J. Endocrinol. Invest. 14: 635-646,1991
Immunodetection of thyroid tumors: role of immuno aggregates AJ. Van Herle*, B. Estour*, G. Juillard**, A Giuliano***, R.A Hawkins****, and K. Van Herle* *UCLA School of Medicine, Department of Medicine, Division of Endocrinology and Metabolism, **Department of Radiation - Oncology, ***Department of General Surgery, ****Department of Radiological Sciences, Division of Nuclear Medicine & Biophysics, Los Angeles, California 90024, USA ABSTRACT. To understand the inconsistent immunodetection of tumors in vivo, a labelled monoclonal antibody (MAb) against a human follicular cancer cell line (UCLA RO 82 W-1) was used as a model for in vitro and in vivo studies. The 131 1 labelled MAb x RO 82 W-1 bound to its target cells (10% to 70%) mainly because of the generation of immunoglobulin aggregates. Aggregates generated by the freezing process were shown by polyacrylamide gel electrophoresis (PAGE) and their removal by filtration. When the aggregated 131 1MAb x
RO 82 W-1 was injected into BALB/c mice bearing UCLA RO 82 W-1 tumors, a high tumor/blood ratio was found in the large tumors. The tracer concentrated in the macroscopically visible necrotic part of the tumor was largely responsible for the scintigraphic detection. Irrelevant 1311-lgG also concentrated in necrotic regions of tumors. Scintigraphic detection of thyroid tumors in this model was related to the degree to which labeled aggregates of IgG, regardless of their specificity, localized in necrotic regions of the tumors.
INTRODUCTION
studies showed that aggregate formation and tumor tissue necrosis were important factors determining immunodetection.
In 1957 Pressman and colleagues proposed the detection of tumors with isotopically labelled antibodies directed against tumor associated antigens (1). Despite the enthusiasm of several groups with regards to the usefulness of such antibodies (2,3), other investigators questioned the specificity of tumor immunodetection (4-6). Certain tumors can be visualized with labelled monoclonal antibodies not directed against an antigen present in the studied tumor cells (6). Tumors releasing a specific antigen in the circulation are not necessarily detected after intravenous administration of the corresponding labelled antibody (3). With the availability of a human thyroid tumor cell line (UCLA RO 82 W-1) we prepared monoclonal antibodies against these cells and studied the binding of one of them to the thyroid tumor cells in vitro and to tumor xenografts after intravenous administration of radiolabelled antibody to the recipients. Antibody controls included nonspecific human and mouse immunoglobulins, specific and nonspecific immunoglobulin aggregates and fragments F (ab') 2, Fab, and Fc. Our
MATERIALS AND METHODS Cell lines The primary tumor cell line used for all our studies, UCLA RO 82 W-1, was derived from a metastatic lesion of a patient with follicular carcinoma of the thyroid (7). The cell line was maintained in RPMI 1640 medium (Irvine Scientific, CA) supplemented with 10% fetal calf serum (FCS), antibiotics and a fungistatic agent (Fungi-Bact solution, Irvine Scientific, CA), in a humidified atmosphere of 95% O2 and 5% CO 2 , The reduplication rate was 4 days, and confluency was reached after 7 days. The cells were harvested by incubation for 3 to 5 minutes with Trypsin-EDTA (1M) (Irvine Scientific) followed by a centrifugation (700 rpm 5 minutes) and washing (2 x) of the pellets with RPMI 1640. The viability of the cells was 95%, or greater, in all in vitro experiments. The cells did not trap iodide either in vitro or in vivo. The other cell lines used as controls included a melanoma cell line (M-14), a lung tumor (100 P3), a prostatic tumor (UCLA RO 84 S-1), a pancreatic tumor (UCLA RO 81 R-4), a colon adenocarcinoma (UCLA RO 83 M-1), a papillary carcinoma of the thyroid (UCLA RO 82 F-2) an anaplastic carcinoma (UCLA RO 81 A-i), and a medullary carcinoma of
Key-words: Thyroid tumors, immunodetection, immune aggregates. tumor necrosis. Correspondence: Andre J. Van Herle. M.D .. UCLA School of Medicine, Department of Medicine, Division of Endocrinology, Los Angeles, California 90024- 1682. Received November 23. 1989; accepted April 11. 1990.
635
AJ Van Her/e , B. Estour, G. Juillard, et al.
IgG immunoglobulins Normal human and mouse IgG ("irrelevant" IgG) were isolated from sera of these respective species by DEAE Sephadex-50 chromatography (Sigma Inc., St. Louis, Mo). The solutions were subsequently concentrated by negative pressure dialysis against PBS pH 7.0. Human Fab, F (ab ')2 , and Fc fragments of human IgG were obtained from Cappel (Worthington Scientific Div, Cooper Biomedicallnc. , Malvern , PA).
the thyroid (UCLA RO 85 D-1). All cell lines were cultured and harvested under identical conditions as the UCLA RO 82 W-1 cell line. Normal lymphocytes and red cells were isolated according to a technique described by Gluckman et al. (8) . Monoe/onal antibody development Two BALB/c female mice (Simmonson Farm, Gilroy, CA) were injected intraperitoneally (ip) with 2 x 107 UCLA RO 82 W-1 cells grown in RPMI 1640 medium supplemented with 10% (FCS) . The cells were harvested while in the exponential growth phase, washed and resuspended in RPMI 1640 without serum and injected . Two weeks after the first injection a booster was given intraperitoneally with the same number of cells. Three days after the booster, the two mice were sacrificed and spleen cells harvested (2 x 108 cells, 99% viability). The S-194 cells (from Dr. M. Cohen of Salk Institute, La Jolla, CA) were incubated with immune spleen cells in a ratio of 1:5 and fused in the presence of 50% polyethyleneglycol (PEG 1500) (Sigma Co., St. Louis , Mo) according to the methods of Oi and Herzenberg (9). The supernatants from the hybridoma cultures were tested in a cellular binding assay with 125 1 labelled Staphylococcal protein A (SpA) against UCLA RO 82 W-1 cells (10). Several positive hybridomas were chosen and cloned by limiting dilution . Cultures yielding positive test for IgG were cloned twice more. A single hybridoma line with the highest antibody activity against the thyroid target cell was chosen for further study. After the antibody from this hybridoma was tested against over 50 cultured human tumor cell lines, it reacted only with UCLA RO 82 W-1 . BALB/c mice were primed with 0.5 mg of pristane intraperitoneally (ip) 10 and 3 days before ip inoculation with 2 x 107 hybridoma cells (from the selected hybridoma cell line). Ascites fluid was collected 7 to 14 days after cell injection , centrifuged at 300 x g, and cell-free supernatants were stored at -20 C. Mouse ascites was precipitated with saturated ammonium sulfate (45%) . The precipitate was dialyzed against phosphate buffered saline (PBS) overnight at 4 C and applied to an Affigel Protein A affinity column ; elution of the antibody was conducted with elution buffer (Biorad , Richmond , CA) . The pooled fractions were brought immediately to a pH 6.5 by addition of phosphate bufffer pH 7.5 (0.5 M) and were concentrated by negative pressure dialysis. The protein was judged homogeneous by polyacrylamide gel electrophoresis (PAGE) in sodium dodecylsulfate (SDS) in nonreducing conditions and by isoelectric focusing .
