E68: Single Cell Expression Analysis of Breast Precancers Identifies Alterations in Epithelial Integrity

Ductal carcinoma in situ (DCIS) is a precursor to invasive breast cancer. Its defining characteristic is an intact basement membrane surrounding the intraductal neoplastic proliferation. Although DCIS cells are imbedded in normal tissue, a clear understanding of DCIS cellular programs as compared to normal cells are needed to understand disease progression.

Methods:

To characterize DCIS cells, single-cell RNA-Sequencing (scRNA-seq) on DCIS (n=16) and synchronous normal breast tissues (n=12) from 15 patients undergoing mastectomy. Epithelial subtypes were inferred based on previously established signatures from normal breast epithelium. Neoplastic epithelial cells within DCIS samples were distinguished from their non-neoplastic epithelial counterparts based on RNA expression inferred copy number variation profiles. Aggregated gene expression and gene set enrichment analyses were performed to identify gene expression patterns between neoplastic and non-neoplastic cell populations . Spatial transcriptomics was used to investigate basement membrane protein expression. Matched and independent bulk RNA-sequencing data sets were used to validate the findings.

Results:

We distinguished 17,454 DCIS epithelial cells (DD) from 13,899 non-neoplastic epithelial cells from the DCIS samples (DN), and identified 14,555 normal epithelial cells from the normal samples (NN). Differential gene expression analyses confirmed the similarity between DN and NN cells, and their distinctions from DD cells. We identified 4,180 differentially expressed in DD compared to the combined DN and NN populations (DNNN). Pathway analyses showed increased proliferation and metabolism in DD compared to DNNN. Examining epithelial cell type compositions, we observed an increase in luminal cells and a concurrent decrease in basal cells in DD compared to DNNN. Spatial transcriptomic and proteomic data show a decrease in expression of basement membrane components by basal cells and myoepithelial cells in those DCIS in close proximity to an invasion event.

Conclusions:

Our observations indicate that the relative expansion of luminal cells and the concurrent reduction of basal cells could potentially weaken basal cell function, given the role of basal cells in maintaining the integrity of the basement membrane. This alteration could result in the loss of contact with an intact basement membrane, leading to subsequent invasion.