Single-Cell RNA Sequencing on Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Identified Multi-Ciliary Cells in Breast Cancer.
Author(s): 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
Publication: Cells, 2025, Vol. 14, Page
PubMed ID: 39936988 PubMed Review Paper? No
Purpose of Paper
This paper compared single-cell RNA sequencing (scRNAseq) profiles of case-matched formalin-fixed, paraffin-embedded (FFPE) and fresh specimens (fixed during workflow) obtained from one invasive ductal carcinoma (IDC) and one invasive lobular carcinoma (ILC). scRNAseq findings were compared with those from immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), electron microscopy and massively parallel sequencing from the same specimens.
Conclusion of Paper
Both tumor specimens were grade 2, estrogen receptor (ER)- and progesterone receptor (PR)-positive, and HER-2 negative (the ILC scored +1 and the IDC +2), with Ki67 proliferation indices of 15% for the ILC and 18% for the IDC specimen. Massively parallel sequencing identified mutations in CDH1 (p.Thr515AsnfsTer22) and PIK3CA (p.His1047Arg) in the ILC specimens and ERBB2 (p.Leu755Ser) in the ILC specimen. Generally, scRNA-Seq libraries that were produced from the fresh-fixed and FFPE specimens were of high quality, with a similar proportion of doublets. The percentage of reads mapping to the mitochondrial genome was slightly higher in FFPE than fixed-fresh specimens but was still low (<20% for almost all). Generally, the median number of genes per cell (after filtering and doublet removal) was similar between FFPE and fixed-fresh specimens for each of the different cell types, but differences were observed in epithelial and fibroblast cells of individual specimens. Importantly, the differences were not systemic, with the number of genes sometimes higher in the FFPE specimen and in other cases higher in the fixed-fresh specimen. More cells were captured from FFPE than fixed-fresh specimens (25,785 versus 21,866). However, the same cell types (five types of epithelial cells, fibroblasts, endothelial cells, pericytes, lymphocytes, myeloid cells, and mast cells) were found in both FFPE and fixed-fresh specimens and each specimen type displayed similar levels of heterogeneity. Further, unified uniform manifold approximation projection (UMAP) plots were comparable between the specimen types. Nevertheless, cell type proportions appeared to differ, with more fibroblasts in FFPE IDC specimen and more epithelial cells 1 and 4 in the fixed-fresh IDC specimen. scRNAseq expression of CDH1, ESR1, PGR, MKI67, and ERBB2 in FFPE and fixed-fresh specimens were consistent with IHC results in the same tissues. Similarly, immune cell typing by scRNAseq generated similar results in FFPE and fixed-fresh specimens but differed between ILC and IDC specimens. scRNAseq identified a chromosome 1q gain and deletions of 16q and 17p in neoplastic epithelial cells, which were later confirmed by FISH analysis of the same specimens. Differences in expression patterns were noted between the ILC and IDC cases, with more heterogeneity in cell-types and expression patterns in the IDC than the ILC case. In the IDC specimen, neoplastic cells expressing genes related to the ciliary machinery of multi-ciliated cells (MCCs) were found at a similar rate to those expressing FOXJ1 by whole slide imaging (1.3% versus 0.7%). The presence of MCCs was confirmed by electron microscopy.
