Spatial localization of RNAs within the breast tumor microenvironment may affect progression and recurrence of DCIS

Ductal carcinoma in situ (DCIS) is a cancerous growth of breast duct cells that may remain indolent or progress to invasive ductal carcinoma (IDC). As screening rates increase, the prevalence of DCIS diagnosis has risen with many women undergoing aggressive treatment with surgery, radiation, and endocrine therapy when diagnosed with DCIS. It is critical to identify factors that predict progression to invasive disease for improved outcomes. Studies involving the breast tumor microenvironment (TME) provide potential understanding of crosstalk between tumor epithelium and stroma to identify factors that influence progression of DCIS lesions. To identify such biomarkers of breast cancer progression, we examined the TME using breast tissue microarrays (TMAs) representing matched benign (normal adjacent), DCIS, and IDC samples. We examined 139 cores which provided data from 47 unique patients. We characterized the expression and spatial distribution patterns of regulatory RNAs (mRNAs and long-noncoding RNAs) including Runt-related transcription factor 1 (RUNX1), Runt-related transcription factor 2 (RUNX2), Mitotically activated long non-coding RNA (MANCR), Cluster of Differentiation 90 (CD90), C-X-C motif chemokine 12 (CXCL12), C-X-C chemokine receptor type 6 (CXCR6), and tumor protein 63 (TP63), using RNAScope fluorescence in situ hybridization (RNA-FISH) and a panel of stromal marker proteins (i.e., Cluster of Differentiation 3 (CD3), Cluster of Differentiation 68 (CD68), Cluster of Differentiation 34 (CD34), Alpha Smooth Muscle Actin (aSMA) and TP63) using multiplex immunofluorescence (IF). We identified several temporal and spatial expression signatures of RNAs and/or proteins throughout breast cancer progression, both in the epithelial and stromal compartment of benign, DCIS or IDC lesions. Spatial proximity analysis to assess location of markers away from epithelial boundary or within the myoepithelial layer of these lesions identified significant association with clinical parameters including tumor recurrence status. The finding that decreased myoepithelial continuity maybe protective against IDC recurrence could help better patient stratification for response to therapy. This work emphasizes the importance of spatial localization of markers in the breast cancer tumor microenvironment and their importance for clinical outcomes.