Preparation of radioiodinated antibody Both purified monoclonal antibodies against UCLA RO 82 W-1 cells (hereafter indicated as MAb x RO 82 W-1) and normal IgG ("irrelevant" mouse IgG) were radioiodinated with various techniques: [1] oxidation of iodide by Chloramine T (11) ; [2] electrolysis (12) or [3] lodogen (Pierce Chemicals Co ., Rockford, IL) . The latter method was selected for all subsequent studies. The labelling procedure was carried out at room temperature by combining 5-100 Ilg of IgG with 1-5 mCi of 13 1 1 or 125 1 (New England Nuclear) for 45 minutes. The reaction was stopped by the addition of 20 III of KI (0.1 M) for 20 minutes. This mixture was then chromatographed on a 20 x 1 cm Biogel P6 column (Bio-Rad, Richmond, CA) to remove unreacted 1311 or 125 1. The specific activities of the radioiodinated products ranged from 5-400 IlCi/llg. They were stored at -20 C unless stated otherwise. In vitro binding studies Binding studies were carried out at room temperature in 50 ml plastic tubes containing 2-5 x 10 7 UCLA RO 82 W-1 cells or an equal number of the tumor cells in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and varying amounts (cpm) of the tracers under analysis. After gentle agitation at 20 C of the tubes at time 0 and at various time intervals thereafter, 400 III of the medium each containing 0.25 x 10 7 cells were transferred to mi crofuge plastic tubes (Tekmar Co ., Cincinnati , Ohio) . For all subsequent studies only experimental points at 0 and 3 hours were obtained. Two control tubes containing usually 5000 cpm/tube for each time point under study were kept and represented the "total counts" added. The experimental set of tubes were then centrifuged in a table top microfuge (Beckman Instruments Inc. , Palo Alto , CA) for 60 seconds at 17,500 rpm in the cold room (4 C) . The supernatant was aspirated and discarded . The pellets were counted in a gamma spectrometer (Nuclear Chicago, IL). Pilot studies had indicated that a washing step was superfluous . The radioactivity in the pellet was expressed as a per-
636
Immunodetection of thyroid tumors
centage of the radioactivity in the control tubes (total counts). When competitive binding studies were done, unlabelled IgG up to 1,000 /lg/tube was added to the 50 ml tube 30 minutes before the tracer addition. The specific binding in competitive studies is expressed as a difference in binding between cells not exposed to unlabelled IgG and those exposed to unlabelled IgG. At the end of each experiment, the viability of the cells were tested by the Trypan Blue Dye exclusion technique, and was greater than 80% even after 18 hours of incubation. In pilot studies the binding of Do tracers (Do tracer refers to a tracer immediately used after labelling) on viable cells (> 95% viability) and on nonviable cells (5% and 8% viability) was measured. The same tracer binding was found on nonviable cells and on viable cells (10%). All labelled proteins were tested in the in vitro binding assay before use in other studies, that is, PAGE electrophoresis and in vivo studies.
ing) on a Kodak X-OMAT film (Eastman Kodak Co., Rochester, NY). The film was processed after a few days of exposure, and the migration distance of the radioactive spots from the top of the gel was compared with those of standard proteins run in similar conditions and stained with Coomassie Blue. High molecular weight standards (Pharmacia, Piscateway, NJ) ranged from 67kd (Albumin) to 660kd (Thyroglobulin).
Immunocytochemical studies The UCLA RO 82 W-1 or M-14 cells were harvested, put on glass slides, fixed with poly-L-Iysine, and dried. Subsequently, the slides were humidified with phosphate buffer saline; after 15 minutes incubation with BSA 1% and 10% goat serum, the excess fluid was removed and MAb RO 82 W-1 was added (dilution 1:4,000) in 1% BSA. A similarly prepared set of slides was treated with heat aggregated (63 C) IgG MAb x RO 82 W-1 (1 :4,000). The slides were then incubated for 60 minutes at room temperature in a moisturized chamber. After they were washed twice with PBS, the second antibody was added: a solution of peroxidase labelled goat anti mouse IgG 1:50. The slides were then reincubated for 60 minutes at room temperature, counterstained with hematoxylin, and subsequently dried, analyzed, and photographed. In each experiment M-14 cells (human melanoma cells) were used as control .
Preparation of immunoglobulin aggregates by heating A monoclonal antibody solution (MAb x RO 82 W1) (2.5 mg/ml) was heated in a thermostatic water bath at 63 C for 10 minutes, which was the optimal time exposure reported by Anderson and Grey (13) for aggregate formation of IgG without visible opalescence. These aggregated immunoglobulins, after labelling with the lodogen method, were used for binding studies. The unlabelled IgG aggregates produced by heating were also utilized in immunocytochemical studies on the thyroid tumor cells UCLA RO 82 W-1 and the M-14 cells (human melanoma).
In vivo immunodetection studies in nude athymic mice Athymic nude female mice BALB/c (Simmonson Farm, Gilroy, CA) were housed in semi-sterile conditions. They were all injected subcutaneously (s.c.) with 1 x 107 UCLA RO 82 W-1 cells in one or more sites. Certain animals (no. 1,7,8,9, Table 1) received at a later date 1 x 107 M-14 cells s.c . The thyroid tumors grew to a sizeable mass and were suitable for scintigraphic studies after 6 to 9 months (> 1.0 cm). The KI (1 .0 M) was then added to the animals' drinking water, 2 days prior to the scintigraphic studies and for the length of the study, except when indicated (Table 1). The 131 1 MAb x RO 82 W-1, or "irrelevant" 131 1 mouse IgG (50-100 /lCi), were injected (004 ml) via the tail vein. Radionuclide imaging was done on anesthetized animals on the day of injection and 3, 5, and 6 days later using a gamma camera fitted with a pinhole collimator. The image data were stored, analyzed, and displayed with a computer interfaced with the gamma camera. Images acquired over a 10-minute interval had about 20-40 thousand counts. After the last scan was done, blood was withdrawn by cardiac puncture, weighed, and
Polyacrylamide gel electrophoresis (PAGE) and autoradiographic studies of unlabelled and 131 1MAb To characterize the unlabelled and labelled MAb immunoglobulins and their aggregates used in the binding studies, PAGE was carried out according to the method of Laemmli (14). Two gradient gels 2/16 PAA (Pharmacia Fine Chemicals, Piscateway, NJ) were run in parallel in non-SOS and nonreducing conditions using TRIS (0.09 M) boric acid (0.08 M), Na2 EOTA 0.0025 M, pH 804. The gels were run at 10 C at 70 V for 30 minutes and subsequently at 400 V for 2 1/2 hours to attain a total of 580 Volt hours. The gels containing nonradioactive IgG were stained with Coomassie Brilliant Blue R-250, (BioRad Laboratories, Richmond , CA) , destained, and photographed. After electrophoresis, the gels containing the labelled preparations were wrapped in a plastic bag and placed in contact (prior to dry-
637
A.J. Van Herle, B. Estour, G. Juillard, et al.
Table 1 - Immunoscintigraphic data and tumor blood ratios in nude mice transplanted with UCLA RO 82 W-1 and M-141 Cells. Mouse 10 no.
Type of tumor implanted
UCLA RO 82 W-1
Type of tracer injected
131 1MAb
x
Scintiscan Macroscopic necrosis day 5 after tracer injection
2
UCLA RO 82 W-1
131 1MAb
x
% Bound SSKI in radioactivity drinking on UCLA RO 82 cells water
MN 2 34 rest of tumor 0.8 0.6
10%
pos
MN212.2 rest of tumor 0.8
50%
+
pos
pos
neg
neg
pos
RO 82 W-1 (00) M-14 1
Tumor/blood ratio
RO 82 W-1 (0 5) 3
UCLA RO 82 W-1
131 1 "Irrelevant" mouse IgG (0 0)
pos
pos
MN227.0 rest of tumor 1.6
1.5%
+
4
UCLA RO 82 W-1
131 1 "Irrelevant" mouse IgG (0 0)
pos
±
MN 2 96 rest of tumor 1.6
1.5% 3.9%
+
5
UCLA RO 82 W-1
131 1MAb x RO 82 W-1 (00)
pos
neg
MN 2 O.3 rest of tumor 0.3
10%
+
6
UCLA RO 82 W-1
131 1 "Irrelevant" mouse IgG (0 0)
pos
neg
MN211.8 rest of tumor 0.7
1.5%
+
7
UCLA RO 82 W-1
131 1MAb x RO 82 W-1 (05)
neg
neg
UCLA RO 82 W-1 0.6
30%
neg
neg
M-14 1 O.5
neg
neg
UCLA RO 82 W-1 0.6
neg
neg
M-14 1 0.3
neg
neg
UCLA RO 82 W-1 0.32 30%
neg
neg
M-14 1 0.30
M-14 1 8
UCLA RO 82 W-1
x RO 82 W-1 (05) 131 1MAb
M-14 1 9
UCLA RO 82 W-1 M-14 1
131 1 "Irrelevant" mouse IgG (0 5)
50%
1M-14 - human melanoma cells 2MN - macroscopic necrosis
counted. The animals were then sacrificed and various organs were removed, weighed and counted. In animals with macroscopic tumor necrosis, the macroscopic necrotic material was separated from the rest of the tumor, both portions were weighed, counted, and tissue/blood ratios were established separately for the macroscopic necrotic and nonnecrotic regions of the tumors. The radioiodinated proteins were either injected on the day of the labelling process (0 0) or 5 days after labelling and storage at -20C (0 5) (Table 1). All tracers were tested in the in vitro binding assay using the UCLA RO 82 W-1 cells on the day of their injection.
