In Vitro Targeting of NG2 Antigen by ²¹³Bi-9.2.27 α-Immunoconjugate Induces Cytotoxicity in Human Uveal Melanoma Cells

Abstract

purpose. To examine uveal melanoma cell lines for the expression of human melanoma proteoglycan (NG2) using monoclonal antibody (mAb) 9.2.27 and subsequently to assess the in vitro specificity and cytotoxicity of mAb 9.2.27 conjugated to the α-particle–emitting radioisotope ²¹³bismuth (²¹³Bi-9.2.27) for uveal melanoma cells.

methods. Immunocytochemistry and flow cytometry were used to examine OCM-1, OCM-3, OCM-8, OMM-1, Mel202 and 92–1 melanoma cell lines for NG2 expression. Melanoma cells were treated with test (²¹³Bi-9.2.27) or control (²¹³Bi-A2) α-immunoconjugates (AICs). The specific cytotoxicity of ²¹³Bi-9.2.27 AIC was evaluated using an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfenyl)-2H-tetrazolium, inner salt) assay. Cell death was also assessed using TUNEL.

results. OCM-1, OCM-8, OMM-1, and Mel202 cells strongly expressed NG2. OCM-3 cells showed moderate expression and 92–1 cells were NG2-negative. ²¹³Bi-9.2.27 specifically killed NG2-positive OCM-1, OCM-8, and OMM-1 cells in a concentration-dependent manner. D ₀ values for 37% cell survival of NG2-positive OCM-1, OCM-8, and OMM-1 cells were 5.8, 5.0, and 5.6 μCi, respectively, and the value was 43.4 μCi for NG2-negative 92–1 cells.

conclusions. The specific cytotoxicity of ²¹³Bi-9.2.27 AIC for NG2-positive, but not NG2-negative, cells suggests NG2 is a suitable target for α-immunotherapy in uveal melanoma. ²¹³Bi-9.2.27 AIC used directly or as adjunct therapy may be a promising new agent for treating NG2-positive uveal melanomas or metastases.

Uveal melanomas affect the iris, ciliary body, and choroid and are the most common primary eye tumors in adults; ciliary body and choroidal melanomas constitute approxi-mately 95% of all uveal melanomas. Primary uveal melanomas may be treated successfully with enucleation, local tumor resection, laser photocoagulation, plaque radiotherapy, proton beam therapy, or combinations of therapies. ^{1 2 3 4 5 6} Earlier studies found minimal benefits using intravenous chemotherapy or immunotherapy for primary and metastatic uveal melanoma, though some success with these modalities has been reported recently. ^{6 7 8 9} Proton beam therapy or local plaque therapy generally produces good clinical responses. ¹⁰ However, radiation-associated morbidity remains a problem and usually involves long-term visual complications. Most patients with uveal melanoma display no detectable evidence of metastases at the time of diagnosis; however, within 5 years of enucleation, metastatic disease, predominantly to the liver, develops in approximately 40% of patients. Once detected clinically, metastatic disease is resistant to most therapies and is usually fatal within 12 months. Local therapies are ineffective for micrometastatic disease, and adjunctive therapies to prevent or treat melanoma metastases are lacking. New approaches to treat uveal melanoma metastases are, therefore, necessary. 

Targeted α-particle therapy (TAT) is an emerging therapeutic modality that uses a labeled antibody or protein to selectively target cancer cells and to deliver a lethal dose of short-range, highly cytotoxic α-radiation. This approach has the capacity to greatly increase the efficacy of tumor cell killing and to reduce damage to the surrounding normal tissue. Stable α-conjugates have been prepared with labeling yields of up to 95% for antibodies and proteins labeled with bismuth-213 (²¹³Bi) using cyclic diethylenetriaminepentaacetic acid anhydride (cDTPA) or DTPA-CHX-A” as chelators. ^{11 213}Bi has a short half-life (t_1/2 = 46 minutes) and emits α-particles with high linear energy transfer radiation and a short range (80 μm). As such, α-particles have several advantages over β-particles. They cause double-strand DNA damage that is not easily repaired by the cell because of the very high linear energy transfer (∼100 times greater than for β-particles); their cytotoxicity is not affected by oxygen; and they are much more cytotoxic, requiring as few as 6 or 7 disintegrations for internalized α-particles and approximately 25 disintegrations for surface-bound α-emitters to kill a cell. ¹² α-Particle therapy has been used in single-cell disorders, such as leukemia, lymphoma, and micrometastatic carcinoma, ^{13 14 15} in which rapid targeting to cancer cells is possible. 

