anti-NCAM Antibody Research Articles

Abstract 31: Organoid derived from human operative sample of NET shows NET characteristics

Abstract Background: Neuroendocrine tumor is a rare neoplasm, then, it was difficult to examine using in vitro or animal models. Recently, patient-derived tumor organoid (PDTO) is considered as a promising method to analyze natural characteristics of human tumor, because PDTO is cultured in near-natural circumstances compared with cell lines. We established PDTO of neuroendocrine tumor (NET) utilizing operative samples from a pancreatic NET patient. Aim: Aim of this study is to establish organoid derived from an operative sample of pancreatic NET patient and to compare its characteristics with original tumor using immuno-histochemistry. Material and Method: A pancreatic NET tissue derived from operative sample was cut into small pieces and was treated by collagenase. Prepared tissues were 3-dimensionally cultured on the Matrigel in a serum-free medium (Advanced MEM/F12) with 4 growth factors (Respondin, EGF, Rock inhibitor, Noggin), then PDTO was established. To evaluate growth rate of PDTO, Major axis of the organoid was measured on the day 1, 2, 3, and 6. One week after primary culture of the organoid, subculture was performed. Then major axis of the 2nd, 6th and 7th filial organoid was also measured and compared with each filial. To evaluate characteristics of PDTO as neuroendocrine tumor, immunohistochemistry was performed for the 1st, 2nd and 3rd filial PDTO using anti-chromogranin-A antibody (Dako, mouse IgG), anti-synaptophysin antibody (Abcam, rabbit IgG), anti-NCAM antibody (Abcam mouse IgG) and Neuro-specific enolase (Dako, rabbit IgG). Results: Growth rate of the 2nd and 3rd filial on the day 6 did not show significant difference compared with that of the 1st filial. On the other hand, Growth rate of the 2nd and 3rd filial was significantly lower than that of the 1st filial. Expression of the 4 factors in the 1st, 2nd and 3rd filial PDTO was kept as much as the primary tumor. Conclusion: PDTO derived from neuroendocrine tumor showed the same characteristics as the primary tumor. The 6th and 7th filial PDTO might show cellular aging. Citation Format: Yasushi Ichikawa, Yukihiko Hiroshima, Noritoshi Kobayashi, Yusaku Masuda, Ayumu Goto, Motohiko Tokuhisa, Yuma Takeda, Takashi Ishikawa, Itaru Endo. Organoid derived from human operative sample of NET shows NET characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 31.

GDNF stimulates the proliferation of cultured mouse immature Sertoli cells via its receptor subunit NCAM and ERK1/2 signaling pathway

BackgroundThe proliferation and final density of Sertoli cells in the testis are regulated by hormones and local factors. Glial cell line-derived neurotrophic factor (GDNF), a distantly related member of the transforming growth factor-β superfamily, and its receptor subunits GDNF family receptor alpha 1 (GFRα1), RET tyrosine kinase, and neural cell adhesion molecule (NCAM) have been reported to be expressed in the testis and involved in the regulation of proliferation of immature Sertoli cells (ISCs). However, the expression patterns of these receptor subunits and the downstream signaling pathways have not been addressed in ISCs.ResultsIn the present study, we have reported that the proliferation of cultured ISCs was significantly enhanced by GDNF. The receptor subunits GFRα1 and NCAM but not RET were expressed in ISCs, and the stimulatory effect of GDNF on the proliferation of ISCs was significantly reduced by anti-NCAM antibody blocking or siRNA that specifically targets NCAM mRNA. Additionally, the ERK1/2 inhibitor, PD98059, completely abolished the mitogenic effect of GDNF on ISCs.ConclusionsGDNF stimulates the proliferation of ISCs via its receptor subunit NCAM and the consequent activation of the ERK1/2 signaling pathway.

Identification of NCAM that interacts with the PHE-CoV spike protein

BackgroundThe spike proteins of coronaviruses associate with cellular molecules to mediate infection of their target cells. The characterization of cellular proteins required for virus infection is essential for understanding viral life cycles and may provide cellular targets for antiviral therapies.ResultsWe identified Neural Cell Adhesion Molecule (NCAM) as a novel interacting partner of the PHE-CoV S protein. A T7 phage display cDNA library from N2a cells was constructed, and the library was screened with the soluble PHE-CoV S glycoproteins. We used a coimmunoprecipitation assay to show that only the NCAM was a binding partner of spike protein. We found that a soluble form of anti-NCAM antibody blocked association of the PHE-CoV with N2a cells. Furthermore, double-stranded siRNA targeted against NCAM inhibited PHE-CoV infection.ConclusionsA novel interaction was identified between NCAM and spike protein and this association is critical during PHE-CoV infection.

