S-type Cells Research Articles

Multiple low-dose radiation-induced neuronal cysteine transporter expression and oxidative stress are rescued by N-acetylcysteine in neuronal SH-SY5Y cells

Recently, several studies have demonstrated that low-dose radiation (LDR) therapy has positively impacts on the treatment of Alzheimer’s disease (AD). LDR suppresses the production of pro-neuroinflammation molecules and improves cognitive function in AD. However, it is unclear whether direct exposure to LDR causes beneficial effects and what mechanism is involved in neuronal cells. In this study, we first determined the effect of high-dose radiation (HDR) alone on C6 cells and SH-SY5Y cells. We found that SH-SY5Y cells were more vulnerable than C6 cells to HDR. Moreover, in neuronal SH-SY5Y cells exposed to single or multiple LDR, N-type cells showed decreased cell viability with increasing radiation exposure time and frequency, but S-type cells were unaffected. Multiple LDR increased proapoptotic molecules such as p53, Bax and cleaved caspase-3, and decreased anti-apoptotic molecule (Bcl2). Multiple LDR also generated free radicals in neuronal SH-SY5Y cells. We detected a change in the expression of the neuronal cysteine transporter EAAC1. Pretreatment with N-acetylcysteine (NAC) rescued the increased in EAAC1 expression and the generation of ROS in neuronal SH-SY5Y cells after multiple LDR. Furthermore, we verified whether the increased in EAAC1 expression induces cell defense or cell death promotion signaling. We showed that transient overexpression of EAAC1 reduced the multiple LDR-induced p53 overexpression in neuronal SH-SY5Y cells. Our results indicate that neuronal cells can be injured by increased production of ROS not only by HDR but also by multiple LDR, which suggests that combination treatment with anti-free radical agents such as NAC may be useful in multiple LDR therapy.

EFFECT OF M2 MACROPHAGE-DERIVED SOLUBLE FACTORS ON DIFFERENTIATION OF SH-SY5Y CELLS

Macrophages play a key role in triggering and regulation of neuroregeneration. The characteristic feature of macrophages is pronounced plasticity, which manifests itself in the ability of macrophages to change their functional phenotype depending on the micromilieu. Apoptotic cell clearance (efferocytosis) is an important inducer of a macrophage polarization to M2 phenotype under pathological settings. Previously, we have developed an original protocol for the generation of M2-like macrophages, polarized by efferocytosis under serum-deprived conditions (M2 (LS), Low Serum). The present study was aimed to assess a neuroregenerative potential of M2 (LS) macrophages. We studied their effect on the differentiation of SH-SY5Y cells in comparison with retinoic acid (RA). As the morphological criteria of differentiation we have assessed the relative content of differentiated cells, i.e., cells with a neurite length exceeding the cell body length, and the average neurite length on days 3, 7, and 13. The ratio of neuron-like (N-type) and epithelial-like (S-type) cells in cultures was also assessed. SH-SY5Y cells were characterized by a low level of spontaneous differentiation, both under standard conditions (10% FBS) and serum deprivation (1% FBS). Upon RA treatment, SH-SY5Y cells stopped proliferating and underwent neuronal differentiation. Cultivation of SH-SY5Y cells in the presence of M2 (LS) conditioned medium also led to a significant increase in the relative content of differentiated cells, the average length of neurite-like processes, as well as a change in the balance of S- and N-type cells towards a pronounced predominance of the latter. The morphological features of differentiation were significantly less pronounced at early stage (day 3) of differentiation as compared with the RA-induced changes and reached the level of positive control only at later stages (day 13) (p < 0.05). In contrast to retinoic acid, M2 (LS) conditioned medium induced neuronal differentiation of SH-SY5Y cells without suppressing their proliferative activity. The data obtained may indicate a high neuroregenerative potential of M2 macrophages in vitro, which is realized through soluble factors and manifests itself in promoting SH-SY5Y differentiation.

