhibit an increased expression of genes implicated in developmental programs and stem cells, are more resistant to chemotherapy, and more readily initiate tumors than adherent cells when injected into mice. Methods: The osteosarcoma cell lines 143B, mHOS, and MG-63 were cultured in low-adherence plates using DMEM or MEM media supplemented with 10% FBS. Spheres were routinely grown for 4-6 days between passages and dissociated using 0.05% trypsin. Relative mRNA expression levels of Nanog, Sox2, Oct4 and Axin2 was assessed using quantitative-PCR (SYBR Green). Resistance to chemotherapy (doxorubicin and cisplatin) was determined by comparing both adherent cells and spheres (in low adherence 96-well plates) grown to at least the second passage. After 72 hours IC50 values were calculated using the CellTitre Glo luminescence assay. To assess in vivo tumorgenicity, adherent and sphere cells were dissociated and injected into the flanks of nude mice at a density of 10,000 cells. Upon tumor formation the mice were euthanized and the tumors re-implanted into a second set of mice to test their ability to serially transplant. Results: Sarcospheres can be successfully and reproducibly grown and passaged in low-adherent culture conditions in media supplemented with 10% FBS. Expression profiling demonstrates increased expression of Sox2, Oct4, Nanog, and Axin2, all genes that have been identified to be associated with developmental programs and stem cells. Sarcospheres also displayed increased chemoresistance compared to adherent cultures to both cisplatin (IC50 change of 1.9, 6.9, and 6.2-fold in 143B, mHOS and MG-63, respectively) and more so to doxorubicin (IC50 change of 12.7, 35 and greater than 74-fold in 143B, mHOS, and MG-63, respectively). Additionally, in a pilot subcutaneous xenograft study, using sphere and adherent cells, differential growth was observed within the TIC-enriched populations demonstrating (1) earlier tumor initiation and (2) a lower overall proliferation rate among the primary and serially transplanted tumors confirming a persistence of the TIC phenotype through in vivo passaging. Conclusions: Osteosarcoma cells cultured in low-adherence conditions in the presence of serum display many of the characteristics of putative tumor-initiating cells including robust sphere formation, upregulation of key stemness mediators, enhanced chemoresistance, and in vivo tumor initiation and serial transplantability. Using this system, we aim to further evaluate these stem-like populations in order to elucidate potential therapies targeting this specialized, chemoresistant niche. Citation Format: Jessica M. Foley, Noel R. Monks, Donald J. Scholten, II, David J. Monsma, Dawna Dylewski, Paula J. Davidson, Matthew R. Steensma. Chemotherapy-resistant subpopulations in a tumor-initiating cell model of human osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A70.