cells (Figure 1). The maximal binding was found to be identical at 4C and 20C and was independent of the oxidative agent used in the labelling process. Despite the rigorous standardization of the binding technique, unexplained variations in maximal binding continued to persist from experiment to experiment. Therefore, the effect of storage of the labelled proteins and the intensity of labelling, i.e., specific activity (SA) on the binding were analyzed. Figure 2 shows a composite binding of the labelled protein as a function of its "age" (storage time), storage temperature (4C, -20C) and specific activity (2.5 versus 20 f..LCi/f..Lg). These studies indicated clearly that the highest binding of 131 1 MAb x RO 82 W-1 occurred after storage at -20C and suggested that the maximal binding is higher for the preparation with higher SA (20 f..LCi/f..Lg versus 2.5 f..LCi/f..Lg). The highest maximal binding (70%) was achieved after 10 days of storage of the tracer at -20C (SA 20 f..LCi/f..Lg). In contrast, the low SA tracer (2.5 f..LCi/f..Lg) kept at -20 C bound maximally after 3 days
RESULTS Binding experiments and effect of tracer storage on binding The binding of 131 1 MAb RO 82 W-1 to RO 82 W-1 cells was dependent on the length of incubation (not shown) up to 3 hours and on the number of
638
Immunodetection of thyroid tumors
10
o no unlabeled IgG •
unlabeled IgG a dded
0..........3--------// r---;'B TIME (HRS)
0 _______
24
0
1.0 x 10 7 cells
20 0_---------0
g' 16 i:i c
0.5 X 10 7 cells
:
:~:~~~~~~~~~~~~~~--------~: ::: ::: :::::
iii 12
C Q)
~ 8 4
o
o
o~'----------~~------------~/~8 TIME (HRS)
(30%). The same tracers when stored at 4C bound less effectively; in contrast to the tracers kept at -20 C, their maximal binding was not substantially affected by their SA (2.5 versus 20 JlCi/Jlg).
Fig. 1 - Effect of incubation time and cell number on the maximal tracer binding and lack of displacement with unlabelled IgG. 131 1MAb x RO 82 W-1 was incubated with increasing numbers of cells. Maximal tracer binding was proportional to the number of cells, and was reached at 3 hours. Unlabelled monoclonal antibody (1000 ~g) incubated with UCLA RO 82 W-1 cells followed by 131 1MAb x RO 82 W1 were non-inhibitory on tracer binding (see inset). In all subsequent figures the maximal binding after 3 hours of incubation is shown except when stated otherwise. Data points represent means of duplicate or triplicate experiments not differing by > 5%. Data are expressed as percentage of total radioactivity added to the tubes.
Similar studies conducted with labelled human F (ab') 2 and Fab fragments showed a similar binding pattern (Figure 3, column sets 2,3). In contrast, 131 1 human Fc fragments did not show a similar binding pattern (Fig. 3, column set 4). Aggregates of labelled human albumin and thyroglobulin did not bind substantially (Figure 3, column sets 5, 6). That the binding was not due to free iodine trapped by the thyroid tumor cells was shown in an experiment using 125 1 tracer (Fig. 3, column set 7). To test the nonspecificity of the binding to other tumor cell lines, as
Specificity of in vitro binding The tracer incubated with UCLA RO 82 W-1 cells on the day of labelling designated (00), bound 3% (Figure 3, column set 1); The 05 tracer stored at -20 C for 5 days showed an increased binding up to 45% for 131-labelled mouse IgG (Figure 3, column set 1).
75 ,/ ,e - _______ ......
65
55 OJ
c
/
i:i 45 c iii
§
"" I•
""
" ""
"
" ""
"
• •
20
.. 2.5
35
~
25
....
....
o
20
t:J.
2.5
15
5
o
o
!
2
4
6
8
10
DAYS
12
14
16
Fig. 2 - Effect of age of tracer storage method and specific activity of the tracer on maximal tracer binding to UCLA ~Ci/fl9 stored at - 20 C RO 82 W-1 cells. The binding of 131 1 flCilfl9 stored at -20 C MAb x RO 82 W-1, labelled at two different SA, high (20 ~Ci;j.l.g) and low (2.5 ~Ci;j.l.g), was tested. The tracers were flCi/fJg stored at 4 C then stored at two temperatures, -20 C and 4 C. The low specific activity tracflCi/fl9 stored at 4 C er (2.5 ~Ci,1.tg) stored at -20 C bound less efficiently to the cells and maximal tracer binding was reached earlier (3 days versus 10 days) than with the high specific activity tracer (20 ~Ci;j.l.g). The ! , SA of the tracers had less effect on the 18 20 maximal tracer binding when they were stored at 4 C.
639
A.J. Van Herle, B. Estour, G. Juillard, et al.
60 1. 2.
50
131
1 Mouse IgG
'3' 1 Human
F (ab') 2 fragments
Fig. 3 - Binding of labelled "Irrelevant" mouse IgG and fragments of human IgG and other labeled aggregated proteins to '3'1 Human Fe fragments UCLA RO 82 W-1 cells. Binding of labeled mouse IgG ("irrelevant" IgG) and other la13' I Human albumin (heat aggregated) belled human IgG fragments (F (ab) '2, Fab, Fc) and proteins are shown The first col1J' I Thyroglobulin (freezer aggregated) umn (open bar) for each set of columns represents the maximal binding of the tracer on the day of labeling (0 0). The second col'25lodine umn (hatched bar) represents the maximal binding on day 5 (0 5) after labeling. The horizontal hatched area represents the range of binding for the 131 1labelled MAb x RO 82 W-1 with a fresh label (0 0 label) to UCLA RO 82 W-1 cells. 131 1mouse IgG and all tested 13111gG fragments, except for the human 131 1 Fc fragment, bound effectively to the UCLA RO 82 W-1 cells, after storage of the labels at -20 C for 5 days. (For further 6 5 7 details see text.)
3. 13'1 Human Fab fragments
4.
40
5.
OJ
.S TI C
iii
C Q)
6.