Tumour‐derived substrate‐adherent cells promote neuroblastoma survival through secreted trophic factors

Neuroblastoma (NB) is the most common extracranial solid tumour in children. NB is highly heterogeneous and is comprised of a mixture of neuroblastic cancer cells and stromal cells. We previously reported that N‐type cells (neuroblastic cells) and S‐type cells (substrate‐adherent cells) in the SK‐N‐SH cell line shared almost identical genetic backgrounds. Sublines of N‐ and S‐type cells were isolated from an early passage (P35) of SK‐N‐SH. Sequencing analysis revealed that all sublines harboured the anaplastic lymphoma kinase (ALK) F1174L mutation, indicating that they were tumour derived. Surprisingly, over 74% resembled S‐type cells. In coculture experiments, S‐type cells protected N‐type cells from apoptosis induced by the oncogenic ALK inhibitor TAE684. Western blotting analyses showed that ALK, protein kinase A (AKT) and STAT3 signalling were stimulated in the cocultures. Furthermore, the conditioned medium from S‐type cells activated these downstream signalling molecules in the N‐type cells. The activation of STAT3 in the N‐type cells was ALK‐independent, while AKT was regulated by the ALK activation status. To identify the responsible soluble factors, we used a combination of transcriptomic and proteomic analysis and found that plasminogen activator inhibitor 1, secreted protein acidic and cysteine rich, periostin and galectin‐1 were potential mediators of STAT3 signalling. The addition of recombinant proteins to the tumour cells treated with the ALK inhibitor partially enhanced cell viability. Overall, the tumour‐derived S‐type cells prevented apoptosis in the N‐type cells via ALK‐independent STAT3 activation triggered by secreted factors. The inhibition of these factors in combination with ALK inhibition could provide a new direction for targeted therapies to treat high‐risk NB.

Evidence for phloem loading via the abaxial bundle sheath cells in maize leaves.

Leaves are asymmetric, with different functions for adaxial and abaxial tissue. The bundle sheath (BS) of C3 barley (Hordeum vulgare) is dorsoventrally differentiated into three types of cells: adaxial structural, lateral S-type, and abaxial L-type BS cells. Based on plasmodesmatal connections between S-type cells and mestome sheath (parenchymatous cell layer below bundle sheath), S-type cells likely transfer assimilates toward the phloem. Here, we used single-cell RNA sequencing to investigate BS differentiation in C4 maize (Zea mays L.) plants. Abaxial BS (abBS) cells of rank-2 intermediate veins specifically expressed three SWEET sucrose uniporters (SWEET13a, b, and c) and UmamiT amino acid efflux transporters. SWEET13a, b, c mRNAs were also detected in the phloem parenchyma (PP). We show that maize has acquired a mechanism for phloem loading in which abBS cells provide the main route for apoplasmic sucrose transfer toward the phloem. This putative route predominates in veins responsible for phloem loading (rank-2 intermediate), whereas rank-1 intermediate and major veins export sucrose from the PP adjacent to the sieve element companion cell complex, as in Arabidopsis thaliana. We surmise that abBS identity is subject to dorsoventral patterning and has components of PP identity. These observations provide insights into the unique transport-specific properties of abBS cells and support a modification to the canonical phloem loading pathway in maize.

Phenotypic niche partitioning and transcriptional responses of Microcystis aeruginosa in a spatially heterogeneous environment

Variant morphotypes (smooth; S-type and wrinkled; W-type cells) of Microcystis aeruginosa KW strain were observed in bloom samples and during the cultivation of cells in static broth cultures. RNA-seq based whole transcriptome sequencing was performed to examine the pattern of gene expression in these morphotypes. In total 493 differentially expressed transcripts were identified between the two morphotypes. Genes involved in photosynthesis, photosynthesis-related antenna proteins and oxidative phosphorylation were significantly upregulated in W-type cells relative to the control (S-type cells). Here we discussed the roles of these genes and showed that M. aeruginosa responds and acclimates to subtle variations in environmental parameters, especially light, through alteration of the transcript levels of relevant genes. Furthermore, additional physiological experiments such as analyses of photosynthetic activity, redox state and exopolysaccharide production provided further evidence and confirmed the validity of the transcriptomic results. The findings of this study provide greater insight into the metabolic pathways and complex mechanisms that may regulate the interaction and niche adaptation of this important bloom-forming organism in fluctuating environmental conditions.