Atypical cell cycle control over neural cell fate

Neuroblastoma is an aggressive pediatric cancer fuelled by inappropriate differentiation of immature cells within the ganglionic lineage. The heterogeneity of the disease, as most cancers, complicates diagnosis and treatment. In neuroblastoma this heterogeneity is well represented in both primary tumors and derived cell lines, and mirrors neural crest plasticity. Multiple studies over the years have attempted to elucidate the molecular basis driving the expansion of the stem-like population within aggressive neuroblastoma; however no study has adequately addressed the role of the core cell cycle machinery. This was the focus of our recent publication in Oncoscience.1 Decreases in activity of the G1/S cyclin dependent kinase (Cdk) Cdk2 and accumulation of the Cdk inhibitor p27Kip,1 supports functional differentiation. Indeed, Cdk2 inhibition is synthetic lethal in MYCN overexpressing neuroblastoma.2 Adult stem cells carefully balance inhibition of the cell cycle with limited expansion in vivo to enable development and regeneration while preventing pathogenesis. The Speedy/RINGO family of ‘cyclin-like’ proteins are capable of binding to and activating the Cdks via a unique mechanism to drive cell growth. Spy1-bound Cdks are not dependent on the classically defined post-translational modifications for activation, nor are they sensitive to suppression by the Cdk inhibitors, quite contrary they can actually promote the degradation of the Cdk inhibitor p27Kip,1 3. Consistent with this guise our group and others have shown that elevated Spy1 levels are capable of overriding numerous forms of senescence.4-6 Why would a cell evolve such a mechanism? We hypothesize that endogenously this may enable expansion of select stem cell populations during development and regeneration, as well as allowing for recovery of a variety of cell types from checkpoint responses. In support of this model, Spy1 has demonstrated roles in spinal cord regeneration and was found to possess stem-like qualities in the developing mammary gland, supporting a general role for Spy1 in select populations of adult stem or progenitor cells.4,7 More recently pathological levels of Spy1 have been implicated in supporting the symmetric expansion of the CD133+ population in human glioma.6 In one of the inaugural issues of Oncoscience we show that Spy1 supports prolonged clonal tumorsphere formation in neuroblastoma cell lines and expands cell populations enriched for markers of multipotency. We find that endogenous Spy1 levels are reduced during guided differentiation of the stem-like population in neuroblastoma and that preventing this downregulation leads to resistance to 13-cis-Retinoic Acid (RA)- induced differentiation. In the mammary gland we have previously reported that Spy1 expression is regulated downstream of c-Myc during normal development. In neuroblastoma we find that c-Myc protein levels correlate with that of Spy1 in all cell lines tested, the relevance for these observations in neuroblastoma pathogenesis remain to be tested. Forced silencing of Spy1 levels in neuroblastoma resulted in a decrease in tumorsphere number, and a reduction in the CD133+ population. Hence, our data supports a novel fundamental role for an atypical cell cycle mechanism in driving expansion of the neural crest stem cells that define the aggressive, drug resistant population in subsets of neuroblastoma. This discovery may represent an important opportunity to design Cdk inhibitor drugs to uniquely target subpopulations of cells within these aggressive neural tumors.

Abstract 3487: CA9 expression highly correlates with cancer stem cell markers during passaging of PDX lines

Abstract Tumor-Initiating Cells (TICs), or Cancer Stem Cells (CSCs), are considered a subpopulation of cells within a tumor that are particularly aggressive because when isolated, these cells can form secondary tumors. Along these lines, experiments in which fragments of tumors, derived from primary patient samples, are re-implanted into nude mice, the majority of cells within the fragment die. However, day 3 after re-implantation, a small subpopulation emerges that is able to seed a new tumor. We hypothesized that these cells are unique and rare within the original tumor and consequently present an opportunity to identify novel markers that can identify these aggressive tumor cells. After sorting live human cells from tumor fragments three days after passaging, we used single cell PCR to determine the extent of heterogeneity within the fragment population. We, in fact, found that there is a subpopulation that highly expresses markers of proliferation and stemness. This is in contrast to the major population within the fragment that is enriched for genes associated with hypoxia and stress response. Interestingly, we found that the hypoxia responsive gene, CA9, does not correlate with other markers of hypoxia, such as VEGF, when analyzing cells derived from the fragment. Instead, CA9 was highly expressed among cells that were also enriched for markers such as Ki67, survivin, and Lgr5. To confirm if CA9 does identify a more proliferative, stem-like population, we have sorted CA9+ and CA9- cells from three day PDX fragments and analyzed using DNA microarrays and real-time PCR. We have consistently found, among several different PDX lines, that passaging of tumors followed by sorting CA9+ cells enriches for cells with increased levels of Lgr5, ASCL2, CD133, and Ki67. This is also validated through IHC analysis in which the majority of the cells that are positive for Ki67 are also positive for CA9. We are continuing to investigate the tumorigenicity of these cells by reinjecting CA9+ and CA9- cells from fragments back into immune-compromised mice. Furthermore, we are building a gene list derived from the microarrays to inspect by single cell PCR if any of these genes associate with Lgr5 in a grown tumor. Thus, we believe this is a functionally relevant and novel method for identify novel TIC markers. Citation Format: Julia Friedman, Wenyan Zhong, Christine Loreth, Veronica Diesl, Xin Han, Justin Lucas, Andrea Hooper, Vlad Buklan, Edward Rosfjord, Danielle Leahy, Judy Lucas, Maximillian Follettie, Kim Arndt. CA9 expression highly correlates with cancer stem cell markers during passaging of PDX lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3487. doi:10.1158/1538-7445.AM2014-3487

Effects of doxorubicin and gemcitabine on the induction of apoptosis in breast cancer cells.

