Imaging DNA damage in vivo using gammaH2AX-targeted immunoconjugates

Abstract

DNA damage responses (DDR) occur during oncogenesis and therapeutic responses to DNA damaging cytotoxic drugs. Thus, a real-time method to image DNA damage in vivo would be useful to diagnose cancer and monitor its treatment. Toward this end, we have developed fluorophore- and radioisotope-labeled immunoconjugates to target a DDR signaling protein, phosphorylated histone H2A variant H2AX (γH2AX), which forms foci at sites of DNA double-strand breaks. Anti-γH2AX antibodies were modified by the addition of diethylenetriaminepentaacetic acid (DTPA) to allow (111)In labeling or the fluorophore Cy3. The cell-penetrating peptide Tat (GRKKRRQRRRPPQGYG) was also added to the immunoconjugate to aid nuclear translocation. In irradiated breast cancer cells, confocal microscopy confirmed the expected colocalization of anti-γH2AX-Tat with γH2AX foci. In comparison with nonspecific antibody conjugates, (111)In-anti-γH2AX-Tat was retained longer in cells. Anti-γH2AX-Tat probes were also used to track in vivo DNA damage, using a mouse xenograft model of human breast cancer. After local X-ray irradiation or bleomycin treatment, the anti-γH2AX-Tat probes produced fluorescent and single photon emission computed tomography signals in the tumors that were proportionate to the delivered radiation dose and the amount of γH2AX present. Taken together, our findings establish the use of radioimmunoconjugates that target γH2AX as a noninvasive imaging method to monitor DNA damage, with many potential applications in preclinical and clinical settings.

Figure 1

A, Schematic overview of the synthesis of ¹¹¹In-DTPA-anti-γH2AX-Tat. B, Radioimmunoassay showing binding of ¹¹¹In-DTPA-anti-γH2AX-Tat to γH2AX, present in whole cell lysates derived from irradiated 231-H2N cells, in competition with increasing concentrations of anti-γH2AX or anti-γH2AX-Tat. C, Internalization of ¹¹¹In-DTPA-anti-γH2AX-Tat and ¹¹¹In-DTPA-mIgG-Tat in irradiated and control MDA-MB-468 whole cells and in, D, nuclei. E, Retention of ¹¹¹In-DTPA-anti-γH2AX-Tat and ¹¹¹In-DTPA-mIgG-Tat in MDA-MB-468 cells, exposed to IR (4 Gy) or in control (unirradiated) cells. Experiments were repeated 3 times with 3 replicates. Error bars show standard deviation (SD).

Figure 2

Co-localization of fluorphore-anti-γH2AX-Tat with γH2AX foci in vitro. MDA-MB-468 cells were exposed to AF488-anti-γH2AX-Tat or AF488-mIgG-Tat and after 1 h were sham-irradiated or irradiated (4 Gy). At (A) 2 h or (B) 24 h after addition of RICs, cells were fixed, permeabilized, stained for γH2AX foci (red) and mounted with Vectashield containing DAPI (blue). Images were acquired using confocal microscopy. Nuclear fluorescence due to AF488 (green) was seen only in irradiated cells exposed to AF488-anti-γH2AX-Tat. Cytofluorograms (CFG) shows co-localization of AF488-anti-γH2AX-Tat with γH2AX foci.

Figure 3

A, MDA-MB-231 cells, exposed to AF555-anti-γH2AX-Tat or AF555-mIgG-Tat for 1 h, were irradiated using 1.5 keV X-Rays (5 Gy), through a gold mask and then incubated for 1 h, fixed, and stained for γH2AX foci and mounted in Vectashield with DAPI. In merged images, co-localization of fluorophore-labeled RIC with γH2AX foci is shown as white. B, WT and H2AX −/− MEFs were exposed to Cy3-anti-γH2AX-Tat or Cy3-mIgG-Tat and after 1 h were sham-irradiated or irradiated (4 Gy). At 2 h after addition of RICs, cells were fixed, permeabilized and stained for γH2AX foci. Co-localization of Cy3-anti-γH2AX-Tat with γH2AX foci is shown in the merged image in WT MEFs and in the cytofluorogram (CFG).

Figure 4

Mice, bearing MDA-MB-468 xenografts, received Cy3-mIgG-Tat or Cy3-anti-γH2AX-Tat i.v. and tumors were sham-irradiated or irradiated (10 Gy). Images were acquired at 24, 48 and 72 h p.i. Cy3-mIgG-Tat did not accumulate in either control or irradiated tumors but Cy3-anti-γH2AX-Tat accumulated in irradiated tumors.

Figure 5

A, SPECT images following ¹¹¹In-DTPA-anti-γH2AX-Tat or ¹¹¹In-DTPA-mIgG-Tat in mice bearing MDA-MB-468 xenografts (white circles). Mice received PBS (control), i.p. bleomycin or the xenograft was irradiated (10 Gy). ¹¹¹In-DTPA-anti-γH2AX-Tat or ¹¹¹In-DTPA-mIgG-Tat (10 μg, 1 MBq/μg) was administered i.v. and SPECT scans performed at 24, 48 and 72 h. Transverse images through the tumor are shown. Coronal images are shown in Supplementary Fig. S4A and S4B. B, Volume of Interest (VOI) analysis of xenografts from (A). Results shown are mean SUV values ± SD (n = 3) (* p < 0.0001).

Figure 6

A, Mice received ¹¹¹In-DTPA-anti-γH2AX-Tat as in Fig. 5A, xenografts (white circles) were sham-irradiated or irradiated (1-4 Gy) and SPECT scans performed at 24 h p.i. Transverse images through the tumor are shown. B, Tumor accumulation of ¹¹¹In-DTPA-anti-γH2AX-Tat expressed as %ID/g and as tumor:muscle ratio. A positive correlation between IR dose and intratumoral accumulation of ¹¹¹In was observed (Spearman r = 0.9; p = 0.042). C, Tumors from (A) were harvested and stained ex vivo for γH2AX foci. Representative 0.8 μm thick slices are shown. The number of γH2AX foci increases with dose of IR.