mAb 9.2.27 is highly specific for the melanoma-associated chondroitin sulfate proteoglycan NG2, ^{16 17} which is expressed on most cutaneous ^{18 19} and uveal melanomas. ²⁰ Stably chelated ²¹³Bi-9.2.27 has recently been found to be highly specific and cytotoxic to skin melanoma cells in vitro ¹¹ and to completely regress tumor growth in a xenograft mouse model of skin melanoma after local injection. ^{14 21} In this study, we examined human uveal melanoma cell lines for NG2 expression and evaluated the in vitro efficacy and specificity of ²¹³Bi-9.2.27 as a cytotoxic agent for these cell lines. 

Materials and Methods

Antibodies

mAb 9.2.27, with a high specificity for human melanoma cells related to a 250-kDa N-linked glycoprotein and a >400-kDa proteoglycan component, was kindly provided by Peter Hersey (Department of Oncology and Immunology, Newcastle Mater Misercordiae Hospital, Newcastle, NSW, Australia). Rabbit anti–NG2 chondroitin sulfate proteoglycan polyclonal antibody (pAb) was obtained from Chemicon International (Temecula, CA). Nonspecific mouse anti–human IgG1 monoclonal antibody (A2) was kindly provided by Andrew Collins (Department of Microbiology, University of New South Wales, Sydney, NSW, Australia). Mouse myeloma IgG2a isotype control and rabbit immunoglobulin isotype control were supplied by Zymed Laboratories Inc. (San Francisco, CA). Goat anti–mouse IgG (Alexa-488) and streptavidin (Alexa-594) conjugates were purchased from Molecular Probes Inc. (Eugene, OR). Goat anti–mouse or sheep anti–rabbit fluorescein isothiocyanate (FITC)–conjugated antibodies were purchased from Chemicon International. 

Cell Culture

Five human uveal melanoma cell lines (OCM-1, OCM-3, OCM-8, Mel202, 92–1) and one cell line derived from a uveal melanoma skin metastasis (OMM-1) were grown in either RPMI 1640 medium (OCM-3, OCM-8, 92–1, Mel202) or DMEM medium (OCM-1 and OMM-1) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 50 IU/mL penicillin, and 50 μg/mL streptomycin. Tissue culture reagents were supplied by ThermoElectron Pty. Ltd. (Noble Park, Victoria, Australia). Cells were maintained in a humidified incubator at 37°C and 5% CO₂. 

Immunocytochemistry

Cells were seeded onto glass coverslips in a 24-well plate at a density of 10⁴cells/well and were cultured overnight in growth medium. After rinsing in PBS (pH 7.4), cells were fixed in cold acetone for 5 minutes, rinsed again in PBS, and blocked in 10% normal goat serum (NGS)/PBS for 20 minutes at room temperature (RT). Cells were incubated overnight at 4°C in mouse anti–human NG2 (IgG2a; 1:100 dilution), rabbit anti–human NG2 pAb (1:200 dilution), or isotype control (mouse IgG2a; 1:100 dilution or rabbit immunoglobulins, 1 μg/mL). After rinsing in PBS, coverslips were incubated for 1 hour at RT in Alexa-488–conjugated goat anti–mouse or goat anti–rabbit antibody (1:1000 dilution). After a final rinse in PBS, coverslips were mounted on slides in glycerol, and viewed using fluorescence microscopy. 

