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Here, we demonstrate a protocol to use 16α-[18F]-fluoro-17β-estradiol (18F-FES) positron emission tomography (PET) as a tool to visualize ERα expression in ERα-positive breast xenografts.
To demonstrate how estrogen receptor alpha (ERα) positive breast cancer xenografts may be visualized in BALB/c nude mice using 16α-[18F]-fluoro-17β-estradiol (18F-FES) positron emission tomography (PET), ovariectomized BALB/c nude mice were injected with ERα-positive breast cancer cells (MCF-7, 3 × 106 cells; shoulder [n = 10] or 4th inguinal mammary fat pad [n = 10]) or ERα-negative breast cancer cells (MDA-MB-231, 1 × 106 cells; mammary fat pad [n = 5]). Mice harboring MCF-7 cells received subcutaneous injections of 20 µg of 17β-estradiol (20 µg/20 µL; corn oil:ethanol, 9:1) in the nape of their necks 2 days prior to cell injection, followed by daily injections five times per week for 5 weeks. Tumor volumes were measured according to the formula: (L*W2)/2 (L; length, W; width). Once tumor volumes reached approximately 100 mm3, 17β-estradiol injections were halted 2 days prior to mice receiving 18F-FES for PET imaging to avoid competitive binding with ERα. Upon 18F-FES administration via the lateral tail vein, PET/MRI was performed for 15 min at 1 h to 1.5 h post-injection. 18F-FES uptake was not observed in ERα-negative, MDA-MB-231 tumor-bearing mice. 18F-FES uptake was most pronounced in mice harboring MCF-7 tumors in the shoulder. In MCF-7 tumors grown in the inguinal mammary fat pad, 18F-FES uptake was less visible, as the intestinal excretion pattern of 18F-FES obscured the radioactivity detectable in these tumors. To use 18F-FES PET as a tool to visualize ERα expression in ERα-positive breast xenografts, we demonstrate that the visibility of 18F-FES uptake is clear in tumors located away from the abdominal region of mice, such as in the shoulder.
Breast cancers (BC) can be stratified into different molecular subtypes1. Breast tumors that are classified as the luminal subtype overexpress estrogen receptor alpha (ERα). As such, this subtype of BC is also referred to as ERα-positive (ERα+). Fortunately, those diagnosed with ERα+ BC experience the highest 10-year survival coupled with low rates of distant metastasis2,3. Due to ERα expression, such patients have access to a collection of hormone therapy options, including selective estrogen receptor modulators (SERMs), anti-estrogen drugs, and aromatase inhibitors4.
To assess whether a breast cancer patient is eligible for hormone therapy, the expression levels of ERα within breast tumors must be determined5,6,7. While the gold standard of testing is conducted using immunohistochemistry (IHC) methods, many reports highlight the issue of both the reproducibility and reliability of the results obtained6,8,9. IHC can give rise to result discordance as the technique is semi-quantitative in nature, where differences in tissue processing and subsequent interpretation can lead to variability6. To rectify this recurring problem, guidelines were set in 2010 and updated in 2020 by the American Society of Clinical Oncology with the intention of reducing interobserver variation10. Currently, the clinically validated cut-off sits at ≥1%, with ERα expression even at very small expression levels demonstrating clinically meaningful benefits using endocrine therapy11.
In advanced BC, ERα expression may differ between metastases and the primary tumor. Some observations report an 18%-55% discrepancy in ERα expression levels between metastatic lesions and the primary tumor, pointing to the importance of determining the ERα status of BC metastases12. To address this, guidelines highlight the importance of confirming hormone receptor status in metastatic lesions to make informed treatment plans13,14. However, the feasibility of this is questionable, particularly through IHC methods, considering that metastases may exist in places that are difficult to take biopsies from.
Molecular imaging methods have emerged to become essential tools for the detection and visualization of tumor lesions within cancer patients. In particular, positron emission tomography (PET) imaging requires the use of tracers or, more specifically, radiopharmaceuticals, which are designed to exploit certain features of tumors with the intention of visualizing these lesions non-invasively. The most common PET tracer used in oncology is 18F-fluorodeoxyglucose (18F-FDG)15. In this study, we explore the use of a radiolabeled form of estradiol, 18F-fluoroestradiol (18F-FES). Estradiol - a ligand for ERα - is a hormone predominantly produced by the ovaries in females16. 18F-FES received recent approval from the Food and Drug Administration (FDA) and is marketed as CeriannaTM. This imaging agent is designed to be used as an adjunct to biopsies in patients with recurrent or metastatic BC17. Whole-body PET imaging with 18F-FES can be used as a non-invasive method to detect ERα levels both in the primary tumor and in distant metastases in regions from which biopsies are difficult to obtain18. The prediction of ERα levels using 18F-FES PET imaging correlates with IHC results, and moreover, little to no detection of ERα using 18F-FES PET imaging is a reliable predictor of tumors that are unlikely to respond to hormone therapy18. To ensure the appropriate use of 18F-FES in the clinic, guidelines have been formulated through consensus by experts in the field19. In this study, we evaluate the use of 18F-FES PET in preclinical models of breast cancer in mice.