Studies
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Study Purpose
This study compared single-cell RNA sequencing (scRNAseq) profiles of case-matched formalin-fixed, paraffin-embedded (FFPE) and fresh specimens (fixed during workflow) obtained from one invasive ductal carcinoma (IDC) and one invasive lobular carcinoma (ILC). scRNAseq findings were compared with those from immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), electron microscopy and massively parallel sequencing from the same specimens. Tumor specimens were obtained from two patients with breast cancer (one ILC and one IDC) during lumpectomy. Contiguous specimens were analyzed fresh, formalin-fixed, paraffin-embedded (details not provided) and stored at room temperature or immersed in OCT compound and snap-frozen in liquid nitrogen for histological analysis. Tumor grade was determined in FFPE sections using the Nottingham histologic grading system. Expression of ER, PR, HER-2, E-Cadherin, P63, CD4, CD8, CD3, CD20, CD68, CD117(c-Kit) and FOXJ1 were evaluated by immunohistochemistry. Chromosomal alterations in HER2/17q and MDM4/1p12 were evaluated by FISH in at least 20 neoplastic cells. Morphology was evaluated by electron microscopy of FFPE sections. Amplification was defined as ≥ 2 times the gene signal than the centromere signal. Mutations were identified by massively parallel sequencing of ten 10 µm-thick FFPE sections. scRNA-Seq libraries were prepared from ten 25 µm-thick FFPE sections and from 50 mg fresh tissue that was fixed (fixative not specified) overnight and dissociated using the Chromium Fixed RNA Profiling Reagent Kits for Singleplexed Samples.
Summary of Findings:
Both tumor specimens were grade 2, estrogen receptor (ER)- and progesterone receptor (PR)-positive, and HER-2 negative (the ILC scored +1 and the IDC +2), with Ki67 proliferation indices of 15% for the ILC and 18% for the IDC specimen. Massively parallel sequencing identified mutations in CDH1 (p.Thr515AsnfsTer22) and PIK3CA (p.His1047Arg) in the ILC specimens and ERBB2 (p.Leu755Ser) in the ILC specimen. Generally, scRNA-Seq libraries that were produced from the fresh-fixed and FFPE specimens were of high quality, with a similar proportion of doublets. The percentage of reads mapping to the mitochondrial genome was slightly higher in FFPE than fixed-fresh specimens but was still low (<20% for almost all). Generally, the median number of genes per cell (after filtering and doublet removal) was similar between FFPE and fixed-fresh specimens for each of the different cell types, but differences were observed in epithelial and fibroblast cells of individual specimens. Importantly, the differences were not systemic, with the number of genes sometimes higher in the FFPE specimen and in other cases higher in the fixed-fresh specimen. More cells were captured from FFPE than fixed-fresh specimens (25,785 versus 21,866). However, the same cell types (five types of epithelial cells, fibroblasts, endothelial cells, pericytes, lymphocytes, myeloid cells, and mast cells) were found in both FFPE and fixed-fresh specimens and each specimen type displayed similar levels of heterogeneity. Further, unified uniform manifold approximation projection (UMAP) plots were comparable between the specimen types. Nevertheless, cell type proportions appeared to differ, with more fibroblasts in FFPE IDC specimen and more epithelial cells 1 and 4 in the fixed-fresh IDC specimen. scRNAseq expression of CDH1, ESR1, PGR, MKI67, and ERBB2 in FFPE and fixed-fresh specimens were consistent with IHC results in the same tissues. Similarly, immune cell typing by scRNAseq generated similar results in FFPE and fixed-fresh specimens but differed between ILC and IDC specimens. scRNAseq identified a chromosome 1q gain and deletions of 16q and 17p in neoplastic epithelial cells, which were later confirmed by FISH analysis of the same specimens. Differences in expression patterns were noted between the ILC and IDC cases, with more heterogeneity in cell-types and expression patterns in the IDC than the ILC case. In the IDC specimen, neoplastic cells expressing genes related to the ciliary machinery of multi-ciliated cells (MCCs) were found at a similar rate to those expressing FOXJ1 by whole slide imaging (1.3% versus 0.7%). The presence of MCCs was confirmed by electron microscopy.
Biospecimens
Preservative Types
- Formalin
- None (Fresh)
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Next generation sequencing RNA Next generation sequencing Morphology Electron microscopy Protein Immunohistochemistry RNA In situ hybridization Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition IDC
ILC
Next generation sequencing Specific Technology platform scRNAseq
FISH
IHC
DNA seq
Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
None (fresh)