Lung-Residing Metastatic and Dormant Neuroblastoma Cells
2011; Elsevier BV; Volume: 179; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2011.03.020
ISSN1525-2191
AutoresLiat Edry Botzer, Shelly Maman, Orit Sagi‐Assif, Tzipi Meshel, Ido Nevo, Tobias Bäuerle, Ilana Yron, Isaac P. Witz,
Tópico(s)Lung Cancer Research Studies
ResumoThe mechanism by which dormant tumor cells can begin growing after long periods of inactivity and accelerate disease recurrence is poorly understood. The present study characterizes dormant neuroblastoma (NB) cells, as well as metastatic cells, which reside in the same organ microenvironment. A xenograft model of human NB consisting of variants that generate nonmetastatic local tumors in the orthotopic inoculation site and variants that generate lung metastatic NB (MetNB) cells was developed in our laboratory. The present study shows that lungs of mice inoculated with nonmetastatic NB variants contain disseminated neuroblastoma (DisNB) human cells. Both DisNB and MetNB variants expressed a similar tumorigenicty phenotype in vivo, whereas the MetNB variants produced a heavy metastatic load and the DisNB variants produced no or little metastasis. A comparative in vitro characterization of MetNB and DisNB cells revealed similarities and differences. DisNB, but not MetNB cells, expressed the minimal residual disease markers PHOX2B and TH. MetNB cells demonstrated higher migratory capacity, an elevated matrix metalloproteinase (MMP) secretion, and a higher constitutive phosphorylation of extracellular signal-regulated kinase (ERK) than DisNB cells. We suggest that characteristics common to both MetNB and DisNB cells were acquired relatively early in the metastatic process and the characteristics that differ between these variants were acquired later. We hypothesize that the DisNB cells are metastasis precursors, which may progress toward metastasis under certain microenvironmental conditions. The mechanism by which dormant tumor cells can begin growing after long periods of inactivity and accelerate disease recurrence is poorly understood. The present study characterizes dormant neuroblastoma (NB) cells, as well as metastatic cells, which reside in the same organ microenvironment. A xenograft model of human NB consisting of variants that generate nonmetastatic local tumors in the orthotopic inoculation site and variants that generate lung metastatic NB (MetNB) cells was developed in our laboratory. The present study shows that lungs of mice inoculated with nonmetastatic NB variants contain disseminated neuroblastoma (DisNB) human cells. Both DisNB and MetNB variants expressed a similar tumorigenicty phenotype in vivo, whereas the MetNB variants produced a heavy metastatic load and the DisNB variants produced no or little metastasis. A comparative in vitro characterization of MetNB and DisNB cells revealed similarities and differences. DisNB, but not MetNB cells, expressed the minimal residual disease markers PHOX2B and TH. MetNB cells demonstrated higher migratory capacity, an elevated matrix metalloproteinase (MMP) secretion, and a higher constitutive phosphorylation of extracellular signal-regulated kinase (ERK) than DisNB cells. We suggest that characteristics common to both MetNB and DisNB cells were acquired relatively early in the metastatic process and the characteristics that differ between these variants were acquired later. We hypothesize that the DisNB cells are metastasis precursors, which may progress toward metastasis under certain microenvironmental conditions. Neuroblastoma (NB) is the most common extracranial solid tumor in children comprising 8% to 10% of all childhood cancers. More than half of these patients have a metastatic disease at diagnosis.1Mullassery D. Dominici C. Jesudason E.C. McDowell H.P. Losty P.D. Neuroblastoma: contemporary management.Arch Dis Childhood-E. 2009; 94: 177-185Crossref PubMed Scopus (27) Google Scholar, 2Modak S. Cheung N.K.V. Neuroblastoma: therapeutic strategies for a clinical enigma.Cancer Treat Rev. 2010; 36: 307-317Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 3Maris J.M. Hogarty M.D. Bagatell R. Cohn S.L. Neuroblastoma.Lancet. 