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10.3390/cells14030197
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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

Single-Cell RNA Sequencing on Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Identified Multi-Ciliary Cells in Breast Cancer

by
Silvia González-Martínez
1,2,3,
José Palacios
2,3,4,5,*,
Irene Carretero-Barrio
2,3,4,
Val F. Lanza
6,7,
Mónica García-Cosío Piqueras
2,3,4,
Tamara Caniego-Casas
2,3,
David Hardisson
3,8,9,
Isabel Esteban-Rodríguez
8,9,
Javier Cortés
1,3,10,11,12,13,14 and
Belén Pérez-Mies
2,3,4,5,*
1
“Contigo Contra el Cáncer de la Mujer” Foundation, 28010 Madrid, Spain
2
Molecular Pathology of Cancer Group, Ramón y Cajal Health Research Institute (IRYCIS), 28034 Madrid, Spain
3
Centre for Biomedical Research in Cancer Networks (CIBERONC), Carlos III Health Institute, 28029 Madrid, Spain
4
Department of Pathology, Ramón y Cajal University Hospital, 28034 Madrid, Spain
5
Faculty of Medicine, University of Alcalá, 28801 Madrid, Spain
6
Centre for Biomedical Research in Infectious Diseases Networks (CIBERINFEC), Carlos III Health Institute, 28029 Madrid, Spain
7
UCA-GTB Unit, Ramón y Cajal Health Research Institute (IRYCIS), 28034 Madrid, Spain
8
Department of Pathology, Hospital Universitario La Paz (IdiPAZ), 28046 Madrid, Spain
9
Faculty of Medicine, Universidad Autónoma de Madrid, 28049 Madrid, Spain
10
International Breast Cancer Center (IBCC), Pangaea Oncology, Quiron-Salud Group, 08017 Barcelona, Spain
11
Medica Scientia Innovation Research, 08007 Barcelona, Spain
12
Medica Scientia Innovation Research, Ridgewood, NJ 07450, USA
13
Department of Medicine, Faculty of Biomedical and Health Sciences, European University of Madrid, 28670 Madrid, Spain
14
IOB Institute of Oncology Madrid, Hospital Beata María Ana de Jesús, 28007 Madrid, Spain
 
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Authors to whom correspondence should be addressed.
Cells 2025, 14(3), 197; https://doi.org/10.3390/cells14030197
Submission received: 23 December 2024 / Revised: 18 January 2025 / Accepted: 26 January 2025 / Published: 29 January 2025
(This article belongs to the Section Cell Methods)
Review Reports Versions Notes

Abstract

The purpose of this study was to evaluate the suitability of formalin-fixed and paraffin-embedded (FFPE) samples and fixed fresh (FF) samples for single-cell RNA sequencing (scRNAseq). To this end, we compared single-cell profiles from FFPE and matched FF tissue samples of one invasive carcinoma of no special type carcinoma (invasive ductal carcinoma–IDC) and one invasive lobular carcinoma (ILC) to assess consistency in cell type distribution and molecular profiles. The results were validated using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and electron microscopy. Additionally, immune cell proportions identified by IHC were quantified using QuPath and compared to the scRNAseq results. FFPE- and FF-derived libraries demonstrated high-quality sequencing metrics, and cellular heterogeneity was similar. No exclusive cell populations were identified by either approach. The four samples analysis identified six types of epithelial cells, as well as tumoral microenvironment populations. The scRNAseq results from epithelial neoplastic cells were concordant with common IHC markers. The proportion of immune cells identified by IHC in FFPE sections were similar to those obtained by scRNAseq. We identified and validated a previously poorly recognized subpopulation of neoplastic multi-ciliated cells (MCCs) (FOXJ1, ROPN1L). Analysis of FOXJ1 in 214 ER-positive invasive carcinomas demonstrated protein expression in one third 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.
Keywords: single-cell RNA sequencing; breast cancer; multi-ciliated cells; fixed fresh tissue; FFPE tissue; tumor heterogeneity single-cell RNA sequencing; breast cancer; multi-ciliated cells; fixed fresh tissue; FFPE tissue; tumor heterogeneity

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MDPI and ACS Style

González-Martínez, S.; Palacios, J.; Carretero-Barrio, I.; Lanza, V.F.; García-Cosío Piqueras, M.; Caniego-Casas, T.; Hardisson, D.; Esteban-Rodríguez, I.; Cortés, J.; Pérez-Mies, B. Single-Cell RNA Sequencing on Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Identified Multi-Ciliary Cells in Breast Cancer. Cells 2025, 14, 197. https://doi.org/10.3390/cells14030197

AMA Style

González-Martínez S, Palacios J, Carretero-Barrio I, Lanza VF, García-Cosío Piqueras M, Caniego-Casas T, Hardisson D, Esteban-Rodríguez I, Cortés J, Pérez-Mies B. Single-Cell RNA Sequencing on Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Identified Multi-Ciliary Cells in Breast Cancer. Cells. 2025; 14(3):197. https://doi.org/10.3390/cells14030197

Chicago/Turabian Style

González-Martínez, Silvia, José Palacios, Irene Carretero-Barrio, Val F. Lanza, Mónica García-Cosío Piqueras, Tamara Caniego-Casas, David Hardisson, Isabel Esteban-Rodríguez, Javier Cortés, and Belén Pérez-Mies. 2025. "Single-Cell RNA Sequencing on Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Identified Multi-Ciliary Cells in Breast Cancer" Cells 14, no. 3: 197. https://doi.org/10.3390/cells14030197

APA Style

González-Martínez, S., Palacios, J., Carretero-Barrio, I., Lanza, V. F., García-Cosío Piqueras, M., Caniego-Casas, T., Hardisson, D., Esteban-Rodríguez, I., Cortés, J., & Pérez-Mies, B. (2025). Single-Cell RNA Sequencing on Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Identified Multi-Ciliary Cells in Breast Cancer. Cells, 14(3), 197. https://doi.org/10.3390/cells14030197

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