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related mortality in females worldwide. The efficacy of chemotherapeutic drugs on breast cancer is hindered by many factors, including that cancer stem-like side population (SP) cells may contribute to tumorigenesis and resistance to chemotherapy. Doxorubicin and gemcitabine are two of the most effective and widely used chemotherapeutic agents for the treatment of various human malignancies. To the best of our knowledge, the effects of doxorubicin and gemcitabine on breast cancer SP cells are poorly understood. In the present study, we examined the potential roles of doxorubicin and gemcitabine in breast cancer main population (MP) and SP cells, and the mechanisms of doxorubicin and gemcitabine. The results showed that doxorubicin and gemcitabine decreasede proliferation, and increased apoptosis in the MCF7 MP and SP cells in vitro. Furthermore, doxorubicin and gemcitabine inhibited tumor growth and improved the survival rate of mice in vivo. Additionally, the mechanisms of doxorubicin and gemcitabine in MCF7 MP and SP cells were determined.

49IN - The Coming Decade

ABSTRACT Epithelial ovarian cancer remains highly lethal regardless of geography, technology, or finances. High-grade serous cancer (HGSC) is associated with p53 loss, defective apoptotic signaling, genomic instability, defective DNA repair, peritoneal dissemination, angiogenic signaling, and drug resistance. Primary therapy with cytoreductive surgery and platinum-based chemotherapy has changed minimally over 30 y, with improved PFS and OS, but without any impact on mortality. Ambitious strategies to restore defective apoptotic signaling (p53), overcome drug resistance, modulate the immune response, and target tumor stem-like populations are needed. Increased prevalence of genomic screening will facilitate decisions regarding lifestyle modifications and risk-reduction surgery. Functional imaging and molecular probes will improve high-risk detection and accuracy of staging, including intra-operative probes to localize microscopic disease. Adjuvant therapy will be reserved for women with HGSC and pre-defined molecular-biologic risk factors. Vaccines with immune response modulation will eventually replace chemotherapy for early-stage disease. Patients with high-volume disease will undergo risk-profiling for neoadjuvant chemotherapy and interval cytoreduction. Intraperitoneal therapy will be offset by targeted agents. Important generic drugs in recurrent disease will receive extended patent protection to assure adequate supply of high-quality reagents. Sequential tumor specimens will be routinely evaluated for clonal evolution and pathway activation. A research foundation will be established through pharmaceutical collaboration with government agencies, eliminating barriers to international research. New agents/combinations will be evaluated in small randomized trials to select promising regimens. Molecular profiles will be standardized and shared to facilitate design and analysis of clinical trials. The accelerated pace of discovery has not yet translated into a reduction in mortality for patients with HGSC, although a number of promising interventions are under development. The next decade will witness a concerted effort to apply technology to address high-priority clinical, scientific, regulatory, information-based, and financial goals. Disclosure: M.A. Bookman: Chair Ovarian Comm NRG-GOG; member independent data monitoring committees industry-sponsored; participant ad-hoc advisory boards clinical trial development investigational agents. Support for attendance. No financial holdings.

A stem cell medium containing neural stimulating factor induces a pancreatic cancer stem-like cell-enriched population.