2007; 369: 2106-2120Abstract Full Text Full Text PDF PubMed Scopus (1670) Google Scholar NB cells disseminate either by hematogenous spread, producing metastasis most frequently in bone marrow, bone, liver, and skin, or by lymphatic spread to regional and distant lymph nodes.4Brodeur G.M. Neuroblastoma.in: Amsterdam edition. Elsevier, New York2000: 1-583Google Scholar Lung metastases are considered a terminal event representing a widely disseminated metastatic disease.5Cowie F. Corbett R. Pinkerton C.R. Lung involvement in neuroblastoma: incidence and characteristics.Med Pediatr Oncol. 1997; 28: 429-432Crossref PubMed Scopus (40) Google Scholar, 6Kammen B.F. Matthay K.K. Pacharn P. Gerbing R. Brasch R.C. Gooding C.A. Pulmonary metastases at diagnosis of neuroblastoma in pediatric patients: CT findings and prognosis.AJR Am J Roentgenol. 2001; 176: 755-759Crossref PubMed Scopus (42) Google Scholar Approximately 50% of children with high-risk NB that complete consolidation therapy develop early or late relapse, often from minimal residual disease in the form of circulating NB cells or micrometastases.7Reynolds C.P. Detection and treatment of minimal residual disease in high-risk neuroblastoma.Pediatr Transplant. 2004; 8: 56-66Crossref PubMed Scopus (47) Google Scholar Most of the children with NB present metastatic disease at diagnosis with poor outcome, despite intensive treatment protocols.8Brodeur G.M. Neuroblastoma: biological insights into a clinical enigma.Nat Rev Cancer. 2003; 3: 203-216Crossref PubMed Scopus (1688) Google Scholar The presence of circulating NB cells and/or NB micrometastasis may indicate a significant high-risk disease.9Kuroda T. Morikawa N. Matsuoka K. Fujino A. Honna T. Nakagawa A. Kumagai M. Masaki H. Saeki M. Prognostic significance of circulating tumor cells and bone marrow micrometastasis in advanced neuroblastoma.J Pediatr Surg. 2008; 43: 2182-2185Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar However, the question whether NB micrometastases develop into metastatic disease is yet to be answered. Previous studies from our laboratory were aimed to identify molecular pathways that are involved in NB metastasis. We focus on the cross talk between metastatic NB cells and components of their microenvironment, and on the downstream effects of such interactions. We suggested that NB cells might use chemokine-chemokine receptor axes in their progression to metastasis. For example the CXCR4–CXCL1210Geminder H. Sagi-Assif O. Goldberg L. Meshel T. Rechavi G. Witz I.P. Ben-Baruch A. A possible role for CXCR4 and its ligand, the CXC chemokine stromal cell-derived factor-1, in the development of bone marrow metastases in neuroblastoma.J Immunol. 2001; 167: 4747-4757PubMed Scopus (345) Google Scholar, 11Nevo I. Sagi-Assif O. Meshel T. Geminder H. Goldberg-Bittman L. Ben-Menachem S. Shalmon B. Goldberg I. Ben-Baruch A. Witz I.P. The tumor microenvironment: cXCR4 is associated with distinct protein expression patterns in neuroblastoma cells.Immunol Lett. 2004; 92: 163-169Crossref PubMed Scopus (23) Google Scholar and the CX3CL1-CX3CR112Nevo I. Sagi-Assif O. Meshel T. Ben-Baruch A. Johrer K. Greil R. Trejo L.E. Kharenko O. Feinmesser M. Yron I. Witz I.P. The involvement of the fractalkine receptor in the transmigration of neuroblastoma cells through bone-marrow endothelial cells.Cancer Lett. 2009; 273: 127-139Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholaraxes take part in extravasation, trans-endothelial migration and invasion thereby promoting progression. CXCR3 on the other hand fulfills antimalignancy functions.13Goldberg-Bittman L. Sagi-Assif O. Meshel T. Nevo I. Levy-Nissenbaum O. Yron I. Witz I.P. Ben-Baruch A. Cellular characteristics of neuroblastoma cells: regulation by the ELR–CXC chemokine CXCL10 and expression of a CXCR3-like receptor.Cytokine. 2005; 29: 105-117Crossref PubMed Scopus (31) Google Scholar To advance the understanding of the molecular mechanisms that promote NB metastasis we developed an orthotopic mouse model for human NB metastasis. An orthotopic implantation of two human NB cell lines (MHH-NB11 and SH-SY5Y) into the adrenal gland of athymic nude mice yielded local adrenal tumors, as well as lung metastases. After repeated cycles of in vivo passages, local and lung metastatic variants were generated.14Nevo I. Sagi-Assif O. Edry Botzer L. Amar D. Maman S. Kariv N. Leider-Trejo L.E. Savelyeva L. Schwab M. Yron I. Witz I.P. Generation and characterization of novel local and metastatic human neuroblastoma variants.Neoplasia. 