Detection of breast cancer microcalcification using ^99mTc-MDP SPECT or Osteosense 750EX FMT imaging

Abstract

Background

In previous work, we demonstrated the presence of hydroxyapetite (type II microcalcification), HAP, in triple negative MDA-MB-231 breast cancer cells. We used ¹⁸F-NaF to detect these types of cancers in mouse models as the free fluorine, ¹⁸F⁻, binds to HAP similar to bone uptake. In this work, we investigate other bone targeting agents and techniques including ^99mTc-MDP SPECT and Osteosense 750EX FMT imaging as alternatives for breast cancer diagnosis via targeting HAP within the tumor microenvironment.

Methods

Thirteen mice were injected subcutaneously in the right flank with 10⁶ MDA-MB-231 cells. When the tumor size reached ~ 0.6 cm³, mice (n = 9) were injected with ~ 37 MBq of ^99mTc-MDP intravenously and then imaged one hour later in a NanoSPECT/CT or injected intravenously with 4 nmol/g of Osetosense 750EX and imaged 24 hours later in an FMT (n = 4). The imaging probe concentration in the tumor was compared to that of muscle. Following SPECT imaging, the tumors were harvested, sectioned into 10 μm slices, and underwent autoradiography or von Kossa staining to correlate ^99mTc-MDP binding with HAP distribution within the tumor. The SPECT images were normalized to the injected dose and regions-of-interest (ROIs) were drawn around bone, tumor, and muscle to obtain the radiotracer concentration in these regions in units of percent injected dose per unit volume. ROIs were drawn around bone and tumor in the FMT images as no FMT signal was observed in normal muscle.

Results

Uptake of ^99mTc-MDP was observed in the bone and tumor with little or no uptake in the muscle with concentrations of 11.34 ± 1.46 (mean ± SD), 2.22 ± 0.95, and 0.05 ± 0.04 %ID/cc, respectively. Uptake of Osteosense 750EX was also observed in the bone and tumor with concentrations of 0.35 ± 0.07 (mean ± SD) and 0.04 ± 0.01 picomoles, respectively. No FMT signal was observed in the normal muscle. There was no significant difference in the bone-to-tumor ratio between the two modalities (5.1 ± 2.3 for SPECT and 8.8 ± 2.2 for FMT) indicating that there is little difference in tumor uptake between these two agents.

Conclusion

This study provides evidence of the accessibility of HAP within the breast tumor microenvironment as an in vivo imaging target for bone-seeking agents. SPECT imaging using ^99mTc-MDP can be rapidly translated to the clinic. FMT imaging using Osteosense 750EX is not currently approved for clinical use and is limited to animal research.

Introduction

Mammograms often reveal small bone-like calcium deposits associated with breast cancer [1], [2], [3], [4], [5]. While there are many theories on the origin of these calcium deposits, more and more evidence in the past years has indicated that they could be the end result of a microcalcification process that begins inside the cell and then migrates to the microenvironment surrounding the tumors [6], [7]. There are two types of microcalcifications: type I contain calcium oxalate, (CO), CaC₂O₄, generally associated with benign disease or non-invasive lobular carcinoma in situ, and type II contain calcium phosphates in the form of carbonated calcium hydroxyapatite (HAP), Ca₁₀(PO₄)₆OH₂, and are mainly associated with malignancy [1], [5], [6], [7], [8], [9], [10], [11], [12]. β-Glycerophosphate (βG) is hydrolyzed to glycerol and inorganic phosphate (Pi) on the malignant cell surface by alkaline phosphatase (ALP). Pi is then transported into the cell by the type II family of Na-Pi cotransporters where Pi then combines with calcium to produce HAP crystals. Finally, HAP leaves the cells and settles into the extracellular matrix [6], [7]. Therefore HAP microcalcifications may be a sign of precancerous cells or early breast cancer including ductal carcinoma in situ (DCIS) [13].

HAP is also the basic component of normal skeletal bone [14], [15]. Single Photon Emission Computed Tomography (SPECT) using [^99mTc]methylene diphosphonate (^99mTc-MDP) and Fluorescence Molecular Tomography (FMT) using Osteosense have been used extensively (and exclusively) for bone imaging. The uptake of ^99mTc-MDP in bone is due to both chemical adsorption of ^99mTc-MDP onto the surface of the HAP in bone and incorporation into the crystalline structure of HAP [16], [17]. Osteosense is a synthetic phosphonate derivative that binds to HAP [18].

We have previously reported on the presence of HAP in the MDA-MB-231 triple negative breast cell line and provided evidence for the use of ¹⁸F-NaF positron emission tomography (PET) imaging for detecting MDA-MB-231 tumors in mouse models [19]. Upon administration, ¹⁸F⁻ dissociates from the sodium ion in the blood and binds to HAP within the tumor microenvironment in a manner similar to bone uptake. In this work, we expand on our previous work and investigate the use of other bone-targeting agents including 99mTc-MDP and Osteosense 750EX in a multimodality approach to image MDA-MB-231 tumors in mouse models of breast cancer via targeting the HAP within the tumor microenvironment. We then compare the results of this work to ¹⁸F-NaF PET results from our previous study.