Flow Cytometry

Cell-surface NG2 expression was detected using flow cytometry. Briefly, confluent adherent cells were rinsed twice with PBS and detached with a scraper. Cells (0.5 ∼ 1.0 × 10⁶) were washed twice in cold Dulbecco’s phosphate-buffered saline (DPBS) with 5% FBS (DPBS/FBS; 200g, 8 minutes) and resuspended in 80 μL DPBS/FBS. Cells were then incubated for 60 minutes at 4°C in either an isotype control (mouse IgG2a or rabbit immunoglobulin), mouse anti–human NG2 (IgG2a), or rabbit anti–human NG2 pAb. After washing with DPBS/FBS, the cells were resuspended and incubated in goat anti–mouse FITC-conjugated antibody (1:80 dilution) or sheep anti–rabbit FITC-conjugated antibody (1:40 dilution) for 45 minutes in the dark at 4°C. The cells were washed again and resuspended in 0.5 mL DPBS/FBS. Autofluorescence was subtracted in all experiments. Ten thousand cells in each sample were counted, and the data were presented as histograms. All data were analyzed (CELLQuest software; Becton-Dickinson, San Jose, CA). 

Radiolabeling of mAbs with Radioisotope

²¹³Bi was produced from the ²²⁵Ac/²¹³Bi generator ²² ; the ²²⁵Ac column was purchased from the United States Department of Energy (Oak Ridge, National Laboratory, Oak Ridge, TN). Using published methods, ¹¹ mAbs 9.2.27 and A2 were conjugated with the chelator, cyclic diethylenetriaminepentaacetic acid anhydride (cDTPA; Sigma-Aldrich Pty., Ltd., Castle Hill, NSW, Australia). Conjugated mAbs 9.2.27 and A2 were measured by plate reader at 280 nm using commercial software (ProMax; Bio-TEC Instruments Inc., Winooski, VT) and were purified on a PD-10 column (Amersham Biosciences Ltd., Bucks, UK). ²¹³Bi was eluted from the ²²⁵Ac column with 250 μL freshly prepared 0.15 M hydriodic acid as the (BiI₅)²- anion species, neutralized to pH 4 to 4.5 with the addition of 3 M ammonium acetate and was immediately used to radiolabel the mAb construct. Two to 3 hours was allowed for ²¹³Bi to grow back in the generator for the next elution. Radiolabeling efficiency was determined by instant thin-layer chromatography, with a 10-μL aliquot of the final reaction mixture applied to silica gel–coated fiber sheets (Gelman Science Inc., Ann Arbor, MI). The paper strips were developed using 0.5 M sodium acetate (pH 5.5) as the solvent. Then the paper strips were cut into four sections, and the γ-emissions from the radioisotope were counted in each section using a 340- to 540-keV window. Radiolabeled protein stays in the section of origin, whereas free radioisotope moves with the solvent front section. Radiolabeling efficiency was 80% to 95% for ²¹³Bi-9.2.27 (test) and ²¹³Bi-A2 (control) AICs. 

In Vitro Cytotoxicity Assay

Three NG2-positive cell lines (OCM-1, OCM-8, OMM-1) and a NG2-negative cell line (92–1) were selected for in vitro AIC treatment. To ensure equal activities, ²¹³Bi-9.2.27 and ²¹³Bi-A2 preparations were measured using a radioisotope calibrator (Atomlab 200; Biodex Medical System, Shirley, NY). Conjugate solutions were neutralized to pH 7.0 by the addition of 10% (vol/vol) 1 M NaHco ₃ (pH 9.0). After this, five serial activities of ²¹³Bi-9.2.27 (1 μCi, 2 μCi, 4 μCi, 8 μCi, 10 μCi) and a dose of ²¹³Bi-A2 at the highest activity (10 μCi) were prepared in 100 μL RPMI 1640/5% FBS and were added to 96-well plates in triplicate containing 2 × 10⁴ OCM-1, OCM-8, OMM-1, or 92–1 cells, respectively. Controls were performed in triplicate in the same 96-well plate for each experiment and consisted of cDTPA-9.2.27, mAb 9.2.27, and RPMI 1640/5% FBS medium alone. Plates were incubated overnight at 37°C, and cell morphology was subsequently assessed before the MTS assay. 

