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Functional Imaging of Estrogen Receptors with Radiolabeled-GAP-EDL in Rabbit Endometriosis Model

Posted Aug 31, 2007 Updated Apr 7, 2023
By Nobukazu Takahashi MD , David J. Yang PhD , Hiroaki Kurihara PhD MD , Agatha Borne MD , Saady Kohanim MD , Chang-Sok Oh PhD , Osama Mawlawi PhD , E. Edmund Kim MD PhD
8 min read

Rationale and Objectives

Endometriosis is a common women’s health problem. Animal models provide an invaluable tool to study the natural history of endometriosis. We previously have reported that 99m Tc-labeled glutamate peptide-estradiol ( 99m Tc-GAP-EDL) is a useful agent for imaging functional estrogen receptor (ER) via an ER-mediated process. This study was to evaluate the feasibility of using radiolabeled GAP-EDL to image ER-positive (ER +) endometriosis in nonprimate animal models.

Materials and Methods

3-Aminoethyl estradiol (EDL) was conjugated to glutamate peptide (GAP) to yield GAP-EDL. In vitro cellular uptake studies of 99m Tc and 68 Ga-GAP-EDL inhibition with cold estrone were conducted in 13,762 rat mammary tumor cells. To create a rabbit model with endometriosis, part of uterine tissue was dissected and grafted in the peritoneal wall. Eight weeks after surgery, scintigraphic images were obtained after intravenous injection of 99m Tc-GAP-EDL (1 mCi/rabbit, intravenous) at 0.5–2.0 hours, and 68 Ga-GAP-EDL at 45 minutes. We also performed 68 Ga-GAP-EDL blocking study in rabbit model by using tamoxifen. The rabbits were sacrificed and the grafts were excised for histologic examination.

Results

In vitro uptake study of 99m Tc- and 68 Ga-GAP-EDL in 13,762 rat breast cancer cells showed gradually increasing uptake of both tracers. Accumulation of 68 Ga-GAP-EDL in 13,762 cells was inhibited with cold estrone in a dose-dependent manner. In the endometriosis model, the grafted uterine tissue could be visualized by 99m Tc-GAP-EDL. Necropsy was performed at 2.5 hours after injection time. Four follicular endometrial lesions in eight implanted endometrial tissues were detected, and all lesions could be detected by 99m Tc-GAP-EDL. Planar scintigraphy of uterus, ovary and implants of necropsy specimen revealed an increased uptake of 99m Tc-GAP-EDL in comparison with surrounding abdominal wall tissue. Microscopic examinations support that 99m Tc-GAP-EDL was accumulated in the microinvasive endometrial tissue. After blocking with tamoxifen, 68 Ga-GAP-EDL accumulation in the endometrial grafts could not be visualized, and endometrial tissue-to-normal tissue count ratios were statistically higher in a nonblocked image than that in the blocked image.

Conclusions

Endometriosis uptake of radiolabeled GAP-EDL was via an estrogen receptor-mediated process. Radiolabeled-GAP-EDLs are useful agents for imaging endometriosis.

Endometriosis is a common women’s health problem that is characterized by the presence of tissue resembling endometrium outside the uterus. The exact prevalence of endometriosis in the population cannot be ascertained because of the need to perform an invasive procedure to determine who is affected. Nevertheless, estimate range from 2% to 22% in asymptomatic women, 40%–60% in women with dysmenorrhea, and 20%–30% in women being investigated for subfertility ( ). The condition causes painful periods, chronic pelvic pain, and subfertility, which are potentially debilitating; and it affects millions of women worldwide. The diagnosis is made on visual inspection of the pelvis, usually at laparoscopy. The natural history is unknown, and well-controlled experiments are difficult to perform because of the need for repeated surgical procedures to assess endometriotic lesions over time. Thus despite more than 50 years’ research, the cause of endometriosis remains unclear, and treatment options are limited ( ).