The neural cell adhesion molecule is involved in the metastatic capacity in a murine model of lung cancer

Neural cell adhesion molecule (NCAM) is involved in cell growth, migration, and differentiation. Its expression and/or polysialylation appear to be deregulated in many different cancer types. We employed the lung tumor cell line LP07, syngeneic in BALB/c mice to investigate the role of NCAM in malignant progression. LP07 cells express the three main NCAM isoforms, all of them polysialylated. This cells line, pretreated with an anti-NCAM antibody and inoculated intravenously (i.v.) into syngeneic mice, developed less and smaller lung metastases. In vitro studies showed that NCAM bound antibody inhibited cell growth, mainly due to an increase in apoptosis, associated with a decrease of cyclin D1 and enhanced expression of active caspase 3 and caspase 9. Anti-NCAM-treated LP07 cells showed impairment in their ability to migrate and adhere to several extracellular matrix components. Secreted uPA activity was also reduced. NCAM-140 knocked-down by siRNA in LP07 cells pretreated or not with anti-NCAM showed an impaired metastasizing ability upon i.v. inoculation into mice. These results suggest that anti-NCAM treatment could be mimicking homophilic trans-interactions and NCAM-140 knocked-down impairs heterophilic interactions, both leading to inhibition of metastatic dissemination. The involvement of NCAM in lung tumor progression was confirmed in human NSCLC tumors. Sixty percent of the cases expressed NCAM at tumor cell level. A multivariate analysis indicated that NCAM expression was associated with a shorter overall survival in this homogeneous series of Stages I and II NSCLC patients. NCAM may be able to modulate mechanisms involved in lung carcinoma progression and represents an attractive target to control metastatic progression.

Smooth muscle metaplasia and innervation in interstitium of endometriotic lesions related to pain

To investigate the pathogenesis of endometriotic pain. Retrospective nonrandomized immunohistochemical study. A university hospital, Department of Gynecology. Twenty human endometriotic specimens were selected from different lesions including ovarian endometrioma, peritoneal lesion, and deep infiltrating lesion. Premenopausal women with histologically diagnosed endometriosis were selected (mean age 39 years; range, 25-53 years). The chief complaint was dysmenorrhea, dyschezia, and dyspareunia. A rat endometriosis model was induced in 10 SLC-Sprague-Dawley rats (8 weeks old) by surgical autotransplantation of the uterus. Immunohistochemical staining of endometriotic specimens for alpha-smooth muscle actin (ASMA), neural cell adhesion molecule (NCAM), and nerve growth factor (NGF) expression. Comparison of the immunoreactive staining of ASMA, NCAM, and NGF expression in human endometriosis and a rat endometriosis model. Morphological analysis revealed thick interstitium in both human and rat endometriotic lesions. The major components of fibrotic interstitium are smooth muscle cells, stained by anti-ASMA antibody, and nerve cells, stained by anti-NCAM antibody. Inflammatory cells are also present (e.g., macrophages and lymphocytes) as revealed by anti-NGF antibody staining. These results suggest that the contraction of smooth muscle cells and the hyperalgia derived from innervation in the interstitial area is related to pain in endometriosis.

Genetic Ablation of Polysialic Acid Causes Severe Neurodevelopmental Defects Rescued by Deletion of the Neural Cell Adhesion Molecule

Poly-alpha2,8-sialic acid (polySia) is a unique modification of the neural cell adhesion molecule, NCAM, tightly associated with neural development and plasticity. However, the vital role attributed to this carbohydrate polymer has been challenged by the mild phenotype of mice lacking polySia due to NCAM-deficiency. To dissect polySia and NCAM functions, we generated polySia-negative but NCAM-positive mice by simultaneous deletion of the two polysialyltransferase genes, St8sia-II and St8sia-IV. Beyond features shared with NCAM-null animals, a severe phenotype with specific brain wiring defects, progressive hydrocephalus, postnatal growth retardation, and precocious death was observed. These drastic defects were selectively rescued by additional deletion of NCAM, demonstrating that they originate from a gain of NCAM functions because of polySia deficiency. The data presented in this study reveal that the essential role of polySia resides in the control and coordination of NCAM interactions during mouse brain development. Moreover, this first demonstration in vivo that a highly specific glycan structure is more important than the glycoconjugate as a whole provides a novel view on the relevance of protein glycosylation for the complex process of building the vertebrate brain.