Differentiation-Induced Remodelling of Store-Operated Calcium Entry Is Independent of Neuronal or Glial Phenotype but Modulated by Cellular Context

Neurogenesis is a complex process leading to the generation of neuronal networks and glial cell types from stem cells or intermediate progenitors. Mapping subcellular and molecular changes accompanying the switch from proliferation to differentiation is vital for developing therapeutic targets for neurological diseases. Neuronal (N-type) and glial (S-type) phenotypes within the SH-SY5Y neuroblastoma cell line have distinct differentiation responses to 9-cis-retinoic acid (9cRA). In both cell phenotypes, these were accompanied at the single cell level by an uncoupling of Ca2+ store release from store-operated Ca2+ entry (SOCE), mediated by changes in the expression of calcium release-activated calcium pore proteins. This remodelling of calcium signalling was moderated by the predominant cell phenotype within the population. N- and S-type cells differed markedly in their phenotypic stability after withdrawal of the differentiation inducer, with the phenotypic stability of S-type cells, both morphologically and with respect to SOCE properties, in marked contrast to the lability of the N-type phenotype. Furthermore, the SOCE response of I-type cells, a presumed precursor to both N- and S-type cells, varied markedly in different cell environments. These results demonstrate the unique biology of neuronal and glial derivatives of common precursors and suggest that direct or indirect interactions between cell types are vital components of neurogenesis that need to be considered in experimental models.

Abstract 4325: The role of tumor cell heterogeneity in the targeted therapy of ALK-positive neuroblastoma

Abstract Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. One of its hallmarks is high histological heterogeneity. The primary tumors are composed of neuroblastic cancer cells, Schwannian stroma and some infiltrating immune cells. The origin and functions of Schwannian stroma still remain controversial. It is suggested that they are tumor-infiltrating normal cells. Studies using Bone Marrow Stromal Cells (BMSCs) or Schwann cells to mimic Schwannian stroma have shown both tumor promoting or suppression effects. An alternative hypothesis is that Schwannian cells are derived from cancer stem-like cells since they shared the same genetic lesions with neuroblastic tumor cells. Our laboratory had reported that in the SK-N-SH cell line, both N-type cells (neuroblastic cells) and S-type cells (Schwann-like cells) shared almost identical genetic background. These cell lines may serve as relevant models to study NB tumor heterogeneity. Genome-wide sequencing for driver oncogenes in familial NB uncovered mutations in Anaplastic Lymphoma Kinase (ALK) gene. ALK point mutations have been found in 8-12% of all NB patients, and are restricted to the kinase domain. Aberrant activation of ALK signaling, and its downstream target, PI3K/AKT, STAT3 and ERK1/2, can contribute to NB tumorigenesis. Thus, ALK is an intensely studied therapeutic target. Sublines of N-type and S-type cells were isolated from an early passage of SK-N-SH. Surprisingly, over 90% have cell morphology that resembled S-type cells. Sequencing analysis revealed that all harbored the same ALK F1174L mutation, indicating they were tumor-derived. Western blotting analysis showed that N-type cells, but not S-type cells, expressed ALK protein. This may explain the insensitivity of S-type cells towards ALK inhibitor, TAE684. Next, co-culture experiments showed that S-type cells protected N-type cells from apoptosis induced by TAE684. Western blotting analysis showed that ALK, AKT and STAT3 signaling were stimulated in the co-culture. Furthermore, conditioned medium (CM) from Schwann-like cells activated these downstream signaling molecules in N-type cells, indicating secreted factors from S-type cells contributed to the pathway stimulations. As AKT and STAT3 were ALK downstream targets, ALK ligand could be a potential factor. We further confirmed these results in two additional ALK WT N-type cell lines, IMR 32 and BE (2)-M17. Co-cultivation and CM experiments had demonstrated robust STAT3 activation even without ALK stimulation. Overall, tumor-derived S-type cells could prevent N-type cells from apoptosis via secreted factors. The signaling involved could be separated into an ALK-independent STAT3 activation and a ligand-activated ALK-AKT pathway. Future work will focus on identifying STAT3 stimulators and ALK ligands in CM of S-type cells. This work was supported by a General Research Fund grant (#14100514) from the Hong Kong University Grants Committee to A.C. Citation Format: Jing LI, Penelope M. OR, Andrew M. CHAN. The role of tumor cell heterogeneity in the targeted therapy of ALK-positive neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4325. doi:10.1158/1538-7445.AM2017-4325