30
600K) are responsible and essential for this binding to occur. Our immunocytochemical studies with heat aggregated IgGs rendered melanoma cells positive even though these cells normally do not recognize the non-aggregated MAb x RO 82 W-1. Because alllgG aggregates bind to the UCLA RO 82 W-1 regardless of the method of formation, immunocytochemical studies should therefore be conducted with MAb, which do not contain large IgG aggregates. Antibody binding data obtained in vitro cannot be transposed to in vivo studies because immuno-targeting of tumors is a multifactorial process. Several investigators have attempted to link the percentage of positive immunoscans with the size of the tumor (6, 16). Mann et al. (6) state that "imaging of tumor deposits appears to involve a nonspecific phenomenon that is largely dependent on tumor weight." Other investigators also believe that nonspecific uptake increases proportionately with the size of tumor (17). Our studies confirm the finding that the larger tumors are the most likely to be detected by immunodetection (60%). However, another factor only occasionally mentioned in the literature seems to be important as well, namely, the presence of tumor necrosis (5, 17). In our study all 4 tumors, which were positive on scintiscan (no.1-no. 4), had a T/B ratio in the "macroscopic necrotic" part of the tumor exceeding 3.4 (range 3.4 - 96). The T/B ratio in the mice with absence of macroscopic tumor necrosis ranged from 0.3 to 0.6 and these tumors indeed were negative on scintiscan. These studies seem to indicate that tumor size may not be as important as the accompanying "macroscopic" tumor necrosis frequently present in large tumors. Moshakis et al. (18) also studied the distribution of CEA in breast tumors implanted in immunosuppressed mice and found that labelled anti-CEA antibody reacted with CEA molecules in the extracellular space away from the tumor and that a paucity of labelled Ab was documentable on the cell membrane. Fab fragments of anti myosin antibody have been shown to localize in the myocardium of patients with myocarditis (19). In addition, immunofluorescent studies of endomyocardial biopsies indicated the nonspecific uptake of IgG on myocytes (20). In a recent study, Rubin et al. were able to demonstrate
111 In-labelled nonspecific immunoglobulin uptake in the detection of focal infections and in metastatic tumors (21). Furthermore, Epstein and associates demonstrated that radiolabeled antinuclear monoclonal antibodies can concentrate within bulky tumors containing necrotic lesions (22). In the latter studies, necrotic regions dissected from the tumors at necropsy gave high tumor to blood ratios similar to what was seen in our studies using immunoaggregates. These studies and our own seem to show that immunodetection may be related to the presence of necrotic tissue and the binding by 131 1 IgG aggregates or 131 1 IgG itself. Because our in-vitro studies have convincingly indicated that the observed tracer binding is primarily due to the presence of immunoglobulin aggregates in the tracer preparations and that the in vivo binding is at least partially due to tumor necrosis, the fundamental question arises whether immunoglobulin binding to tumor necrosis areas is the basic mechanism of immunodetection? Indirect evidence supportive of this concept is provided by the following findings: Pilot studies have indicated that necrotic cells bind immunoaggregated IgG better than nonaggregated IgG's in vitro. Because the "macroscopic" necrotic part of the tumor has a T/B ratio higher than the rest of the tumor, 131 1 MAb aggregates used in these binding studies may bind more effectively to the necrosis or to elements present in necrotic tissue. If highly specific Abs are used, as was the case in studies reported by Lundy et al. (23), then the visualization may be due to specific IgG. Additional studies in our laboratory (not shown) have also indicated that tumor extracts from scintigraphically detectable tumors contain high molecular weight aggregates, which are retained by Biorad A 15 columns. Based on these in vivo studies, binding of labelled aggregated IgGs to tumor necrosis represents a plausible and important mechanism of immuno-detection. Furthermore our studies clearly indicate that 131 1 "irrelevant" mouse IgG can lead to tumor targeting (mouse no. 2, Fig. 9, panel B) and provides a possible explanation for the data reported by Mann et al. (6) and find confirmation in the recent data of Rubin et al. (21). We have shown that specific 131 1 MAb and "irrelevant" 131 1 mouse IgG can bind in vitro to tumor cells and that in vivo positive images can be obtained with both labels, which confirms earlier data reported by Bullard et al. using intracranial human glioma xenografts (24). The in vitro binding is directly related to tracer aggregate formation which occurs during the freezing process. Many investigators do not state the preservation method of the tracers used for in vitro or in vivo binding studies and it is
644
Immunodetection of thyroid tumors
thus difficult to assess whether similar nonspecific binding of labelled immunoaggregates played a role in their studies. The formation of immunoaggregates during the freezing process are certainly not the only factor involved in obtaining positive scintigraphies; tumor size and the "macroscopic" tumor necrosis, which accompanies increased tumor size, may be equally important. Our studies suggest that development of immunoscintigraphic methods, with "irrelevant" labeled aggregated immunoglobulins, may be a direction worthwhile to explore in the field of tumor immunodetection.
8.
9.
10.
ACKNOWLEDGMENTS The authors thank K. Kellett, B. Cheng, T. Totanes, D. Padua, and D. Canlapan for their excellent technical assistance. This work was supported in part by a grant from the California Cancer Research Coordinating Committee A850702.
11.
REFERENCES
12.
1. Pressman D., Day ED., Blau M. The use of paired labelling in the determination of tumor-localizing antibodies. Cancer Res. 17: 845,1957. 2. Goldenberg D.M., DeLand F., Euishin K., Bennett S., Primus F.J., Van Nagell Jr J.R., Estes N., DeSimone P., Rayburn P. Use of radiolabelled antibodies to carcinoembryonic antigen for the detection and localization of diverse cancers by external photoscanning. N. Engl. J. Med. 298: 1384,1978. 3. Mach J.P., Carrel S., Forni M., Ritschard J., Donath A, Alberto P. Tumor localization of radiolabelled antibodies against carcinoembryonic antigen in patients with carcinoma. N. Engl. J. Med. 303: 5, 1980. 4. Goldenberg D.M., Preston D.F., Primus F.J., Hansen H.J. Photoscan localization of GH-39 tumors in hamsters using radiolabelled anticarcinoembryonic antigen immunoglobulin G. Cancer Res. 34: 1, 1974. 5. Stuhlmiller G.M., Sullivan D.C., Vervaert C., Croker B.P., Harris C.C., Seigler H.J. In vivo tumor localization using tumor-specific monkey xenoantibody and murine monoclonal xenoantibody. Ann. Surg. 194:592,1981. 6. Mann BD., Cohen M.B., Saxton R.E., Morton D.L., Benedict W.F., Korn E.L., Spolter L., Graham L., Chang C.C., Burk MW. Imaging of human tumor xenografts in nude mice with radiolabelled monoclonal antibodies. Limitations of specificity due to nonspecific uptake of antibody. Cancer 54: 1318,1984. 7. Estour B., Van Herle AJ., Juillard G.J.F., Totanes
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T.L., Sparkes R.S., Giuliano AE, Klandorf H. Characterization of a human follicular thyroid carcinoma cell line (UCLA RO 82 W-1). Virchows Archiv, B. Cell Pathology 57: 167,1989. Gluckman E., Parr MD., Michelson E., Schroeder M.L. Mixed leukocyte cultures in dogs. A microtechnique using gradient purified cells. Transplantation 15: 642, 1973. Oi V.T., Herzenberg LA Immunoglobulin producing hybrid cell lines. In: Mishell B.B., Shigii S.M. (Eds.), Selected methods in cellular immunology. W.H. Freeman, San Francisco, 1980, p. 351. Brown J.P., Tamerius JD., Hellstrom I. Indirect 125 1labelled protein. An assay for monoclonal antibodies to cell-surface antigens. J. Immunol. Methods 31: 201,1979. Greenwood F.C., Hunter W.M., Glover J.S. The preparation of 1311-labelled human growth hormone of high specific radioactivity. Biochem. J. 89: 114,1963. Gautvik K.M., Svindahl K., Skretting A, Stenberg B., Aas M., Myhre L., Ekeland A, Johannesen J.v. Uptake and localization of 131 1labelled anti-calcitonin immunoglobulins in rat medullary thyroid carcinoma tissue. Cancer 50: 1107, 1982. Anderson C.L., Grey H.M. Receptors for aggregated IgG on mouse lymphocytes. Their presence on thymocytes, thymusderived, and bone marrow-derived lymphocytes. J. Exp. Med. 139: 1175,1974. Laemmli V.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 227: 680,1970. Krishnan E.C., Krishnan L., Jewell WR. Characterization of Fc receptors associated with human malignant tumors. J. Surg. Oncol. 16: 179,1981. Moshakis V., Mcllhinney RAJ., Raghavan D., Neville AM. Monoclonal antibodies to detect human tumors: an experimental approach. J. Clin. Pathol. 34:314, 1981. Epenetos AA, Nimmon C.C., Arklie J., Elliott A.T., Hawkins LA, Knowles RW., Britton K.E., Bodmer WF. Detection of human cancer in an animal model using radio-labelled tumour-associated monoclonal antibodies. Br. J. Cancer 46: 18,1982. Moshakis V., Mcllhinney RAJ., Raghava D., Neville AM. Localization of human tumor xenografts after i.v. administration of radiolabelled monoclonal antibodies. Br. J. Cancer 44: 91, 1981. Yasuda T., Balacios I.F., Khaw BA, Dec W., Gold
A.J. Van Herle, B. Estour, G. Juillard, et al.