2008; 10: 816-827Abstract Full Text PDF PubMed Scopus (23) Google Scholar The local variants form tumors at the orthotopic inoculation site and do not form lung metastasis (as judged by histopathology), whereas the metastatic variants from the same NB cell lines form local tumors as well as macroscopic lung metastasis after orthotopic inoculation into the adrenal gland.14Nevo I. Sagi-Assif O. Edry Botzer L. Amar D. Maman S. Kariv N. Leider-Trejo L.E. Savelyeva L. Schwab M. Yron I. Witz I.P. Generation and characterization of novel local and metastatic human neuroblastoma variants.Neoplasia. 2008; 10: 816-827Abstract Full Text PDF PubMed Scopus (23) Google Scholar Originating in the same tumors, these variants have an identical genetic background. Genomic, proteomic, or transcriptomic differences between these variants can thus be ascribed to the differences in their metastatic phenotype. This model system was primarily established to gain additional understanding of biological mechanisms leading to metastasis. The model system was used as a generic metastasis model rather than as a tool to characterize specific clinical manifestations of NB metastasis. Nonetheless, a small set of genes that were differentially expressed in the metastatic and the local variants could segregate stage 4 and stage 1 NB patients.15Nevo I. Oberthuer A. Botzer E. Sagi-Assif O. Maman S. Pasmanik-Chor M. Kariv N. Fischer M. Yron I. Witz I.P. Gene-expression-based analysis of local and metastatic neuroblastoma variants reveals a set of genes associated with tumor progression in neuroblastoma patients.Int J Cancer. 2010; 126: 1570-1581PubMed Google Scholar The molecular signatures shared by metastatic NB variants and stage 4 NB patients, and the signature shared by local NB variants and stage 1 NB patients, highlights the translational significance of the orthotopic mouse model for human NB metastasis. In the present study, we used the previously mentioned model of NB metastasis to examine the tumorogenicity and metastatic capacity of the local and metastatic NB variants. We show that nude mice orthotopically inoculated with local NB variants that do not form metastasis in the lungs, nonetheless, harbor disseminated NB (DisNB) cells in their bone marrow and lungs. Two NB cell populations sharing the same genetic background, invading the same distant organ, but differing in their ability to create metastasis in this organ were thus obtained. The characteristics differentiating these populations may be those required from dormant micrometastatic tumor cells to progress toward metastasis. In this study, we performed a comparative phenotypic analysis of these two NB cell populations. NB variants generated from the parental cell line MHH-NB-1116Pietsch T. Gottert E. Meese E. Blin N. Feickert H.J. Riehm H. Kovacs G. Characterization of a continuous cell line (MHH-NB-11) derived from advanced neuroblastoma.Anticancer Res. 1988; 8: 1329-1333PubMed Google Scholar (kindly provided by Dr. Torsten Pietsch, Department of Neuropathology, University of Bonn Medical Center, Bonn, Germany) and SH-SY5Y, as well as murine DA3 cells, were maintained in culture as previously described.14Nevo I. Sagi-Assif O. Edry Botzer L. Amar D. Maman S. Kariv N. Leider-Trejo L.E. Savelyeva L. Schwab M. Yron I. Witz I.P. Generation and characterization of novel local and metastatic human neuroblastoma variants.Neoplasia. 2008; 10: 816-827Abstract Full Text PDF PubMed Scopus (23) Google Scholar Generation of metastatic neuroblastoma (MetNB) and disseminated neuroblastoma (DisNB) variants was performed in male athymic nude mice (BALB/c background) purchased from Harlan Laboratories Ltd. (Jerusalem, Israel) or from Charles River (Sulzfeld, Germany) and used for tumorigenicity and metastasis experiments. Mice (7- to 10-weeks-old) were housed and maintained for approximately 6 months in laminar flow cabinets under specific pathogen-free conditions at the animal quarters of Tel-Aviv University or at the central animal facility of the German Cancer Research Center (DKFZ). Experiments were performed in accordance with current regulations and standards of the Tel-Aviv University Institutional Animal Care and Use Committee