Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

mRNA-Seq of single prostate cancer circulating tumor cells reveals recapitulation of gene expression and pathways found in prostate cancer.

Author(s): Cann GM, Gulzar ZG, Cooper S, Li R, Luo S, Tat M, Stuart S, Schroth G, Srinivas S, Ronaghi M, Brooks JD, Talasaz AH

Publication: PLoS One, 2012, Vol. 7, Page e49144

PubMed ID: 23145101 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the effectiveness and efficiency of two different CTC isolation methods, MagSweeper and CellSearch. CTC isolation using MagSweeper was also examined for effects on downstream applications. Blood from an unspecified number of patients with metastatic prostate cancer was collected in a 10 mL EDTA tube for MagSweeper isolation and in a CellSave tube for CellSearch enumeration of CTCs.  For MagSweeper isolation, 3.75 mL of blood was subjected to red cell lysis for 15 minutes at RT, cell pellet was resuspended in a 1% BSA/PBS/5 mM EDTA solution containing anti-human CD45 and anti-EpCAM beads, rotated for 30 minutes at 4°C followed by addition of Phycoerythrin (PE) anti-human EpCAM antibody, then rotated at 4°C for an additional 30 minutes, transferred to a 6-well plate, and then incubated for 15 minutes at 4°C prior to two rounds of MagSweeper isolation. Collected cells were snap-frozen on dry ice and stored at -80°C. CTC isolation and enumeration by CellSearch was performed by Quest diagnostics and details were not provided. Single cells isolated by MagSweeper were lysed and RNA was reverse transcribed using the SMARTer Ultra Low Input RNA for Illumina Sequencing kit, cDNA was amplified by PCR prior to conversion into DNA sequencing libraries that were quantified using a bioanalyzer and qPCR. To assess whether the MagSweeper isolation process affected the global transcriptional profile of isolated cells, single cell mRNA-Seq was performed on four fresh LNCaP cells just prior to spiking into blood and four LNCaPs after MagSweeper isolation from spiked normal blood after frozen storage for at least one month.

   Summary of Findings:

   Recovery of CTCs from blood collected from patients with metastatic prostate cancer was comparable between MagSweeper isolation and CellSearch methods, although MagSweeper demonstrated a slightly higher recovery in specimens with low CTC numbers. When amplified cDNA from a fresh LNCaP cell and a MagSweeper isolated cell were compared using a bioanalyzer, similar molecular weight products and differential expression analysis of RNA-seq expression profiles revealed only one differentially expressed gene at a false discovery rate (FDR) cutoff of 0.05 and none at a cutoff of 0.01.

   Amplification products of 67 CTCs from 13 prostate cancer patients collected by MagSweeper isolation displayed a range of sizes for amplified cDNAs that correlated to RNA quality, with 21% of traces characterized as high quality RNA that demonstrated amplification peaks centered around 1000 bps, 37% of traces characterized as partially degraded RNA with intermediate length amplification products, and 42% of traces characterized as degraded RNA with predominantly low molecular weight amplification products. Single cell mRNA-Seq data analysis revealed CTCs displayed a wider range of coverage bias than prostate cancer cell lines or normal prostate tissue.  Specifically, CTCs displayed a 3’ bias indicative of RNA degradation. The number of RefSeq transcripts (a subset of quality control genes) with greater or equal to a fragments per kilobase of exon per million fragments mapped (FPKM) value of 10 were also lower in MagSweeper isolated CTCs compared to those obtained using a prostate cancer cell line (4,622 vs. 2,362 transcripts, respectively).  The authors attribute the heterogeneity in RNA quality to short half-lives and apoptosis of CTCs rather than the MagSweeper isolation.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Fluorescent microscopy
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Cell capture method	MagSweeper CellSearch
   Preaquisition	Diagnosis/ patient condition	Prostate Cancer Normal

   View more