22. Epstein AL., Chen F.-M., Taylor C.R. A novel method for the detection of necrotic lesions in human cancers. Cancer Res. 4&5842,1988.
H.K., Leinbach R.C., Fallon J.T., Barlai-Kovach M., Strauss H.w., Haber E. Monoclonal indium-III anti myosin antibody imaging versus right ventricular biopsy in diagnosing acute myocarditis. Circulation 72: (Suppl. III), 11A, 1985 (Abstract).
23. Lundy J., Mornex F., Keenan A.M., Greiner J.w., Colcher D. Radioimmunodetection of human colon carcinoma xenografts in visceral organs of congenitally athymic mice. Cancer 57: 503, 1986.
20. Scully R.E., Mark E.J., McNeely B.U. Case Records of the Massachusetts General Hospital. N. Engl. J. Med. 314: 1240,1986. 21. Rubin R.H., Fischman AJ., Callahan R.J., Khaw B.A, Keech F., Ahmad M., Wilkinson R., Strauss H.w. 111ln-labeled nonspecific immunoglobulin scanning in the detection of focal infection. N. Engl. J. Med. 321: 935,1989.
24. Bullard D.E., Adams C.J., Coleman R.E., Bigner D.o. In vivo imaging of intracranial human glioma xenografts comparing specific with nonspecific radiolabeled monoclonal antibodies. J. Neurosurg. 64: 257, 1986.
646
Immunodetection of thyroid tumors: role of immuno aggregates AJ. Van Herle*, B. Estour*, G. Juillard**, A Giuliano***, R.A Hawkins****, and K. Van Herle* *UCLA School of Medicine, Department of Medicine, Division of Endocrinology and Metabolism, **Department of Radiation - Oncology, ***Department of General Surgery, ****Department of Radiological Sciences, Division of Nuclear Medicine & Biophysics, Los Angeles, California 90024, USA ABSTRACT. To understand the inconsistent immunodetection of tumors in vivo, a labelled monoclonal antibody (MAb) against a human follicular cancer cell line (UCLA RO 82 W-1) was used as a model for in vitro and in vivo studies. The 131 1 labelled MAb x RO 82 W-1 bound to its target cells (10% to 70%) mainly because of the generation of immunoglobulin aggregates. Aggregates generated by the freezing process were shown by polyacrylamide gel electrophoresis (PAGE) and their removal by filtration. When the aggregated 131 1MAb x
RO 82 W-1 was injected into BALB/c mice bearing UCLA RO 82 W-1 tumors, a high tumor/blood ratio was found in the large tumors. The tracer concentrated in the macroscopically visible necrotic part of the tumor was largely responsible for the scintigraphic detection. Irrelevant 1311-lgG also concentrated in necrotic regions of tumors. Scintigraphic detection of thyroid tumors in this model was related to the degree to which labeled aggregates of IgG, regardless of their specificity, localized in necrotic regions of the tumors.
INTRODUCTION
studies showed that aggregate formation and tumor tissue necrosis were important factors determining immunodetection.
In 1957 Pressman and colleagues proposed the detection of tumors with isotopically labelled antibodies directed against tumor associated antigens (1). Despite the enthusiasm of several groups with regards to the usefulness of such antibodies (2,3), other investigators questioned the specificity of tumor immunodetection (4-6). Certain tumors can be visualized with labelled monoclonal antibodies not directed against an antigen present in the studied tumor cells (6). Tumors releasing a specific antigen in the circulation are not necessarily detected after intravenous administration of the corresponding labelled antibody (3). With the availability of a human thyroid tumor cell line (UCLA RO 82 W-1) we prepared monoclonal antibodies against these cells and studied the binding of one of them to the thyroid tumor cells in vitro and to tumor xenografts after intravenous administration of radiolabelled antibody to the recipients. Antibody controls included nonspecific human and mouse immunoglobulins, specific and nonspecific immunoglobulin aggregates and fragments F (ab') 2, Fab, and Fc. Our
MATERIALS AND METHODS Cell lines The primary tumor cell line used for all our studies, UCLA RO 82 W-1, was derived from a metastatic lesion of a patient with follicular carcinoma of the thyroid (7). The cell line was maintained in RPMI 1640 medium (Irvine Scientific, CA) supplemented with 10% fetal calf serum (FCS), antibiotics and a fungistatic agent (Fungi-Bact solution, Irvine Scientific, CA), in a humidified atmosphere of 95% O2 and 5% CO 2 , The reduplication rate was 4 days, and confluency was reached after 7 days. The cells were harvested by incubation for 3 to 5 minutes with Trypsin-EDTA (1M) (Irvine Scientific) followed by a centrifugation (700 rpm 5 minutes) and washing (2 x) of the pellets with RPMI 1640. The viability of the cells was 95%, or greater, in all in vitro experiments. The cells did not trap iodide either in vitro or in vivo. The other cell lines used as controls included a melanoma cell line (M-14), a lung tumor (100 P3), a prostatic tumor (UCLA RO 84 S-1), a pancreatic tumor (UCLA RO 81 R-4), a colon adenocarcinoma (UCLA RO 83 M-1), a papillary carcinoma of the thyroid (UCLA RO 82 F-2) an anaplastic carcinoma (UCLA RO 81 A-i), and a medullary carcinoma of
Key-words: Thyroid tumors, immunodetection, immune aggregates. tumor necrosis. Correspondence: Andre J. Van Herle. M.D .. UCLA School of Medicine, Department of Medicine, Division of Endocrinology, Los Angeles, California 90024- 1682. Received November 23. 1989; accepted April 11. 1990.
635
AJ Van Her/e , B. Estour, G. Juillard, et al.
IgG immunoglobulins Normal human and mouse IgG ("irrelevant" IgG) were isolated from sera of these respective species by DEAE Sephadex-50 chromatography (Sigma Inc., St. Louis, Mo). The solutions were subsequently concentrated by negative pressure dialysis against PBS pH 7.0. Human Fab, F (ab ')2 , and Fc fragments of human IgG were obtained from Cappel (Worthington Scientific Div, Cooper Biomedicallnc. , Malvern , PA).
