Quantification of CCL2 in paired human breast tissue samples

To investigate the relationship between mammographic density and CCL2, immunohistochemistry to detect CCL2 was conducted on paired breast tissue samples of high mammographic density (HMD) and low mammographic density (LMD). This approach has been described previously [23, 25], and offers excellent scope to study histological parameters associated with HMD in small sample sizes, as paired-sample statistical analysis can be applied.

Briefly, ethics approval from the Peter MacCallum Human Research Ethics Committee (number 08/21) and St. Vincent’s Hospital, Victoria was obtained and the study conducted in accordance with the Australian National Statement on Ethical Conduct in Human Research. Breast tissue was collected from women undergoing prophylactic mastectomy for breast cancer prevention consented to the study through the Victorian Cancer Biobank (VCB 10010). These women had a confirmed BRCA1/2 carrier status, a past history of breast cancer in the other breast or a family history of two or more first-degree or second-degree relatives with breast cancer who were diagnosed before the age of 50 years. Resected breast tissue was transferred immediately on ice to the pathology department upon completion of mastectomy, where pathologists resected slices of breast tissue using a sterile technique. X-rays of those slices were taken by a breast radiographer using uniform radiological parameters and were assessed against a calibration ruler for selection of HMD and LMD regions. The tissue slice was transferred to a biosafety level-2 hood, where areas that appeared white on X-rays were selected and removed using sterile blades and defined as HMD regions, and areas that appeared black were similarly selected, removed and classified as LMD regions. The HMD and LMD regions were subjected to routine formalin fixation and paraffin embedding.

Paraffin-embedded human breast tissue sections were placed on a hotplate at 60 °C for 60 minutes before dewaxing in two 5-minute washes in Safsolv and gradually passed through 100%, 95%, 80% and 70% ethanol for rehydration as described previously [23, 25]. Before sections were incubated with mouse anti-human CCL2 antibody (R&D systems) for 30 minutes at room temperature, sections were placed in Dako EnVision™ low pH antigen retrieval solution (Dako) and brought to 90 °C in a water bath for 20 minutes followed by Envision™ wash buffer and endogenous peroxidase block. After primary antibody incubation, sections were incubated with Dako EnVision™ HRP (ready-to-use, Dako) for 30 minutes at room temperature and washed in Envision™ wash buffer for 5 minutes. The detection of bound antibody was performed according to the manufacturer’s instructions. Tissue sections were counterstained with haematoxylin prior to dehydration, and cleared and mounted as described above. Slides stained with isotype-matched primary antibody were included as negative controls.

Stained tissue sections were captured as a digital image using a Nanozoomer 1.0 (Hamamatsu, Shizouka, Japan) at a zoom equivalent to a × 40 objective lens. All quantification analysis was performed blinded. Three epithelium clusters were randomly selected from each section for quantification. To determine the intensity of CCL2-positive staining within the epithelium of each cluster, staining was quantified using the IHC profiler within Image J analysis software. The percentage of positive staining was determined by combining percentage of high, medium and low positive staining within each selected epithelium measured by the IHC profiler and the mean value of three clusters per patient was calculated.