Table 1.
Patient demographics.
Fig 1.
Epic Sciences single cell NGS-based CNV analysis pipeline.
FASTQ files were aligned to hg38 human reference genome from UCSC using BWA. BAM files with a MAPQ quality score greater than 30 were kept for further analysis with two separate pipelines: analysis pipeline 1 for genomic instabilities estimation using 1M bp window; analysis pipeline 2 for determining copy number alterations of individual genes.
Fig 2.
Representative images of CTCs identified.
Representative CTC images from cell lines (A) LNCaP, (B) PC3, and (C) VCaP. Slides were stained with CK, AR, CD45, and DAPI. Individual CTCs were identified by Epic Sciences’ algorithm and visually confirmed.
Fig 3.
Whole Genome Amplification, library preparation quality control, read quality, output, and alignment quality.
(A) DNA concentrations and total yield for each single cell WGA as measured by UV/Vis. Libraries were constructed from independent replicates of single cells: 8 from LNCaP, 8 from PC3, 5 from VCaP, and 4 from WBCs. Overall, we achieved an average 100% success rate during the single cell whole genome amplification procedure: 8/8 single cells (100%) successfully amplified for PC3 and LNCaP cancer cell lines, while 5/5 (100%) single VCaP cells and 4/4 (100%) single WBCs amplified. An average yield of 578 ng (range of 227–1190 ng) was obtained from the single cell WGA reactions. (B) Concentrations of the next-generation sequencing libraries as measured by PicoGreen. All of the NGS libraries passed QC with adequate yield except for two samples that failed to render any detectable amount of library DNA product (one PC3 and WBC replica samples) (23/25; 92% success rate). An average yield of 581 ng (range of 397–1216 ng) was obtained among the single cell NGS libraries. (C) Assessment of NGS read quality. >99% of the NGS reads had an average PHRED score greater than Q30 pre-alignment, indicating high quality reads. An average of 17 million reads/sample (in each direction) were obtained. 99% of the reads mapped to the reference genome (hg38) with 79% of the reads mapping with a MAPQ score greater than 30. *Failed library preparation.
Fig 4.
Prostate cancer cell line single cell CNV profiles, genomic instability scores and AR, PTEN copy number status.
Whole genome copy number plots from prostate cancer cell lines (A) LNCaP, (B) PC3, and (C) VCaP, and (D) WBC controls. (E) Absolute Pearson correlation values (0–100%) were calculated across samples and viewed using Circos Table Viewer (http://circos.ca/presentations/articles/vis_tables1/). For visualization purposes, the top 25% highest correlations are displayed. Each color-coded segment represents a cell line replicate. Correlations between replicates are denoted by links or ribbons, the width of which is proportional to the degree of correlation. Much higher correlations were observed in intra-cell line comparisons than inter-cell line comparisons, indicating that the assay has good reproducibility regardless of cell line used. (F) Box-whisker plot of LST scores for prostate cancer cell lines and WBCs. All 3 cell lines had high LST scores compared to the WBCs, with PC3 and VCaP having the highest scores. (G) Box-whisker plot of log2 normalized DNA copy number in AR. Amplification of the AR gene was observed in the VCaP single cells reproducibly (5/5, 100%). This amplification was not observed in PC3 (0/7), LNCaP (0/8), or WBC controls (0/3). (H) Box-whisker plot of log2 normalized DNA copy number in PTEN. The VCaP cell line has non-deleted PTEN (0/5, 0%), while PTEN loss was detected in PC3 (6/7, 86%), LNCaP (1/8, 13%), and 1/3 WBC controls (1/3, 33%).
Table 2.
Prostate Cancer Cell Line Genomic Instability Scores.
Fig 5.
Genomic instability and CNVs in mCRPC patient CTCs.
(A) Box-whisker plot of LST scores for patient CTCs. High LST scores were observed in 5/7 (71%) patients. (B) Dot plot of log2 normalized DNA copy number in AR. Amplification of the AR gene was observed in 5/7 (71%) patients. (C) Dot plot of AR N-Terminal protein expression status in each single CTC as detected by IF, 5/7 (71%) patients had amplified AR protein, where AR amplification is observed in both CK positive and CK negative CTCs. (D) Box-whisker plot of AR N-Terminal protein expression in AR copy number gain and copy number neutral CTCs. Higher AR protein expression was observed in the AR copy number gain group, p = 0.0025 by Student’s t-test. (E) Dot plot of log2 normalized DNA copy number in PTEN. Loss of PTEN was observed in 5/7 (71%) patients. In each figure, one dot represents a single CTC.
Table 3.
PTEN status detected by CNV and FISH in patient CTCs.