Cells were then washed in DPBS, incubated in 100 μL serum-free, phenol-red free RPMI 1640 containing 20 μL reagent (Cell Titer 96 Aqueous One Solution; Promega, Madison, WI) for 3 hours at 37°C. The reaction was stopped by the addition of 10% sodium dodecyl sulfate, and the absorbance of each well was recorded at 490 nm using a plate reader (Spectro Max; Bio-Rad, Hercules, CA). The absorbance reflects the number of surviving cells. Blanks were subtracted from all data, and results were analyzed (Prism software; GraphPad Software Inc., San Diego, CA). 

Assessment of Cell Death

TUNEL Technique.

To assess whether AIC treatment induced apoptotic cell death, cultured OCM-1, OCM-8, OMM-1, and 92–1 cells were treated with a low or a high concentration of ²¹³Bi-9.2.27 (2 μCi or 10 μCi, respectively) and with ²¹³Bi-A2, cDTPA-9.2.27, or medium alone at 37°C overnight. After treatment, cells were washed with DPBS and were harvested by scraping. Cell cytospins using 3 × 10⁴ cells/100 μL were prepared using a centrifuge (Heraeus Megafuge 1.0R; DJB Labcare, Buckinghamshire, UK) and were fixed in 2% paraformaldehyde at RT for 30 minutes. Apoptosis was detected using a modified TUNEL method. ^{23 24} Briefly, cytospins were rinsed in terminal deoxynucleotidyl transferase (TdT; pH 7.2) buffer for 10 minutes at RT and then incubated at 37°C for 1 hour in reaction mixture containing 10.5% TdT, 0.42% biotin-16 to 2′-deoxyuridine-5′-triphosphate (dUTP; Roche Molecular Sciences Pty. Ltd., Sydney, NSW, Australia), and 0.13% terminal transferase enzyme (Roche Molecular Sciences) in Milli-Q water. The reaction was stopped by immersion in 2× SSC (3 M sodium chloride and 0.3 M sodium citrate, pH 7.0) for 15 minutes at RT, followed by rinsing in 1% bovine serum albumin (BSA) in PBS. To label DNA fragments, cytospins were then incubated in streptavidin-Alexa 594 conjugate (1:1000 dilution) for 1 hour at RT, rinsed in PBS, coverslipped (Universal Mount; Invitrogen Life Technologies, Carlsbad, CA), and examined using a fluorescence microscope (Diaplan; Leitz, Wetzlar, Germany) at 50× magnification. 

The specificity of TUNEL reactivity was confirmed in parallel negative (omitting terminal transferase enzyme from the reaction mixture) and positive (human retina) controls. Apoptotic cells were identified by TUNEL labeling and the presence of nuclear chromatin fragments. 

Acridine Orange/Ethidium Bromide Staining.

Acridine orange/ethidium bromide (AO/EB) staining was used to identify apoptotic cells, as described previously. ²⁵ Briefly, cultured OCM-1, OCM-8, OMM-1, and 92–1 cells were treated with a low or a high concentration of ²¹³Bi-9.2.27 (2 μCi and 10 μCi, respectively) or with ²¹³Bi-A2, cDTPA-9.2.27 or medium alone at 37°C overnight. At 24 and 48 hours, 25 μL floating control or treated cells were stained with 1 μL 100 μg/mL AO/EB in PBS; 10 μL stained cells was placed onto a glass slide, coverslipped, and examined immediately using a fluorescence microscope (Diaplan; Leitz). 

Results

Expression of NG2 on Uveal Melanoma Cell Lines

Immunocytochemistry.

