Hammoudeh, SM;
Hammoudeh, AM;
Venkatachalam, T;
Rawat, S;
Jayakumar, MN;
Rahmani, M;
Hamoudi, R;
(2021)
Enriched transcriptome analysis of laser capture microdissected populations of single cells to investigate intracellular heterogeneity in immunostained FFPE sections.
Computational and Structural Biotechnology Journal
, 19
pp. 5198-5209.
10.1016/j.csbj.2021.09.010.
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Abstract
To investigate intracellular heterogeneity, cell capture of particular cell populations followed by transcriptome analysis has been highly effective in freshly isolated tissues. However, this approach has been quite challenging in immunostained formalin-fixed paraffin-embedded (FFPE) sections. This study aimed at combining the standard pathology techniques, immunostaining and laser capture microdissection, with whole RNA-sequencing and bioinformatics analysis to characterize FFPE breast cancer cell populations with heterogeneous expression of progesterone receptor (PR). Immunocytochemical analysis revealed that 60% of MCF-7 cells admixture highly express PR. Immunocytochemistry-based targeted RNA-seq (ICC-RNAseq) and in silico functional analysis revealed that the PR-high cell population is associated with upregulation in transcripts implicated in immunomodulatory and inflammatory pathways (e.g. NF-κB and interferon signaling). In contrast, the PR-low cell population is associated with upregulation of genes involved in metabolism and mitochondrial processes as well as EGFR and MAPK signaling. These findings were cross-validated and confirmed in FACS-sorted PR high and PR-low MCF-7 cells and in MDA-MB-231 cells ectopically overexpressing PR. Significantly, ICC-RNAseq could be extended to analyze samples captured at specific spatio-temporal states to investigate gene expression profiles using diverse biomarkers. This would also facilitate our understanding of cell population-specific molecular events driving cancer and potentially other diseases.
| Type: | Article |
|---|---|
| Title: | Enriched transcriptome analysis of laser capture microdissected populations of single cells to investigate intracellular heterogeneity in immunostained FFPE sections |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1016/j.csbj.2021.09.010 |
| Publisher version: | https://doi.org/10.1016/j.csbj.2021.09.010 |
| Language: | English |
| Additional information: | © 2021 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/). |
| Keywords: | Intracellular heterogeneity, RNA sequencing, Breast cancer, Immunocytochemistry, Progesterone receptor, NF-jB signaling |
| UCL classification: | UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Surgery and Interventional Sci UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10148947 |
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