and with the institutional guidelines of the local animal ethics committee of the central animal facility of the German Cancer Research Center (DKFZ). Cells were harvested and transferred to RPMI 1640 medium supplemented with 5% to 10% Fetal Calf Serum (FCS). Only single-cell suspensions of greater than 90% viability (trypan blue exclusion) were used for injection. Anesthesia was induced either by ketamine (100 mg/kg body mass; Kepro Deventer, The Netherlands) and 2% xylazine (10 mg/kg body mass; Medical Market, Tel Izhak, Israel) administered intraperitoneally or by a mixture of laughing gas (nitrous oxide; 1 L/min), oxygen (0.5 L/min), and isoflurane (1 to 1.5 vol. %). Tumor cells (1 × 106/50 μL) were injected orthotopically into the adrenal gland. The injected volume was small enough to keep the mechanical pressure low to prevent leakage of cells out of the gland. Furthermore, a real-time PCR assay to determine the presence of human cells in nude mice (see as follows) indicated that nude mice injected intravenously with human NB cells contained such cells in their lungs 2 hours after inoculation. On the other hand, no such cells could be detected in the lungs of mice that received an intra-adrenal inoculation of the same cells, 2 hours as well as 24 hours after inoculation. These results clearly showed that there was no NB cell leakage into the asculature after an intra-adrenal inoculation. The intra-adrenal inoculation required surgical exposure of the left adrenal gland under anesthesia, as previously described.14Nevo I. Sagi-Assif O. Edry Botzer L. Amar D. Maman S. Kariv N. Leider-Trejo L.E. Savelyeva L. Schwab M. Yron I. Witz I.P. Generation and characterization of novel local and metastatic human neuroblastoma variants.Neoplasia. 2008; 10: 816-827Abstract Full Text PDF PubMed Scopus (23) Google Scholar Briefly, a left-side, high-paracostal approach to the abdomen allowed visualization of the cranial tip of the left kidney. A 27-gauge needle was introduced through the left adrenal fat pad into the adrenal gland after retraction of the left kidney. Special care was taken so as not to penetrate the back side of the adrenal gland. The peritoneum was closed by surgical stitching and the skin was adapted using clips. We monitored and verified the development of local tumors in the adrenal gland using an ultrasound imaging in vivo. Mice were anesthetized by 2% inhalation of isoflurane (Halocarbon Products, River Edge, NJ) delivered with oxygen, using a breathing anesthetic delivery system. The high-resolution small animal ultrasound system (Vevo 770; VisualSonic, Toronto, Canada) was used for the visualization of adrenal tumors. Imaging settings were standardized and unchanged throughout the experiment. All mice inoculated with the both the local, as well as the metastatic variants developed adrenal tumors. Tumor volume was calculated using the ellipsoid equation: π/6 × length × width × height (Figure 1). Mice were sacrificed and the local (adrenal) tumor, lungs, bone marrow, and other peritoneal organs suspected to harbor metastases were removed. Tumor lesions and organs were either fixed in 4% buffered formalin and embedded in paraffin for histopathological examination, or cultured for the isolation of human NB cells. For the culturing of bone marrow cells, bone marrow was isolated from the tibias and femurs. The bone marrow was triturated using a 25-gauge needle in RPMI 1640 medium. The cells were then washed twice in PBS and resuspended in RPMI 1640 medium. Total RNA was isolated from cells or organs using the EZ-RNA kit (Biological Industries, Beit Haemek, Israel), according to the manufacturer's instructions. Pieces of lungs and sternum of inoculated mice were harvested and immediately placed on dry ice. RNA was isolated from frozen tissues and used for generation of first-strand cDNA synthesis, using the M-MLV Reverse Transcriptase (Ambion, Inc., Austin, TX) according to the manufacturer's instructions. PCR oligonucleotide primers were designed according to sequences derived from the National Center for Biotechnology Information database, and described in Table 1. Primers for this reaction were designed to react specifically with the human plasminogen activator receptor, urokinase type (PLAUR) sequence and have no cross reactivity with