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Experimental Therapeutics |
Clinic for General and Thoracic Surgery, Christian-Albrechts-University, D-24105 Kiel, [S. E., C. T., K. F.], Department of Molecular Biology and Biochemistry, Institute of Chemistry, University of Hamburg, D-20251 Hamburg, Germany [K. D., R. B.], and Lombardi Cancer Center, Georgetown University, Washington, DC 20007 [H. J.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, natural human cytotoxic anti-NB IgM antibodies have been discovered in the serum of healthy humans. High levels of these antibodies were reported in 
30% of blood donors (7)
. Interestingly, these antibodies are barely found in children suffering from neuroblastoma, suggesting a protective role in neuroblastoma development (8)
.
Natural human anti-NB IgM antibodies recognize a Mr 260,000 surface protein on human neuroblastoma cells (7 , 9) . A pilot study has demonstrated the efficacy of these antibodies in eradicating s.c. xenografts of human NB in nude rats (10) . Despite the use of an extremely small amount of active IgM antibody and the large size of the antibody, which theoretically hinders its tissue distribution, a dramatic tumor regression was achieved. This is unlikely to solely rely on an antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activation, and indeed it was found that these antibodies also induce apoptosis, which significantly contributes to their cytotoxicity (9 , 10) .
The principal mechanism of human anti-NB IgM antibody function in vivo has been determined in s.c. xenograft models. However, comparative studies of s.c. and orthotopically injected tumor cells demonstrate that the resulting tumors differ regarding their invasive and metastatic behavior (11, 12, 13) . A more realistic view of the clinical potential of anti-NB antibodies could be obtained if a tumor model that better reflects the growth pattern of advanced NB in children (i.e., large adrenal gland tumors and multiple small metastatic lesions preferentially in the liver and bones) were available.
In this study we have characterized a new human NB animal model that reflects the common growth pattern of NB in children. Using this model we demonstrate the ability of natural human cytotoxic anti-NB IgM antibodies to eradicate human NB cells.
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MATERIALS AND METHODS |
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Purification of Human Anti-NB IgM.
Serum of blood donors was obtained from the Transfusion Medical Center of the University Hospital Kiel and screened for the presence of cytotoxic anti-NB antibodies using an in vitro cytotoxicity assay as previously described (7)
. Two donors were identified who showed anti-NB cytotoxicity of >90% cell killing (donors 005 and 062). The IgM fraction of the serum of these donors was purified, and cytotoxicity was confirmed. In this study we exclusively used the serum from donor 005, which showed a cytotoxicity level of 94% LAN-1 cell lysis.
Purification of the IgM fraction was performed by a combination of size exclusion (Sephacryl S-300 HR; Amersham Pharmacia Biotech, Freiburg, Germany) and anion exchange chromatography (Macro-Prep High Q; Bio-Rad, Munich, Germany) as previously described (7) . Using this procedure, IgM was purified to homogeneity as confirmed by SDS-PAGE and Western blotting as previously described (8) . Purified fractions were concentrated to 1 mg/ml PBS by ultrafiltration with stirred ultrafiltration cells (Amicon, Beverly, MA). Aliquots were stored at 4°C after sterile filtration through a 0.2-µm cellulose acetate filter. Antibody binding and the cytotoxicity against NB cells were confirmed by FACS analysis using dichlorotriacinylamino-fluorescein-conjugated goat antihuman IgM (Dianova, Hamburg, Germany) and propidium iodide, respectively (7) .
Animal Experiments.
Four-week-old male nude rats (rnu/rnu) were obtained from Harlan Winkelmann (Borchen, Germany). To induce metastatic NB, rats were anesthetized, and 1 x 107 LAN-1 NB cells in a 50-µl volume were injected in the tail vein or after laparotomy, in the capsule of the left adrenal gland or in the aorta abdominalis next to the diaphragm. The lethality of either method was 0%. Bleeding occurred in 5% of rats receiving an intra-aortal cell injection and was controlled intraoperatively by local compression. Antibodies were injected i.p. as described in detail in "Results." At the end of the experiments rats were killed and inspected, and adrenal glands, kidney, femur, liver, and lung were removed, snap-frozen in liquid nitrogen, and stored at -80°C until further studied. Adrenal gland tumors were measured in three dimensions, and the volume was calculated by the formula 
/2 x (d1 x d2 x d3).
