3.5 SPECT scans
3.5.2 Tumor visualization with 99m Tc-Cetuximab
Since the labeling of Cetuximab with reduced 99mTc-pertechnetate was successful,
99mTc-Cetuximab was used for the visualization of primary 231 and MDA-MB-468 mammary carcinomas that developed after orthotopic cell implantation. Before using the 99mTc-Cetuximab in SPECT, the clearance from blood pool was determined in a non-tumor bearing mouse to estimate the best time point for SPECT scans, i.e. when the remaining activity in the blood reached its plateau (Figure 28). A serum half-life of 180 min with a plateau of 6.1% ID/g ± 0.89 were identified for 99mTc-Cetuximab allowing imaging from approx. 16 h post injection. For organizational matters SPECT scans were performed with 99mTc-Cetuximab approx. 24 h post injection.
Figure 28: Blood clearance of 99mTc-Cetuximab
The blood clearance and half-life in blood were determined in a non-tumor bearing mouse for IgG1 99mTc-Cetuximab (half-life 180 min, N=1) by non-linear regression applying a of a one phase exponential decay fitting. A remaining activity of 6.1% ID/g ± 0.89 was determined as plateau for 99mTc-Cetuximab.
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Validation of the specific in vivo tumor visualization with 99mTc-Cetuximab
The specificity of the binding of 99mTc-Cetuximab to EGFR in vivo was validated by use of an 99mTc-isotype antibody. 15 µg (98 pmol, 103 MBq ± 14) of the radiolabeled 99m Tc-isotype antibody were injected i.v. in MDA-MB-468 tumor bearing mice (N = 3) and in vivo SPECT scans were performed 24 h post injection. The next day, the same mice received 15 µg (98 pmol, 95 MBq ± 5) of the radiolabeled 99mTc-Cetuximab i.v. and underwent in vivo SPECT scans 24 h post injection (Figure 29A). A significant tumor uptake of 99mTc-Cetuximab of 6.4% ID/cm3 ± 2.0 (p < 0.05) was determined compared to unspecific tissue uptake (area on the contra lateral side, consisting presumably of muscle, fat and bone) of 0.6% ID/cm3 ± 0.2 (Figure 29B). The 99mTc-isotype antibody revealed a significant tumor uptake of 2.1% ID/cm3 ± 0.1 (p < 0.005) compared to unspecific tissue uptake of 0.75% ID/cm3 ± 0.05. Nevertheless, the tumor uptake of
99mTc-Cetuximab was significantly higher compared to the 99mTc-isotype (p < 0.05) concluding a specific uptake of 99mTc-Cetuximab to MDA-MB-468 tumors. Values for unspecific uptake of 99mTc-IgG1 and 99mTc-Cetuximab in tissues did not differ significantly.
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Figure 29: Uptake of 99mTc-Cetuximab to MDA-MB-468 tumors was specific
In vivo validation of specific binding of 99mTc-Cetuximab to EGFR by SPECT. MDA-MB-468 tumor bearing mice (N = 3) received (A) 15 µg (98 pmol, 103 MBq ± 14) of the radiolabeled 99mTc-isotype antibody and (B) 15 µg (98 pmol, 95 MBq ± 5) of the radiolabeled 99mTc-Cetuximab i.v. on consecutive days. In vivo SPECT scans were performed 24 h post each injection. A significant tumor uptake of 99mTc-Cetuximab of 6.4% ID/cm3 ± 2.0 was determined compared to unspecific tissue uptake (area on the contra lateral side, consisting presumably of muscle, fat and bone) of 0.6% ID/cm3 ± 0.2. The 99mTc-isotype antibody revealed a significant tumor uptake of 2.1% ID/cm3 ± 0.1 compared to an unspecific tissue uptake of 0.75% ID/cm3 ± 0.05. The tumor uptake of 99mTc-Cetuximab was significantly higher compared to the
99mTc-isotype concluding a specific uptake of 99mTc-Cetuximab to MDA-MB-468 tumors. Unspecific tissue uptakes of both radiolabeled antibodies did not differ significantly. 360° SPECT scans were performed in 10 frames with an acquisition of 180 sec per frame; total scan time were 30 min. Tumor derived signals were segmented and compared to an area of the same volume of the contra lateral side. Data are shown as mean ± SD in % ID/cm3. Tumor and tissue uptakes were compared applying a ratio paired t test. *: p < 0.05; **: p < 0.005. Bar represents 10 mm.
