Cells Of Neuroectodermal Origin Research Articles

Abstract 4650: Quantitative assessment of the intracellular uptake of chlorotoxin in a U87 human glioma mouse model for the targeted drug delivery system

Abstract This study quantitatively evaluates the tumor cell-targeting capabilities and intracellular uptake pattern of chlorotoxin (CTX) when delivered intratumorally (IT) and intravenously (IV) using subcutaneous (SC) U87 human glioblastoma mouse model. CTX is a scorpion-derived polypeptide that selectively binds to tumor cells of neuroectodermal origin, such as glioma and a wide range of other tumor cells, but not normal cells. The quantification of CTX cellular uptake presented here will assist in the further development of our tumor-targeting drug delivery system (DDS) consisting of a chemotherapeutic encapsulated in nonionic surfactant vesicles embedded in a thermosensitive cross-linked chitosan hydrogel, which has affinity for MUC1 receptor overexpressed on many tumor cells, including low-differentiated glioma. CTX addition to the DDS will serve as a second tumor-targeting molecule. In this study, U87 human glioma cells were implanted SC in the flank of nude athymic/ncr/nus mice. When tumors reached approximately the volume of 0.25 cm3, mice were injected either IT or IV (tail vein) with 50 μl of 100 μg CTX or phosphate buffer saline (PBS) as a control. The tumors were harvested at 5 minutes post IT injection or 7 hours post IV injection, fixed, sectioned at 5 μm, and followed by either immunofluorescence or immunocytochemistry using anti-CTX primary antibody and AlexaFluor-594 conjugated secondary antibody or immunoperoxidase- AEC kit, and evaluated by confocal or scanning microscopy, respectively. CTX levels were compared between three different tumor regions (A, B, and C; A = injection site marked by methylene blue dye, C = most distant from injection site, B = equidistant between these two) after direct IT injection and compared to CTX levels in tumor tissues following IV injection. Quantitative evaluation of CTX levels was performed with ImageJ software, while further intracellular analysis of CTX presence utilized MATLAB system. CTX was found to be evenly distributed between cells within each region post IT injection, but decreased in intensity from the injection site (A) toward the tumor edge. With region A set at 100% CTX intensity, the levels decreased to 23.7% (in region B), and 2.1% (in region C), compared to 1.8% post IV injection. Our previous in vitro studies showed CTX localization near the nucleus in U87 glioma cells but not in normal human astrocytes. Current evaluation of tumor z-sections quantified levels of CTX inside U87 glioma cells in vivo. These results confirm the specificity of CTX uptake by U87 human glioma cells in vitro and in vivo with CTX tissue levels post IV injection, which is comparable to those post IT injection in the distant C tumor region. This proves CTX is an important second tumor-targeting molecule for our DDS system. Citation Format: Alan D. Roberts, Joseph O. Johnson, Aleksandra Karolak, Norma Alcantar, Katarzyna A. Rejniak, Marzenna Wiranowska. Quantitative assessment of the intracellular uptake of chlorotoxin in a U87 human glioma mouse model for the targeted drug delivery system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4650.

Novel drug - dinutuximab for high-risk neuroblastoma

Neuroblastoma is most common extracranialtumor in childrens of less than 5 year. Prognosis of high risk neuroblastoma is not good even after use of aggressive chemotherapy. Newer drug anti-GD2 monoclonal antibodies, dinutuximab in combination with granulocyte–macrophage colony-stimulating factor (GM-CSF) or interleukin-2 has been recently approved by FDA for the treatment of high risk neuroblastoma. Dinutuximab binds to the glycolipid GD2 present on neuroblastoma cells and on cells of neuroectodermal origin, and enhances cell lysis through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

Abstract 3909: Interactions of glioma cells with chlorotoxin: an ATR-FTIR spectroscopy study