Induction of apoptosis in human salivary gland tumor cells by anti-NCAM antibody

Neural cell adhesion molecule (NCAM) is a type of cell surface glycoprotein and a member of the immunoglobulin superfamily. It has been reported that NCAM may be associated with perineural invasion by malignant salivary gland tumors such as adenoid cystic carcinoma. We have previously demonstrated that NCAM is constitutively expressed in the human salivary gland tumor cell line HSG, in vitro. In the present study, we have aimed to clarify the hypothesis that NCAM-mediated inhibition of salivary gland tumor proliferation is caused by homophilic binding and involves the prevention of signal transduction for perineural invasion using HSG cells. NCAM mRNA and protein expression was found to decrease in a dose-dependent manner upon treatment with the anti-NCAM antibody (MAb NCAM) for 24 h. The MTT assay showed a significant reduction in the number of viable HSG cells. Confocal laser microscopy showed that HSG cells underwent apoptosis after treatment with MAb NCAM. The activation of caspases 3, 7 and 9 was observed in HSG cells after treatment with MAb NCAM, thus confirming that apoptosis was induced by the activated caspases. Apaf-1 activity was also detected in HSG cells in a dose-dependent manner after treatment with MAb NCAM. The up-regulation of TGF-beta1-mediated NCAM expression appeared to lead to the activation of homophilic NCAM binding, further accelerating HSG cell proliferation. In addition, the localization of NCAM in adenoid cystic carcinomas (ACCs) was examined using an immunohistochemical method. NCAM was slightly to moderately positive in 9 of 13 cases (69.2%) of ACC. These findings suggest that NCAM is associated not only with a cell-to-cell adhesion mechanism, but also with tumorigenesis, including growth, development and perineural invasion in human salivary gland tumors.

Specific expression of an HNK-1 carbohydrate epitope and NCAM on femoral nerve Schwann cells in mice

Schwann cells are glial cells of the peripheral nervous system. There are two known subtypes of Schwann cells: those that are myelin-forming; and those that are non-myelin-forming. In this study, we looked at the expression of cell adhesion molecules in Schwann cells to determine whether other subtypes might exist. We used immunohistological analysis of femoral nerve segments containing sensory and motor fascicles, stained with anti-HNK-1, M6749 and anti-neural cell adhesion molecule (NCAM) monoclonal antibodies. Anti-HNK-1 and M6749 were positive in the motor fascicle, while anti-NCAM was positive in the sensory fascicle. Immunoblot analysis with the anti-HNK-1 and M6749 antibodies showed stronger immunoreactivity in the motor fraction than in the sensory fraction in the 100 kDa band. With the anti-NCAM antibody, the 140 and 120 kDa bands were seen in the sensory fascicle fraction, but not in the motor fascicle fraction. HNK-1-positive-cells were seen in motor fascicles 7 days after transection. However, the level of immunoreactivity diminished at 14 days, and no immunoreactivity was seen at 21 days. NCAM-positive cells were not observed 3 days after transection. In development, HNK-1-positive-cells and NCAM-positive cells were seen after P- 21. These results suggest that the Schwann cells from the motor and the sensory fascicles have different subtypes. The motor and sensory Schwann cells may play different roles and function in a different way during peripheral nerve regeneration. In addition, there could be more stages of Schwann cell differentiation than previously thought; it is possible that myelin-forming Schwann cells differentiate into HNK-1-positive-cells (motor myelin-forming Schwann cells) and HNK-1-negative-cells (sensory myelin-forming Schwann cells), and non-myelin-forming Schwann cells differentiate into NCAM-positive cells (sensory non-myelin-forming Schwann cells) and NCAM-negative cells (autonomic non-myelin-forming Schwann cells).

