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uPAR-targeted multimodal tracer for pre- and intraoperative imaging in cancer surgery.

Boonstra MC, van Driel PB, van Willigen DM, Stammes MA, Prevoo HA, Tummers QR, Mazar AP, Beekman FJ, Kuppen PJ, van de Velde CJ, Löwik CW, Frangioni JV, van Leeuwen FW, Sier CF, Vahrmeijer AL - Oncotarget (2015)

Bottom Line: The urokinase receptor (uPAR) plays an important role in the development of cancer, tumor invasion, angiogenesis, and metastasis and over-expression is found in the majority of carcinomas.This study aims to develop the first clinically relevant anti-uPAR antibody-based imaging agent that combines nuclear (111In) and real-time near-infrared (NIR) fluorescent imaging (ZW800-1).This strategy can assist in surgical planning and subsequent precision surgery to reduce the number of incomplete resections.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Leiden University Medical Center, Leiden, Netherlands.

ABSTRACT
Pre- and intraoperative diagnostic techniques facilitating tumor staging are of paramount importance in colorectal cancer surgery. The urokinase receptor (uPAR) plays an important role in the development of cancer, tumor invasion, angiogenesis, and metastasis and over-expression is found in the majority of carcinomas. This study aims to develop the first clinically relevant anti-uPAR antibody-based imaging agent that combines nuclear (111In) and real-time near-infrared (NIR) fluorescent imaging (ZW800-1). Conjugation and binding capacities were investigated and validated in vitro using spectrophotometry and cell-based assays. In vivo, three human colorectal xenograft models were used including an orthotopic peritoneal carcinomatosis model to image small tumors. Nuclear and NIR fluorescent signals showed clear tumor delineation between 24h and 72h post-injection, with highest tumor-to-background ratios of 5.0 ± 1.3 at 72h using fluorescence and 4.2 ± 0.1 at 24h with radioactivity. 1-2 mm sized tumors could be clearly recognized by their fluorescent rim. This study showed the feasibility of an uPAR-recognizing multimodal agent to visualize tumors during image-guided resections using NIR fluorescence, whereas its nuclear component assisted in the pre-operative non-invasive recognition of tumors using SPECT imaging. This strategy can assist in surgical planning and subsequent precision surgery to reduce the number of incomplete resections.

No MeSH data available.


Related in: MedlinePlus

In vitro agent validationA) Flow cytometer analyses show high uPAR expression on HT-29 cells while no expression is detectable on the Caco-2 cell line. B) Graph shows the serum stability of hybrid ATN-658. An increase in aggregates and albumin bound agents is seen over time, with 60% of the agent still free after 48 h. C) Cell based plate assay analyses show the specific binding of hybrid ATN-658 on uPAR expressing HT-29 cells. Hybrid ATN-658 signal intensities differed significantly from the control hybrid MOPC-21 at all dose groups except 0 nM. D) No specific binding on the control cell line Caco-2 and there were no significant differences between both tracers at all dose groups. A.U.= arbitrary units.
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Figure 1: In vitro agent validationA) Flow cytometer analyses show high uPAR expression on HT-29 cells while no expression is detectable on the Caco-2 cell line. B) Graph shows the serum stability of hybrid ATN-658. An increase in aggregates and albumin bound agents is seen over time, with 60% of the agent still free after 48 h. C) Cell based plate assay analyses show the specific binding of hybrid ATN-658 on uPAR expressing HT-29 cells. Hybrid ATN-658 signal intensities differed significantly from the control hybrid MOPC-21 at all dose groups except 0 nM. D) No specific binding on the control cell line Caco-2 and there were no significant differences between both tracers at all dose groups. A.U.= arbitrary units.

Mentions: uPAR was confirmed to be expressed on HT-29 colorectal cancer cells with around 20,000 copies per cell, which is considered moderate compared to previously reported values between 50,000-200,000 on monocytoid cells and neo-angiogenic endothelial cells. Caco-2 colorectal cancer cells showed minimal expression (<1000 copies per cell) and was used as a control cell line (Figure 1A). ATN-658 and isotype antibody control MOPC-21 were conjugated to the hybrid label (DTPA-Lys(ZW800)-Cys-NHS) in mean ratios (dye:antibody) of 1.7:1 and 2.2:1 respectively. Cell based plate assay analyses were performed to evaluate retained binding capacity of the agents after conjugation. On HT-29 cells a dose-dependent fluorescent signal was detected with hybrid ATN-658, whereas with hybrid MOPC-21 no specific signals were obtained, except at the highest concentrations (Figure 1C). Single NIR dye ZW800-1 showed no signals at all. As expected, on the Caco-2 cells no specific signals were observed with either hybrid ATN-658 or hybrid MOPC-21 (Figure 1D). HPLC analysis showed that hybrid ATN-658 monoclonal antibody was moderately stable in human serum: 60% of the agent was still free after 48h, while the remaining 40% was aggregated or bound to serum albumin (Figure 1B).


uPAR-targeted multimodal tracer for pre- and intraoperative imaging in cancer surgery.