Abstract Chlorotoxin is a scorpion-derived peptide that preferentially binds to tumor cells of neuroectodermal origin, like glioma, but not to normal non-transformed cells. We have previously reported the development of a targeted nanodelivery system using chlorotoxin. Chlorotoxin has been studied as an imaging and targeting agent for drug and radioisotope delivery, but the mechanism of interaction of chlorotoxin with cancer cells has not been well understood and it needs to be further evaluated in order to optimize its use as a targeting compound for cancer cells. In this study, we used U87 human glioma cell line to evaluate the binding kinetics of chlorotoxin by Attenuated Total Reflection Fourier Transform Infra-Red (ATR-FTIR) spectroscopy. As a sensitive and label free technique, ATR-FTIR is a powerful diagnostic tool for the comparison of cancer cells with normal cells. First, we characterized the signature spectra of chlorotoxin and U87 cells by assigning and evaluating the infrared absorption bands and their second derivatives. Next, we studied the spectral differences between U87 cells incubated with and without chlorotoxin for incremental time periods ranging from 15 minutes to 24 hours. We also examined the metabolic changes in U87 cells induced with chlorotoxin at different incubation time points by measuring the ratios of integrated areas corresponding to different chemical conformations of proteins, lipids, carbohydrates, and nucleic acids. Our results revealed spectral changes in U87 cells at different stages of incubation with chlorotoxin. The most notable change occurred after 30 minutes of incubation, where the band assigned to CH3 bending of lipid membranes shifted from 1456 cm-1 to 1450 cm-1. Consequently, a new shoulder appeared at wavenumber of 1466 cm-1 assigned to CH2 bending of lipid membranes for U87 cells treated with chlorotoxin. These changes indicate a direct interaction due to the presence of a molecular binding site between chlorotoxin and the lipids of the cell membrane. Another significant alternation occurred after 1 hour of incubation of U87 cells with chlorotoxin in the spectral region assigned for nucleic acids. This interaction included a large upshift of the band at 1036 cm-1 to 1051 cm-1 assigned to C-O stretching vibrations of ribose ring in RNA due to the hydrogen bonding with chlorotoxin. These findings suggest at least two different interactions of chlorotoxin with U87 human glioma cells that can be further explored for improving chlorotoxin containing drug delivery systems. We are currently using the ATR-FTIR method to conduct control experiments by measuring possible interactions of chlorotoxin with normal human astrocytes. Citation Format: Rana Falahat, Marzenna Wiranowska, Ryan Toomey, Norma Alcantar. Interactions of glioma cells with chlorotoxin: an ATR-FTIR spectroscopy study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3909.

Abstract 3677: Enhanced targeted delivery of paclitaxel to tumor cells of epithelial and neuroectodermal origin using chlorotoxin-chitosan nanodelivery system

Abstract We report a development of a novel localized nanodelivery system featuring an enhanced targeted and controlled paclitaxel (PTX) delivery to epithelial and neuroectodermal origin tumor cells. The presented drug delivery system consists of a chitosan hydrogel embedded with PTX loaded non-ionic surfactant vesicles (PTX-niosomes) and chlorotoxin (CTX). We found a higher accumulation of chitosan on the surface of ovarian epithelial carcinoma cells known to overexpress MUC1 antigen as compared to normal ovarian epithelial cells. We therefore hypothesized that besides serving as a localized drug delivery platform, chitosan hydrogel can actively target MUC1 overexpressing tumor cells. To further improve the tumor-specific delivery of PTX, we have incorporated CTX, a 36-amino acid peptide, which binds to tumor cells of neuroectodermal origin, but not to non-transformed cells. Using a High Performance Liquid Chromatography (HPLC) assay, we have measured the in-vitro release profiles of PTX from chitosan hydrogel embedded with PTX-niosomes and CTX. We have assessed the morphology of CTX and PTX-nisomes as well as chitosan hydrogel embedded with PTX-niosomes and CTX using Transmission Electron Microscopy (TEM). We have used Attenuated Total Reflectance-Fourier Transform Infra-Red (ATR-FTIR) spectroscopy to investigate the possible molecular interactions between chitosan hydrogel and various tumor cells. To better understand the mechanism involved in interactions between chitosan and specific tumor cells, we have developed a new Cell based Enzyme Linked Immunosorbent Assay (Cell ELISA) allowing us to quantify the expression level of MUC1 in epithelial and neuroectodermal origin tumor cells. Our Cell ELISA results revealed higher expression level of MUC1 (around 1.5 fold) in ovarian epithelial carcinoma cell line (OV2008) when compared to normal ovarian epithelial cell line (MCC3). The ATR-FTIR results showed a specific interaction of chitosan with OV2008 cells implying the mucoadhesive property of chitosan and its potential in targeting of MUC1 overexpressing tumor cells. We observed a sustained PTX release from chitosan hydrogel embedded with PTX-niosomes (< 40% of cumulative release occurred within two weeks). Release studies in cell-free system also indicated that embedding CTX along with the niosomes does not have any adverse effect on the controlled release of PTX from the chitosan network. In fact, attachment of CTX to the surface of PTX-niosomes shown by TEM imaging improves the stability of PTX-niosomes resulting in extended release rates. Together these findings indicate that the proposed drug delivery system with the distinctive tumor targeting features and extended release profiles would improve the specific delivery of PTX to epithelial and neuroectodermal origin tumor cells. Citation Format: Rana Falahat, Eva Williams, Marzenna Wiranowska, Ryan Toomey, Norma Alcantar. Enhanced targeted delivery of paclitaxel to tumor cells of epithelial and neuroectodermal origin using chlorotoxin-chitosan nanodelivery system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3677. doi:10.1158/1538-7445.AM2015-3677

Neuroectoderm-specific deletion of cathepsin D in mice models human inherited neuronal ceroid lipofuscinosis type 10.