OCM-1, OMM-1, OCM-3, OCM-8, and Mel202 uveal melanoma cell lines displayed strong immunoreactivity for NG2 antigen compared with the isotype control (Fig. 1A 1B 1C 1D 1E) . The 92–1 cell line displayed minimal NG2 immunoreactivity (Fig. 1F) , similar to the isotype control (Fig. 1G) . NG2-immunolabeled cells displayed obvious punctate staining clearly localized to cell membranes (Fig. 1A 1B 1C 1D 1E 1H ). Similar patterns of immunolabeling were seen with mAb 9.2.27 and pAb human NG2 (Fig. 1H) . Flow cytometry confirmed these observations. 

Flow Cytometry.

OCM-1, OMM-1, OCM-8, and Mel202 cell lines strongly expressed NG2 immunolabeling (98%, 98%, 99%, and 94% positive cells, respectively; Figs. 2A 2C 2D 2F ) compared with the isotype control, and OCM-3 cells showed moderate immunolabeling (85% positive cells; Fig. 2E ). NG2 immunolabeling of 92–1 cells was similar to that of the isotype control (2% positive cells; Fig. 2B ). Similar results were found using rabbit anti–human NG2 pAb (not shown). 

Cytotoxicity

²¹³Bi-9.2.27 AIC was specifically cytotoxic to NG2-positive melanoma cells (OCM-1, OMM-1, and OCM-8) in a concentration-dependent fashion (Fig. 3A 3B 3C)but not to 92–1 NG2-negative melanoma cells (Fig. 3D) . For 37% cell survival, the corresponding D₀ values for the NG2-positive cell lines—OCM-1, OMM-1, and OCM-8—were 5.8 μCi, 5.0 μCi, and 5.6 μCi, respectively, and 43.4 μCi for NG2-negative 92–1 melanoma cells. A nonspecific control α-immunoconjugate (²¹³Bi-A2) that did not specifically target the NG2 protein induced minimal cytotoxicity in all cell lines (Fig. 3A 3B 3C 3D) . 

Morphology

After 24-hour treatment with ²¹³Bi-9.2.27 (2-μCi and 10-μCi doses), NG2-positive cells (OCM-1, OCM-8, OMM-1) detached from the plate and rounded up in a concentration-dependent fashion. Pyknotic cells and some necrotic cells were seen in the floating cell population (Figs. 4C 4D 4G 4H) ; adherent cells were also visible, though much reduced in numbers compared with controls (Figs. 4D 4H) . Cells cultured with isotype control AIC (²¹³Bi-A2), cDTPA-9.2.27, or medium alone remained adherent to the plate, showed little evidence of cell death, and displayed the spindle or epithelioid morphology characteristic of the respective cell lines (Figs. 4A 4B 4E 4F) . 

Cell Death

Cells treated with nonspecific AIC or medium alone displayed no obvious TUNEL labeling (Figs. 5A 5B) . Some TUNEL-positive OCM-1, OMM-1, and OCM-8 cells were observed after treatment with ²¹³Bi-9.2.27 AIC (Figs. 5C 5D) . However, when the terminal transferase enzyme was omitted, no TUNEL-positive cells were seen (Fig. 5E) . AO/EB staining of floating cells 24 hours after treatment with ²¹³Bi-9.2.27 AIC showed some cells with nuclear chromatin condensation and fragmentation—morphologic features consistent with apoptotic cell death (Figs. 5F 5H) . Viable cells were also seen in control cultures (Fig. 5G) . We also observed degenerate cells ²⁵ that stained with EB in ²¹³Bi-9.2.27 AIC–treated cultures (not shown). 