Immunohistochemical detection of neurotrophin-3 and -4, and their receptors in mouse taste bud cells

Neurotrophin-3 (NT3) and neurotrophin-4 (NT4) affect the survival and maintenance of central and peripheral neurons. Using an immunohistochemical method, we examined whether the taste bud cells in the circumvallate papillae of normal mice expressed NT3, NT4, and their respective receptors TrkC and TrkB, and if so, what type of cells in the taste buds expressed them. Double immunostaining for either of them and PGP 9.5, NCAM, or gustducin was used to determine which cell types expressed which neurotrophins and receptors. Normal taste bud cells expressed NT3, NT4, and the TrkB receptor, but not TrkC. The percentage of NT3-immunoreactive cells among all taste bud cells was 89.0%, that of NT4-immunoreactive cells, 58.6%, and that of TrkB-immunoreactive cells, 80.8%. Almost none of the NT4-immunoreactive cells were reactive with anti-PGP 9.5 or the anti-NCAM antibody, but they could be stained with anti-gustducin, revealing that NT4-immunoreactive cells were contained only in the type-II--and possibly type-I--cell population. On the other hand, NT3-, and TrkB-immunoreactive cells included type-III cells, together with type-II, -I, and basal cells, because they were positive for PGP 9.5 and gustducin. We conclude that NT4 may exert trophic actions on all types of taste bud cells by binding to their TrkB receptors, and NT3 may also have a similar, though negligible role.

唾液腺腺様嚢胞癌の増殖・進展メカニズムに関する研究―ＮＣＡＭの発現とその機能について―

Neural cell adhesion molecule (NCAM) is one cell surface glycoprotein and a member of the immunoglobulin superfamily. It has been reported that NCAM may be associated with perineural invasion by malignant glandular tumors, such as bile duct carcinoma or gallbladder carcinoma. However, it remains unclear whether NCAM is really associated with the development of salivary gland tumors. Previous studies have demonstrated that NCAM is constitutively expressed in the human salivary gland tumor cell line HSG in vitro. In this study, molecular mechanisms of perineural invasion were examined using HSG cells in which NCAM-mediated inhibition of salivary gland tumor proliferation is caused by homophilic binding. In addition, the localization of NCAM in adenoid cystic carcinomas (ACCs) was examined immunohistochemically. The levels of NCAM mRNA and protein expression were found to decrease in a dose-dependent manner on treatment with anti-NCAM antibody. The MTT assay revealed a significant reduction in the number of viable HSG cells. Confocal laser microscopy showed that HSG cells undergo apoptosis in response to treatment with anti-NCAM antibody. The activation of caspases 3, 7, and 9 was observed in HSG cells after treatment with anti-NCAM antibody, thus confirming the induction of activated caspase-mediated apoptosis. The up-regulation of TGF-β1-mediated NCAM expression seemed to lead to the activation of homophilic NCAM binding, further accelerating HSG cell proliferation. Immunohistochemical examinations revealed that NCAM was slightly to moderately positive in 9 of 13 cases (69.2%) of ACC.These findings suggest that NCAM is associated not only with a cell-to-cell adhesion mechanism but also with tumorigenesis, including growth, development, and perineural invasion in human ACCs.

Overexpression of neural cell adhesion molecule in Chagas' myocarditis

The expression of the neural cell adhesion molecule (NCAM) was studied in normal human myocardium and in Chagas' disease myocarditis. We found that NCAM is expressed in the conduction system as well as the myocardium in the fetal heart, but its expression is restricted to the conduction system and absent in the adult myocardium. Chagas' disease is an American endemic disease caused by the Trypanosoma cruzi parasite, which produces myocarditis and a blockade of the conduction system, resulting in cardiac dysfunction. We studied the expression of NCAM in paraffin-embedded human heart tissues from 34 autopsies of patients with Chagas' myocarditis and from murine and canine experimental acute Chagas' myocarditis, using a polyclonal anti-NCAM antibody and immunohistochemistry. Our results show a dramatic upregulation of NCAM expression in the intercalated discs of cardiomyocytes in acute and chronic Chagas' myocarditis. Surprisingly, the NCAM signal was detected in intracellular nests of amastigote forms of the parasite, within infected cardiomyocytes of human and experimental Chagas' myocarditis. In contrast, cardiac cell-cell adhesion proteins, N-cadherin and β-catenin, were found in intercalated discs distorted by the infection but absent from the amastigote nests. Proteins reactive to several antibodies against NCAM were detected by Western immunoblotting in cultured T cruzi parasites and in trypomastigote forms of T cruzi extracted from the blood of infected mice. The upregulation of NCAM in Chagas' myocarditis and the expression of NCAM or a NCAM-like protein by T cruzi suggest that NCAM may act as a receptor for tissue targeting and cellular invasion by T cruzi in Chagas' disease. HUM PATHOL 32;149-155. Copyright © 2001 by W.B. Saunders Company

Specific cell targeting for delivery of toxins into small-cell lung cancer using a streptavidin fusion protein complex.