Cathepsin D (Ctsd) is a ubiquitously expressed aspartic protease functioning primarily in the acidic endosomal/lysosomal cell compartment. At an age of 26±1 days, mice with constitutive Ctsd deficiency (Ctsd(-/-)) die from a neurodegenerative lysosomal storage disease equivalent to the congenital neuronal ceroid lipofuscinosis (NCL) type 10 in humans. In addition to neurodegeneration, Ctsd(-/-) mice exhibit a loss of CD4(+)/CD8(+)-double-positive thymocytes and an atrophy of the intestinal mucosa. To date, it is not understood if and how these phenotypes are triggering each other. In addition, the cell type causing initiation of NCL in Ctsd(-/-) mice has not been identified yet. To investigate the tissue- and cell type-specific functions of Ctsd, we generated a novel conditional Ctsd allele by flanking the second exon with loxP sites. We compared a ubiquitous Ctsd deletion with a deletion of the protease by a Nestin-promoter controlled Cre-recombinase expression in cells of neuroectodermal origin, e.g. in neurons and astroglia, but not in microglia. First, we confirmed absence of Ctsd in the respective cell- and tissue types. The neuroectoderm specific knock-out mice survived about 5.5 days longer than the mice with ubiquitous Ctsd deletion, which was in line with the progress in brain histopathology. Atrophies of thymus and small intestine were delayed to similar extend. The conditional Ctsd knock-out mouse model established in this study not only demonstrates that this type of NCL is initiated by cells of neuroectodermal origin, but will also help to further study tissue-specific functions of Ctsd invivo.

Extraosseous Ewing′s tumor of larynx: A rare presentation

Primitive neuroectodermal tumors (PNETs) are a group of highly malignant tumors composed of small round cells of neuroectodermal origin that affect soft tissue and bone. PNET of the larynx is extremely rare. We report a case of a 41-year-old male who presented with the complaints of progressively increasing stridor of 3 months duration, which was diagnosed as a case of neuroectodermal tumor in the subglottis. Patient was subjected to microlaryngeal surgery and the tumor was excised. Postoperatively, patient was given three cycles of chemotherapy comprising of ifosfamide, etoposide, and mesna, along with granulocyte colony stimulating factor, with 21 days interval. After chemotherapy repeat computed tomography scan showed no evidence of the tumor and no lymphadenopathy. Patient is symptom free for 18 months following completion of treatment. He is under regular follow-up and is undergoing monthly serial endoscopic evaluation.

Abstract 5410: Enhanced targeting delivery to tumor cells using mucoadhesive chitosan and chlorotoxin

Abstract Specific drug delivery to tumor cells without affecting normal cells remains a major challenge in cancer treatment. We present a localized drug delivery system with enhanced targeting ability consisting of non-ionic surfactant vesicles (niosomes) with chlorotoxin (CTX) embedded in a chitosan hydrogel. This system represents a novel approach in cancer therapy through the controlled and targeted delivery of drugs to tumor cells. We have found the specific accumulation of chitosan on the surface of ovarian epithelial carcinoma cells known to have high expression of mucin antigen MUC1, but not on the normal ovarian epithelial cells. This finding demonstrates the capability of chitosan to target tumor cells expressing MUC1. Similarly, the incorporation of CTX (a 36-amino acid peptide capable of binding preferentially to tumor cells of neuroectodermal origin but not to normal cells) along with niosomes in the chitosan hydrogel has been used as the second targeting strategy to further improve the specific delivery of drugs to tumor cells such as glioma. To investigate the possible molecular interaction between the chitosan hydrogel and various cell lines, Attenuated Total Reflectance-Fourier Transform Infra-Red (ATR-FTIR) spectroscopy has been used. The expression level of MUC1 in different cell lines was quantitatively evaluated using Cell based Enzyme Linked Immunosorbent Assay (Cell-ELISA) to better understand the mechanism involved in interactions between chitosan and specific tumor cells. In vitro release studies were performed to examine the effect of CTX on the release rates. Transmission Electron Microscopy (TEM) is being used for morphology assessments of CTX and nisomes as well as chitosan hydrogel embedded with niosomes and CTX. Cell-ELISA data revealed high expression level of MUC1 in ovarian epithelial carcinoma cell line (OV2008) and a moderate expression level in normal ovarian epithelial cell line (MCC3) implicating higher affinity of chitosan for OV2008 than MCC3 cell lines. This is consistent with ATR-FTIR results as well as the previously observed higher chitosan accumulation on the surface of OV2008 compared to MCC3, confirming the mucoadhesive property of chitosan and indicating its specificity in targeting MUC1 overexpressing tumor cells. In vitro release studies show that embedding CTX along with the niosomes does not disturb the controlled release from the chitosan network. In fact, attachment of CTX to the surface of niosomes shown by TEM imaging improves the stability of niosomes resulting in extended release rates. Based on these findings, we anticipate that the drug delivery system described here would improve tumor cell uptake of drugs due to the enhanced tumor targeting and its controlled, sustained and localized drug delivery to cancer cells. Citation Format: Rana Falahat, Eva Williams, Marzenna Wiranowska, Ryan Toomey, Norma Alcantar. Enhanced targeting delivery to tumor cells using mucoadhesive chitosan and chlorotoxin. [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 5410. doi:10.1158/1538-7445.AM2014-5410