the thyroid (UCLA RO 85 D-1). All cell lines were cultured and harvested under identical conditions as the UCLA RO 82 W-1 cell line. Normal lymphocytes and red cells were isolated according to a technique described by Gluckman et al. (8) . Monoe/onal antibody development Two BALB/c female mice (Simmonson Farm, Gilroy, CA) were injected intraperitoneally (ip) with 2 x 107 UCLA RO 82 W-1 cells grown in RPMI 1640 medium supplemented with 10% (FCS) . The cells were harvested while in the exponential growth phase, washed and resuspended in RPMI 1640 without serum and injected . Two weeks after the first injection a booster was given intraperitoneally with the same number of cells. Three days after the booster, the two mice were sacrificed and spleen cells harvested (2 x 108 cells, 99% viability). The S-194 cells (from Dr. M. Cohen of Salk Institute, La Jolla, CA) were incubated with immune spleen cells in a ratio of 1:5 and fused in the presence of 50% polyethyleneglycol (PEG 1500) (Sigma Co., St. Louis , Mo) according to the methods of Oi and Herzenberg (9). The supernatants from the hybridoma cultures were tested in a cellular binding assay with 125 1 labelled Staphylococcal protein A (SpA) against UCLA RO 82 W-1 cells (10). Several positive hybridomas were chosen and cloned by limiting dilution . Cultures yielding positive test for IgG were cloned twice more. A single hybridoma line with the highest antibody activity against the thyroid target cell was chosen for further study. After the antibody from this hybridoma was tested against over 50 cultured human tumor cell lines, it reacted only with UCLA RO 82 W-1 . BALB/c mice were primed with 0.5 mg of pristane intraperitoneally (ip) 10 and 3 days before ip inoculation with 2 x 107 hybridoma cells (from the selected hybridoma cell line). Ascites fluid was collected 7 to 14 days after cell injection , centrifuged at 300 x g, and cell-free supernatants were stored at -20 C. Mouse ascites was precipitated with saturated ammonium sulfate (45%) . The precipitate was dialyzed against phosphate buffered saline (PBS) overnight at 4 C and applied to an Affigel Protein A affinity column ; elution of the antibody was conducted with elution buffer (Biorad , Richmond , CA) . The pooled fractions were brought immediately to a pH 6.5 by addition of phosphate bufffer pH 7.5 (0.5 M) and were concentrated by negative pressure dialysis. The protein was judged homogeneous by polyacrylamide gel electrophoresis (PAGE) in sodium dodecylsulfate (SDS) in nonreducing conditions and by isoelectric focusing .
Preparation of radioiodinated antibody Both purified monoclonal antibodies against UCLA RO 82 W-1 cells (hereafter indicated as MAb x RO 82 W-1) and normal IgG ("irrelevant" mouse IgG) were radioiodinated with various techniques: [1] oxidation of iodide by Chloramine T (11) ; [2] electrolysis (12) or [3] lodogen (Pierce Chemicals Co ., Rockford, IL) . The latter method was selected for all subsequent studies. The labelling procedure was carried out at room temperature by combining 5-100 Ilg of IgG with 1-5 mCi of 13 1 1 or 125 1 (New England Nuclear) for 45 minutes. The reaction was stopped by the addition of 20 III of KI (0.1 M) for 20 minutes. This mixture was then chromatographed on a 20 x 1 cm Biogel P6 column (Bio-Rad, Richmond, CA) to remove unreacted 1311 or 125 1. The specific activities of the radioiodinated products ranged from 5-400 IlCi/llg. They were stored at -20 C unless stated otherwise. In vitro binding studies Binding studies were carried out at room temperature in 50 ml plastic tubes containing 2-5 x 10 7 UCLA RO 82 W-1 cells or an equal number of the tumor cells in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and varying amounts (cpm) of the tracers under analysis. After gentle agitation at 20 C of the tubes at time 0 and at various time intervals thereafter, 400 III of the medium each containing 0.25 x 10 7 cells were transferred to mi crofuge plastic tubes (Tekmar Co ., Cincinnati , Ohio) . For all subsequent studies only experimental points at 0 and 3 hours were obtained. Two control tubes containing usually 5000 cpm/tube for each time point under study were kept and represented the "total counts" added. The experimental set of tubes were then centrifuged in a table top microfuge (Beckman Instruments Inc. , Palo Alto , CA) for 60 seconds at 17,500 rpm in the cold room (4 C) . The supernatant was aspirated and discarded . The pellets were counted in a gamma spectrometer (Nuclear Chicago, IL). Pilot studies had indicated that a washing step was superfluous . The radioactivity in the pellet was expressed as a per-
636
Immunodetection of thyroid tumors
centage of the radioactivity in the control tubes (total counts). When competitive binding studies were done, unlabelled IgG up to 1,000 /lg/tube was added to the 50 ml tube 30 minutes before the tracer addition. The specific binding in competitive studies is expressed as a difference in binding between cells not exposed to unlabelled IgG and those exposed to unlabelled IgG. At the end of each experiment, the viability of the cells were tested by the Trypan Blue Dye exclusion technique, and was greater than 80% even after 18 hours of incubation. In pilot studies the binding of Do tracers (Do tracer refers to a tracer immediately used after labelling) on viable cells (> 95% viability) and on nonviable cells (5% and 8% viability) was measured. The same tracer binding was found on nonviable cells and on viable cells (10%). All labelled proteins were tested in the in vitro binding assay before use in other studies, that is, PAGE electrophoresis and in vivo studies.
ing) on a Kodak X-OMAT film (Eastman Kodak Co., Rochester, NY). The film was processed after a few days of exposure, and the migration distance of the radioactive spots from the top of the gel was compared with those of standard proteins run in similar conditions and stained with Coomassie Blue. High molecular weight standards (Pharmacia, Piscateway, NJ) ranged from 67kd (Albumin) to 660kd (Thyroglobulin).
Immunocytochemical studies The UCLA RO 82 W-1 or M-14 cells were harvested, put on glass slides, fixed with poly-L-Iysine, and dried. Subsequently, the slides were humidified with phosphate buffer saline; after 15 minutes incubation with BSA 1% and 10% goat serum, the excess fluid was removed and MAb RO 82 W-1 was added (dilution 1:4,000) in 1% BSA. A similarly prepared set of slides was treated with heat aggregated (63 C) IgG MAb x RO 82 W-1 (1 :4,000). The slides were then incubated for 60 minutes at room temperature in a moisturized chamber. After they were washed twice with PBS, the second antibody was added: a solution of peroxidase labelled goat anti mouse IgG 1:50. The slides were then reincubated for 60 minutes at room temperature, counterstained with hematoxylin, and subsequently dried, analyzed, and photographed. In each experiment M-14 cells (human melanoma cells) were used as control .
Preparation of immunoglobulin aggregates by heating A monoclonal antibody solution (MAb x RO 82 W1) (2.5 mg/ml) was heated in a thermostatic water bath at 63 C for 10 minutes, which was the optimal time exposure reported by Anderson and Grey (13) for aggregate formation of IgG without visible opalescence. These aggregated immunoglobulins, after labelling with the lodogen method, were used for binding studies. The unlabelled IgG aggregates produced by heating were also utilized in immunocytochemical studies on the thyroid tumor cells UCLA RO 82 W-1 and the M-14 cells (human melanoma).
In vivo immunodetection studies in nude athymic mice Athymic nude female mice BALB/c (Simmonson Farm, Gilroy, CA) were housed in semi-sterile conditions. They were all injected subcutaneously (s.c.) with 1 x 107 UCLA RO 82 W-1 cells in one or more sites. Certain animals (no. 1,7,8,9, Table 1) received at a later date 1 x 107 M-14 cells s.c . The thyroid tumors grew to a sizeable mass and were suitable for scintigraphic studies after 6 to 9 months (> 1.0 cm). The KI (1 .0 M) was then added to the animals' drinking water, 2 days prior to the scintigraphic studies and for the length of the study, except when indicated (Table 1). The 131 1 MAb x RO 82 W-1, or "irrelevant" 131 1 mouse IgG (50-100 /lCi), were injected (004 ml) via the tail vein. Radionuclide imaging was done on anesthetized animals on the day of injection and 3, 5, and 6 days later using a gamma camera fitted with a pinhole collimator. The image data were stored, analyzed, and displayed with a computer interfaced with the gamma camera. Images acquired over a 10-minute interval had about 20-40 thousand counts. After the last scan was done, blood was withdrawn by cardiac puncture, weighed, and
Polyacrylamide gel electrophoresis (PAGE) and autoradiographic studies of unlabelled and 131 1MAb To characterize the unlabelled and labelled MAb immunoglobulins and their aggregates used in the binding studies, PAGE was carried out according to the method of Laemmli (14). Two gradient gels 2/16 PAA (Pharmacia Fine Chemicals, Piscateway, NJ) were run in parallel in non-SOS and nonreducing conditions using TRIS (0.09 M) boric acid (0.08 M), Na2 EOTA 0.0025 M, pH 804. The gels were run at 10 C at 70 V for 30 minutes and subsequently at 400 V for 2 1/2 hours to attain a total of 580 Volt hours. The gels containing nonradioactive IgG were stained with Coomassie Brilliant Blue R-250, (BioRad Laboratories, Richmond , CA) , destained, and photographed. After electrophoresis, the gels containing the labelled preparations were wrapped in a plastic bag and placed in contact (prior to dry-
637
A.J. Van Herle, B. Estour, G. Juillard, et al.