New modalities of treatment for small-cell lung cancer (SCLC) are needed, because the majority of patients continue to die of disseminated disease despite an initial response to conventional chemotherapy. Abnormal surface expression of the neural-cell adhesion molecule (NCAM) has been noted to be highly associated with SCLC. We examined the ability and efficiency of a streptavidin-Protein A (ST-PA) fusion protein complexed with an anti-NCAM monoclonal antibody (Mab) to transfer biotinylated beta-galactosidase into human SCLC cell lines NCI-H69, NCI-H526, and NCI-H446. When the surface molecule NCAM was targeted with this system, more than 99% of the targeted cells internalized and exhibited beta-galactosidase activity. In addition, we evaluated cytotoxic activity against SCLC lines NCI-H69 and NCI-H526 by efficient delivery of biotinylated glucose oxidase using the same ST-PA/anti-NCAM Mab complex. Cytotoxicity of the transduced cells (SCLC) was 10-fold and 100-fold greater, respectively, than the glucose oxidase control. This system could be widely applied for specific therapy of cancer cells by targeting unique surface molecules (antigens) using the corresponding Mab/ST-PA complex to transfer a variety of effector molecules; e.g., immunotoxic compounds, into target cells with a high degree of efficiency and specificity.

Neural cell adhesion molecule expression in adenoid cystic carcinoma of the head and neck.

To investigate whether there is a correlation between neural cell adhesion molecule (NCAM) expression and perineural spread in patients with adenoid cystic carcinoma of the head and neck (ACCHN). Retrospective review of medical records and immunohistochemical staining of specimens from 37 patients treated at the University of Arkansas in Little Rock from 1987 to 1997. Sections from paraffin-embedded specimens were examined for the presence of NCAM using monoclonal anti-NCAM antibody by avidin-biotin-peroxidase immunohistochemical staining. NCAM staining was scored in each specimen and correlated with the data obtained from patient charts. Twenty-five of 37 specimens (68%) showed histopathological evidence of perineural spread. All 37 specimens (100%) stained positive for NCAM, regardless of perineural spread status. Our results suggested that the use of NCAM expression as a predictor of perineural spread is highly unlikely.

Expression of the neural cell adhesion molecule in mouse taste buds after denervation.

Expression of the neural cell adhesion molecule (NCAM) was studied by use of an immunocytochemical technique in the taste buds of mouse circumvallate papillae after bilateral transection of the glossopharyngeal nerves. In untreated mice, innervated type-III cells reacted with anti-NCAM antibody. After denervation the taste buds gradually decreased in number and size, and were practically absent within 11 days. In parallel, NCAM-reactive cells decreased at 3 and 8 days after surgery and at 11 days they were no longer found. Three days after denervation, synaptic contacts between type-III cells and nerve fibres were not found because of the disappearance of nerve fibres. However, remaining type-III cells, characterized with dense-cored vesicles, still maintained NCAM expression on their plasma membrane until day 8.

Preparation of a conjugate of mitomycin C and anti-neural cell adhesion molecule monoclonal antibody for specific chemotherapy against biliary tract carcinoma.

To develop a specific chemotherapy modality for the perineural invasion of neural cell adhesion molecule (NCAM) positive biliary tract cancer, an anticancer drug, mitomycin C (MMC), was covalently bound to anti-NCAM monoclonal antibody (anti-NCAM MoAb) to form a conjugate using the cyanogen bromide method. The substitution ratio of the conjugate determined spectrophotometrically was 3.5 (MMC mol/immunoglobulin G mol). The cytotoxic activity of the conjugate, which was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test in the growth inhibition of the neuroblastoma cell line with NCAM expression, was maintained at 65.8% to 94.5% compared with the same concentration of MMC solution. The binding activity of the conjugate to the NCAM-positive bile duct cancer was examined by immunohistochemical staining and proved to be the same level as that of free anti-NCAM MoAb. The conjugate prepared in this study therefore appeared to be a potentially useful tool for immunotargeting specific chemotherapy against biliary tract cancer with NCAM expression.