Dacarbazine, a chemotherapeutic against metastatic melanoma and a reference drug for new treatment modalities

Melanoma is a tumour derived from melanocytes, cells of neuroectodermal origin. Melanoma treatment represents a challenge to oncologists due to its aggressive course and early and multiple metastases. Surgical excision of lesions is a highly effective intervention, but only in early stages. In contrast, median survival of patients with metastatic melanoma is still below one year. In 2011 the FDA and EMA have approved new drugs, ipilimumab and vemurafenib, that might be a major breakthrough in treating patients with advanced melanoma. However, time is needed to conclude whether they replace dacarbazine, a drug used for over 30 years in the therapy of metastatic melanoma, even if the response rate was only 10-15%. The mechanism of dacarbazine action is not clear but it is probably based on methylation of purine bases in DNA. The low therapeutic efficacy of dacarbazine might be the consequence of rapid removal of DNA lesions by repair systems. A high melanoma chemoresistance is also driven by the extent and nature of alterations in signal transductions in tumour cells. None of the previously conducted trials proved superiority of any treatment modality over monotherapy with dacarbazine. Higher response rates did not correlate with survival benefit, and more intense adverse effects were frequently observed. There are some expectations for targeted therapy and immunotherapy, which have already demonstrated some efficacy in clinical studies. This review aims at providing the current knowledge on dacarbazine and its analogue, temozolomide, including the latest results of clinical studies combining these drugs with other treatment protocols.

The Immunohistochemical Characterization of Devil Facial Tumor Disease (DFTD) in the Tasmanian Devil (Sarcophilus harrisii)

Immunohistochemical techniques were used to characterize the disfiguring and debilitating fatal neoplastic disease, devil facial tumor disease (DFTD), which has recently affected a significant proportion of the wild population of Tasmanian Devils (Sarcophilus harrisii). The diagnostic values of a number of immunohistochemical stains were employed to further characterize 50 representative cases. The neoplasms were negative for cytokeratin (0/48), epithelial membrane antigen (0/42), von Willebrand factor (vWF) (0/11), smooth muscle actin (SMA) (0/26), desmin (0/47), glial fibrillary acid protein (0/13), CD16 (0/13), CD57 (0/43), CD3 (0/18), and LSP1 (0/16). DFTD cells were positive for vimentin (50/50), S-100 (41/48), melan A (11/39), neuron specific enolase (35/35), chromogranin A (12/12) and synaptophysin (29/30). The cells were negative for amyloid (0/30) and stained negatively with Singh's silver (0/34) but were weakly argyrophilic (3/40) using Grimelius histochemical stain. These staining characteristics are consistent with cells of neuroectodermal origin.

Human fibroblast-derived cell lines have characteristics of embryonic stem cells and cells of neuro-ectodermal origin

Fibroblasts are the most ubiquitous cells in complex organisms. They are the main cells of stromal tissue and play an important role in repair and healing of damaged organs. Here we report new data—initially serendipitous findings—that fibroblast-derived cell line (human fetal lung derived cells, MRC-5) have the morphology, growth rate and gene expression pattern characteristic of embryonic stem cells and cells of neuro-ectodermal origin. We have developed a serum-free culture system to maintain these cells in proliferative state. We discovered that, at proliferative state, these cells express transcription factors of pluripotent cells, OCT-3/4 and REX-1, and embryonic cell surface antigens SSEA-1, SSEA-3, and SSEA-4, as well as TRA-1-60 and TRA-1-81. In addition to embryonic cell markers, the fibroblasts expressed neuroectodermal genes: Musashi-1, nestin, medium neurofilament, and β-III tubulin. RT-PCR data revealed that mesencephalic transcription factors, Nurr-1 and PTX-3, were also expressed in MRC-5 cells, and that these cells could be induced to express tyrosine hydroxylase (TH). Expression of TH followed down-regulation of genes associated with cell proliferation, OCT-3/4, REX-1, and β-catenin. These data indicate that the cells commonly known as fibroblasts have some of the characteristics of stem cells, and can be induced to become neuroectodermal cells and perhaps even mature neurons.