Abstract 1711: Detection of circulating tumor cells for use as prognostic, predictive and pharmacodynamic biomarkers in neuroblastoma

Abstract Introduction: Neuroblastoma (NB) is the commonest extracranial childhood solid tumour. New treatments and a better understanding of drug resistance are needed to improve survival. Circulating tumour cells (CTCs) may provide a source of tumour cells for genetic biomarkers, give insights into tumour load and serve as pharmacodynamic (PD) biomarkers for new treatments. In clinical diagnostics, slide based FISH is used to detect MYCN amplification in neuroblastoma tumours. As it is not possible to conduct slide based FISH on an entire blood sample due to the infrequency of CTCs in peripheral blood (1 in 106 leucocytes), an alternative method FISH-IS (in suspension) was used in the present study to detect MYCN gene amplification using the Imagestream imaging flow cytometer (ISx). Aims: 1) To detect CTCs from blood and disseminated tumour cells (DTC) from bone marrow (BM) from NB patients using the (ISx) 2) To use these cells for genetic and PD biomarker studies for novel targeted therapies. Methods: A panel of NB cell lines (n=6) cloned into neuronal (N) and substrate-adherent (S) types were used to determine expression of NB cell surface markers (GD2 and NCAM) using the ISx. Western blotting and fluorescence activated cell sorting (FACS) were used to validate NCAM and GD2 expression respectively. 25 paired NB patient blood and BM samples were analysed for CTCs and DTCs, 4 unpaired blood samples were analysed for CTCs and one unpaired BM sample was for DTCs using the ISx to detect GD2 +ve and CD45-ve cells. Results: Using the ISx 80-90% of N-type cells immunostained positive for GD2 compared to 0.8 -10% of S-type cells confirmed by FACS. CTCs were detected in 21/29 patient samples and DTCs in 19/26 cases. The numbers of CTCs ranged from 10-522/ml blood and DTCs from 57-35,515/ml of bone marrow. After validating FISH-IS protocol on ISx using NGP and SHSY5Y cell lines, 4 NB patient DTC samples were analysed for MYCN amplification. 2/4 cases showed MYCN gene amplification consistent with results obtained on the primary tumour. Conclusion: This the first study to show that DTCs and CTCs are detectable in NB patient samples using the ISx. Future work will involve further genetic characterisation and development of PD biomarkers for Phase I clinical trials. Citation Format: Swathi Merugu, Deborah Tweddle. Detection of circulating tumor cells for use as prognostic, predictive and pharmacodynamic biomarkers in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1711. doi:10.1158/1538-7445.AM2017-1711

Cooperative cell invasion: matrix metalloproteinase-mediated incorporation between cells.

Progression to metastatic disease is a leading cause of cancer death. Tumors are a complex mixture of cell types, both genetically heterogeneous malignant cells and associated nonmalignant cells. Models mimicking this heterogeneous cell environment have revealed that invasive cell populations can induce dissemination by otherwise poorly/noninvasive tumor cells, known as cooperative invasion. Neuroblastoma tumors arise in children and are characterized by mixed cellular populations in vivo, consisting chiefly of neuronal (N)-type and substrate (S)-type cells. The S-type cells have all the hallmarks of invasive leader cell populations and have been coisolated with N-type cells from metastatic bone lesions, but to date their ability to induce cooperative invasion has not been investigated. Therefore, in the present study, we analyzed the invasive behavior of mixed N-type and S-type multicellular spheroids embedded in three-dimensional collagen gels. Our analyses show that S-type cells induce invasion of either single cells or small cell clusters of N-type cells. In contrast to other reports of cooperative invasion in which mixed cultures exhibit a follow-the-leader mechanism, we show coincident emergence of S- and N-type cells from mixed spheroids. Our data suggest mutual effects between the two cell types. Thus, whereas coculture with S-type cells induces N-type invasion, coculture with N-type cells slows S-type invasion. Using matrix metalloproteinase (MMP) inhibitors and cell incorporation assays, we demonstrate that MMP activity is required for S-type cells to insert into layers of N-type cells. Our study therefore highlights an important role for S-type neuroblastoma cells in the invasion process and reveals a new mechanism of cooperative invasion.