Two polysialic acid synthases, mouse ST8Sia II and IV, synthesize different degrees of polysialic acids on different substrate glycoproteins in mouse neuroblastoma Neuro2a cells.

We previously cloned cDNAs encoding two different polysialic acid (PSA) synthases, ST8Sia II and IV, from mouse, and showed that both mouse ST8Sia II and IV can synthesize PSA on the neural cell adhesion molecule (NCAM) as well as other glycoproteins such as fetuin, at least in vitro (Kojima, N., Tachida, Y., Yoshida, Y., and Tsuji, S. (1996) J. Biol. Chem. 271, 19457-19463]. In the present study, to clarify how the two PSA synthases act differently in vivo, we first cloned PSA-expressing cell lines (N2a-II and N2a-IV) by stable transfection of the cDNA encoding either mST8Sia II or IV into mouse neuroblastoma Neuro2a cells, which do not express PSA but express NCAM, then compared the expression of the PSA and NCAM isoforms and de novo synthesis of PSA between N2a-II and N2a-IV. Western blotting with an anti-NCAM polyclonal antibody showed that NCAM was expressed as the polysialylated form in both ST8Sia II cDNA-transfected and ST8Sia IV cDNA-transfected Neuro2a cells, but that the polysialylated NCAMs expressed in ST8Sia IV cDNA-transfected clones migrated much slower on SDS-PAGE than those expressed in ST8Sia II cDNA-transfected clones. The slower migration of polysialylated NCAM of the ST8Sia IV cDNA-transfected clone (N2a-IV) than that of the ST8Sia II cDNA-transfected clone (N2a-II) was also observed when cells were metabolically labeled with [3H]glucosamine or pulse-chase labeled with [35S] methionine followed by immunoprecipitation with anti-PSA antibody or anti-NCAM monoclonal antibody. In addition, polysialylated N-glycans of PSA-carrying glycoproteins prepared from [3H] glucosamine-labeled N2a-IV by immunoprecipitation with anti-PSA monoclonal antibody were eluted at a much higher salt concentration than those from [3H] glucosamine-labeled N2a-II on an anion-exchange column. These results indicated that the degree of de novo polysialylation of NCAM by mST8Sia IV was much higher than that by mST8Sia II. In N2a-IV, NCAM-120, -140, and -180 were expressed as polysialylated forms, while polysialylation was restricted to NCAM-140 and -180, i.e., not NCAM-120, in N2a-ST8Sia II. Metabolic labeling of the cells with [3H] glucosamine, pulse-chase labeling with [35S] methionine followed by immunoprecipitation with anti-PSA antibody, and subsequent sialidase treatment revealed that NCAM-140 and -180 were specifically polysialylated in N2a-II, whereas not only NCAM but also other glycoproteins were de novo polysialylated in N2a-IV. The above results demonstrated that the two different PSA synthases, mST8Sia II and IV, synthesize PSA of different lengths on different substrate glycoproteins in vivo when the enzymes are expressed in neuroblastoma Neuro2a cells. These differences suggest that mST8Sia II and IV play different roles in the biosynthesis and expression of PSA.

Pharmacokinetics, biodistribution and dosimetry of anti-NCAM monoclonal antibody RNL-1 and its fragments in experimental lung cancer: Mijnheere EP, Boerman OC, Poels LG, Slober RL, Van Eys GJJM, Ramaekers FCS. Dept. Molecular Cell Biol./Genetics, University of Limburg, P.O. Box 616, 6200 Maastricht. J Exp Clin Cancer Res 1996;15:11–18

Pharmacokinetics, biodistribution and dosimetry of anti-NCAM monoclonal antibody RNL-1 and its fragments in experimental lung cancer: Mijnheere EP, Boerman OC, Poels LG, Slober RL, Van Eys GJJM, Ramaekers FCS. Dept. Molecular Cell Biol./Genetics, University of Limburg, P.O. Box 616, 6200 Maastricht. J Exp Clin Cancer Res 1996;15:11–18

Radioimmunotherapy of small-cell lung cancer xenografts using 131I-labelled anti-NCAM monoclonal antibody 123C3.

