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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\u2013IDC) 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.<\/jats:p>","DOI":"10.3390\/cells14030197","type":"journal-article","created":{"date-parts":[[2025,1,29]],"date-time":"2025-01-29T10:36:28Z","timestamp":1738146988000},"page":"197","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Single-Cell RNA Sequencing on Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Identified Multi-Ciliary Cells in Breast Cancer"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8139-8369","authenticated-orcid":false,"given":"Silvia","family":"Gonz\u00e1lez-Mart\u00ednez","sequence":"first","affiliation":[{"name":"\u201cContigo Contra el C\u00e1ncer de la Mujer\u201d Foundation, 28010 Madrid, Spain"},{"name":"Molecular Pathology of Cancer Group, Ram\u00f3n y Cajal Health Research Institute (IRYCIS), 28034 Madrid, Spain"},{"name":"Centre for Biomedical Research in Cancer Networks (CIBERONC), Carlos III Health Institute, 28029 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6730-5066","authenticated-orcid":false,"given":"Jos\u00e9","family":"Palacios","sequence":"additional","affiliation":[{"name":"Molecular Pathology of Cancer Group, Ram\u00f3n y Cajal Health Research Institute (IRYCIS), 28034 Madrid, Spain"},{"name":"Centre for Biomedical Research in Cancer Networks (CIBERONC), Carlos III Health Institute, 28029 Madrid, Spain"},{"name":"Department of Pathology, Ram\u00f3n y Cajal University Hospital, 28034 Madrid, Spain"},{"name":"Faculty of Medicine, University of Alcal\u00e1, 28801 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1986-2965","authenticated-orcid":false,"given":"Irene","family":"Carretero-Barrio","sequence":"additional","affiliation":[{"name":"Molecular Pathology of Cancer Group, Ram\u00f3n y Cajal Health Research Institute (IRYCIS), 28034 Madrid, Spain"},{"name":"Centre for Biomedical Research in Cancer Networks (CIBERONC), Carlos III Health Institute, 28029 Madrid, Spain"},{"name":"Department of Pathology, Ram\u00f3n y Cajal University Hospital, 28034 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Val F.","family":"Lanza","sequence":"additional","affiliation":[{"name":"Centre for Biomedical Research in Infectious Diseases Networks (CIBERINFEC), Carlos III Health Institute, 28029 Madrid, Spain"},{"name":"UCA-GTB Unit, Ram\u00f3n y Cajal Health Research Institute (IRYCIS), 28034 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0293-6323","authenticated-orcid":false,"given":"M\u00f3nica","family":"Garc\u00eda-Cos\u00edo Piqueras","sequence":"additional","affiliation":[{"name":"Molecular Pathology of Cancer Group, Ram\u00f3n y Cajal Health Research Institute (IRYCIS), 28034 Madrid, Spain"},{"name":"Centre for Biomedical Research in Cancer Networks (CIBERONC), Carlos III Health Institute, 28029 Madrid, Spain"},{"name":"Department of Pathology, Ram\u00f3n y Cajal University Hospital, 28034 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-9302-9519","authenticated-orcid":false,"given":"Tamara","family":"Caniego-Casas","sequence":"additional","affiliation":[{"name":"Molecular Pathology of Cancer Group, Ram\u00f3n y Cajal Health Research Institute (IRYCIS), 28034 Madrid, Spain"},{"name":"Centre for Biomedical Research in Cancer Networks (CIBERONC), Carlos III Health Institute, 28029 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2183-3699","authenticated-orcid":false,"given":"David","family":"Hardisson","sequence":"additional","affiliation":[{"name":"Centre for Biomedical Research in Cancer Networks (CIBERONC), Carlos III Health Institute, 28029 Madrid, Spain"},{"name":"Department of Pathology, Hospital Universitario La Paz (IdiPAZ), 28046 Madrid, Spain"},{"name":"Faculty of Medicine, Universidad Aut\u00f3noma de Madrid, 28049 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isabel","family":"Esteban-Rodr\u00edguez","sequence":"additional","affiliation":[{"name":"Department of Pathology, Hospital Universitario La Paz (IdiPAZ), 28046 Madrid, Spain"},{"name":"Faculty of Medicine, Universidad Aut\u00f3noma de Madrid, 28049 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Javier","family":"Cort\u00e9s","sequence":"additional","affiliation":[{"name":"\u201cContigo Contra el C\u00e1ncer de la Mujer\u201d Foundation, 28010 Madrid, Spain"},{"name":"Centre for Biomedical Research in Cancer Networks (CIBERONC), Carlos III Health Institute, 28029 Madrid, Spain"},{"name":"International Breast Cancer Center (IBCC), Pangaea Oncology, Quiron-Salud Group, 08017 Barcelona, Spain"},{"name":"Medica Scientia Innovation Research, 08007 Barcelona, Spain"},{"name":"Medica Scientia Innovation Research, Ridgewood, NJ 07450, USA"},{"name":"Department of Medicine, Faculty of Biomedical and Health Sciences, European University of Madrid, 28670 Madrid, Spain"},{"name":"IOB Institute of Oncology Madrid, Hospital Beata Mar\u00eda Ana de Jes\u00fas, 28007 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bel\u00e9n","family":"P\u00e9rez-Mies","sequence":"additional","affiliation":[{"name":"Molecular Pathology of Cancer Group, Ram\u00f3n y Cajal Health Research Institute (IRYCIS), 28034 Madrid, Spain"},{"name":"Centre for Biomedical Research in Cancer Networks (CIBERONC), Carlos III Health Institute, 28029 Madrid, Spain"},{"name":"Department of Pathology, Ram\u00f3n y Cajal University Hospital, 28034 Madrid, Spain"},{"name":"Faculty of Medicine, University of Alcal\u00e1, 28801 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,1,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Wang, S., Sun, S.-T., Zhang, X.-Y., Ding, H.-R., Yuan, Y., He, J.-J., Wang, M.-S., Yang, B., and Li, Y.-B. 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