Genome-wide transcriptional comparison of MPP<sup>+</sup> treated human neuroblastoma cells with the state space model

This study compared a parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP+) response in two distinct phenotypes of human neuroblastoma cell lines: neuronal N-type SH-SY5Y cells and flat substrate-adherent S-type SH-EP cells. SH-SY5Y and SH-EP cells shared only 14% of their own MPP+ response genes, and their gene ontology (GO) analysis revealed significant endoplasmic reticulum (ER) stress by misfolded proteins. Gene modules, which are groups of transcriptionally co-expressed genes with similar biological functions, were identified for SH-SY5Y and SH-EP cells by using time-series microarray data with the state space model (SSM). All modules of SH-SY5Y and SH-EP cells showed strong positive auto-regulation that was often mediated via signal molecules and may cause bi-stability. Interactions in gene levels were calculated by using SSM parameters obtained in the process of module identification. Gene networks that were constructed from the gene interaction matrix showed different hub genes with high node degrees between SH-SY5Y and SH-EP cells. That is, key hub genes of SH-SY5Y cells were DCN, HIST1H2BK, and C5orf40, whereas those of SH-EP cells were MSH6, RBCK1, MTHFD2, ZNF26, CTH, and CARS. These results suggest that inhibition of the mitochondrial complex I by MPP+ might induce different downstream processes that are cell type dependent.

SOX2 promotes tumorigenicity and inhibits the differentiation of I-type neuroblastoma cells.

SOX2 is a transcription factor associated with the pluripotency, proliferative potential, and self-renewing properties observed with embryonic stem cells and germ cells. SOX2 expression has been reported in several cancers and is implicated in tumorigenesis. We previously found that SOX2 expression was correlated to the clinical stage of neuroblastoma. Recently, we found that SOX2 overexpression occurs in I-type neuroblastoma cells (BE(2)-C cells). To elucidate the tumorigenic function of SOX2, we established a SOX2 overexpressed BE(2)-C cell line. SOX2 overexpressed cells showed higher tumorigenicity than control cells and exhibited decreased expression levels of marker proteins of N- or S-type cells after agent-induced differetiation. By contrast, in cells where SOX2 mRNA expression was knocked down by gene-specific siRNA, tumorigenicty was significantly decreased and the expression levels of marker proteins of N- or S-type cells were upregulated. In conclusion, our findings indicate an important function for SOX2 in promoting tumorigenicity of I-type neuroblastoma cells and in inhibiting their differentiation, suggesting that SOX2 might be a potential therapeutic target in neuroblastoma.

Downregulation of OCT4 promotes differentiation and inhibits growth of BE (2)-C human neuroblastoma I-type cells

In invitro continuous culture, N (neuroblastic)-, S(substrate adherent)- and I (intermediate)-type cells were identified in human neuroblastoma (NB), where I-type is recognized as a stem cell type. Octamer-binding protein 4 (OCT4) is a cell marker used to identify the stemness of cells, whose roles in regulating I-type NB cells have yet to be fully elucidated. In the present study, we assessed the differentiation regulation role of OCT4 in BE (2)-C of I-type cells. We demonstrated that downregulation of OCT4 expression in BE (2)-C was associated with reduced cell proliferation and loss of colony formation ability on soft agar, and prompted BE (2)-C cells to differentiate to S-type cells. By contrast, overexpression of OCT4 increased cell proliferation and colony formation ratio, but no obvious differentiation promotion was observed. Furthermore, induced differentiation of BE (2)-C cells to S-type by 5-bromo-2'-deoxyuridine (BrdUrd) was accompanied by reduced expression of OCT4.

Kidins220/ARMS depletion is associated with the neural-to Schwann-like transition in a human neuroblastoma cell line model