Table 1 - Immunoscintigraphic data and tumor blood ratios in nude mice transplanted with UCLA RO 82 W-1 and M-141 Cells. Mouse 10 no.
Type of tumor implanted
UCLA RO 82 W-1
Type of tracer injected
131 1MAb
x
Scintiscan Macroscopic necrosis day 5 after tracer injection
2
UCLA RO 82 W-1
131 1MAb
x
% Bound SSKI in radioactivity drinking on UCLA RO 82 cells water
MN 2 34 rest of tumor 0.8 0.6
10%
pos
MN212.2 rest of tumor 0.8
50%
+
pos
pos
neg
neg
pos
RO 82 W-1 (00) M-14 1
Tumor/blood ratio
RO 82 W-1 (0 5) 3
UCLA RO 82 W-1
131 1 "Irrelevant" mouse IgG (0 0)
pos
pos
MN227.0 rest of tumor 1.6
1.5%
+
4
UCLA RO 82 W-1
131 1 "Irrelevant" mouse IgG (0 0)
pos
±
MN 2 96 rest of tumor 1.6
1.5% 3.9%
+
5
UCLA RO 82 W-1
131 1MAb x RO 82 W-1 (00)
pos
neg
MN 2 O.3 rest of tumor 0.3
10%
+
6
UCLA RO 82 W-1
131 1 "Irrelevant" mouse IgG (0 0)
pos
neg
MN211.8 rest of tumor 0.7
1.5%
+
7
UCLA RO 82 W-1
131 1MAb x RO 82 W-1 (05)
neg
neg
UCLA RO 82 W-1 0.6
30%
neg
neg
M-14 1 O.5
neg
neg
UCLA RO 82 W-1 0.6
neg
neg
M-14 1 0.3
neg
neg
UCLA RO 82 W-1 0.32 30%
neg
neg
M-14 1 0.30
M-14 1 8
UCLA RO 82 W-1
x RO 82 W-1 (05) 131 1MAb
M-14 1 9
UCLA RO 82 W-1 M-14 1
131 1 "Irrelevant" mouse IgG (0 5)
50%
1M-14 - human melanoma cells 2MN - macroscopic necrosis
counted. The animals were then sacrificed and various organs were removed, weighed and counted. In animals with macroscopic tumor necrosis, the macroscopic necrotic material was separated from the rest of the tumor, both portions were weighed, counted, and tissue/blood ratios were established separately for the macroscopic necrotic and nonnecrotic regions of the tumors. The radioiodinated proteins were either injected on the day of the labelling process (0 0) or 5 days after labelling and storage at -20C (0 5) (Table 1). All tracers were tested in the in vitro binding assay using the UCLA RO 82 W-1 cells on the day of their injection.
cells (Figure 1). The maximal binding was found to be identical at 4C and 20C and was independent of the oxidative agent used in the labelling process. Despite the rigorous standardization of the binding technique, unexplained variations in maximal binding continued to persist from experiment to experiment. Therefore, the effect of storage of the labelled proteins and the intensity of labelling, i.e., specific activity (SA) on the binding were analyzed. Figure 2 shows a composite binding of the labelled protein as a function of its "age" (storage time), storage temperature (4C, -20C) and specific activity (2.5 versus 20 f..LCi/f..Lg). These studies indicated clearly that the highest binding of 131 1 MAb x RO 82 W-1 occurred after storage at -20C and suggested that the maximal binding is higher for the preparation with higher SA (20 f..LCi/f..Lg versus 2.5 f..LCi/f..Lg). The highest maximal binding (70%) was achieved after 10 days of storage of the tracer at -20C (SA 20 f..LCi/f..Lg). In contrast, the low SA tracer (2.5 f..LCi/f..Lg) kept at -20 C bound maximally after 3 days
RESULTS Binding experiments and effect of tracer storage on binding The binding of 131 1 MAb RO 82 W-1 to RO 82 W-1 cells was dependent on the length of incubation (not shown) up to 3 hours and on the number of
638
Immunodetection of thyroid tumors
10
o no unlabeled IgG •
unlabeled IgG a dded
0..........3--------// r---;'B TIME (HRS)
0 _______
24
0
1.0 x 10 7 cells
20 0_---------0
g' 16 i:i c
0.5 X 10 7 cells
:
:~:~~~~~~~~~~~~~~--------~: ::: ::: :::::
iii 12
C Q)
~ 8 4
o
o
o~'----------~~------------~/~8 TIME (HRS)
(30%). The same tracers when stored at 4C bound less effectively; in contrast to the tracers kept at -20 C, their maximal binding was not substantially affected by their SA (2.5 versus 20 JlCi/Jlg).
Fig. 1 - Effect of incubation time and cell number on the maximal tracer binding and lack of displacement with unlabelled IgG. 131 1MAb x RO 82 W-1 was incubated with increasing numbers of cells. Maximal tracer binding was proportional to the number of cells, and was reached at 3 hours. Unlabelled monoclonal antibody (1000 ~g) incubated with UCLA RO 82 W-1 cells followed by 131 1MAb x RO 82 W1 were non-inhibitory on tracer binding (see inset). In all subsequent figures the maximal binding after 3 hours of incubation is shown except when stated otherwise. Data points represent means of duplicate or triplicate experiments not differing by > 5%. Data are expressed as percentage of total radioactivity added to the tubes.
Similar studies conducted with labelled human F (ab') 2 and Fab fragments showed a similar binding pattern (Figure 3, column sets 2,3). In contrast, 131 1 human Fc fragments did not show a similar binding pattern (Fig. 3, column set 4). Aggregates of labelled human albumin and thyroglobulin did not bind substantially (Figure 3, column sets 5, 6). That the binding was not due to free iodine trapped by the thyroid tumor cells was shown in an experiment using 125 1 tracer (Fig. 3, column set 7). To test the nonspecificity of the binding to other tumor cell lines, as
Specificity of in vitro binding The tracer incubated with UCLA RO 82 W-1 cells on the day of labelling designated (00), bound 3% (Figure 3, column set 1); The 05 tracer stored at -20 C for 5 days showed an increased binding up to 45% for 131-labelled mouse IgG (Figure 3, column set 1).
75 ,/ ,e - _______ ......
65
55 OJ
c
/
i:i 45 c iii
§
"" I•
""
" ""
"
" ""
"
• •
20
.. 2.5
35
~
25
....
....
o
20
t:J.
2.5
15
5
o
o
!
2
4
6
8
10
DAYS
12
14
16
Fig. 2 - Effect of age of tracer storage method and specific activity of the tracer on maximal tracer binding to UCLA ~Ci/fl9 stored at - 20 C RO 82 W-1 cells. The binding of 131 1 flCilfl9 stored at -20 C MAb x RO 82 W-1, labelled at two different SA, high (20 ~Ci;j.l.g) and low (2.5 ~Ci;j.l.g), was tested. The tracers were flCi/fJg stored at 4 C then stored at two temperatures, -20 C and 4 C. The low specific activity tracflCi/fl9 stored at 4 C er (2.5 ~Ci,1.tg) stored at -20 C bound less efficiently to the cells and maximal tracer binding was reached earlier (3 days versus 10 days) than with the high specific activity tracer (20 ~Ci;j.l.g). The ! , SA of the tracers had less effect on the 18 20 maximal tracer binding when they were stored at 4 C.
639
A.J. Van Herle, B. Estour, G. Juillard, et al.
60 1. 2.