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Visualization of tumors by in vivo SPECT and biodistribution analysis applying 99mTc-Cetuximab
After demonstrating specific tumor visualization of of high EGFR expressing MDA-MB-468 tumors by the use of 99mTc-Cetuximab in combination with in vivo SPECT, the approach was adapted to the low EGFR expressing MDA-MB-231 tumors. A dose finding study figured out 10 µg (65 pmol) as best concentration for tumor visualization applying multi-pinhole SPECT. In this study in vivo SPECT scans and ex vivo biodistribution analysis was performed on different cohorts.
Mice bearing the high and low EGFR expressing MDA-MB-468 and MDA-MB-231 tumors, respectively received each 65 pmol (10 µg; 87 MBq ± 12) 99mTc-Cetuximab i.v.
For biodistribution analysis two MDA-MB-468 tumor bearing mice received 65 pmol (10 µg; 82 MBq ± 4) 99mTc-IgG1 isotype for biodistribution analysis. In vivo SPECT scans (tumor volumes: 115.2 mm3 ± 73.8; N = 10) or ex vivo biodistribution analysis (tumor weights: 32 mg ± 20; N = 7) of MDA-MB-468 tumor bearing mice were performed 24 post probe injection. In vivo SPECT scans revealed a high tumor uptake of 99m Tc-Cetuximab with values of 5.5% ID/cm3 ± 2.2 compared to an unspecific tissue uptake (area of the contra lateral side) of 0.7% ID/cm3 ± 0.2 resulting in a tumor to tissue ratio of 7.8 ± 3.2 (Table 19). Besides the distinct tumor derived signal a high accumulation in the liver could be detected assuming hepatic excretion of 99mTc-Cetuximab.
Furthermore, unspecific accumulation in the armpits of the forelegs suggest unspecific uptake in the lymph nodes, especially within the axial or brachial lymph nodes.
A high tumor uptake of 99mTc-Cetuximab of 14.6% ID/g ± 6.9 was determined ex vivo for MDA-MB-468 tumors compared to a remaining activity of 5.5% ID/g ± 1.9 in the blood and an muscle uptake of 0.5% ID/g ± 0.1 resulting in tumor to blood and tumor to muscle ratios of 3.1 ± 2.5 and 30.7 ± 18.3, respectively (Figure 31 and Table 20).
Application of the 99mTc-IgG1 isotype resulted in a low MDA-MB-468 tumor uptake of 1.1% ID/g ± 0.2 determined by ex vivo biodistribution analysis. Unspecific uptake to the muscle and remaining activity in the blood were comparable to 99mTc-Cetuximab (Figure 31 and Table 19).
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Table 19: In vivo determined tumor and tissue uptake of 99mTc-Cetuximab
Tumor model N Volume mm3
Tumor uptake
% ID/cm3
Tissue uptake
% ID/cm3
Tumor to tissue ratio MDA-MB-468 10 115.2 ± 73.8 5.49 ± 2.20 0.74 ± 0.25 7.8 ± 3.2 MDA-MB-231 5 1247 ± 1199 2.13 ± 0.37 0.56 ± 0.25 4.3 ± 1.8 In vivo tumor and tissue uptakes were determined 24 h post 99mTc-Cetuximab intravenous injection in tumor bearing mice by in vivo SPECT scans. Volumes of tumor derived signals were displayed in mm3. Tissue uptake was determined by segmentation of a region with the same size as the tumor containing tissue only on the contra lateral side and used for determination of the in vivo tumor to tissue ratios.
Data are shown as mean ± standard deviation in % ID/cm3.
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Figure 30: Tumor uptake of 99mTc-Cetuximab was dependent on EGFR expression
Mice bearing the high and low EGFR expressing MDA-MB-468 (N = 10) and MDA-MB-231 (N = 5) tumors, respectively received 65 pmol (10 µg; 87 MBq ± 12) 99mTc-Cetuximab i.v. (A) In vivo SPECT scans were performed 24 post injection and revealed a significant tumor uptake of 99mTc-Cetuximab of 5.5% ID/cm3 ± 2.2 and 2.1% ID/cm3 ± 0.4 to MDA-MB-468 and MDA-MB-231 tumors compared to tissue uptakes (area of the contra lateral side) of 0.7% ID/cm3 ± 0.2 (MDA-MB-468) and 0.6% ID/cm3 ± 0.2 (MDA-MB-231). Furthermore, the uptake to MDA-MB-468 tumors was significantly higher than to MDA-MB-231 tumors but unspecific tissue uptakes of 99mTc-Cetuximab did not differ significantly. A high accumulation in the liver could be detected, assuming hepatic excretion of 99mTc-Cetuximab. Also, accumulation in the armpits of the forelegs was detected suggesting unspecific uptake of 99m Tc-Cetuximab to the lymph nodes. 360° SPECT scans were performed in 10 frames with an acquisition of 180 sec per frame; total scan time were 30 min. Tumor derived signals were segmented and compared to an area of the same volume of the contra lateral side. Data are presented as mean ± SD in % ID/cm3. Tumor and tissue uptakes were compared applying a ratio paired t test. An unpaired two-tailed t test was used for comparison between MDA-MB-468 and MDA-MB-231. **: p < 0.05; ****: p < 0.0001.