Peripheral neuroblastic tumors exist as a heterogeneous mixture of neuroblastic (N-type) cells and Schwannian stromal (S-type) cells. These stromal cells not only represent a differentiated and less aggressive fraction of the tumor, but also have properties that can influence the further differentiation of nearby malignant cells. In vitro neuroblastoma cultures exhibit similar heterogeneity with N-type and S-type cells representing the neuroblastic and stromal portions of the tumor, respectively, in behavior, morphology, and molecular expression patterns. In this study, we deplete kinase D-interacting substrate of 220kD (Kidins220) with an shRNA construct and thereby cause morphologic transition of the human SH-SY5Y neuroblastoma cell line from N-type to S-type. The resulting cells have similar morphology and expression profile to SH-EP1 cells, a native S-type cell line from the same parent cell line, and to SH-SY5Y cells treated with BrdU, a treatment that induces S-type morphology. Specifically, both Kidins220-deficient SH-SY5Y cells and native SH-EP1 cells demonstrate down-regulation of the genes DCX and STMN2, markers for the neuronal lineage. We further show that Kidins220, DCX and STMN2 are co-down-regulated in cells of S-type morphology generated by methods other than Kidins220 depletion. Finally, we report that the association of low Kidins220 expression with S-type morphology and low DCX and STMN2 expression is demonstrated in spontaneously occurring human peripheral neuroblastic tumors. We propose that Kidins220 is critical in N- to S-type transition of neural crest tumor cells.

Store-operated Ca2 + entry in proliferating and retinoic acid-differentiated N- and S-type neuroblastoma cells

Neuroblastoma cell lines are heterogeneous, comprised of at least three distinct cell phenotypes; neuroblastic N-type cells, non-neuronal substrate-adherent S-type cells and intermediate I-type cells. N- and S-type cell populations were enriched from the parental SH-SY5Y neuroblastoma cell line and induced to differentiate by the addition of retinoic acid (RA), a drug used in the treatment of neuroblastoma. N- and S-type cells were identified based on their differential expression of β-tubulin III, vimentin and Bcl-2. Store-operated Ca2+ entry (SOCE) was then measured in proliferating and differentiated N- and S-type cell populations and the expression of STIM1, Orai1 and TRPC1, three proteins reported to play a key role in SOCE, was determined. In N-type cells the RA-induced switch from proliferation to differentiation was accompanied by a down-regulation in SOCE. STIM1 and Orai1 expression became down-regulated in differentiated cells, consistent with their respective roles as ER Ca2+ sensor and store-operated Ca2+ channel (SOC). TRPC1 became up-regulated suggesting that TRPC1 is not involved in SOCE, at least in differentiated N-type cells. In S-type cells SOCE remained active following the RA-induced switch from proliferation to differentiation and the expression of STIM1 and Orai1 remained unchanged. TRPC1 was not expressed in S-type cells. Our results indicate that differentiation of neuronal cells is associated with a remodelling of SOCE. Therapeutic targeting of SOCE proteins could potentially be a means of promoting neuronal differentiation in the treatment of neuroblastoma.

The Role of p75NTR and Its Signaling Pathways in Fenretinide (4HPR)-Induced Apoptosis in Neuroblastoma Cells (P06.003)

Objective: The overall goal is to understand the role of p75NTR and its signaling pathways in Fenretinide (4-hydroxyphenyl retinamide - 4HPR) induced apoptosis in neuroblastoma cells and determine some of the signaling pathways that mediate this effect. Understanding the role of p75NTR in mediating 4HPR-induced apoptosis will facilitate development of targeted therapies for the treatment of neuroblastoma by providing insight into pathways that could be targeted for combination chemotherapy. Background 4HPR is well tolerated in clinical trials and has been shown to cause apoptosis by increasing ROS generation in cancer cells. Targeting minimal residual disease in neuroblastoma involves sensitizing these tumors to chemotherapeutics by manipulating receptor signaling. p75NTR has a pro/anti-apoptotic role depending on the intracellular conditions. Design/Methods: S-type neuroblastoma cells (SH-EP1, SK-N-AS, SK-N-MC) with varying amounts of p75NTR are used to determine the effect of p75NTR signaling on the 4HPR-concentration curve. Changes in oxidative stress as a result of 4HPR treatment are recorded using western blotting and fluorescent microscopy. Results: 4HPR reduces cell viability in a variety of neuroblastoma cell lines when they are treated for 24(15%)-72 hrs (40%). p75NTR expression is pro-apoptotic and sensitizes SH-EP1 cells by decreasing viability, metabolic health and upregulating 4HPR-induced apoptosis. Treatment of SH-EP1 cells with 4HPR for 72 hours revealed no change in production of cellular OH- or cytoslic O2- but a sharp increase was observed in mitochondrial O2- production(Scr>p75shRNA transfected SH-EP1). Scavenging oxygen radicals in the mitochondria or inhibiting Complex II inhibits the pro-apoptotic effect of Fenretinide. Akt signaling is upregulated in SH-EP1 cells with p75shRNA compared to native SH-EP1 cells. Conclusions: This will facilitate the sensitization of minimal residual disease and preventing recurrence using combination chemotherapeutics for treatment by creating conditions of increased oxidative stress and enhance response by tumors to conventional chemotherapeutics causing apoptosis. Disclosure: Dr. Ganeshan has nothing to disclose. Dr. Schor has nothing to disclose.