the mouse PLAUR sequence.Table 1PCR Oligonucleotide PrimersReaction specificityAccession no.Primer designationSequencePCRHumanNM_001005376PLAUR (365)-S5′-GTCACCTATTCCCGAAGCCG-3'PLAUR (954)-AS5′-CGGTACTGGACATCCAGGTCT-3'RT-PCRHumanNM_000360TH (1218)-S5′-TGTACTGGTTCACGGTGGAGTT-3'TH (1339)-AS5′-AATCTCAGGCTCCTCAGACA-3'NM_003924.3PHOX2B (592)-S5′-TACGCCGCAGTTCCTTACAA-3'PHOX2B (711)-AS5′-GAAGACCCTTTCCAGCTCTTT-3'HumanNM_004048β2 mol/L(415)-S5′-ATGTAAGCAGCATCATGGAG-3'β2 mol/L(484)-AS5′-AAGCAAGCAGAATTTGGAAT-3'Human and miceNM_001013RS9(341)-S5′-TTACATCCTGGGCCTGAAGAT-3'RS9(490)-AS5′-GGGATGTTCACCACCTGCTT-3'The primer designation indicates the first nucleotide according to sequences derived from the National Center for Biotechnology Information database and the direction of the primer, sense (S) or anti-sense (AS).PLAUR, plasminogen activator receptor, urokinase type; TH, tyrosine hydroxylase. Open table in a new tab The primer designation indicates the first nucleotide according to sequences derived from the National Center for Biotechnology Information database and the direction of the primer, sense (S) or anti-sense (AS). PLAUR, plasminogen activator receptor, urokinase type; TH, tyrosine hydroxylase. PLAUR oligonucleotides were used for the amplification of 589 bp. MHH-NB-11 cells were used as source for human cells, serving as positive control. Murine DA3 cells were used as negative control. The PCR reaction for human PLAUR in mouse tissues was: 94°C for 3 minutes, followed by 45 cycles of: 94°C for 40 seconds, 64°C for 1 minute, and 72°C for 40 seconds. Quantification of cDNA targets was performed on Rotor-gene 6000TM (Corbett life science, Sydney, Australia), using Rotor gene 6000 series software. All reactions were performed in duplicates. Transcripts were detected using SYBR Green I (Thermo Fisher scientific, Abgene, England) according to the manufacturer's instructions. Optimal reaction conditions for amplification of target genes were performed according to manufacturer's recommendation. PCR amplification was performed over 40 cycles (95°C for 15 seconds, 59°C for 20 seconds, and 72° for 15 seconds). Dissociation curves for each primer set indicated a single product and no-template controls were negative after 40 cycles. The sequence of the primers used is detailed in Table 1. For the expression level of paired-like Homeobox 2B (PHOX2B) and tyrosine hydroxylase (TH) results were normalized to ribosomal RNA (rS9) as an internal control. For the detection of human NB cells in mouse tissues, two pairs of specific primers that recognize human mRNA were used in each reaction. A set of primers for β2M, which does not react with mouse cDNA and a set of primers for the normalizing RS9 mRNA, which reacts with human as well as mouse cells, generating an identical product, enabling to compare a relative amount of mRNA copies in organs harvested from mice that were inoculated with DisNB or MetNB variants. The following human specific antibodies were used for flow cytometry: phycoerythrin conjugated anti-CD56, 0.03 μg/sample (DakoCytomation, Glostrup, Denmark); anti-CX3CL1, 1 μg/sample (Torrey Pines Biolabs, Inc., East Orange, NJ); anti CX3CR1, 1 μg/sample and anti-c-kit, 0.5 μg/sample (YB5.B8, eBioscience, San Diego, CA); anti-CXCR3, 2.5 μg/sample (49801), anti-CXCR5, 2.5 μg/sample (51505), anti-CXCR6, 2.5 μg/sample (56811), (R&D Systems); anti-CD44, 0.25 μg/sample (IM7, BioLegend, San Diego, CA); anti-CXCR4, 0.5 μg/sample (B-R24; Diaclone, Stamford, CT), anti-CD133 0.05 μg/sample (293C3, Miltenyi Biotec), anti-CD34 0.5 μg/sample (581, BD Biosciences Pharmingen, San Diego, CA). Anti–Human Leukocyte Antigen (HLA)-A–B–C, mAb (W6/32)17Parham P. Barnstable C.J. Bodmer W.F. Use of a monoclonal antibody (W6/32) in structural studies of HLA-A,B,C, antigens.J Immunol. 1979; 123: 342-349PubMed Google Scholarand anti–H-2 mAb (20-8-4S)18Ozato K. Sachs D.H. Monoclonal antibodies to mouse MHC antigens III Hybridoma antibodies reacting to antigens of the H-2b haplotype reveal genetic control of isotype expression.J Immunol. 