Multiomic single cell analysis of primary pancreatic ductal adenocarcinoma enhanced by fixation

Will Hwang, Katie Kennedy, Joe Dahl, Jamie Remington, Jay West

We have developed a method for improved retrieval of multiomic details from challenging cell types. Application of a non-formaldehyde fixation protocol enabled us to preserve single cells for genomic and whole transcriptomic amplification. We have married this process to ResolveDNA and ResolveOME technologies to recover 99% of the genome and full-length mRNA transcripts. We have demonstrated that the quality of data from fixed and live single cells is indistinguishable in a NA12878/HG001 cell line. We have applied this technique to enhance recovery of genomic data from pancreatic ductal adenocarcinoma (PDAC) primary cells. For live single cells, that were not fixed, recovery of DNA and RNA for amplification from pan-cytokeratin (CK+) enriched tumor cells was severely impaired. We believe this to be driven by rapid cell death after isolation. For live non-tumor (CK-) cells retrieval of genetic material without fixation was more robust. None the less, fixation enhanced the probability of amplification from both CK+ and CK- single PDAC cells. Analysis of the individual CK+ and CK- cells by low pass whole genome sequencing demonstrated a defined copy number variation profile for the CK+ cells while the CK- cells that were isolated exhibited normal ploidy across the genome. In addition to copy number alterations commonly seen in PDAC, CK+ cells exhibited transcription profiles consistent with tumor biology – including upregulation of the MAP-K and double-stranded break repair pathways (DSBR). Key genes included ERBB3 and Met in the MAP-K pathway, and CIB1 and TENT4A in the DSBR pathway. Transcriptional profiling of these two populations by principal component analysis segregated single tumor cells from non-tumor cells across both components of the analysis. Indicating that in CK+ cells there is an activation of DNA damage response and an attempt to mitigate the overactive MAP-K pathway. Interestingly, while mitochondrial pathways are up regulated in CK+ relative to CK- cells, a closer look reveals the most frequently upregulated gene families are cytochrome c oxidases and NADHs. The increased expression indicates a tumor-specific response to increased energy needs within the cells. Furthermore, cell identification through transcriptional analysis demonstrates the majority of CK- single cells isolated were tumor infiltrating immune cells. Overall, the data showcase the ability to study an extremely challenging sample type without the limitations of formalin-derived fixation methods and associated artifacts.