Imaging of tumor spheroids, dual-isotope SPECT, and autoradiographic analysis to assess the tumor uptake and distribution of different nanobodies

Irati Beltrán Hernández, Rene Rompen, Raffaella Rossin, Katerina T. Xenaki, Eugene A. Katrukha, Klaas Nicolay, Paul van Bergen en Henegouwen, Holger Grüll (Corresponding author), Sabrina Oliveira (Corresponding author)

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Abstract

Purpose: Recent studies have shown rapid accumulation of nanobodies (NBs) in tumors and fast clearance of the unbound fraction, making NBs exceptional tracers for cancer imaging. In this study, we investigate the combination of in vitro imaging of tumor spheroids, in vivo dual-isotope single-photon emission computed tomography (SPECT), and ex vivo autoradiographic analysis of tumors to efficiently, and with few mice, assess the tumor uptake and distribution of different NBs. Procedures: The irrelevant NB R2 (16 kDa) and the EGFR-targeted NBs 7D12 (16 kDa) and 7D12-R2 (32 kDa) were investigated. Confocal microscopy was used to study the penetration of the NBs into A431 tumor spheroids over time, using the anti-EGFR monoclonal antibody (mAb) cetuximab (150 kDa) as a reference. Dual-isotope [ 111 In]DOTA-NB/[ 177 Lu]DOTA-NB SPECT was used for longitudinal imaging of multiple tracers in the same animal bearing A431 tumor xenografts. Tumor sections were analyzed using autoradiography. Results: No binding of the irrelevant NB was observed in spheroids, whereas for the specific tracers an increase in the spheroid’s covered area was observed over time. The NB 7D12 saturated the spheroid earlier than the larger, 7D12-R2. Even slower penetration was observed for the large mAb. In vivo, the tumor uptake of 7D12 was 19-fold higher than R2 after co-injection in the same animal, and 2.5-fold higher than 7D12-R2 when co-injected. 7D12-R2 was mainly localized at the rim of tumors, while 7D12 was found to be more evenly distributed. Conclusions: This study demonstrates that the combination of imaging of tumor spheroids, dual-isotope SPECT, and autoradiography of tumors is effective in comparing tumor uptake and distribution of different NBs. Results were in agreement with published data, highlighting the value of monomeric NBs for tumor imaging, and re-enforcing the value of these techniques to accurately assess the most optimal format for tumor imaging. This combination of techniques requires a lower number of animals to obtain significant data and can accelerate the design of novel tracers.

Original languageEnglish
Pages (from-to)1079-1088
Number of pages10
JournalMolecular Imaging and Biology
Volume21
Issue number6
Early online date11 Mar 2019
DOIs
Publication statusPublished - Dec 2019

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Single-Domain Antibodies
Single-Photon Emission-Computed Tomography
Isotopes
Neoplasms
Autoradiography
Monoclonal Antibodies

Keywords

  • Dual-isotope SPECT
  • Nanobodies
  • Spheroids
  • Tumor imaging

Cite this

Beltrán Hernández, Irati ; Rompen, Rene ; Rossin, Raffaella ; Xenaki, Katerina T. ; Katrukha, Eugene A. ; Nicolay, Klaas ; van Bergen en Henegouwen, Paul ; Grüll, Holger ; Oliveira, Sabrina. / Imaging of tumor spheroids, dual-isotope SPECT, and autoradiographic analysis to assess the tumor uptake and distribution of different nanobodies. In: Molecular Imaging and Biology. 2019 ; Vol. 21, No. 6. pp. 1079-1088.
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abstract = "Purpose: Recent studies have shown rapid accumulation of nanobodies (NBs) in tumors and fast clearance of the unbound fraction, making NBs exceptional tracers for cancer imaging. In this study, we investigate the combination of in vitro imaging of tumor spheroids, in vivo dual-isotope single-photon emission computed tomography (SPECT), and ex vivo autoradiographic analysis of tumors to efficiently, and with few mice, assess the tumor uptake and distribution of different NBs. Procedures: The irrelevant NB R2 (16 kDa) and the EGFR-targeted NBs 7D12 (16 kDa) and 7D12-R2 (32 kDa) were investigated. Confocal microscopy was used to study the penetration of the NBs into A431 tumor spheroids over time, using the anti-EGFR monoclonal antibody (mAb) cetuximab (150 kDa) as a reference. Dual-isotope [ 111 In]DOTA-NB/[ 177 Lu]DOTA-NB SPECT was used for longitudinal imaging of multiple tracers in the same animal bearing A431 tumor xenografts. Tumor sections were analyzed using autoradiography. Results: No binding of the irrelevant NB was observed in spheroids, whereas for the specific tracers an increase in the spheroid’s covered area was observed over time. The NB 7D12 saturated the spheroid earlier than the larger, 7D12-R2. Even slower penetration was observed for the large mAb. In vivo, the tumor uptake of 7D12 was 19-fold higher than R2 after co-injection in the same animal, and 2.5-fold higher than 7D12-R2 when co-injected. 7D12-R2 was mainly localized at the rim of tumors, while 7D12 was found to be more evenly distributed. Conclusions: This study demonstrates that the combination of imaging of tumor spheroids, dual-isotope SPECT, and autoradiography of tumors is effective in comparing tumor uptake and distribution of different NBs. Results were in agreement with published data, highlighting the value of monomeric NBs for tumor imaging, and re-enforcing the value of these techniques to accurately assess the most optimal format for tumor imaging. This combination of techniques requires a lower number of animals to obtain significant data and can accelerate the design of novel tracers.",
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Beltrán Hernández, I, Rompen, R, Rossin, R, Xenaki, KT, Katrukha, EA, Nicolay, K, van Bergen en Henegouwen, P, Grüll, H & Oliveira, S 2019, 'Imaging of tumor spheroids, dual-isotope SPECT, and autoradiographic analysis to assess the tumor uptake and distribution of different nanobodies', Molecular Imaging and Biology, vol. 21, no. 6, pp. 1079-1088. https://doi.org/10.1007/s11307-019-01320-x