Cancer stem cells (CSCs) have been studied for their self-renewal capacity and pluripotency, as well as their resistance to anticancer therapy and their ability to metastasize to distant organs. CSCs are difficult to study because their population is quite low in tumor specimens. To overcome this problem, we established a culture method to induce a pancreatic cancer stem-like cell (P-CSLC)-enriched population from human pancreatic cancer cell lines. Human pancreatic cancer cell lines established at our department were cultured in CSC-inducing media containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), neural cell survivor factor-1 (NSF-1), and N-acetylcysteine. Sphere cells were obtained and then transferred to a laminin-coated dish and cultured for approximately two months. The surface markers, gene expression, aldehyde dehydrogenase (ALDH) activity, cell cycle, and tumorigenicity of these induced cells were examined for their stem cell-like characteristics. The population of these induced cells expanded within a few months. The ratio of CD24high, CD44high, epithelial specific antigen (ESA) high, and CD44variant (CD44v) high cells in the induced cells was greatly enriched. The induced cells stayed in the G0/G1 phase and demonstrated mesenchymal and stemness properties. The induced cells had high tumorigenic potential. Thus, we established a culture method to induce a P-CSLCenriched population from human pancreatic cancer cell lines. The CSLC population was enriched approximately 100-fold with this method. Our culture method may contribute to the precise analysis of CSCs and thus support the establishment of CSC-targeting therapy.

Differentiation therapy: sesamin as an effective agent in targeting cancer stem-like side population cells of human gallbladder carcinoma

BackgroundRecent studies have demonstrated that side population (SP) cells isolated from various cancer cell lines and primary tumors possess stem cell-like properties. Sesamin, a food-derived agent, possesses anti-cancer activities both in vitro and in vivo. The present study was designed to determine whether sesamin also have effects on cancer stem-like SP cells from gallbladder cancer (GBC).MethodsIn this study, we sorted SP cells by flow cytometry. SP cells were cultured and treated with sesamin. Tumor-sphere formation, colony formation, Matrigel invasion and tumorigenic potential were determined. Expression of nuclear NF-κB, IL-6, p-Stat3, Twist, E-cadherin and Vimentin was measured by Western blot, immunofluorescence staining or RT-PCR analysis. Nuclear NF-κB activity and IL-6 protein level were assessed with ELISA. Xenograft tumors were generated in nude mice.ResultsAfter treated with sesamin, SP cells differentiated into cells expressing the epithelial marker (E-cadherin). Sesamin effectively affected SP cells stem cell-like characteristics (i.e., tumor-sphere formation, colony-formation, Matrigel invasion), weakened the drug-resistance of SP cells and inhibited tumor growth both in vitro and in vivo. Treatment with sesamin significantly reduced the expression of nuclear NF-κB, IL-6, p-Stat3, Twist and Vimentin (a mesenchymal marker) in SP cells. Nuclear NF-κB activity and IL-6 level were also decreased after treatment with sesamin.ConclusionFood-derived sesamin directs the epithelial differentiation of cancer stem-like SP cells from GBC, which is associated with attenuation of NF-κB-IL-6-Stat3-Twist signal pathway.

GLIOMA SPECIFIC PEPTIDES: A PLATFORM FOR MOLECULAR IMAGING AND THERAPEUTIC TARGETING

BACKGROUND: Despite intense investigation, the ability to treat high-grade glioma (HGG) has remained dismal, in part due to molecular and phenotypic heterogeneity. The cancer stem cell, a cell believed to be the clonogenic core of tumors including human glioma, has been a major focus for the development of new therapies but emerging evidence has exposed the dynamic and heterogenous characteristics of this stem-like population that facilitates evasion from current therapies. METHODS: To capture and target the complexity and heterogeneity of glioma, we employed a combinatorial phage-display biopanning strategy to isolate peptides that specifically bind and home in vivo to key disease reservoirs within glioma; namely the invasive and stem-like populations of glioma cells (GSC). Synthetic peptides, individually or in combination, were conjugated to gadolinium or a chemotherapeutic agent and administered to animals bearing orthotopic tumors established from human glioma-like-stem cells. RESULTS: Using this approach we identified a panel of peptides with the ability to detect the heterogeneity of patient gliomas in vivo successfully imaging a range of tumors including diffuse infiltrating tumours that are otherwise invisible by conventional imaging technologies. In addition, we identified peptides with multifunctional capabilities including peptides that functionally block glioma invasion, define a subpopulation of GSCs, and target chemotherapeutic agents in vivo. CONCLUSIONS: These data highlight the utility of the identified peptides as platforms for the precise and sensitive imaging of these heterogenous tumors and for the development of molecularly targeted therapeutics. SECONDARY CATEGORY: Tumor Biology.