Tissue Immunostaining.
Cryostat sections (5 µm) of the respective organs were prepared. As a staining method, we used the immunoperoxidase technique as previously described (14)
. NB cells were identified by murine monoclonal antibodies directed against the NB84 antigen (DAKO, Glostrup, Denmark) and the GD2 antigen (BW 704; Behringwerke, Marburg/Lahn, Germany). Additional staining included monoclonal antibodies directed against p53 (DAKO), Fas-receptor (Immunotech, Marseilles, France), and Fas-ligand (Alexis Corp., Grünberg, Germany). In parallel to an immunostaining we performed H&E staining for morphological evaluation.
Determination of the Proliferation Rate.
Tumors were immunostained using the MIB-1 antibody purchased from Dianova (Hamburg, Germany) and the immunoperoxidase technique. In a blinded manner two independent investigators counted stained tumor cells of 20 microscopic fields to determine the proliferation rate (= percentage of proliferating cells within a tumor).
Determination of the Apoptotic Rate.
Tumors were stained using the Apop Tag TUNEL staining kit obtained from Oncor (Gaithersburg, MD). Two independent investigators counted stained tumor cells of 20 microscopic fields to determine the apoptotic rate (= percentage of apoptotic cells within a tumor).
Detection of IgM Antibodies within NB Tissue.
Tumor cryostat sections of IgM antibody-treated and untreated (buffer control) animals were stained by immunofluorescence technique using a fluorescent Cy3-labeled rabbit antihuman IgM antibody (Dianova, Hamburg, Germany) as previously described (9)
.
Detection of Complement Accumulation in Adrenal Gland Tumors.
Cryostat slices from adrenal glands of IgM-treated and control animals were immunostained using polyclonal goat anti-C3 antibodies (Quidel, San Diego, CA) and alkaline phosphatase-labeled rabbit antigoat IgG antibody (Sigma Chemical, Deisenhofen, Germany) as previously described (9)
.
Data Analysis.
For statistical analysis we used the unpaired t test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After 3 weeks a laparotomy was performed and showed large adrenal gland tumors of 5,300 ± 1,100 mm3 (mean ± SD) in orthotopically injected rats and 1,570 ± 450 mm3 in the intra-aortal group. Tail vein injection did not cause visible tumors at this time interval. Five weeks after orthotopic injection, extremely large encapsulated adrenal gland tumors 25,132 ± 1,570 mm3 were found in all of the animals of the orthotopically treated animals. One hundred percent of the rats that were injected in the tail vein or in the aorta developed infiltrative adrenal gland tumors. Although tail vein injection resulted in tumors of 42 ± 12 mm3, intra-aortal injection caused tumors of 5300 ± 1300 mm3. Regardless of the type of injection, adrenal tumors showed the typical appearance of human NB with a gray to yellow color, hemorrhage, and foci of calcification.
Macroscopic metastases were not apparent in either group, but femur fracture occurred frequently in rats that had bone metastases identified histologically. We prepared tissue sections of various organs (femur, liver, kidney, lung, spleen) and searched for micrometastatic tumors using immunohistochemistry (GD2 and NB84 antibodies). None of the rats receiving orthotopic injection of LAN-1 cells presented micrometastatic tumor growth or even single disseminated NB cells. After tail vein injection we found in one rat a single cluster of micrometastatic NB cells in the femur whereas all of the other organs remained tumor cell free. Most frequently metastases appeared in rats receiving intra-aortal cell injection. Multiple micrometastases (cell clusters of >10 cells) were found in 50% (3 of 6) of the livers and in 60% (4 of 6) of the femurs. The femur of all four mice showed both bone marrow involvement and bone-infiltrating tumor growth. Neither micrometastases nor single cells were found in the lung, kidney, and spleen of this group. Figure 1
shows a NB tumor in situ, the histopathological picture of the adrenal gland and femur metastases (H&E staining), and immunostaining of a liver micrometastasis by anti-GD2 antibodies.
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Time Course of Tumor Growth
In the next set of experiments we analyzed the time course of metastatic NB growth. The data are summarized in Fig. 2
. After intra-aortal injection of 1 x 107 LAN-1 cells, animals were killed after 1, 3, 7, 14, and 21 days. The adrenal glands, liver, and femur were asservated, and cryostat sections were prepared and immunostained using NB84 and GD2 antibodies. After 24 h, 3 days, and 7 days we found increasing numbers of tumor cells in the adrenal glands and to a lesser extent in the liver and bone. Fourteen days after tumor cell injection small adrenal tumors became visible, which grew to a size of 6–8 mm in diameter after 21 days. In the femur and the liver multiple micrometastases appeared after 21 days.