Imaging of tumor spheroids, dual-isotope SPECT, and autoradiographic analysis to assess the tumor uptake and distribution of different nanobodies. / Beltrán Hernández, Irati; Rompen, Rene; Rossin, Raffaella; Xenaki, Katerina T.; Katrukha, Eugene A.; Nicolay, Klaas; van Bergen en Henegouwen, Paul; Grüll, Holger (Corresponding author); Oliveira, Sabrina (Corresponding author).

In: Molecular Imaging and Biology, Vol. 21, No. 6, 12.2019, p. 1079-1088.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Imaging of tumor spheroids, dual-isotope SPECT, and autoradiographic analysis to assess the tumor uptake and distribution of different nanobodies

AU - Beltrán Hernández, Irati

AU - Rompen, Rene

AU - Rossin, Raffaella

AU - Xenaki, Katerina T.

AU - Katrukha, Eugene A.

AU - Nicolay, Klaas

AU - van Bergen en Henegouwen, Paul

AU - Grüll, Holger

AU - Oliveira, Sabrina

PY - 2019/12

Y1 - 2019/12

N2 - Purpose: Recent studies have shown rapid accumulation of nanobodies (NBs) in tumors and fast clearance of the unbound fraction, making NBs exceptional tracers for cancer imaging. In this study, we investigate the combination of in vitro imaging of tumor spheroids, in vivo dual-isotope single-photon emission computed tomography (SPECT), and ex vivo autoradiographic analysis of tumors to efficiently, and with few mice, assess the tumor uptake and distribution of different NBs. Procedures: The irrelevant NB R2 (16 kDa) and the EGFR-targeted NBs 7D12 (16 kDa) and 7D12-R2 (32 kDa) were investigated. Confocal microscopy was used to study the penetration of the NBs into A431 tumor spheroids over time, using the anti-EGFR monoclonal antibody (mAb) cetuximab (150 kDa) as a reference. Dual-isotope [ 111 In]DOTA-NB/[ 177 Lu]DOTA-NB SPECT was used for longitudinal imaging of multiple tracers in the same animal bearing A431 tumor xenografts. Tumor sections were analyzed using autoradiography. Results: No binding of the irrelevant NB was observed in spheroids, whereas for the specific tracers an increase in the spheroid’s covered area was observed over time. The NB 7D12 saturated the spheroid earlier than the larger, 7D12-R2. Even slower penetration was observed for the large mAb. In vivo, the tumor uptake of 7D12 was 19-fold higher than R2 after co-injection in the same animal, and 2.5-fold higher than 7D12-R2 when co-injected. 7D12-R2 was mainly localized at the rim of tumors, while 7D12 was found to be more evenly distributed. Conclusions: This study demonstrates that the combination of imaging of tumor spheroids, dual-isotope SPECT, and autoradiography of tumors is effective in comparing tumor uptake and distribution of different NBs. Results were in agreement with published data, highlighting the value of monomeric NBs for tumor imaging, and re-enforcing the value of these techniques to accurately assess the most optimal format for tumor imaging. This combination of techniques requires a lower number of animals to obtain significant data and can accelerate the design of novel tracers.

AB - Purpose: Recent studies have shown rapid accumulation of nanobodies (NBs) in tumors and fast clearance of the unbound fraction, making NBs exceptional tracers for cancer imaging. In this study, we investigate the combination of in vitro imaging of tumor spheroids, in vivo dual-isotope single-photon emission computed tomography (SPECT), and ex vivo autoradiographic analysis of tumors to efficiently, and with few mice, assess the tumor uptake and distribution of different NBs. Procedures: The irrelevant NB R2 (16 kDa) and the EGFR-targeted NBs 7D12 (16 kDa) and 7D12-R2 (32 kDa) were investigated. Confocal microscopy was used to study the penetration of the NBs into A431 tumor spheroids over time, using the anti-EGFR monoclonal antibody (mAb) cetuximab (150 kDa) as a reference. Dual-isotope [ 111 In]DOTA-NB/[ 177 Lu]DOTA-NB SPECT was used for longitudinal imaging of multiple tracers in the same animal bearing A431 tumor xenografts. Tumor sections were analyzed using autoradiography. Results: No binding of the irrelevant NB was observed in spheroids, whereas for the specific tracers an increase in the spheroid’s covered area was observed over time. The NB 7D12 saturated the spheroid earlier than the larger, 7D12-R2. Even slower penetration was observed for the large mAb. In vivo, the tumor uptake of 7D12 was 19-fold higher than R2 after co-injection in the same animal, and 2.5-fold higher than 7D12-R2 when co-injected. 7D12-R2 was mainly localized at the rim of tumors, while 7D12 was found to be more evenly distributed. Conclusions: This study demonstrates that the combination of imaging of tumor spheroids, dual-isotope SPECT, and autoradiography of tumors is effective in comparing tumor uptake and distribution of different NBs. Results were in agreement with published data, highlighting the value of monomeric NBs for tumor imaging, and re-enforcing the value of these techniques to accurately assess the most optimal format for tumor imaging. This combination of techniques requires a lower number of animals to obtain significant data and can accelerate the design of novel tracers.

KW - Dual-isotope SPECT

KW - Nanobodies

KW - Spheroids

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