Discussion

Monoclonal antibodies are being increasingly recognized for their potential in anticancer therapeutics, with strategies that include mAbs combined with cytotoxic drugs or conjugated with radionuclides or immunologic effector cells. The selection of target antigens and targeting vectors requires high specificity and affinity to cancer tissue. To date, one of the most widely explored strategies for enhancing the efficacy of antitumor antibodies is direct arming by chelator linkage to toxins or radionuclides. ²⁶ Targeted radiotherapy that uses radioactive isotopes linked to mAbs or proteins specific for cancer cells has been proposed for various tumors. ^{12 27 28 29}  

We recently described NG2 expression in a series of primary uveal melanomas and normal control eyes. ²⁰ In that study, 95% (18/19) of uveal melanoma specimens expressed moderate to high levels of tumor cell surface mAb 9.2.27 immunoreactivity (NG2). ²⁰ However, in most melanoma-affected eyes, the retina, retinal pigmented epithelium, and choroid displayed low-level immunostaining. Furthermore, in control eye specimens (without known disease; n = 5), the retina and choroid appeared negative for mAb 9.2.27. ²⁰ We did observe evidence of moderate immunoreactivity in optic nerve axon bundles of control and melanoma-affected eyes; however, this immunostaining is not cellular but is associated with the myelin (unpublished observation, 2004). The present study also demonstrates high levels of in vitro cell surface NG2 expression on uveal melanoma cell lines OCM-1, OCM-3, OCM-8, Mel202, and OMM-1 (but not 92–1 cells) using flow cytometry and immunocytochemistry with mAb 9.2.27 and a polyclonal NG2 antibody. Taken together with earlier observations on primary uveal melanoma and the apparent absence of mAb 9.2.27 immunoreactivity on normal ocular tissues, ²⁰ these results support the use of NG2 as a potential effective tumor cell surface marker for targeting primary uveal melanoma, similar to observations in cutaneous melanoma. ^{11 14 21}  

In the present study, we also observed similar levels of NG2 immunoreactivity on OCM-1 and OMM-1 cell lines—uveal and metastatic melanoma cell lines, respectively. A recent study ³⁰ comparing mRNA expression profiling of cancer-related genes in uveal melanoma and liver metastases from the same patient found some similarities in patterns of gene expression. These observations suggest that circulating cancer clones from primary uveal melanomas may not necessarily lose NG2 expression, but this remains to be confirmed in primary metastatic uveal melanomas. Application of ²¹³Bi-9.2.27 AIC to target metastatic uveal melanoma may be possible either systemically or through isolated hepatic perfusion (IHP). A recent study using IHP of high-dose chemotherapy in a small group of patients with uveal melanoma with metastases confined to the liver indicates that this approach may produce tumor response in some patients. ³¹  

The in vitro cytotoxicity of ²¹³Bi-9.2.27 to uveal melanoma cell lines in the present study was specific, concentration dependent, and directly related to the level of NG2 expressed on these cells. The D ₀ values for the NG2-positive cell lines (OCM-1, OMM-1, and OCM-8) were 5.8 μCi, 5.0 μCi, and 5.6 μCi, respectively, and 43.4 μCi for the NG2-negative 92–1 melanoma cell line. A similar pattern of cell killing was observed for OCM-1, OMM-1, and OCM-8 cells, consistent with their similar levels of NG2 expression. Significantly, there was no cell killing for cDTPA-9.2.27 cold conjugate or mAb 9.2.27 alone groups. From these observations, <10% of melanoma cells would be expected to survive after 10 μCi ²¹³Bi-9.2.27 AIC compared with survival of >90% melanoma cells for the same activity of nonspecific ²¹³Bi-A2. Clearly, ²¹³Bi-9.2.27 can specifically target and kill NG2-positive cells while sparing NG2-negative (92–1) uveal melanoma cells. 