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94% cell killing). Analysis of the isolated IgM fraction by SDS-PAGE and Western blot revealed highly purified antibodies (Fig. 3A)
, which efficiently bind to NB cells (Fig. 3B)
and lyse >90% of LAN-1 cells (Fig. 3C)
. We also confirmed binding to the Mr 260,000 antigen using Western blot (data not shown).
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Experiment 1 (Early Adjuvant Therapy).
In this experiment we started the therapy of rats (n = 5) directly after tumor cell injection. Every other day, rats received 1 mg of purified IgM antibodies eight times i.p. The treatment was discontinued 16 days after cell injection, and on the 22nd day animals were killed and inspected, and organs were removed for analysis.
None of the antibody-treated rats showed enlarged adrenal glands. Not even a single tumor cell in the gland was detectable, as determined by immunostaining, which suggests a complete eradication of the injected tumor cells (Fig. 4A)
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. In control experiments (n = 4) rats were treated with buffer, and tumors reached a mean volume of 870 ± 460 mm3. Micrometastatic cell clusters were found in one liver and two femurs. The difference in tumor sizes of experiment 1 and experiment 2 compared with the control group was statistically significant (P < 0.05). The number of positive liver and femur samples was too small to allow statistical evaluation.
Experiment 3 (Treatment of Advanced Tumors).
Two weeks after intra-aortal tumor cell injection, rats (n = 4) with visible adrenal gland tumors received 1 mg of anti-NB IgM antibodies for 5 consecutive days. A control group (n = 4) was similarly treated with buffer solution. The rats were killed 24 days after tumor cell injection (i.e., 5 days after the final antibody injection).
All of the rats of the control group developed large NBs in both adrenal glands, with an average tumor volume of 2821 ± 1200 mm3. Significantly smaller tumors (P < 0.05) were found in animals treated with IgM antibodies, showing an average tumor volume of 318 ± 109 mm3 (Fig. 4B)
. In both the treatment group and the control group, liver micrometastases were present in one rat of each group. One animal of the treated versus three rats of the control group showed micrometastases in the femur.
Evaluation of the Cytotoxic Antibody Effects
Detection of IgM Antibodies in Adrenal Gland Tumors
To determine whether the injected antibodies reached their target, we performed cryostat sections of adrenal gland tumors of the treatment group and the control group of experiment 2. Slides were stained using a fluorescent Cy3-labeled rabbit antihuman IgM antibody. Our analysis show accumulation of IgM antibodies within the tumor tissue and tumor cell membrane staining in animals treated with purified anti-NB IgM antibodies. IgM staining was not found in tumor-free regions of the adrenal gland and was also undetected in adrenal gland tumors of untreated animals (Fig. 5)
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, shows the results of the immunostaining, which is representative for the comprehensive analysis of all of the tumors.
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, untreated rats showed an apoptotic rate of 5.1 ± 0.51% in adrenal gland tumors. In contrast, the apoptotic rate in IgM antibody-treated animals was 29 ± 6.4% (P < 0.05). |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nude rats were chosen because rat and human complement share comparable properties when activated by human antibodies (15) . Furthermore, the size of nude rats simplifies surgical manipulation compared with nude mice. A disadvantage of nude rats is that T cells, which may cause tumor rejection, begin to recover after 12 weeks (16) . This limits the time frame of therapeutic studies and makes it important to develop a model in which metastatic NB appears within a short period of time.
In our study, orthotopic injection led to large adrenal gland tumors but did not cause metastatic growth. In contrast, adrenal gland tumors and metastases appeared when LAN-1 cells were injected either in the aorta or, though significantly smaller, in the tail vein. The appearance of large adrenal gland tumors is remarkable because approximately <1% of tumor cells may have directly reached the adrenal gland after intra-aortal injection and even less after tail vein injection. Various mechanisms may contribute to the development of adrenal gland tumors, such as optimal homing conditions of the endothelium and a microenvironment that supports tumor growth. The finding of an additional micrometastatic spread exclusively in the liver and bones (femur) supports such mechanisms that appear to be similar in rats and humans, where children with NB typically present huge adrenal gland tumors and multiple small metastases preferentially in the bones and liver (17 , 18) .