Abstract 1528: Cooperative cross-talk between neuroblastoma subtypes confers resistance to anaplastic lymphoma kinase inhibition

Abstract Neuroblastoma is a pediatric solid tumor that can be stratified into stroma-rich and stroma-poor histological subgroups. The stromal compartment of neuroblastoma is composed mostly of Schwann cells and they play critical roles in the differentiation, survival and angiogenic responses of tumor cells. In certain neuroblastoma cell lines, the coexistence of neuroblastic N-type and substrate-adherent S-type is frequently observed. One such cell line, SK-N-SH, harbors a F1174L oncogenic mutation in the Anaplastic Lymphoma Kinase (ALK) gene. Treatment of SK-N-SH with an ALK chemical inhibitor, TAE684, resulted in the outgrowth of S-type cells that expressed the Schwann cell marker, S100α6. Nucleotide sequencing analysis of these TAE684-resistant (TR) sublines revealed the presence of the ALK F1174L mutation suggesting their tumor origin, although ALK protein was not detected. Consistent with these findings, TR cells displayed ∼9-fold higher IC50 values than N-type cells. Also, unlike N-type cells, TR cells have readily detectable phosphorylated STAT3 but weaker phosphorylated AKT. Under co-culture conditions, TR cells conferred survival to N-type cells against the apoptotic effect of TAE684. Co-cultivation also greatly enhanced the overall phosphorylation of STAT3 and its transcriptional activity in N-type cells. Finally, conditioned medium from TR clones enhanced cell viability of N-type cells and this effect was Phosphatidylinositol 3-Kinase-dependent. Taken together, these results demonstrate the ability of tumor-derived S-type cells in protecting N-type cells against the apoptotic effect of an ALK kinase inhibitor through upregulating pro-survival signaling. This project was supported by MACC funds, Advancing a Healthier Wisconsin, Wisconsin Breast Cancer Showhouse (O.W.), NIH CA133669 (H.G. and A.M.C.) and the Children's Research Institutes of the Children's Hospital of Wisconsin. 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 1528. doi:1538-7445.AM2012-1528

Outcome of the p53-mediated DNA damage response in neuroblastoma is determined by morphological subtype and MYCN expression

Background: MYCN oncogene amplification occurs in 20-25% of neuroblastoma and is associated with a poor prognosis. We previously reported that MYCN amplified (MNA) p53 wild-type neuroblastoma cell lines failed to G1 arrest in response to irradiation, but this could not be attributed to MYCN alone. Hypothesis: Genes co-amplified with MYCN and/or the predominant cell type, neuronal (N) or substrate adherent (S) phenotypes determine the downstream response to DNA damage in neuroblastoma cell lines. Methods: The MYCN amplicons of five MNA and two non-MNA cell line were mapped using 50K Single Nucleotide Polymorphism (SNP) arrays. One MNA (NBL-W) and one non-MNA neuroblastoma cell line (SKNSH) were sub-cloned into N and S-type cells and the p53 pathway investigated after irradiation induced DNA damage. To determine the role of p53 it was knocked down using siRNA. Results: No genes with a potential role in cell cycle regulation were consistently co-amplified in the MNA cell lines studied. High MYCN expressing NBLW-N cells failed to G1 arrest following irradiation and showed impaired induction of p21 and MDM2, whereas low MYCN expressing NBLW-S cells underwent a G1 arrest with induction of p21 and MDM2. Conversely N type cells underwent higher levels of apoptosis than S type cells. Following p53 knockdown in SHSY5Y N-type cells there was a decrease in apoptosis. Conclusions: The downstream response to DNA damage in p53 wild-type neuroblastoma cell lines is p53 dependent, and determined both by the morphological sub-type and MYCN expression.

