DNA damage measurements within tissue samples with Repair Assisted Damage Detection (RADD)

Abstract

Exposures to genotoxic carcinogens and reactive species result in strand breaks and a spectrum of covalent modifications to DNA that can induce mutations and contribute to the initiation and progression of cancer. Measurements of DNA damage within tissue or tumor samples can serve as a biomarker for exposures or assess changes in DNA repair capacity relevant in cancer development and treatment. Numerous methods to characterize DNA damage exist. However, these methods are primarily applicable to isolated DNA or cultured cells, often require a substantial amount of material, and may be limited to the detection and quantification of only a handful of DNA adducts. Here, we used the Repair Assisted Damage Detection (RADD) assay to detect and excise DNA adducts using a cocktail of DNA repair enzymes, then the damage site within the genome are tagged for detection using a modified nucleotide. We previously demonstrated the RADD assay can detect lesions within isolated DNA and fixed cells, and now RADD can be used to detect DNA adducts and DNA strand breaks in formalin-fixed paraffin-embedded (FFPE) tissue samples. We verified the ability of the RADD assay to detect DNA damage in tissue by exogenously inducing DNA damage with X-rays and restriction enzymes. We also showed that RADD can be multiplexed with antibodies to detect cell cycle markers or other proteins of interest. Finally, we showed that RADD can detect DNA damage within clinically relevant ovarian tumor samples. RADD is a flexible and easy-to-use assay that allows relative damage levels to be determined within FFPE samples and allows the heterogeneity of DNA adducts and strand breaks within clinically relevant samples to be measured.

Figure 1.. RADD detection of DNA damage within FFPE samples.

A. RADD workflow. FFPE tissue samples are deparaffinized and rehydrated. The RADD assay is performed with a cocktail of DNA repair enzymes that detects and removes damaged DNA leaving gaps where damaged bases were encountered. Lesion filling occurs with a DNA polymerase and a tagged dUTP molecule allows for fluorescent detection of the damage site. B. RADD detection of DNA lesions within FFPE immortalized cell lines. C. Strand break markers for γH2AX and 53BP-1 confirm presence of DNA damage within the FFPE samples. Scale bars 100 μm.

Figure 2.. RADD signal in FFPE xenograft tumors correlates with higher proliferation.

TOV112D xenografts from two separate mice are co-stained for DNA damage using (A) RADD (red) and cell proliferation wth (B) Ki67 (purple). C. Merged image with nuclear staining. D. H&E stain of adjacent tumor slice.

Figure 3.. RADD detection of DNA adducts within human tumors and adjacent normal tissues.

Stitched image of RADD performed on a single lung squamous cell carcinoma and adjacent normal tissue (A) and on a single breast ductal infiltrative carcinoma and adjacent normal tissue (B). Brightness and contrast are optimized for visualization in this image. C. Tissue slices were imaged using constant laser power and gain, and the fluorescent intensity of individual tissue images measured. Graphed is the summed fluorescent intensity from each individual tissue section imaged at 10x.

Figure 4.. RADD measures DNA damage induced after fixation in normal mouse lung.

A. Fluorescent intensity of RADD staining after exposure to increasing amounts of X-ray irradiation. B. Fluorescent intensity of RADD staining after exposure to 5 units of the restriction enzymes VspI, EcoRI, VspI + EcoRI, or VspI + SalI for 1 h prior to performing the RADD assay.

Figure 5.. RADD measures DNA levels in ovarian patient tumor samples.

A. Basal levels of DNA damage were detected in tumor samples collected in a de-bulking surgery before the start of chemotherapy (pre) and at a second de-bulking surgery when the tumor recurred after treatment with carboplatin and paclitaxel (post). A two-tailed t-test is used to determine significance between pre- and post-treatment tumors for a single patient. B. Representative images of a tumor section imaged from patient 1 and patient 4 pre- and post-treatment are shown. C. Fluorescent intensity of DNA damage staining in the pre-treatment tumor is divided into low, moderate, and high staining categories, so the trend in DNA damage staining intensity pre- and post-treatment can be observed across the eight patient samples.