NG2 proteoglycan has been implicated in the growth and invasion of cutaneous melanoma, though its function in uveal melanoma is unclear. As a membrane-spanning molecule, NG2 can interact with extracellular and intracellular components and may trigger cytoskeleton-dependent changes in cell morphology and motility in response to the extracellular environment. ^{32 33 34} Both the growth control and the cell motility aspects of NG2 function have been observed in studies of cutaneous melanoma progression, with NG2-expressing mouse melanoma cells reported to grow faster and to be more metastatic than their NG2-negative counterparts. ^{18 35} Overexpression of NG2 has also been found to increase tumor initiation, growth rates, neovascularization, and cellular proliferation, factors that predispose to poorer survival outcome. ³⁶  

Perivascular cells (mature and immature smooth muscle cells and pericytes) on arterioles and capillaries have been observed to express NG2 immunoreactivity during normal development and in pathologic vascular remodeling, as seen, for example, during tumor growth. ^{36 37 38} Smooth muscle cells and pericytes have also been observed to be NG2-immunoreactive in developing and adult rat retina ^{39 40} and in diabetic human retina. ⁴¹ However, in our earlier study, obvious NG2 immunolabeling of perivascular cells was not apparent in human retina, choroid, or primary uveal melanoma specimens. ²⁰ These differences may be related to the use of different antibodies, detection systems, or immunolabeling techniques. However, it is important to establish whether normal adult human choroidal and retinal perivascular cells do express NG2 antigen, particularly if local therapy is to be considered. Furthermore, NG2-expressing pericytes and smooth muscle cells may represent targets for therapy (including ²¹³Bi-9.2.27) in uveal melanoma, as proposed in a recent study of orthotopic human uveal melanomas induced in immunosuppressed wild-type and NG2-knockout mice (Ozerdem U. IOVS 2005;46:ARVO E-Abstract 4620). Pericytes were observed throughout these uveal melanomas, with a significant decrease in tumor microvascular density in NG2-knockout mice; the importance of pericytes and NG2 proteoglycan in tumor vascularization suggests potential therapeutic targets in controlling tumor growth (Ozerdem U. IOVS 2005;46:ARVO E-Abstract 4620). Further studies of NG2 antigen expression on vascular-associated cells in normal and tumor-affected human retina and choroid and in uveal melanoma are being pursued. 

Our recent studies of prostate cancer cells found a high percentage of TUNEL-positive cells after treatment with AICs, indicating the induction of cell death predominantly by apoptosis. ^{42 43 44} An earlier electron microscopy study of AIC-treated murine lymphoma cells reported bizarre blebbing patterns, condensation of chromosomal material, and internucleosomal DNA fragmentation, also consistent with the induction of apoptotic cell death. ⁴⁵ In the present study, TUNEL-positive cells were seen 24 hours after ²¹³Bi-9.2.27 AIC treatment of NG-2 positive cell lines; however, apoptosis was not the only mode of cell death observed. Many factors, including antigen affinity and antigen density, play important roles in the killing of targeted antigen-positive cells. For example, after binding the cell NG2 antigen, ²¹³Bi-9.2.27 may form ²¹³Bi-9.2.27-NG2 complexes at the cell membrane, emitting α-particles that can kill uveal melanoma cells by causing double-DNA strand breaks. ¹² Alternatively, surface-bound ²¹³Bi-9.2.27-NG2 complexes may be internalized by the cell with increased cell killing efficiency, ^{14 15} as suggested in ²¹³Bi-Herceptin–mediated killing of prostate cancer cells. ⁴⁴ The relative importance of these factors (antigen density, antigen affinity, and internalization) in AIC-mediated killing of uveal melanoma cells remains to be determined. 

We are conducting a phase 1 clinical trial for secondary or recurrent skin melanoma using intralesional injection of ²¹³Bi-9.2.27 AIC. To date, this study shows almost complete tumor cell kill in a diffuse area around the injection site compared with lesions injected with antibody alone, consistent with specific targeting of this AIC (unpublished data, 2004). The present study shows that ²¹³Bi-9.2.27 AIC can also selectively kill NG2-positive uveal melanoma cells and that it has the potential to target human primary uveal melanoma cells that express high levels of NG2. These results support further investigations into the efficacy of this AIC for local and systemic therapy in animal models of uveal melanoma, particularly with liver metastases. 

 Supported in part by grants from the Sydney Foundation for Medical Research (MCM), the Ophthalmic Research Institute of Australia, and the US Department of Defense (YL).

