Transcriptome profiling of the microdissected breast tissue compartments

In addition to the accumulation of pro-oncogenic mutations in the epithelial cells, the tumorigenic process involves the dysregulation of the interactions between the epithelial cells and their microenvironment, as well as alterations within the microenvironment itself. Therefore, investigating the causes of the loss of the normal tissue homeostasis in the earliest stages of breast cancer carcinogenesis is the necessary underpinning of an effective breast cancer prevention strategy. In order to comprehensively capture the transcriptomic alterations occurring in the breast in a precancerous stage, we examined a unique resource present in our institute including breast tissue biopsies donated by women two to six years prior to the clinical manifestation of breast cancer (labeled “susceptible normal tissue”) as well as specimens from healthy women. Donors in the two cohorts were at a premenopausal status and were matched according to age, racial background and menstrual phase. Both the susceptible and healthy breast tissues appear histologically normal. Upon microdissection of the three breast tissue compartments including epithelial, stromal and adipose tissue, transcriptomic analysis was performed. Upregulation of genes involved in lipid metabolism and fatty acid transport was observed also in the susceptible stroma and adipose tissue compartments, respectively. The study cohorts included 16 healthy women and 7 additional individuals, who were clinically healthy at the time of tissue donation but later had a diagnosis of breast cancer (labeled “Susceptible”). The subjects in these two cohorts were matched to the cases according to age, racial background, body mass index and follow-up time. To comprehensively capture the transcriptomic alterations occurring in the breast cancerization field, we used laser microdissection microscopy to isolate the epithelial (or terminal duct luminal unit)-, stromal- and adipose tissue compartments separately from each of 23 fresh-frozen breast biopsies. However, while all the 23 breast tissue cores included enough epithelial and stromal area, 7 samples lacked of sufficient adipose tissue for the following step. RNA was isolated from the captured cells and interrogated with next generation RNA sequencing. Moreover, breast epithelial compartment from paired susceptible, adjacent normal and contralateral normal breasts donated by two women was also analyzed through RNA-sequencing.