We have studied the therapeutic efficacy of 131I-labelled monoclonal antibody 123C3 in human small-cell lung carcinoma xenografts established from the NCI-H69 cell line in nude mice. Several radiation doses were administered intraperitoneally and different treatment schedules were tested. The maximal tolerated dose, 2 x 500 microCi, resulted in complete remission of tumours smaller than 200 mm3 and long-lasting remission (more than 135 days) of the larger tumours. In control experiments, treatment with unlabelled monoclonal antibody 123C3 did not affect the tumour growth rate, while the effect of radiolabelled non-relevant, isotype-matched, monoclonal antibody M6/1 was minor and transient. Regrowth of the tumours occurred in all cases and could not be attributed to loss of neural cell adhesion molecule (NCAM) expression. Tumour recurrence is probably caused by insufficient radiation dosage. Radiation-induced toxicity was monitored by assessment of weight and bone marrow examination. Weight loss was observed in all treatment groups, but the mice regained their initial weight within 14 days, except for the group receiving the highest radiation dose (3 x 600 microCi). In this group all mice died as a result of radiotoxicity. Of the mice injected with 600 microCi radiolabelled control antibody, 50% died within 2 weeks after administration. Apparently the higher uptake of the radiolabelled monoclonal antibody in the tumour reduced systemic radiation toxicity.

Magnetic Affinity Cell Sorting (MACS) Separation and Flow Cytometric Characterization of Neural Cell Adhesion Molecule-Positive, Cultured Myogenic Cells from Normal and Dystrophic Dogs

We developed a magnetic affinity cell sorting (MACS) assay based on differential expression of neural cell adhesion molecule (NCAM) isoforms in muscle cell cultures from normal and dystrophic dogs. NCAM is expressed during normal muscle differentiation, but has not been extensively examined within the context of muscle disease. A myogenic MACS assay could potentially maximize chances of obtaining normal nonsenescent, low-passage myogenic cells capable of proliferating in vitro and in vivo following transplantation. Myoblast-specific anti-NCAM polyclonal antibody directed against the NCAM isoform associated with muscle cell proliferation more effectively separated mixed canine cultures than did monoclonal antibodies directed against differentiated NCAM isoforms in the MACS assay. Flow cytometry using 5.1H11 anti-NCAM monoclonal antibody was then performed on normal and dystrophic fractionated cells and the results from these two groups were compared to each other and to nonfractionated cell populations. Normal canine cell cultures that had not been separated contained a larger percentage of FACscan-positive cells than did corresponding dystrophic canine cell cultures. Prior polyclonal anti-NCAM MACS separation of dystrophic cultures yielded higher numbers of adherent cells and higher gating percentages of 5.1H11-positive cell populations than did normal cultures. However, cells from dystrophic animals exhibited lower mean fluorescent expression of NCAM than normal cells.

Detection of bone marrow metastases in small cell lung cancer patients: Comparison of immunologic and morphologic methods : Beiske K, Myklebust AT, Aamdal S, Langholm R, Jakobsen E, Fodstad O. Institute for Cancer Research, Norwegian Radium Hospital, 0310 Oslo. Am J Pathol 1992;141:531–538

An immunocytochemical method, involving four monoclonal antibodies (MAbs) previously selected for their specific binding to small cell lung cancer (SCLC) cells in human bone marrow, was used for detection of bone marrow metastases in 81 patients with diagnosed SCLC. This procedure was compared with two routine morphologic methods with regard to diagnostic efficiency and sensitivity. Bone marrow involvement was found in 26 patients (32%), one of which had limited disease according to conventional clinical criteria. Eight of the positive cases were exclusively diagnosed by immunocytochemistry, whereas the histologic and cytologic methods separately identified two patients each. Immunocytochemistry had a detection level of tumor cells in the mononuclear cell fraction of approximately 1-2%, whereas no patients with less than 10% immunocytologically detectable tumor cells were diagnosed by cytomorphologic examination of bone marrow aspirates. Evidence was obtained that the diagnostic efficiency of any method increased with the number of samples examined. Of the four MAbs used, the anti-NCAM antibody, MOC-1, labeled tumor cells in all immunologically positive patients, and in all but one of these patients all cytologically confirmed tumor cells were stained. The antibodies MOC-31, which recognize a cluster-2 antigen, and NrLu10 bound nearly all tumor cells in most cases, whereas MLuC1 only diagnosed tumor cells in a fraction of the patients. The results show that the immunocytochemical application of these antibodies is superior to morphologic techniques in detecting SCLC bone marrow metastases. Further use of the method might provide prognostically and therapeutically useful information.