 Submitted for publication May 9, 2005; revised August 12, 2005; accepted October 18, 2005.

 Disclosure: Y. Li, None; J. Wang, None; S.M. Abbas Rizvi, None; M.J. Jager, None; R.M. Conway, None; F.A. Billson, None; B.J. Allen, None; M.C. Madigan, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement ” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

NG2 immunoreactivity in melanoma cell lines. (A) OCM-1, (B) OMM-1, (C) OCM-3, (D) OCM-8, and (E) Mel202 cell lines display strong immunostaining for NG2, with obvious punctate staining clearly localized to cell membranes. (F) 92–1 cells are negative for NG2 antigen, similar to the isotype control (G). (H) Distinct cell membrane immunolabeling is also obvious using polyclonal human anti–NG2 antibody, seen in this example of OMM-1 melanoma cells.

Figure 2.

 

Flow cytometric analysis of NG2 immunoreactivity on melanoma cell lines using mAb 9.2.27. Data are shown as histograms using a mouse IgG2a-negative control to determine background fluorescence and to set the marker (M1). All cell lines except 92–1 show high levels of NG2 expression. (A) OCM-1, 98%; (B) 92–1, 2%; (C) OMM-1, 98%; (D) Mel202, 99%; (E) OCM-3, 85%; and (F) OCM-8, 94%; FL1-H log fluorescence intensity.

Figure 3.

 

Representative cytotoxicity of (A) OCM-1, (B) OMM-1, (C) OCM-8, and (D) 92–1 melanoma cell lines. Twenty thousand cells are seeded in triplicate and treated with a range of concentrations of ²¹³Bi-9.2.27 or a maximum concentration (10 μCi) of ²¹³Bi-A2 nonspecific isotype control, respectively. After overnight incubation, cell survival is measured by MTS assay at 24 hours and is expressed as a percentage of controls. Each point represents a mean of three experiments.

Figure 4.

 

Morphologic features of OCM-1 (A–D) and OCM-8 (E–H) cells 24 hours after AIC treatment. Cells cultured with isotype control AIC (²¹³Bi-A2), cDTPA-9.2.27, or medium alone remain adherent to the plate, show little evidence of cell death, and display the characteristic spindle/epithelioid morphology of the respective cell lines (A, B, E, F). After treatment with ²¹³Bi-9.2.27 AIC [(C, G) 2 μCi; (D, H) 10 μCi], many NG2-positive cells detach from the plate and round up, more obviously with 10 μCi ²¹³Bi-9.2.27 AIC (C, D, G, H). Some apoptotic and necrotic cells are seen in these detached cell populations (C, D, G, H). Adherent cells are also visible 24 hours after treatment (D, H).

Figure 5.

 

TUNEL assay of OCM-8 (A–C) and OCM-1 (D, E) melanoma cells. No TUNEL-positive cells are seen after treatment with (A) nonspecific AIC or (B) medium alone. Some TUNEL-positive cells (arrows) are seen in cultures 24 hours after treatment with (C) 2 μCi and (D) 10 μCi ²¹³Bi-9.2.27 AIC. (E) No labeling is seen when terminal transferase enzyme is omitted from the reaction mixture. AO/EB staining of OCM-1 cells is also shown (F–H). After treatment with ²¹³Bi-9.2.27 AIC, some cells display nuclear chromatin fragmentation and condensation, characteristic of apoptosis (arrows, F, H). Viable control cells are shown (G).

The authors thank John Kearsley, Director, St. George Cancer Services, for ongoing support and David Zhang for assistance with flow cytometry experiments. OCM-1, OCM-3, and OCM-8 cell lines were kindly provided by June Kan-Mitchell (University of California at San Diego, CA); OMM-1 cells were kindly provided by Gregorius P. M. Luyten (Erasmus University, Rotterdam, The Netherlands); and Mel202 cells were kindly provided by Bruce R. Ksander (Schepens Eye Research Institute, Harvard Medical School, Boston, MA).