Comparison of genomic profiles in human neuroblastic SH-SY5Y and substrate-adherent SH-EP cells using array comparative genomic hybridization

Human neuroblastoma is the most commonly diagnosed solid tumor in children. The existence and development of S-type cells is important for the prognosis and malignant properties in neuroblastoma patients. However, their origin is controversial and the relationship between S- and N-type cells in neuroblastoma has not yet been clarified. To investigate the feasibility of inter-conversion and characteristic features between S-type cells lacking malignant potential and N-type cells having metastatic potential, the genomic profiles of neuroblastoma SH-EP (S-type) and SH-SY5Y (N-type) cells were compared at high resolution. Common gain segments (>10 Mb) between SH-EP and SH-SY5Y cells were observed at 1q21.1–q44 and 17q21.32–q25.3. The results of the fluorescent in situ hybridization (FISH) analysis showed good agreement with the array-CGH data. Genome-wide inspection of SH-EP and SH-SY5Y cells successfully identified not only common chromosomal aberrations but also genomic variations, suggesting that interconversion could not take place between S- and Ntype cells. The identified differences between less aggressive S-type cells (SH-EP) and highly aggressive neuroblastic N-type cells (SH-SY5Y) might be useful for understanding tumorigenicity and discovery of potential new markers in neuroblastoma. This is the first effort to compare genomic profiles between less aggressive S-type cells (SH-EP) and highly aggressive neuroblastic N-type cells (SH-SY5Y) at high resolution.

Cooperative Cross-Talk between Neuroblastoma Subtypes Confers Resistance to Anaplastic Lymphoma Kinase Inhibition

Neuroblastoma is a pediatric solid tumor that can be stratified into stroma-rich and stroma-poor histological subgroups. The stromal compartment of neuroblastoma is composed mostly of Schwann cells, and they play critical roles in the differentiation, survival, and angiogenic responses of tumor cells. In certain neuroblastoma cell lines, the coexistence of neuroblastic N-type and substrate-adherent S-type is frequently observed. One such cell line, SK-N-SH, harbors a F1174L oncogenic mutation in the anaplastic lymphoma kinase (ALK) gene. Treatment of SK-N-SH with an ALK chemical inhibitor, TAE684, resulted in the outgrowth of S-type cells that expressed the Schwann cell marker, S100α6. Nucleotide sequencing analysis of these TAE684-resistant (TR) sublines revealed the presence of the ALK F1174L mutation, suggesting their tumor origin, although ALK protein was not detected. Consistent with these findings, TR cells displayed approximately 9-fold higher IC(50) values than N-type cells. Also, unlike N-type cells, TR cells have readily detectable phosphorylated STAT3 but weaker phosphorylated AKT. Under coculture conditions, TR cells conferred survival to N-type cells against the apoptotic effect of TAE684. Cocultivation also greatly enhanced the overall phosphorylation of STAT3 and its transcriptional activity in N-type cells. Finally, conditioned medium from TR clones enhanced cell viability of N-type cells, and this effect was phosphatidylinositol 3-kinase dependent. Taken together, these results demonstrate the ability of tumor-derived S-type cells in protecting N-type cells against the apoptotic effect of an ALK kinase inhibitor through upregulating prosurvival signaling.

High-resolution morphological identification and characterization of living neuroblastoma SK-N-SH cells by hopping probe ion conductance microscopy

It is well known that heterogeneous neuroblastoma (NB) has three different phenotypic variants; the differentiation and transdifferentiation among them increase the complexity of this cancer. However, high-resolution characterization of their morphology has not been addressed by conventional microscopy. Under the help of high-resolution hopping probe ion conductance microscopy, we demonstrated that three morphologically distinct cell populations exist in live NB SK-N-SH cell line. Among these three types cells, N-type cells had the smallest cell volume and the biggest height, S-type owned the biggest cell volume and the smallest height, and I-type was intermediate between N- and S-type. The difference of microvilli morphology rendered I- and N-type cells with a higher RMS than S-type cells. These high-resolution morphologic criteria can provide more direct information to distinguish different cell types in NB tumor, and may give a hand to the development of new therapies and diagnosis.