Generation and characterization of dimeric small immunoproteins specific for neuroblastoma associated antigen GD2

GD2 is a disialoganglioside expressed at high density on the surface of malignant cells of neuroectodermal origin, especially in neuroblastoma (NB) and melanoma. Since its expression in normal tissues is very restricted, GD2 represents an excellent target for neuroectodermal tumor targeting. Mini-antibody technology allows the production of dimeric single-chain antibodies, also called small immunoproteins (SIPs), which are composed of a scFv fused to a dimerizing domain of immunoglobulin heavy chains. Dimerization results in an increase of the total apparent affinity and a slower clearance in vivo than scFvs. These properties make SIPs very attractive molecules for tumor targeting. We isolated the variable regions from an anti-GD2 monoclonal antibody and exploited the SIP technology to generate two novel anti-GD2 SIPs. The first anti-GD2 SIP is a fully murine molecule containing the CH3 domain of mouse IgG1, whereas the second construct is a hybrid mouse-human molecule containing the CH4 domain of human IgE. Both mini-antibodies were successfully produced and shown to retain binding specificity as well as an affinity similar to that of the original antibody.

Potentiality of Anti-idiotypic Antibodies Mimicking GD2 to Induce Cellular Immunity

Background: Disialoganglioside GD2 is a tumor-associated antigen that is overexpressed on tumor cells of neuroectodermal origin, such as melanoma, small cell lung carcinoma and neuroblastoma. Immunity against GD2 has anti-tumor activities, but GD2 is poorly immunogenic. Anti-idiotypic antibodies that mimic GD2 may induce more effective immune responses than GD2 antigen itself, because they are protein antigens and are known to be able to break immune tolerance. In our previous study, we produced anti-idiotypic antibodies mimicking GD2 (3A4 and 3H9), which induced humoral immunity. However, cellular immunity is essential to eradicate tumor cells in vivo as well as humoral immunity. In the present study, we investigated whether these anti-idiotypic antibodies 3A4 and 3H9 could induce cellular immunes responses. Methods: BALB/C mice were immunized with anti-idiotypic antibody 3A4 or 3H9, or normal mouse IgG as a negative control. Lymphoproliferative responses, cytokine production responses, and delayed-type hypersensitivity reactions were measured in mice immunized with the anti-idiotypic antibodies. Results: Both the anti-idiotypic antibody 3A4 and 3H9 induced GD2-specific lymphoproliferative responses and IFN-γ production of lymph node lymphocytes in BALB/C mice. Only anti-idiotypic antibody 3H9 induced significant GD2-specific delayed-type hypersensitivity in the mice. Conclusion: These results show that anti-idiotypic antibodies 3A4 and 3H9 have the potentiality of inducing GD2-specific cellular immune responses that cannot be induced by the native antigen GD2 itself. (Immune Network 2004;4(4):229-236)

E-45. Small cell lung cancer: Targeting minimal residual disease

The outcome for patients with small cell lung cancer has improved marginally over the last 20 years. Standard chemotherapy regimens yield high response rates and some cures when combined with thoracic irradiation in limited stage patients. Nonetheless, SCLC has a high relapse rate due to drug resistance. This has resulted in continued poor survival for most patients. Developing ways to maintain response after induction chemotherapy or chemoradiotherapy has been a major research focus. The lack of an assay capable of measuring residual disease limits our ability to gauge the risk for relapse or to assess the efficacy of therapy in this setting. At this time all patients are considered at high risk and the challenge is to maintain their remission status. The chemosensitivity of SCLC logically supports the use of maintenance chemotherapy or high dose treatments as a way to attack residual metastatic disease. However, trials using more chemotherapy have generally resulted in more toxicity with no improvement in survival. In 13 randomized trials of maintenance chemotherapy, only one showed an overall survival advantage, five showed improvements only in subgroups of patients, and one showed a shorter survival with continued treatment [ 11. At least 36 studies of high dose chemotherapy with autologous bone marrow or stem cell rescue have been reported [2,3]. In these trials, only a small fraction of patients can complete the high dose chemotherapy due to co-morbid illnesses or toxicity, treatment related mortality is significant, and survival remains poor. In order to change outcomes in SCLC substantially, new approaches are needed. The matrix metalloproteinase (MMP) inhibitors are a novel class of drugs that were tested in SCLC patients as a potential therapy aimed at maintaining remission, but unfortunately yielded disappointing results in phase III trials. MMPs are a class of over 30 extracellular enzymes that degrade connective tissue and play an important role in bone development, wound healing, and vascular remodeling. Several inhibitors of these enzymes have been developed to decrease the cell migration responsible for metastatic spread. Dr. Shepherd reported the results of a phase III NCICEORTC trial in which 532 patients with small cell lung cancer who completed initial therapy were randomized to receive marimastat, a broad-spectrum MMP inhibitor, or placebo [4]. There was no difference between the marimastat and placebo groups with regards to overall survival or progression free survival. Furthermore, myalgias and arthralgias necessitated dose reduction or drug discontinuation in 33% and 32% of the patients receiving marimastat. The results of this trial, as well as the early discontinuation of a similar trial with BAY 12-9566, raise doubts as to the efficacy of this class of agents in SCLC, though specific inhibition of other MMPs could be explored. At Memorial Sloan-Kettering, we are attempting to take advantage of unique epitopes present on SCLC to develop vaccines. One potential target identified is the ganglioside GD3 expressed on cells of neuroectodermal origin. Using an anti-idiotype approach, the mouse monoclonal antibody BEC2 was developed against the binding region of the mouse monoclonal antibody R24 which binds to GD3. A pilot study was performed in which 1.5 patients with SCLC who had a major response to induction chemotherapy were immunized with BEC2. BCG was also added as an immunologic adjuvant [5]. Of the 13 evaluable patients, all developed anti-BEC2 antibodies (HAMA). Five patients had measurable anti-GD3 IgM antibody and 3 of those patients developed IgG antibodies. Median overall survival was 21 months; median time to relapse was 11 months in the extensive stage group and had not been reached in the limited stage group (>47 months). These results were a significant improvement when compared to two historical control groups treated at our center, and prompted an international phase III trial in SCLC patients with limited disease and a complete or partial response after initial therapy (the SILVA trial, lead by the EORTC). This trial completed accrual in October, 2002. Currently, we are also testing the immunogenicity of vaccines against other targets in SCLC such as GM2, Fucosyl GM1 [6,7], Globo H, and polysialic acid [S]. In order to optimize the likelihood of immunologic effect, we plan to combine the vaccines into a polyvalent vaccine. Only new strategies will improve outcomes for SCLC patients. Eligible individuals should enroll in clinical trials after chemotherapy and irradiation.

