mRNA-Seq of Single Prostate Cancer Circulating Tumor Cells Reveals Recapitulation of Gene Expression and Pathways Found in Prostate Cancer

<div><p>Circulating tumor cells (CTC) mediate metastatic spread of many solid tumors and enumeration of CTCs is currently used as a prognostic indicator of survival in metastatic prostate cancer patients. Some evidence suggests that it is possible to derive additional information about tumors from expression analysis of CTCs, but the technical difficulty of isolating and analyzing individual CTCs has limited progress in this area. To assess the ability of a new generation of MagSweeper to isolate intact CTCs for downstream analysis, we performed mRNA-Seq on single CTCs isolated from the blood of patients with metastatic prostate cancer and on single prostate cancer cell line LNCaP cells spiked into the blood of healthy donors. We found that the MagSweeper effectively isolated CTCs with a capture efficiency that matched the CellSearch platform. However, unlike CellSearch, the MagSweeper facilitates isolation of individual live CTCs without contaminating leukocytes. Importantly, mRNA-Seq analysis showed that the MagSweeper isolation process did not have a discernible impact on the transcriptional profile of single LNCaPs isolated from spiked human blood, suggesting that any perturbations caused by the MagSweeper process on the transcriptional signature of isolated cells are modest. Although the RNA from patient CTCs showed signs of significant degradation, consistent with reports of short half-lives and apoptosis amongst CTCs, transcriptional signatures of prostate tissue and of cancer were readily detectable with single CTC mRNA-Seq. These results demonstrate that the MagSweeper provides access to intact CTCs and that these CTCs can potentially supply clinically relevant information.</p> </div

Expression, clustering, and functional classification of genes expressed in human prostate CTCs.

<p>(A) Expression of prostate cancer associated genes. For each gene, fragments per kilobase of exon per million fragments mapped (FPKM) for each CTC and controls are shown. FPKM values greater than 5 are shaded green and those with values between 1 and 5 are shaded light green. AR (androgen receptor), KLK3 (prostate specific antigen), and TMPRSS2are markers of prostate tissue. CD45 is a white blood cell marker. Prostate cancer cell lines include LNCaP and PC-3 while T24 is a bladder cancer, and WBC is a single white blood cell. Normal denotes normal prostate tissue. (B) Unsupervised clustering of over-expressed genes in patient CTCs. Colored bars across the top of the figure indicate different patients while individual patient CTCs are listed at the bottom of the cluster. (C) Functional classification of genes overexpressed in CTC using Gene Ontology (GO) classifications. For each functional grouping the % of genes over-expressed in each GO category is indicated.</p

MagSweeper isolation has minimal effects on single cell transcriptomes.

<p>(A) Bioanalyzer traces of amplified cDNAs from single LNCaP cells pre (Single LNCaP(pre)) and post MagSweeping (Single LNCaP (post)), and positive control (12 pg of LNCaP total RNA) and negative control (Negative control). (B) Heatmap of correlations between fresh and MagSwept single-cell RNA-Seq data and table of correlations between fresh and MagSwept samples. Yellow indicates higher correlations and red lower correlations. (C) Representative bioanalyzer traces of good, intermediate, and poor CTC cDNA amplification products. (D) Percent breakout of CTC RNA quality based on classification of cDNA amplification products – green indicates good quality, yellow samples are partially degraded RNA and red indicates degraded RNA samples. (E) Sequenced CTCs, their RNA quality and % alignment of passing filter mRNA-Seq reads to the human genome build hg19. Patient CTC ID indicates single patient CTCs identified as patient number. CTC number (P1.1). RNA Quality is based on bioanalyzer traces of amplified cDNA and Align% is alignment % of mRNA-Seq reads.</p

Alignment metrics of human prostate CTC mRNA-Seq sequences.

<p>(A) Percentage of passing filter (PF) reads that aligned, percentage of alignments that map to coding regions, median coverage CV, and percentage of reads that map to the five genes with the highest number of mapped reads. To calculate the “Median CV”, first the CV of coverage was calculated across each of 600 genes in a hand-picked set of quality control genes expressed in universal human RNA (Agilent), and then the median of these CVs is taken (considering only the genes with at least 1× coverage). The “% aligned” is the percentage of PF reads that align to the genome or to a splice site, excluding mitochondria and ribosomal RNA. Based on historical data, expected values for median CV and % aligned are <65% and >60%, respectively. Coding alignments are the % of reads that map to exons. The % of reads aligned to the top 5 genes for each sample are shown (B) Examples of the average length-normalized coverage across the 600 quality control genes, from samples LNCaP.3, N.1, P2.1, P3.4. Position 0 is the 5′-end of the transcripts and 100 is the extreme 3′-end of the transcript.</p

Metrics of MagSweeper circulating tumor cell (CTC) isolation.

<p>(A) Image of hood prototype of the MagSweeper. (B) Percent capture of 100 LNCaP cells spiked into 3.75 ml of normal blood (N = 54 experiments and 11 donors). Blue circles show mean percent recoveries of live EpCAM (+)/CD45 (−)/DAPI(−) cells and red circles show mean recoveries of membrane compromised EpCAM (+)/CD45 (−)/DAPI(+) cells. Error bars represent +/−1 S.D. (C) Percentage capture and purity of 10 LNCaP cells isolated following spiking into 7.5 ml of normal blood. Blue bars are the mean percent recovery of cells after MagSweeper isolation (Cell Capture) and pick and manual place single cell isolation (Cell Isolation) while purple bars show purity of LNCaP cells after MagSweeper and single cell isolation (N = 6 experiments and 4 donors). Cell purity was calculated as the number of spiked cells isolated divided by the number of spiked cells counted plus white blood cells counted. Error bars represent +/−1 S.D. (D) Bright field (BF) and images of fluorescently stained CTCs isolated from a prostate cancer patient blood sample, and contaminating WBC found after MagSweeper isolation. Scale bar = 20 microns. (E) MagSweeper versus CellSearch comparison of patient samples. Samples with 0 CTC were assigned a value of 1 for plotting purposes.</p