Animal models provide an invaluable tool to study risk factors, prevalence, and the natural history of endometriosis. It has been reported that estrogen receptors (ERs) are overexpressed in uterine endometrium and endometriotic lesions of patients with endometriosis. Laparoscopy observation is generally performed to detect endometriosis. The decision to perform laparoscopy on patients with infertility is very complex owing to a number of factors, such as maternal age, semen parameters, tubal patency, pelvic symptoms, insurance coverage, surgical risks, and availability of surgical expertise. Moreover, the procedure is invasive and the information obtained is morphologic and anatomic changes. Thus it would be desirable to develop radiolabeled estradiol for noninvasive diagnosis and monitoring of the therapy for endometriosis.

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Material and methods

Chemical Analysis

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Synthesis of 99m Tc and 68 Ga-Glutamate Peptide-Estradiol

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Figure 1, The synthetic scheme of 99m Tc-GAP-EDL.

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In Vitro Cellular Uptake Studies

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Gamma Scintigraphic Studies in Rabbits with Endometriosis

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Positron Emission Tomography Studies in Rabbits with Endometriosis

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Statistical Analysis

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Results

Chemistry

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In Vitro Cellular Uptake Studies

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Figure 2, In vitro uptake study of 99m Tc-GAP and 99m Tc-GAP-EDL in 13,762 cells. A total of 100,000 rat mammary tumor cells per well were incubated with 99m Tc-GAP and 99m Tc-GAP-EDL (three wells per tracer). Cells were harvested at 90 minutes of incubation. 99m Tc-GAP-EDL had higher uptake than 99m Tc-GAP at 2 and 4 hours. * P < .05 compared between 99m Tc-GAP and 99m Tc-GAP-EDL.

Figure 3, In vitro uptake study of 68 Ga-GAP and 68 Ga-GAP-EDL in 13762 cells. A total of 100,000 rat mammary tumor cells per well were incubated with 68 Ga-tracers (three wells per tracer). Cells were harvested at 90 minutes of incubation. 68 Ga-GAP-EDL had higher uptake than 68 Ga-GAP at 2 and 4 hours. * P < .05, ** P < .005 compared between 68 Ga-GAP and 68 Ga-GAP-EDL.

Figure 4, In vitro uptake study of 99m Tc-GAP in RBA CRL-1747 cells and 99m Tc-GAP-EDL in MCF-7 and RBA CRL-1747 cells. 99m Tc-GAP-EDL was statistically higher in MCF-7 cell line than RBA CRL-1747 cell line.

Figure 5, In vitro uptake study of 68 Ga-GAP-EDL with cold estrone in 13,762 cells. A total of 100,000 rat mammary tumor cells were incubated with 68 Ga-GAP-EDL in the presence of cold estrone (15–300 umol/L). Cells were harvested at 90 minutes of incubation. Results expressed as % uptake relative to control group. There was a decreased uptake after adding cold estrone in a dose-dependent manner. * P < .005 compared with control group.

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Gamma Scintigraphic Studies in Rabbits with Endometriosis

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Figure 6, X-ray image (a) and planar scintigraphic image of 99m Tc-GAP-EDL (b) and 99m Tc-GAP (c) in the endometriosis rabbit model. Grafts were macroscopically visible at 8 weeks. The cystlike implant was correlated with increased 99m Tc-GAP-EDL uptake (indicated by arrows), but without 99m Tc-GAP uptake.

Figure 7, Necropsy tissue (a, b) and planar image of 99m Tc-GAP-EDL (c) in an endometriosis rabbit model. Necropsy was performed 2.5 hours after injection time. Planar scintigraphy imaging of uterus, ovary, and implants (indicated as arrows) reveals increased uptake of 99m Tc-GAP-EDL.

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Figure 8, X-ray image (a) and positron emission tomography image of 68 Ga-GAP-EDL without blocking (b) and with blocking (c) and 68 Ga-GAP as a control (d) in the endometriosis rabbit model. A rabbit was pre-treated with tamoxifen (2 mg, intravenously). After 20 minutes, the rabbit was administrated with 68 Ga-GAP-EDL (0.785 mCi, intravenously). The select coronal images were obtained at 45 minutes after injection of radio labeled compounds. Foci of endometriosis could not be visualized with blocking and control (indicated as arrows).

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Discussion

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Academic Radiology, Volume 14, Issue 9
Journals General Radiology
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