Single-cell RNA sequencing on formalin-fixed and paraffin-embedded (FFPE) tissue identified multi-ciliary cells in breast cancer

The purpose of this study was to evaluate the suitability of paraffin-embedded (FFPE) samples and fixed fresh (FF) samples for single-cell RNA sequencing (scRNAseq). To this end, we compared whether single-cell profiles from FFPE and matched FF tissue samples from one invasive carcinoma of no especial type carcinoma (invasive ductal carcinoma –IDC) and one invasive lobular carcinoma (ILC) were similar when captured using the common immunohistochemical (IHC) and immune features of the tumors.

FF and FFPE tissue derived libraries showed high-quality parameters. The cell heterogeneity obtained was similar, although, there were some differences in the number of cells captured, but there were no specific cell populations captured exclusively by one of the two different approaches. The combined analysis of the four samples identified six types of epithelial cells (normal basal cells, four subpopulations of neoplastic cells in the IDC and one in the ILC), and different tumoral microenvironment populations.

scRNAseq results from epithelial neoplastic cells were concordant with common IHC markers. In addition, after quantitative assessment of the whole slide images using QuPath, the proportion of immune cells identified by IHQ in FFPE sections were similar to those obtained by scRNAseq. Confirming the suitability of scRNAseq using FFPE tissue, this technique identified one subpopulation of neoplastic cells present only in the IDC, characterized by the expression of genes related with multi-ciliated cell (MCC) differentiation ( FOXJ1, ROPN1L, TPPP3, CFAP45 ). IHC analysis of FOXJ1 expression confirmed the presence of positive cells only in the IDC in a similar proportion to that obtained by scRNAseq. Analysis of FOXJ1 in a cohort of 214 ER-positive invasive carcinomas demonstrated protein expression in at least 1% of cells in 33% of tumors, suggesting frequent focal MCC differentiation. Our results support the suitability of scRNAseq analysis using FFPE tissue and identified a subpopulation of neoplastic MCC in breast cancer.