UNLABELLED: Noninvasive monitoring of gene transfer will benefit basic research and patient care. Most gene-transfer imaging systems do not directly detect the gene of interest, and most do not exploit radiopharmaceuticals that have Food and Drug Administration approval for total-body use. (111)In-Octreotide is used clinically to locate tumors overexpressing primarily somatostatin receptor type 2 (SSTR2). We report the in vitro and in vivo detection of SSTR2 chimeric gene transfer with this radiopharmaceutical. METHODS: Full-length SSTR2A was ligated into a vector downstream of a 5' Igkappa leader sequence and the hemagglutinin A (HA) sequence. The vector plus insert was then introduced into HT1080 cells. Igkappa and HA domain functions were confirmed by immunologic methods. Receptor binding was studied in transfected cells incubated with (111)In-octreotide with and without somatostatin-28. Mice bearing tumors produced by transfected cells were injected with (111)In-octreotide for biodistribution and imaging studies. RESULTS: Cell-membrane localization by the amino-terminal Igkappa domain was confirmed by immunofluorescence. The HA domain was identified by enzyme-linked immunosorbent assay, immunofluorescence, and Western blotting analysis with anti-HA antibodies. (111)In-Octreotide detected the SSTR2 portion of the fusion protein in vitro (receptor-binding assay) and in vivo (biodistribution studies and gamma-camera imaging). In addition, in vitro studies using either the anti-HA antibody or (111)In-octreotide correlated with biodistribution and imaging studies when cell clones expressing different levels of the fusion protein were tested. This approach may be feasible clinically because we were able to discern chimeric gene transfer in tumor-bearing animals with (111)In-octreotide at doses similar to those already used in humans. CONCLUSION: With this method it may be possible to monitor transfer of a gene of interest directly and noninvasively.
UNLABELLED: Noninvasive monitoring of gene transfer will benefit basic research and patient care. Most gene-transfer imaging systems do not directly detect the gene of interest, and most do not exploit radiopharmaceuticals that have Food and Drug Administration approval for total-body use. (111)In-Octreotide is used clinically to locate tumors overexpressing primarily somatostatin receptor type 2 (SSTR2). We report the in vitro and in vivo detection of SSTR2 chimeric gene transfer with this radiopharmaceutical. METHODS: Full-length SSTR2A was ligated into a vector downstream of a 5' Igkappa leader sequence and the hemagglutinin A (HA) sequence. The vector plus insert was then introduced into HT1080 cells. Igkappa and HA domain functions were confirmed by immunologic methods. Receptor binding was studied in transfected cells incubated with (111)In-octreotide with and without somatostatin-28. Mice bearing tumors produced by transfected cells were injected with (111)In-octreotide for biodistribution and imaging studies. RESULTS: Cell-membrane localization by the amino-terminal Igkappa domain was confirmed by immunofluorescence. The HA domain was identified by enzyme-linked immunosorbent assay, immunofluorescence, and Western blotting analysis with anti-HA antibodies. (111)In-Octreotide detected the SSTR2 portion of the fusion protein in vitro (receptor-binding assay) and in vivo (biodistribution studies and gamma-camera imaging). In addition, in vitro studies using either the anti-HA antibody or (111)In-octreotide correlated with biodistribution and imaging studies when cell clones expressing different levels of the fusion protein were tested. This approach may be feasible clinically because we were able to discern chimeric gene transfer in tumor-bearing animals with (111)In-octreotide at doses similar to those already used in humans. CONCLUSION: With this method it may be possible to monitor transfer of a gene of interest directly and noninvasively.
Authors: David M Harris; Inbal Hazan-Haley; Kevin Coombes; Carlos Bueso-Ramos; Jie Liu; Zhiming Liu; Ping Li; Murali Ravoori; Lynne Abruzzo; Lin Han; Sheela Singh; Michael Sun; Vikas Kundra; Razelle Kurzrock; Zeev Estrov Journal: PLoS One Date: 2011-06-22 Impact factor: 3.240