Characterization of Anti-anti-idiotypic Antibodies (Ab3) Induced by Immunization of Anti-idiotypic Antibodies (Ab2) Mimicking Disialoganglioside GD2

Background: Disialoganglioside GD2 is a tumor-associated antigen that is overexpressed on tumor cells of neuroectodermal origin, such as melanoma and neuroblastoma. Anti-idiotypic antibodies that mimic GD2 may induce more effective immune responses than GD2 antigen itself, because they are protein antigens and are known to be able to break immune tolerance. In this study, to explore the potential of anti-idiotypic antibodies as tumor vaccines, the ability of anti-idiotypic antibodies (Ab2) to induce anti-anti-idiotypic antibodies (Ab3) that bind to the original antigen GD2 was investigated. Methods: Six monoclonal anti-idiotypic antibodies (1A8, 1G5, 2B6, 3A4, 3D6, 3H9) to monoclonal antibody M2058, which is a monoclonal antibody to GD2, were produced in mice. Three (1A8, 3A4, 3H9) of them were selected based on their ability to inhibit the binding of Ab1 to D142.34 (murine melanoma cell expressing GD2). These 3 different Ab2 were injected into rabbits, and rabbit Ab3 induced by each of them were characterized. Results: Ab3-containing sera from two rabbits immunized with 1A8, 3A4, or 3H9 bound significantly (P<0.05) to D142.34 but not to B78.96 (GD2-negative cell), and bound significantly (P<0.05) to isolated GD2 but not to GD1a. Ab3-containing sera from two rabbits immunized with 3A4 or 3H9 inhibited significantly (P<0.05) the binding of Ab1 M2058 to D142.34, and inhibited significantly (P<0.05) the binding of Ab1 M2058 to the Ab2. Conclusion: These results suggest that anti-idiotypic antibodies 3A4 and 3H9 have a potential to be used as vaccines against tumors expressing GD2 by inducing GD2-specific antibodies (Ab3). (Im m un e N etw ork 2003;3(2):118-125)

A Morphological Study of the Developmentally Regulated Transport of Iron into the Brain

The distribution of transferrin, transferrin receptor, ferritin and ferric iron was studied in the developing rat brain. Transferrin immunoreactivity (IR) was observed diffusely in the brain from E16 until P10 from where it gradually decreased. The subcellular distribution of transferrin-IR in neurons was compatible with receptor-mediated uptake from P21 and onwards. Transferrin receptor-IR was observed prenatally on cells of neuroectodermal origin in the ventricular zone and in brain capillary endothelial cells (BCECs). In postnatal rats, transferrin receptor-IR in BCECs was most pronounced in rats aged P10–P21 but thereafter decreased in intensity. The neuronal transferrin-receptor IR in postnatal brains was not consistently expressed on neurons until from P21 and onwards. Transferrin receptor-IR was not observed in astrocytes, oligodendrocytes or ramified microglial cells at any age. Ferric iron and ferritin were present in BCECs already from E16, declined from P3–P5, and was absent by P10. There results are discussed with emphasis on the age-dependent transport of transferrin into the developing brain. The upregulated expression of transferrin receptors on BCECs in the second and third postnatal week is compatible with a high need for iron at this age. The neuronal transferrin receptor expression by P21 coincides with a drop in transferrin-IR and iron transport into the brain at this age, suggesting that neuronal transferrin receptor mRNA is posttranscriptionally regulated by the lowered iron availability from this developmental stage onwards.