50
131
1 Mouse IgG
'3' 1 Human
F (ab') 2 fragments
Fig. 3 - Binding of labelled "Irrelevant" mouse IgG and fragments of human IgG and other labeled aggregated proteins to '3'1 Human Fe fragments UCLA RO 82 W-1 cells. Binding of labeled mouse IgG ("irrelevant" IgG) and other la13' I Human albumin (heat aggregated) belled human IgG fragments (F (ab) '2, Fab, Fc) and proteins are shown The first col1J' I Thyroglobulin (freezer aggregated) umn (open bar) for each set of columns represents the maximal binding of the tracer on the day of labeling (0 0). The second col'25lodine umn (hatched bar) represents the maximal binding on day 5 (0 5) after labeling. The horizontal hatched area represents the range of binding for the 131 1labelled MAb x RO 82 W-1 with a fresh label (0 0 label) to UCLA RO 82 W-1 cells. 131 1mouse IgG and all tested 13111gG fragments, except for the human 131 1 Fc fragment, bound effectively to the UCLA RO 82 W-1 cells, after storage of the labels at -20 C for 5 days. (For further 6 5 7 details see text.)
3. 13'1 Human Fab fragments
4.
40
5.
OJ
.S TI C
iii
C Q)
6.
30
600K) are responsible and essential for this binding to occur. Our immunocytochemical studies with heat aggregated IgGs rendered melanoma cells positive even though these cells normally do not recognize the non-aggregated MAb x RO 82 W-1. Because alllgG aggregates bind to the UCLA RO 82 W-1 regardless of the method of formation, immunocytochemical studies should therefore be conducted with MAb, which do not contain large IgG aggregates. Antibody binding data obtained in vitro cannot be transposed to in vivo studies because immuno-targeting of tumors is a multifactorial process. Several investigators have attempted to link the percentage of positive immunoscans with the size of the tumor (6, 16). Mann et al. (6) state that "imaging of tumor deposits appears to involve a nonspecific phenomenon that is largely dependent on tumor weight." Other investigators also believe that nonspecific uptake increases proportionately with the size of tumor (17). Our studies confirm the finding that the larger tumors are the most likely to be detected by immunodetection (60%). However, another factor only occasionally mentioned in the literature seems to be important as well, namely, the presence of tumor necrosis (5, 17). In our study all 4 tumors, which were positive on scintiscan (no.1-no. 4), had a T/B ratio in the "macroscopic necrotic" part of the tumor exceeding 3.4 (range 3.4 - 96). The T/B ratio in the mice with absence of macroscopic tumor necrosis ranged from 0.3 to 0.6 and these tumors indeed were negative on scintiscan. These studies seem to indicate that tumor size may not be as important as the accompanying "macroscopic" tumor necrosis frequently present in large tumors. Moshakis et al. (18) also studied the distribution of CEA in breast tumors implanted in immunosuppressed mice and found that labelled anti-CEA antibody reacted with CEA molecules in the extracellular space away from the tumor and that a paucity of labelled Ab was documentable on the cell membrane. Fab fragments of anti myosin antibody have been shown to localize in the myocardium of patients with myocarditis (19). In addition, immunofluorescent studies of endomyocardial biopsies indicated the nonspecific uptake of IgG on myocytes (20). In a recent study, Rubin et al. were able to demonstrate
111 In-labelled nonspecific immunoglobulin uptake in the detection of focal infections and in metastatic tumors (21). Furthermore, Epstein and associates demonstrated that radiolabeled antinuclear monoclonal antibodies can concentrate within bulky tumors containing necrotic lesions (22). In the latter studies, necrotic regions dissected from the tumors at necropsy gave high tumor to blood ratios similar to what was seen in our studies using immunoaggregates. These studies and our own seem to show that immunodetection may be related to the presence of necrotic tissue and the binding by 131 1 IgG aggregates or 131 1 IgG itself. Because our in-vitro studies have convincingly indicated that the observed tracer binding is primarily due to the presence of immunoglobulin aggregates in the tracer preparations and that the in vivo binding is at least partially due to tumor necrosis, the fundamental question arises whether immunoglobulin binding to tumor necrosis areas is the basic mechanism of immunodetection? Indirect evidence supportive of this concept is provided by the following findings: Pilot studies have indicated that necrotic cells bind immunoaggregated IgG better than nonaggregated IgG's in vitro. Because the "macroscopic" necrotic part of the tumor has a T/B ratio higher than the rest of the tumor, 131 1 MAb aggregates used in these binding studies may bind more effectively to the necrosis or to elements present in necrotic tissue. If highly specific Abs are used, as was the case in studies reported by Lundy et al. (23), then the visualization may be due to specific IgG. Additional studies in our laboratory (not shown) have also indicated that tumor extracts from scintigraphically detectable tumors contain high molecular weight aggregates, which are retained by Biorad A 15 columns. Based on these in vivo studies, binding of labelled aggregated IgGs to tumor necrosis represents a plausible and important mechanism of immuno-detection. Furthermore our studies clearly indicate that 131 1 "irrelevant" mouse IgG can lead to tumor targeting (mouse no. 2, Fig. 9, panel B) and provides a possible explanation for the data reported by Mann et al. (6) and find confirmation in the recent data of Rubin et al. (21). We have shown that specific 131 1 MAb and "irrelevant" 131 1 mouse IgG can bind in vitro to tumor cells and that in vivo positive images can be obtained with both labels, which confirms earlier data reported by Bullard et al. using intracranial human glioma xenografts (24). The in vitro binding is directly related to tracer aggregate formation which occurs during the freezing process. Many investigators do not state the preservation method of the tracers used for in vitro or in vivo binding studies and it is
644
Immunodetection of thyroid tumors
thus difficult to assess whether similar nonspecific binding of labelled immunoaggregates played a role in their studies. The formation of immunoaggregates during the freezing process are certainly not the only factor involved in obtaining positive scintigraphies; tumor size and the "macroscopic" tumor necrosis, which accompanies increased tumor size, may be equally important. Our studies suggest that development of immunoscintigraphic methods, with "irrelevant" labeled aggregated immunoglobulins, may be a direction worthwhile to explore in the field of tumor immunodetection.
8.
9.
10.
ACKNOWLEDGMENTS The authors thank K. Kellett, B. Cheng, T. Totanes, D. Padua, and D. Canlapan for their excellent technical assistance. This work was supported in part by a grant from the California Cancer Research Coordinating Committee A850702.
11.
REFERENCES
12.
1. Pressman D., Day ED., Blau M. The use of paired labelling in the determination of tumor-localizing antibodies. Cancer Res. 17: 845,1957. 2. Goldenberg D.M., DeLand F., Euishin K., Bennett S., Primus F.J., Van Nagell Jr J.R., Estes N., DeSimone P., Rayburn P. Use of radiolabelled antibodies to carcinoembryonic antigen for the detection and localization of diverse cancers by external photoscanning. N. Engl. J. Med. 298: 1384,1978. 3. Mach J.P., Carrel S., Forni M., Ritschard J., Donath A, Alberto P. Tumor localization of radiolabelled antibodies against carcinoembryonic antigen in patients with carcinoma. N. Engl. J. Med. 303: 5, 1980. 4. Goldenberg D.M., Preston D.F., Primus F.J., Hansen H.J. Photoscan localization of GH-39 tumors in hamsters using radiolabelled anticarcinoembryonic antigen immunoglobulin G. Cancer Res. 34: 1, 1974. 5. Stuhlmiller G.M., Sullivan D.C., Vervaert C., Croker B.P., Harris C.C., Seigler H.J. In vivo tumor localization using tumor-specific monkey xenoantibody and murine monoclonal xenoantibody. Ann. Surg. 194:592,1981. 6. Mann BD., Cohen M.B., Saxton R.E., Morton D.L., Benedict W.F., Korn E.L., Spolter L., Graham L., Chang C.C., Burk MW. Imaging of human tumor xenografts in nude mice with radiolabelled monoclonal antibodies. Limitations of specificity due to nonspecific uptake of antibody. Cancer 54: 1318,1984. 7. Estour B., Van Herle AJ., Juillard G.J.F., Totanes
13.
14.
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