Abstract
Tumor samples are conserved in clinical practice in formalin-fixed paraffin-embedded (FFPE) blocks. Formalin fixation chemically alters nucleic acids, rendering transcriptomic analysis challenging. RNA-sequencing is usually performed on tumor bulk, without distinction of cell subtypes or location. Here we describe the development of a robust method for RNA extraction and exome-capture RNA-sequencing of laser-capture microdissected tumor cells (TC) and stromal immune cells (TIL) based on their morphology. We applied this method on 7 tumor samples (surgical or core needle biopsy) of triple-negative breast cancer (TNBC) stored in FFPE blocks over 3-10 years. Unsupervised clustering and principal component analysis showed a clear separation between gene-expression profile of TIL and TC. TIL were enriched in markers of B cells (CD79B, PAX5 and BLNK) and T cells (CD2, CD3D and CD8B) whereas tumor cells expressed epithelial markers (EPCAM, MUC1 and KRT8). Microenvironment cell populations-counter (MCP)-counter deconvolution showed an enrichment in adaptive immune cell signatures in microdissected TIL. Transcripts of immune checkpoints were differentially expressed in TIL and TC. We further validated our results by qRT-PCR and multispectral immunohistochemistry. In conclusion, we showed that combining laser-capture microdissection and RNA-sequencing on archived FFPE blocks is feasible and allows spatial transcriptional characterization of tumor microenvironment.
Footnotes
Conflict of interest: All the authors disclose no potential conflict of interest
Financial support: ILG is supported by funding from the Swiss National Science Foundation (Ambizione n° PZ00P3_173959), la Ligue Genevoise Contre le Cancer (grant n°1712), la Fondation pour la lutte contre le cancer et pour des recherches médico-biologiques and The Novartis Biomedical Research Foundation (#17C150). PT and PC are supported by funding from La Ligue Genevoise Contre le Cancer.








