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Single-Cell RNA Sequencing on Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Identified Multi-Ciliary Cells in
Silvia González-Martínez1,2,3, José Palacios2,3,4,5, Irene Carretero-Barrio2,3,4
1"Contigo Contra el Cáncer de la Mujer" Foundation, 28010 Madrid, Spain.
Cells
|February 12, 2025
Summary
Formalin-fixed paraffin-embedded (FFPE) tissue is suitable for single-cell RNA sequencing (scRNAseq), offering consistent cell type distribution and molecular profiles compared to fresh-frozen (FF) samples. This method also identified a novel neoplastic multi-ciliated cell (MCC) subpopulation in breast cancer.
Area of Science:
- Oncology
- Genomics
- Molecular Biology
Background:
- Single-cell RNA sequencing (scRNAseq) is a powerful tool for dissecting cellular heterogeneity in tumors.
- The utility of formalin-fixed paraffin-embedded (FFPE) tissues, a widely available resource, for scRNAseq remains under investigation.
- Comparing FFPE and fresh-frozen (FF) samples is crucial for validating scRNAseq methodologies in cancer research.
Purpose of the Study:
- To evaluate the suitability of FFPE and FF samples for scRNAseq.
- To compare cell type distribution and molecular profiles between FFPE- and FF-derived scRNAseq data.
- To identify and validate novel cell subpopulations in breast cancer using scRNAseq.
Main Methods:
- Comparative analysis of scRNAseq data from matched FFPE and FF breast cancer tissues (invasive ductal carcinoma and invasive lobular carcinoma).
- Validation of scRNAseq findings using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and electron microscopy.
- Quantification and comparison of immune cell proportions using QuPath and scRNAseq.
Main Results:
- FFPE- and FF-derived scRNAseq libraries yielded high-quality data with similar cellular heterogeneity and cell type distributions.
- No exclusive cell populations were identified between FFPE and FF samples.
- scRNAseq results for epithelial neoplastic cells were concordant with IHC markers, and immune cell proportions were comparable between methods.
- A previously under-recognized subpopulation of neoplastic multi-ciliated cells (MCCs), expressing FOXJ1 and RPN1L, was identified and validated.
- FOXL1 protein expression was observed in approximately one-third of ER-positive invasive carcinomas, indicating frequent focal MCC differentiation.
Conclusions:
- FFPE tissues are suitable for scRNAseq, providing reliable data comparable to FF tissues.
- scRNAseq of FFPE samples can effectively characterize tumor heterogeneity and identify novel cell subpopulations.
- The identification of neoplastic MCCs in breast cancer opens new avenues for understanding tumor biology and potential therapeutic targets.