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Mice bearing low EGFR expressing MDA-MB-231 tumors underwent in vivo SPECT scans (tumor volumes: 1247 mm3 ± 1199; N = 5) or ex vivo biodistribution analysis (tumor weights: 164 mg ± 200; N = 8) 24 h post injection. Compared to high EGFR expressing MDA-MB-468 tumors, in vivo SPECT scans revealed a relatively low tumor uptake of 99mTc-Cetuximab to MDA-MB-231 tumors of 2.1% ID/cm3 ± 0.4 compared to an unspecific tissue uptake (area of the contra lateral side) of 0.6% ID/cm3 ± 0.2 resulting in an in vivo tumor to tissue ratio of 4.4 ± 1.8 (Figure 30 and Table 19).
Noteworthy, the MB-231 tumors were approx. 10 fold larger compared to MDA-MB-468 tumors. Similar to the results obtained in MDA-MDA-MB-468 tumor bearing mice, a high accumulation in the liver and unspecific accumulations in the armpits of the forelegs were detected.
A tumor uptake of 99mTc-Cetuximab of 6.2% ID/g ± 2.7 was determined ex vivo for MDA-MB-231 tumors compared to a remaining activity of 5.2% ID/g ± 1.3 in the blood and an muscle uptake of 0.5% ID/g ± 0.1 resulting in tumor to blood and tumor to muscle ratios of 1.2 ± 0.4 and 11.2 ± 4.4, respectively (Figure 31 and Table 20).
Figure 31: Ex vivo biodistribution analysis of 99mTc-Cetuximab in tumor bearing mice
Ex vivo biodistribution was carried out 24 h post i.v. injection of 65 pmol (10 µg) in MDA-MB-468 (N = 7) or MDA-MB-231 (N = 8) tumor bearing mice. The 99mTc-Isotype IgG was administered to MDA-MB-468 tumor bearing mice (N = 2). The tumor uptake of 99mTc-Cetuximab was dependent on the EGFR expression level with values of 14.6% ID/g ± 6.9 for the high EGFR expressing MDA-MB-468 and 6.2% ID/g ± 2.7 for the low EGFR expressing MDA-MB-231 tumor. Administration of the isotype control IgG resulted in an low uptake of 1.1% ID/g ± 0.3 in a MDA-MB-468 tumor. Both antibodies had an equally high remaining activity in the blood (5.5% ID/g ± 1.9) and were removed from the body via hepatic excretion (liver uptake: 16.8% ID/g ± 4.3). Data are presented as mean ± SD in % ID/g.
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Table 20: Ex vivo determined tumor and tissue uptake of 99mTc-Cetuximab
Uptake Ratio
Tumor model Antibody N Tumor weight mg
Tumor
% ID/g
Blood
% ID/g
Muscle
% ID/g Tumor to blood Tumor to tissue MDA-MB-468 99mTc-Cetuximab 7 32 ± 20 14.60 ± 6.89 5.54 ± 1.90 0.50 ± 0.12 3.1 ± 2.5 30.7 ± 18.3 MDA-MB-231 99mTc-Cetuximab 8 164 ± 200 6.19 ± 2.71 5.20 ± 1.28 0.54 ± 0.08 1.2 ± 0.4 11.2 ± 4.4 MDA-MB-468 99mTc-IgG1 Isotype 2 22 ± 7 1.14 ± 0.22 4.99 ± 0.41 0.32 ± 0.06 0.2 ± 0.03 3.4 ± 0.04
Ex vivo tumor and tissue uptakes were determined after dissection 25 h post 99mTc-Cetuximab i.v. injection. Muscle was used for calculating ex vivo tissue uptake. Ex vivo uptakes are expressed as mean ± SD in % ID/g.
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