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Am J Transl Res 2013;5(3):291-302
Original Article
HaloTag as a reporter gene: positron emission tomography imaging
with 64Cu-labeled second generation HaloTag ligands
Hao Hong, Hélène A Benink, H Tetsuo Uyeda, Hector F Valdovinos, Yin Zhang, Poncho Meisenheimer, Todd E
Barnhart, Frank Fan, Weibo Cai
Department of Radiology, University of Wisconsin - Madison, Madison, WI, USA; Promega Corporation, Madison,
WI, USA; Promega Biosciences, LLC, San Luis Obispo, CA, USA; Department of Medical Physics, University of
Wisconsin - Madison, Madison, WI, USA; University of Wisconsin Carbone Cancer Center, Madison, WI, USA
Received March 19, 2013; Accepted April 3, 2013; Epub April 19, 2013; Published April 30, 2013
Abstract: The goal of this study is to employ the HaloTag technology for positron emission tomography (PET),
which involves two components: the HaloTag protein (a modified hydrolase which covalently binds to synthetic
ligands) and HaloTag ligands (HTLs). 4T1 murine breast cancer cells were stably transfected to express
HaloTag protein on the surface (termed as 4T1-HaloTag-ECS, ECS denotes extracellular surface). Two new
HTLs were synthesized and termed NOTA-HTL2G-S and NOTA-HTL2G-L (2G indicates second generation, S
stands for short, L stands for long, NOTA denotes 1,4,7-triazacyclononane-N,N’N’’-triacetic acid). Microscopy
studies confirmed surface expression of HaloTag in 4T1-HaloTag-ECS cells, which specifically bind NOTA-
HTL2G-S/L. Uptake of 64Cu-NOTA-HTL2G-L in 4T1-HaloTag-ECS tumors (4.3 ± 0.5, 4.1± 0.2, 4.0 ± 0.2, 2.3 ± 0.1,
and 2.2 ± 0.1 %ID/g at 0.5, 3, 6, 18, and 24 h post-injection respectively; n = 4) was significantly higher than that in
the 4T1 tumors (3.0 ± 0.3, 3.0± 0.1, 3.0 ± 0.2, 2.0 ± 0.4, and 2.4 ± 0.3 %ID/g at 0.5, 3, 6, 18, and 24 h post-injection
respectively; n = 4) at early time points. In comparison, 64Cu-NOTA-HTL2G-S did not demonstrate significant
uptake in either 4T1-HaloTag-ECS or 4T1 tumors. Blocking studies and autoradiography of tumor lysates
confirmed that 64Cu-NOTA-HTL2G-L binds specifically to HaloTag protein in the 4T1-HaloTag-ECS tumors,
corroborated by histology. HaloTag protein-specific targeting and PET imaging in vivo with 64Cu-NOTA-HTL2G-L
serves as a proof-of-principle for future non-invasive and sensitive tracking of HaloTag-transfected cells with PET,
as well as many other studies of gene/protein/cell function in vivo. (AJTR1303006).
Keywords: HaloTag, positron emission tomography (PET), reporter gene, 64Cu, cancer, molecular imaging
Address correspondence to: Dr. Weibo Cai, Departments of Radiology and Medical Physics, University of
Wisconsin - Madison, Room 7137, 1111 Highland Avenue, Madison, WI 53705-2275, USA. Tel: 608-262-1749;
Fax: 608-265-0614; E-mail: wcai@uwhealth.org
Original Article
HaloTag as a reporter gene: positron emission tomography imaging
with 64Cu-labeled second generation HaloTag ligands
Hao Hong, Hélène A Benink, H Tetsuo Uyeda, Hector F Valdovinos, Yin Zhang, Poncho Meisenheimer, Todd E
Barnhart, Frank Fan, Weibo Cai
Department of Radiology, University of Wisconsin - Madison, Madison, WI, USA; Promega Corporation, Madison,
WI, USA; Promega Biosciences, LLC, San Luis Obispo, CA, USA; Department of Medical Physics, University of
Wisconsin - Madison, Madison, WI, USA; University of Wisconsin Carbone Cancer Center, Madison, WI, USA
Received March 19, 2013; Accepted April 3, 2013; Epub April 19, 2013; Published April 30, 2013
Abstract: The goal of this study is to employ the HaloTag technology for positron emission tomography (PET),
which involves two components: the HaloTag protein (a modified hydrolase which covalently binds to synthetic
ligands) and HaloTag ligands (HTLs). 4T1 murine breast cancer cells were stably transfected to express
HaloTag protein on the surface (termed as 4T1-HaloTag-ECS, ECS denotes extracellular surface). Two new
HTLs were synthesized and termed NOTA-HTL2G-S and NOTA-HTL2G-L (2G indicates second generation, S
stands for short, L stands for long, NOTA denotes 1,4,7-triazacyclononane-N,N’N’’-triacetic acid). Microscopy
studies confirmed surface expression of HaloTag in 4T1-HaloTag-ECS cells, which specifically bind NOTA-
HTL2G-S/L. Uptake of 64Cu-NOTA-HTL2G-L in 4T1-HaloTag-ECS tumors (4.3 ± 0.5, 4.1± 0.2, 4.0 ± 0.2, 2.3 ± 0.1,
and 2.2 ± 0.1 %ID/g at 0.5, 3, 6, 18, and 24 h post-injection respectively; n = 4) was significantly higher than that in
the 4T1 tumors (3.0 ± 0.3, 3.0± 0.1, 3.0 ± 0.2, 2.0 ± 0.4, and 2.4 ± 0.3 %ID/g at 0.5, 3, 6, 18, and 24 h post-injection
respectively; n = 4) at early time points. In comparison, 64Cu-NOTA-HTL2G-S did not demonstrate significant
uptake in either 4T1-HaloTag-ECS or 4T1 tumors. Blocking studies and autoradiography of tumor lysates
confirmed that 64Cu-NOTA-HTL2G-L binds specifically to HaloTag protein in the 4T1-HaloTag-ECS tumors,
corroborated by histology. HaloTag protein-specific targeting and PET imaging in vivo with 64Cu-NOTA-HTL2G-L
serves as a proof-of-principle for future non-invasive and sensitive tracking of HaloTag-transfected cells with PET,
as well as many other studies of gene/protein/cell function in vivo. (AJTR1303006).
Keywords: HaloTag, positron emission tomography (PET), reporter gene, 64Cu, cancer, molecular imaging
Address correspondence to: Dr. Weibo Cai, Departments of Radiology and Medical Physics, University of
Wisconsin - Madison, Room 7137, 1111 Highland Avenue, Madison, WI 53705-2275, USA. Tel: 608-262-1749;
Fax: 608-265-0614; E-mail: wcai@uwhealth.org
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