Imaging DNA Damage Allows Detection of Preneoplasia in the BALB-neuT Model of Breast Cancer

Abstract

A prominent feature of many human cancers is oncogene-driven activation of the DNA damage response (DDR) during early tumorigenesis. It has been shown previously that noninvasive imaging of the phosphorylated histone H2A variant H2AX, γH2AX, a DNA damage signaling protein, is possible using 111 In-labeled anti-γH2AX antibody conjugated to the cell-penetrating peptide transactivator of transcription (TAT). The purpose of this study was to investigate whether 111 Inanti-γH2AX-TAT detects the DDR during mammary oncogenesis in BALB-neuT mice. Methods: Mammary fat pads from BALB-neuT and wild-type mice (age, 40-106 d) were immunostained for γH2AX. 111 In-anti-γH2AX-TAT or a control probe was administered intravenously to BALB-neuT mice. SPECT was performed weekly and compared with tumor detection using palpation and dynamic contrastenhanced MR imaging. Results: γH2AX expression was elevated in hyperplastic lesions in the mammary fat pads of BALB-neuT mice aged 76-106 d, compared with normal fat pads from younger mice and carcinomas from older mice (13.5 ± 1.2 γH2AX foci/cell vs. 5.2 ± 1.5 [P , 0.05] and 3.4 ± 1.1 [P , 0.001], respectively). Serial SPECT imaging revealed a 2.5-fold increase in 111 In-anti-γH2AX-TAT accumulation in the mammary fat pads of mice aged 76-106 d, compared with control probe (P 5 0.01). The median time to detection of neoplastic lesions by 111 In-anti-γH2AX-TAT (defined as .5% injected dose per gram of tissue) was 96 d, compared with 120 and 131 d for dynamic contrast-enhanced MR imaging and palpation, respectively (P , 0.001). Conclusion: DDR imaging using 111 In-anti-γH2AX-TAT identified mammary tumors significantly earlier than MR imaging. Imaging the DDR holds promise for the detection of preneoplasia and as a technique for screening cancer-prone individuals.

PET imaging of DNA damage using 89Zr-labelled anti-γH2AX-TAT immunoconjugates

“…High concentrations of unlabelled anti-γH2AX-TAT slow down γH2AX foci formation, but have no effect on the resolution of DSBs, as shown by the neutral comet assay (data not shown). We previously showed that 111 In-anti-γH2AX-TAT, labelled with low amounts of 111 In necessary for imaging (<1 MBq/μg), similar to 89 Zr-anti-γH2AX-TAT, also does not affect DSB repair in vitro [11] or cause tumour growth delay in vivo [12]. In this manuscript, we show that this is also the case for the 89 Zr-labelled version, even though the spectrum of emitted particles includes more energetic gamma rays and a beta particle as well as low-energy electrons.…”

“…Firstly, applications of DNA damage imaging could be envisioned in early detection of tumours, which had been anticipated nearly a decade ago [8]. We have used the DNA damage repair response to oncogenic stress in a mmtv-driven neuT-overexpressing mouse model of breast cancer [12]. Furthermore, DNA damage imaging could aid tumour diagnosis.…”

“…Here, we describe the use of 89 Zr-anti-γH2AX-TAT, a positron-emitter-labelled variant of the previously described 111 In-labelled agent used for SPECT imaging [12,3]. The need for an alternative for PET imaging is motivated by the increased use of PET versus SPECT in oncology research and the acceptance of PET/CT as the standard of care [24].…”

“…At higher specific activities (the amount of 111 In per gram of compound) the compound can amplify the extent of DSBs and thereby causes inhibition of tumour growth [11]. Furthermore, we applied γH2AX imaging for the very early detection of aplastic lesions in a mouse model of HER2-overexpressing breast cancer [12]. The conjugate used in all of these studies was based on anti-γH2AX antibodies conjugated to the cell-penetrating peptide, TAT (GRKKRRQRRRPPQGYG), which also harbours a nuclear localization sequence [13].…”

The Use of Novel PET Tracers to Image Breast Cancer Biologic Processes Such as Proliferation, DNA Damage and Repair, and Angiogenesis

“…Antibodies to gH2AX labeled with fluorescent probes are commonly used for laboratory assays of cell death. 111 In-anti-gH2AX-TAT immunoconjugate was used as a SPECT probe for detecting DNA damage in mice during mammary oncogenesis and showed changes before MR imaging (66). 89 Zr-anti-gH2AX-TAT immunoconjugates have also shown promising results in a preclinical breast cancer model with 8-fold-higher uptake in irradiated gH2AX cells than nonirradiated controls (67).…”