1981; 126: 317-321PubMed Google Scholar were kindly provided by Dr. R. Ehrlich, Department of Cell Research and Immunology, Tel-Aviv University, and were used at 1:500 and 1:3000, respectively. Fluorescein isothiocyanate–conjugated goat anti-mouse IgG and goat anti-rabbit IgG (Jackson ImmunoResearchLaboratories, West Grove, PA) were used at 0.5 μg/sample as secondary antibodies for flow cytometry. For Western blot analysis: anti-extracellular signal-regulated kinase2 (C-14, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-phosphorylated-ERK1/2 (R&D Systems) at 1:1000; anti-phosphorylated AKT (S473, R&D Systems) at 1:2000; anti AKT1 (B-1, Santa Cruz Biotechnology at 1:1000; anti-paracingulin (CGNL1, Zymed Laboratories Inc., San Francisco, CA) at 1:500; anti-matrix metalloproteinase (MMP)-2 and anti-MM9 at 1:500 μg/mL (EMD Chemicals, Inc., San Diego, CA). Horseradish peroxidase-conjugated goat anti-mouse antibodies and rabbit anti-goat antibodies were used according to the manufacturer's instructions (Jackson ImmunoResearch Laboratories). The 5 × 105 cells were washed with FACS medium (RPMI 1640 medium supplemented with 5% FCS and 0.01% sodium azide). Samples were incubated for 1 hour at 4°C with the relevant primary antibody. After a wash with FACS medium, cells were incubated for 1 hour at 4°C with fluorescein isothiocyanate-conjugated secondary antibody. For double staining, we preformed another wash with FACS medium; cells were then incubated for an additional 1 hour at 4°C with another relevant antibody (conjugated with phycoerythrin). After an additional wash, antigen expression was determined using FACSort (Becton Dickinson, Mountain View, CA) and CellQuest (Becton Dickinson) software. Baseline staining was obtained by labeling the cells with secondary antibodies alone. Statistical analysis was performed using the Student's t-test. NB cells were lysed with RIPA buffer (20 mmol/L Tris, pH 8, 150 mmol/L NaCl, 1%NP-40, 0.1% SDS, 0.75% deoxycholate, 5 mmol/L EDTA, pH 8, 3 mmol/L EGTA, pH 8, 20 mmol/L sodium phosphate, pH 7.6, 2 mmol/L sodium orthovanadate, 5 mmol/L NaF, 5 mmol/L sodium pyrophosphate, pH 7.6, 2 ng/mL aprotinin, 2 ng/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride). Cell lysates were incubated for 20 minutes on ice, and cleared by centrifugation for 20 minutes at 16,000 g, 4°C. After the addition of Laemmli sample buffer, the lysates were boiled for 10 minutes, resolved on SDS-polyacrylamide gel electrophoresis (PAGE), and transferred onto nitrocellulose membrane. The target proteins were detected by using a relevant primary antibody and suitable horseradish peroxidase-conjugated secondary antibodies, which are described in the Antibodies section. Bands were visualized by enhanced chemiluminescence (ECL) (Amersham, Buckinghamshire, United Kingdom) and autoradiographed by exposure to Fuji film. The amount of the relevant protein in the lanes was estimated by densitometry and was calculated in reference to the loading control in the lane using Scion Image software (Scion, Frederick, MD). Cells were grown in six-well plates coated with 10 μg/mL fibronectin (Biological Industries). On confluence, the cell monolayer was wounded with a plastic tip, then washed twice with RPMI 1640 medium, and replaced with fresh growth medium. Closure of the denuded area was monitored using an inverted microscope (Eclipse TE 2000-S; Nikon, Enfield, CT) and fitted with a digital camera (DXM1200F; Nikon). Photograph documentation was taken at days 1, 2, and 3 after the wounding. NB cells were incubated overnight in growth medium. After 24 hours the medium was replaced by serum-free RPMI 1640 medium for an additional 24 hours. The presence of matrix metalloproteinases in the conditioned medium was determined by separation on 7.5% SDS-PAGE containing 0.1% gelatin as substrate. After electrophoresis, gels were washed three times in 50 mmol/L Tris–HCl, pH 7.4, containing 2.5% Triton X-100. The gels were then washed three times in 50 mM Tris–HCl buffer, pH 7.4, followed by incubation in a buffer, consisting of 50 mmol/L Tris–HCl, pH 7.4, 0.02% sodium azide, and 10 mmol/L CaCl2 for 48 hours at 37°C. After three additional washes in double-distilled H2O, gels were stained with 0.25% Coomassie blue and de-stained in 20% methanol and 10% glacial acetic acid, and clear bands of protein degradation were visualized. NB cells were harvested from 80% confluent monolayer cultures and seeded at a density of 1 × 104 to 105 cells per well in a 96-well, flat-bottomed, tissue culture plate. Proliferation and