Production and molecular characterization of clinical phase i anti-melanoma mouse IgG3 monoclonal antibody R24.

R24 is a mouse IgG3 monoclonal antibody (mab) that reacts with the ganglioside GD3 expressed by cells of neuroectodermal origin. The anti-tumor activity of R24 has been demonstrated in initial phase I and pilot trials in patients suffering from metastatic melanoma. The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important antibody. Growth, metabolism, and IgG production of R24 secreting hybridoma cells were analyzed on 1 L bioreactor bench scale using repeated-batch mode. The amount of 57 mg of pure mab was obtained from 1.6 L crude supernatant by protein A chromatography. Western blot binding assays with sugar-specific lectins revealed glycosylation of the heavy chains, whereas no carbohydrates were detectable on the light chains. Because glycosylation is essential for antibody effector functions in vivo (such as complement fixation or binding to macrophage Fc receptors), mab R24 was subjected to both enzymatic deglycosylation using PNGase F and chemical deglycosylation by hydrazinolysis. Released glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry. Six major biantennary chains of the complex glycosylation phenotype were found with variations in galactosylation and core fucosylation. The predominant N-linked structure, indicating the high degree of agalactosyl glycoforms, was the agalacto biantennary chain with a relative percentage of 57% (51% core-fucosylated, 6% nonfucosylated). The second most abundant oligosaccharide was the monogalacto biantennary chain amounting to 30% (26% core- and 4% nonfucosylated). The antibody contained 0.46 microg sialic acid per mg protein, which splits into 0.243 microg Neu5Gc and 0.217 microg Neu5Ac, corresponding to a Neu5Ac:Neu5Gc ratio of 1:1.06. Furthermore, the antigen specificity of R24 was determined by immunodetection of GD3 on thin-layer chromatograms, and real time GD3-antibody binding interactions were measured with an optical biosensor (BIAcore). From the structural data obtained in this study it is concluded that glycosylation of the antibody may be important in the clinical outcome of targeted anti-cancer immunotherapy.

Transcriptional regulation of the leptin gene promoter in rat GH3 pituitary and C6 glioma cells

Leptin was originally believed to be an exclusively adipocyte-derived hormone regulating appetite and energy balance. It has recently become apparent that leptin is actively expressed in a number of other tissues including the CNS and pituitary, as well as brain- and pituitary-derived cell lines. However, the factors controlling leptin expression in cells of neuroectodermal origin are unknown. The mouse leptin gene 5′-flanking DNA contains multiple AP-1 and SRF-1 binding sites as well as a consensus CRE site at −491 to −482 bp. In addition, a number of potential PIT1 and Oct-1 binding sites may contribute to leptin gene transcription in pituitary and brain. We have used leptin promoter-luciferase reporter constructs to examine the regulation of the leptin promoter in 3T3-L1 preadipocytes, C6 glioma cells, and GH3 pituitary cells in response to serum and hormonal stimuli. Cells were transiently transfected with reporter constructs containing either the proximal 500 bp of the leptin promoter (−500-luc) or 6000 bp of the leptin gene 5′ flanking region (−6000-luc). Functional analysis indicates that the leptin promoter is constitutively active in all 3 cell lines. Transcriptional activity was significantly higher with a −500 to +9 promoter than with a construct containing −6000 to +9 bp of 5′ flanking DNA, indicating the presence of repressor elements which may contribute to the tissue-specific regulation of leptin expression. However, qualitatively similar results were observed with both constructs in response to serum and hormonal manipulation. Leptin promoter activity was significantly stimulated by serum in all cell lines, although to varying extents. In contrast, the response of the leptin promoter to insulin, IGF-1 and dibutyryl cAMP was cell-type specific and dependent on the presence or absence of FBS in the culture medium. Insulin, IGF-1 and dibutyryl cAMP each caused an approximately two-fold stimulation of leptin promoter activity in 3T3-L1 cells under serum-free conditions, but had no significant effect in the presence of 10% FBS. In contrast, dibutyryl cAMP markedly stimulated leptin promoter activity (5–8-fold) in C6 or GH3 cells in the presence or absence of FBS, whereas insulin or IGF-1 had minimal effects. These findings support our previous studies on the regulation of leptin steady state mRNA levels in C6 cells and demonstrate tissue-specific differences in the regulation of leptin gene transcription in adipose vs. neuroectodermal tissues.

Vimentin immunohistochemistry in human fetal brain: methods of standard incubation versus thermal intensification achieve different objectives.