In our study, adrenal gland, liver, and femur tumors showed the same proliferation rate (80%), but liver and bone tumors presented a 6-fold higher apoptotic rate than the adrenal gland tumors. This strongly suggests that the stromal microenvironment promotes progression of NB cells in the adrenal glands because comparable numbers of NB cells were able to reach the stroma of adrenal glands, liver, and bones. The low apoptotic rate in adrenal glands suggests most favorable growth conditions for NB cells, whereas the liver and femur may present a more hostile environment, which still enables survival but slows down tumor growth. Although the factors that are responsible for these differences are not known, our model may help to elucidate them in future studies.
We used the intra-aortal injection modus to study the therapeutic effect of natural anti-NB IgM antibodies because it reflects the growth pattern of metastatic human NB and offers the longest observation period. Together with a low mortality and the absence of lethality, this approach justifies to us the slightly increased stress for the animals.
Because NB cells preferentially formed large metastatic tumors in the adrenal gland, our main interest was the eradication of NB cells from this location because antibodies have to attack tumor cells in a place that offers them most favorable growth conditions.
We demonstrated a remarkable therapeutic effect against established NB, which in the adrenal glands shrank by almost 90%. Additionally, we tested the ability of anti-NB IgM antibodies to block the development of metastatic disease in early stages of progression. We applied antibodies before homing of tumor cells to the adrenal glands (directly after tumor cell injection) and when tumor cells had reached the stroma of target organs (6 days after cell injection). Metastatic growth was completely inhibited in the first group, but even after micrometastases had formed within adrenal gland stroma, the growth significantly slowed down, and tumors reached 10% of the size of the untreated control group.
The high potential of anti-NB IgM antibodies is fully appreciated when one considers the injected amount of active antibody. The antibody doses of 1 mg/day is comparable with other studies using anti-GD2 antibodies (2
, 3)
. However, although these studies applied monoclonal antibodies, we used the total IgM fraction, which is assumed to contain only 0.1–1% (=1–10 µg) of specific IgM, in other words 100- to 1000-fold less antibody than other antibody studies. This strongly suggests that anti-NB IgM antibodies activate not only common complement pathways but have further specific cytotoxic properties. We confirmed previous studies (10)
and showed that in addition to complement activation, the apoptotic rate was increased 6-fold in antibody-treated rats. Thus far, neither the antigen nor the apoptotic pathway of anti-NB IgM antibodies have been characterized, and further research will focus on their elucidation.
From a theoretical point of view these antibodies have two major advantages compared with murine antibodies derived against tumor-associated antigens. Because of their human origin they are not immunogenic, and more importantly, the chance of major cytotoxic side effects is remote because they naturally occur in humans.
Natural human cytotoxic anti-NB IgM antibodies have a high potential to improve current therapy of human NB. To us it becomes obligatory to bring them into clinic. However, for clinical purposes antibodies must be prepared in a large scale. Therefore, we currently prepare monoclonal antibodies by recombination techniques, which can be applied in clinical studies and help in understanding their function as highly effective anti-NB agents.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Schleswig-Holsteinische Krebsgesellschaft e.V., 24103 Kiel, Germany. 
2 To whom requests for reprints should be addressed, at Department of Oncology and Surgery, Lombardi Cancer Center, NRB Room E316, Georgetown University Medical Center, 3970 Reservoir Road, NW, Washington DC 20007. Phone: (202) 687-1235; Fax: (202) 687-4821; E-mail: juhlh{at}georgetown.edu
3 The abbreviations used are: NB, neuroblastoma; FACS, fluorescence-activated cell sorter; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling.
Received 5/ 5/00. Accepted 1/26/01.
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REFERENCES |
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chain of the IgM molecule. B, the binding of anti-NB IgM antibodies (blood donor 005) to LAN-1 NB cells in a FACS analysis. As a control, the IgM fraction of a blood donor with low cytotoxicity (003) was measured. C, results from an in vitro cytotoxicity assay of LAN-1 cells incubated with human anti-NB IgM antibody (blood donor 005) and control antibodies (blood donor 003). Cytotoxicity was determined by propidium iodide staining and FACS analysis. B and C, results from one representative experiment of three.