Boonstra MC, van Driel PB, van Willigen DM, Stammes MA, Prevoo HA, Tummers QR, Mazar AP, Beekman FJ, Kuppen PJ, van de Velde CJ, Löwik CW, Frangioni JV, van Leeuwen FW, Sier CF, Vahrmeijer AL - Oncotarget (2015)

In vitro agent validationA) Flow cytometer analyses show high uPAR expression on HT-29 cells while no expression is detectable on the Caco-2 cell line. B) Graph shows the serum stability of hybrid ATN-658. An increase in aggregates and albumin bound agents is seen over time, with 60% of the agent still free after 48 h. C) Cell based plate assay analyses show the specific binding of hybrid ATN-658 on uPAR expressing HT-29 cells. Hybrid ATN-658 signal intensities differed significantly from the control hybrid MOPC-21 at all dose groups except 0 nM. D) No specific binding on the control cell line Caco-2 and there were no significant differences between both tracers at all dose groups. A.U.= arbitrary units.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4546465&req=5

Figure 1: In vitro agent validationA) Flow cytometer analyses show high uPAR expression on HT-29 cells while no expression is detectable on the Caco-2 cell line. B) Graph shows the serum stability of hybrid ATN-658. An increase in aggregates and albumin bound agents is seen over time, with 60% of the agent still free after 48 h. C) Cell based plate assay analyses show the specific binding of hybrid ATN-658 on uPAR expressing HT-29 cells. Hybrid ATN-658 signal intensities differed significantly from the control hybrid MOPC-21 at all dose groups except 0 nM. D) No specific binding on the control cell line Caco-2 and there were no significant differences between both tracers at all dose groups. A.U.= arbitrary units.
Mentions: uPAR was confirmed to be expressed on HT-29 colorectal cancer cells with around 20,000 copies per cell, which is considered moderate compared to previously reported values between 50,000-200,000 on monocytoid cells and neo-angiogenic endothelial cells. Caco-2 colorectal cancer cells showed minimal expression (<1000 copies per cell) and was used as a control cell line (Figure 1A). ATN-658 and isotype antibody control MOPC-21 were conjugated to the hybrid label (DTPA-Lys(ZW800)-Cys-NHS) in mean ratios (dye:antibody) of 1.7:1 and 2.2:1 respectively. Cell based plate assay analyses were performed to evaluate retained binding capacity of the agents after conjugation. On HT-29 cells a dose-dependent fluorescent signal was detected with hybrid ATN-658, whereas with hybrid MOPC-21 no specific signals were obtained, except at the highest concentrations (Figure 1C). Single NIR dye ZW800-1 showed no signals at all. As expected, on the Caco-2 cells no specific signals were observed with either hybrid ATN-658 or hybrid MOPC-21 (Figure 1D). HPLC analysis showed that hybrid ATN-658 monoclonal antibody was moderately stable in human serum: 60% of the agent was still free after 48h, while the remaining 40% was aggregated or bound to serum albumin (Figure 1B).

Bottom Line: The urokinase receptor (uPAR) plays an important role in the development of cancer, tumor invasion, angiogenesis, and metastasis and over-expression is found in the majority of carcinomas.This study aims to develop the first clinically relevant anti-uPAR antibody-based imaging agent that combines nuclear (111In) and real-time near-infrared (NIR) fluorescent imaging (ZW800-1).This strategy can assist in surgical planning and subsequent precision surgery to reduce the number of incomplete resections.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Leiden University Medical Center, Leiden, Netherlands.

ABSTRACT
Pre- and intraoperative diagnostic techniques facilitating tumor staging are of paramount importance in colorectal cancer surgery. The urokinase receptor (uPAR) plays an important role in the development of cancer, tumor invasion, angiogenesis, and metastasis and over-expression is found in the majority of carcinomas. This study aims to develop the first clinically relevant anti-uPAR antibody-based imaging agent that combines nuclear (111In) and real-time near-infrared (NIR) fluorescent imaging (ZW800-1). Conjugation and binding capacities were investigated and validated in vitro using spectrophotometry and cell-based assays. In vivo, three human colorectal xenograft models were used including an orthotopic peritoneal carcinomatosis model to image small tumors. Nuclear and NIR fluorescent signals showed clear tumor delineation between 24h and 72h post-injection, with highest tumor-to-background ratios of 5.0 ± 1.3 at 72h using fluorescence and 4.2 ± 0.1 at 24h with radioactivity. 1-2 mm sized tumors could be clearly recognized by their fluorescent rim. This study showed the feasibility of an uPAR-recognizing multimodal agent to visualize tumors during image-guided resections using NIR fluorescence, whereas its nuclear component assisted in the pre-operative non-invasive recognition of tumors using SPECT imaging. This strategy can assist in surgical planning and subsequent precision surgery to reduce the number of incomplete resections.

No MeSH data available.


Related in: MedlinePlus