Vimentin is transiently expressed in many cells of neuroectodermal origin in the fetal central nervous system and may be demonstrated by immunohistochemistry in paraffin sections. Traditional methods incubate antibodies with tissue sections at room temperature, but boiling the incubating solution in a microwave oven enhances antigen retrieval and greatly intensifies immunoreactivity. This study compares vimentin immunoreactivity using various dilutions of a commercial monoclonal antibody and various incubation conditions including room temperature, overnight at 4 degrees C, and after heating to boiling either in a microwave oven or on a hot plate. Fifteen fetal brains and spinal cords were examined, ranging in age from 8 to 24 wk gestation. Normal brains of four term or near-term neonates and two with hypoxic/ischemic lesions were also studied. Hot plate and microwave heating were equivalent. No differences in immunoreactivity could be attributed to gestational age per se. Boiling for 10 min in a microwave oven at a dilution of 1:200 is recommended for the enhanced demonstration of fetal histological structures such as radial glial fibers, but it is not recommended for studies of maturational gradients of cellular vimentin that differ at each gestational age, as in fetal ependymal cells or during the conversion of radial glia to mature astrocytes. For semiquantitative developmental studies, vimentin incubation at room temperature or overnight in the refrigerator is recommended at a dilution of 1:25 or 1:50 to avoid misinterpretation of apparent excessive staining after thermal intensification. Recommendations of commercial suppliers focus on the identification of neoplastic cells and must be modified to demonstrate subtle developmental changes in fetal tissues.

Targeting Liposomes Through Immunoglobulin Superfamily Domains: P0 Protein as a Model

The objective of this study was to evaluate the potential of P0 protein, a cell adhesion molecule from peripheral nerve myelin, as a targeting ligand for liposomes. To evaluate binding characteristics and identify possible binding domains, cell-interaction studies were carried out with P0 protein reconstituted into liposomes (P0 liposomes) under various conditions. P0 liposomes with intact P0 protein were tested after endoglycosidase F treatment (cleaves the carbohydrate moiety) or trypsin digestion (removes the hydrophobic portion, residues 1-79, leaving the carbohydrate portion intact). The cellular uptake was quantitated using radioactive lipids in the liposome bilayer and a liposome-entrapped water soluble compound (inulin). The presence of intact P0 protein in the liposome bilayer increased the rate of interaction of liposomes 3-4 times with M21 melanoma and HTB-11 neuroblastoma cells (cells of neuroectodermal origin), two times with Caki-1 renal carcinoma cells, and marginally with 3T3 fibroblasts (mesodermal origin). This binding was inhibited by anti-chick P0 antibodies and Fab fragments. A control transmembrane glycoprotein, glycophorin A., when reconstituted in liposomes had no effect on the binding of liposomes with M21 cells. The results indicate that P0 protein plays a specific role in the binding of liposomes to cells. Removal of the N-asparagine linked carbohydrate from the P0 protein in the liposomes resulted in an increase of their association with M21 cells five times that of control liposomes (no protein) and two times that of the non-endoglycosidase F-treated P0 liposomes. To further characterize the binding domains of P0 reconstituted into liposomes, competition studies were carried out in the presence of synthetic P0-peptides. The competition studies indicated that both the extracellular (residues 90-96) and intracellular (residues 201-207) domain of P0 protein may be involved in the interaction with cell membranes. The results suggest that P0 protein is capable of mediating specific heterophilic interactions with various cell lines and targeting to cells of neuroectodermal origin may be achieved.

Cell scattering of SK-N-MC neuroepithelioma cells in response to Ret and FGF receptor tyrosine kinase activation is correlated with sustained ERK2 activation.

The c-ret proto-oncogene encodes a receptor tyrosine kinase which plays an important role in kidney and enteric nervous system development. Germline mutations in c-ret are responsible for the dominantly inherited cancer syndromes, multiple endocrine neoplasia types 2A and 2B and familial medullary thyroid carcinoma as well as the developmental disorder Hirschsprung's disease. Using SK-N-MC neuroepithelioma cells stably transfected with an EGFR/Ret chimeric receptor, we have studied cellular consequences and signalling events following activation of exogenous EGFR/Ret and endogenous FGF and PDGF receptor tyrosine kinases in cells of neuroectodermal origin. Here we report that Ret activation led to cell scattering, growth inhibition and loss of anchorage-independent growth. Basic FGF, but not PDGF, evoked similar responses in those cells. Nevertheless, activation of all three receptor tyrosine kinases led to ERK2 activation. Analysis of the kinetics of ERK2 activation and downstream events revealed that Ret and FGF receptor activation led to sustained ERK2 activation and SRE transactivation, while PDGF treatment led to transient ERK2 activation and failed to induce SRE transactivation. Our results suggest that sustained, but not transient ERK2 activation may be involved in cell scattering.