viability under normal conditions or under doxorubicin/desferrioxamine (DOX/DFX) treatment were monitored in triplicates using a XTT-based assay after 0, 24, 48, 72, and 96 hours, according to the manufacturer's instructions (Biological Industries). Absorbance at 450 nm (OD450) was determined for each well using an automated microplate reader (SpectraMax 190; Molecular Devices Corp., Sunnyvale, CA). Subtraction of nonspecific readings (measured at 630 nm) was automatically performed. To obtain the percentage of cell growth or cell viability, the OD450 of the cells (in each time point of Dox/DFX treatment) was divided by the OD450 of the cells at the initial time point of the experiment. NB cells were plated for 24 hours in growth medium. After 24 hours the medium was replaced with serum-free medium and cells were incubated for additional 24 hours. Cells were washed with ice-cold PBS and lysed. The lysates were resolved on SDS-PAGE and transferred onto nitrocellulose membrane as previously described. Total ERK2 or AKT, and phosphorylated ERK1/2 or AKT proteins were detected by using the relevant primary antibodies and suitable horseradish peroxidase-conjugated secondary antibodies (previously described). The amount of total ERK2/AKT was detected after stripping the membrane from the phosphorylated antibodies and exposing it to the relevant antibodies. Phosphorylation was calculated in reference to total protein in the lane as measured by densitometry using Scion Image software (Scion). An unpaired Student's t-test was used to compare in vitro results. The analysis of mouse survival was computed by the Kaplan-Meier method. We have previously demonstrated that 50% of mice inoculated orthotopically with cells of the metastatic variants (either MHH.Lu3 or SY5Y.Lu2) formed lung metastasis within a period of 6 to 10 weeks after inoculation. However, none of the mice inoculated orthotopically with the nonmetastatic variants (either MHH.Ad or SY5Y.Ad originating from NB MHH or SY5Y tumors, respectively) formed such metastasis up to 16-weeks postinoculation. Immunohistochemical examination using synaptophysin (an NB cell marker) did not detect NB cells in the lungs or bone marrow of these mice.14Nevo I. Sagi-Assif O. Edry Botzer L. Amar D. Maman S. Kariv N. Leider-Trejo L.E. Savelyeva L. Schwab M. Yron I. Witz I.P. Generation and characterization of novel local and metastatic human neuroblastoma variants.Neoplasia. 2008; 10: 816-827Abstract Full Text PDF PubMed Scopus (23) Google Scholar The possibility was raised that the nonmetastatic cells can reach the metastatic target organs, but are unable to propagate and form detectable metastases. To test this possibility, we cultured target organs and searched these cultures for human NB cell outgrowth, as well as for human RNA (described as follows). Lungs and bone marrow of nude mice orthotopically inoculated with nonmetastatic cells (either MHH.Ad or SY5Y.Ad variants) were put in culture in an attempt to possibly discover DisNB cells in these organs. Indeed, after a culture period of 6 to 12 weeks, an outgrowth of human NB cells was detected in the culture flasks. These cells were immunophenotyped by flow cytometry for the expression of human and mouse major histocompatibility complex class 1 antigens and for the expression of CD56, a marker for human NB.19Warzynski M.J. Graham D.M. Axtell R.A. Higgins J.V. Hanmers Y.A. Flow cytometric immunophenotyping test for staging/monitoring neuroblastoma patients.Cytometry. 2002; 50: 298-304Crossref PubMed Scopus (34) Google Scholar, 20Warzynski M.J. Graham D.M. Axtell R.A. Higgins J.V. Hammers Y.A. Flow cytometric immunophenotyping test for staging/monitoring neuroblastoma patients.Cytometry. 2000; 42: 324-325Google Scholar The results demonstrated that lungs and bone marrow of nude mice inoculated with nonmetastatic MHH or SY5Y NB cells harbored DisNB human cells. It should be noted that the bone marrow of mice inoculated with metastatic cells that developed only lung macrometastases did harbor DisNB cells (Figure 2), although no overt metastasis was detected in this compartment. Similar results were obtained with the bone marrow and lung metastasizing SY5Y.Lu2 variant (data not shown). RNA of cells cultured from
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