Histone Modifications, Stem Cells and Prostate Cancer

Prostate cancer (PCa) is a very common neoplasm, which is generally treated by chemo-, radio-, and/or hormonal-therapy. After a variable time, PCa becomes resistant to conventional treatment, leading to patient death. Prostate tumor-initiating cells (TICs) and cancer repopulating cells (CRCs) are stem-like populations, driving respectively cancer initiation and progression. Histone modifiers (HMs) control gene expression in normal and cancer cells, thereby orchestrating key physiological and pathological processes. In particular, Polycomb group genes (PcGs) are a set of HMs crucial for lineage-specific gene silencing and stem cell self renewal. PcG products are organized into two main Polycomb Repressive Complexes (PRCs). At specific loci, PRC2 catalyzes histone H3 Lys27 trimethylation, which triggers gene silencing by recruiting PRC1, histone deacetylases and DNA methyl transferases. PRC1 catalyzes addition of the repressive mark histone H2A ubiquitination. Recently, the catalytic component of PRC1 (BMI1) was shown to play critical roles in prostate CRC self-renewal and resistance to chemotherapy, resulting in poorer prognosis. Similarly, pharmacological disruption of PRC2 by a small molecule inhibitor reduced the tumorigenicity and metastatic potential of prostate CRCs. Along with PcGs, some histone lysine demethylases (KDMs) are emerging as critical regulators of TIC/CRC biology. KDMs may be inhibited by specific small molecules, some of which display antitumor activity in PCa cells at micromolar concentrations. Since epigenetic gene regulation is crucial for stem cell biology, exploring the role of HMs in prostate cancer is a promising path that may lead to novel treatments.

Protein kinase C-delta inactivation inhibits the proliferation and survival of cancer stem cells in culture and in vivo

BackgroundA subpopulation of tumor cells with distinct stem-like properties (cancer stem-like cells, CSCs) may be responsible for tumor initiation, invasive growth, and possibly dissemination to distant organ sites. CSCs exhibit a spectrum of biological, biochemical, and molecular features that are consistent with a stem-like phenotype, including growth as non-adherent spheres (clonogenic potential), ability to form a new tumor in xenograft assays, unlimited self-renewal, and the capacity for multipotency and lineage-specific differentiation. PKCδ is a novel class serine/threonine kinase of the PKC family, and functions in a number of cellular activities including cell proliferation, survival or apoptosis. PKCδ has previously been validated as a synthetic lethal target in cancer cells of multiple types with aberrant activation of Ras signaling, using both genetic (shRNA and dominant-negative PKCδ mutants) and small molecule inhibitors. In contrast, PKCδ is not required for the proliferation or survival of normal cells, suggesting the potential tumor-specificity of a PKCδ-targeted approach.MethodsshRNA knockdown was used validate PKCδ as a target in primary cancer stem cell lines and stem-like cells derived from human tumor cell lines, including breast, pancreatic, prostate and melanoma tumor cells. Novel and potent small molecule PKCδ inhibitors were employed in assays monitoring apoptosis, proliferation and clonogenic capacity of these cancer stem-like populations. Significant differences among data sets were determined using two-tailed Student’s t tests or ANOVA.ResultsWe demonstrate that CSC-like populations derived from multiple types of human primary tumors, from human cancer cell lines, and from transformed human cells, require PKCδ activity and are susceptible to agents which deplete PKCδ protein or activity. Inhibition of PKCδ by specific genetic strategies (shRNA) or by novel small molecule inhibitors is growth inhibitory and cytotoxic to multiple types of human CSCs in culture. PKCδ inhibition efficiently prevents tumor sphere outgrowth from tumor cell cultures, with exposure times as short as six hours. Small-molecule PKCδ inhibitors also inhibit human CSC growth in vivo in a mouse xenograft model.ConclusionsThese findings suggest that the novel PKC isozyme PKCδ may represent a new molecular target for cancer stem cell populations.