All animal studies were approved by the Austin Hospital Animal Ethics Committee (A2023/05812) and conducted in compliance with the Australian Code for the care and use of animals for scientific purposes.
1. Cell preparation
2. Cell injection into ovariectomized mice
3. Preparation of estradiol solution
4. Subcutaneous injection of estradiol solution
5. 18F-FES PET and MRI imaging of ovariectomized mice
CAUTION: Use protective equipment when handling radioactivity. Follow all applicable regulatory procedures when handling radioactivity.
6. Harvesting and fixing tumour tissue for immunohistochemistry
7. Immunohistochemistry for the detection of estrogen receptor alpha (ERα)
To determine the location for which ERα positive tumors can be clearly visualized using 18F-FES PET, three cohorts of ovariectomized mice were used in this study (Figure 1). Two groups of mice were injected with MCF-7 cells - an ERα positive breast cancer cell line - either IMF or in the shoulder. As a negative control, another cohort of mice was injected with MDA-MB-231 cells, a commonly used triple-negative breast cancer cell line that does not express ERα (
Here, we describe the utility of 18F-FES PET/MRI in the detection of breast tumors characterized by ERα expression. As an example, we demonstrate that one location at which ERα positive tumors can be visualized is in the shoulder of mice - these tumors can be clearly identified by 18F-FES uptake, compared to tumors located within the 4th inguinal mammary fat pad (Figure 4). 18F-FES uptake was not visible in MDA-MB-231 tumors, confirming i...
The authors have nothing to disclose.
This work was supported by the National Breast Cancer Foundation (IIRS-22-071). We acknowledge the Operational Infrastructure Support program of the Victorian State Government. This research was also undertaken using the Solid Target Laboratory, an ANSTO-Austin-LICR Partnership, also supported by the National Imaging Facility and the Victorian Government. The authors acknowledge the scientific and technical assistance of the National Imaging Facility, a National Collaborative Research Infrastructure Strategy (NCRIS) capability, at the La Trobe-ONJCRI node, Olivia Newton-John Cancer Research Institute (ONJCRI). Figures 1 and 3 have been made with BioRender.
Name | Company | Catalog Number | Comments |
2.5% Trypsin (10x) | Gibco | 15090-046 | |
27 G x 13 mm 0.5 mL insulin syringe | Terumo | SS*05M2713KA | For cell injections |
29 G x 13 mm 0.5 mL insulin syringe | Terumo | SS*05M2913KA | For estradiol injections |
30% H2O2 | Chem-Supply | HA154 | Diluted to a 3% working solution with distilled water |
Corn oil | Sigma | C8267 | |
DAB Substrate Kit | Abcam | ab64238 | |
Dako anti-rabbit-HRP, 110 mL | Aligent-Dako | K4003 | Secondary antibody used for IHC |
DMEM/F-12 Medium | Gibco | 11320033 | |
Dose calibrator | Capintec | 5130-3216 | |
Estradiol | Sigma | E2758 | |
Estrogen Receptor α (D8H8) Rabbit mAb | Cell Signalling Technology | #8644 | Primary antibody used for IHC |
FBS | Bovogen | SFBS | |
Heat element (Infra Red Lamp) | Amcal | 12400 | For tail vein dilation |
Matrigel | Corning | 356225 | |
MultiCell 4 Channel Monitoring kit for triple- or quadruple-mouse imaging chamber | Mediso | PR-MC900200 | For monitoring of mouse respiration |
NanoScan PET/MRI 3T System | Mediso | PR-RD000000 | For PET/MRI acquistion |
PBS (1x) | Gibco | 14190-144 | |
TBST | ThermoFisher | #28360 | Wash buffer for IHC |
Three mice imaging chamber | Mediso | PR-MC407300 | For PET/MRI acquistion |
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