Using 16α-[18F]-Fluoro-17β-Estradiol PET to Visualize Estrogen Receptor α Expression in Human Breast Cancer Xenografts in Female Ovariectomized Mice

Summary

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.

Abstract

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 4^th 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.

Introduction

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 metastasis^2,3. Due to ERα expression, such patients have access to a collection of hormone therapy options, including selective estrogen receptor modulators (SERMs), anti-estrogen drug....

Protocol

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

1. Using routine cell culture procedures, maintain MDA-MB-231 and MCF-7 cells in DMEM/F-12 supplemented with penicillin/streptomycin and 10% fetal bovine serum (FBS) in T175 tissue culture flasks. Prior to preparing cells for injection, passage cells at least 3x following revival from liquid nitrogen stores. Grow the cells in a humidified incubator at 37 °C with 5% CO₂.

Results

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 (.......

Discussion

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 4^th inguinal mammary fat pad (Figure 4). ¹⁸F-FES uptake was not visible in MDA-MB-231 tumors, confirming i.......

Materials

List of materials used in this article

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