High-resolution array comparative genomic hybridization of single micrometastatic tumor cells

Abstract

Only few selected cancer cells drive tumor progression and are responsible for therapy resistance. Their specific genomic characteristics, however, are largely unknown because high-resolution genome analysis is currently limited to DNA pooled from many cells. Here, we describe a protocol for array comparative genomic hybridization (array CGH), which enables the detection of DNA copy number changes in single cells. Combining a PCR-based whole genome amplification method with arrays of highly purified BAC clones we could accurately determine known chromosomal changes such as trisomy 21 in single leukocytes as well as complex genomic imbalances of single cell line cells. In single T47D cells aberrant regions as small as 1-2 Mb were identified in most cases when compared to non-amplified DNA from 10(6) cells. Most importantly, in single micrometastatic cancer cells isolated from bone marrow of breast cancer patients, we retrieved and confirmed amplifications as small as 4.4 and 5 Mb. Thus, high-resolution genome analysis of single metastatic precursor cells is now possible and may be used for the identification of novel therapy target genes.

Figure 1.

Contamination of amplified DNA samples and PFGE purification of BAC DNA from E. coli DNA. (A) Amplified DNA from empty-control for the amplification PCR, 1 cell, 100 cells and 40 000 cells (from left to right) were spotted onto a membrane and hybridized with Dig-labeled bacterial contamination. The more human cellular DNA was added, the weaker the hybridization signal of the labeled contamination. The PCR-mock-control contains the highest amount of contamination. (B) Schematic illustration of the pulsed-field-gel-electrophoresis (PFGE) of PI-SceI digested RP11-BAC clones. A PI-SceI site is located in the pBACe3.6 vector sequence, while no restriction site is present in the genomic DNA of E. coli. The linearized BAC DNA is recovered after gel-electrophoresis by gel extraction (BAC clones 86c20, 106m3). (C) and (D) E. coli contamination of BAC clone preparations determined by qPCR before and after adapter-linker-amplification, respectively. The same 15 BAC clones were prepared by PFGE- and conventional purification. The contamination with E. coli DNA is expressed as percentage of TRPE sequence in comparison to pBACe3.6 sequence.

Figure 2.

Hybridization of the test-array using DNA from pooled cells and amplified single cell DNA from a patient with trisomy 21. The six different BAC DNA preparations were spotted onto one array and hybridized with Cy3- and Cy5-labeled reference and test DNA probes. The log2 transformed ratios of test-to-reference DNA fluorescence intensities are plotted against the chromosomal locations of the BAC clones. Dashed gray and black/blue lines represent the base line and the upper/lower significance thresholds defined by the three-times standard deviation, respectively. (A–F) Non-amplified DNA from 10⁶ cells (G–L) Amplified single cell DNA. Note that the trisomy 21 is only detected by the PFGE-amplified BAC-clones (G–I). Red dots represent the experiment in which probe and reference were labeled with Cy5 and Cy3, respectively, the green dots represent the color switch experiment and ‘+’ represents the mean log2 ratio of both experiments. The 3-fold standard deviation is calculated individually from the balanced clones in each experiment and as threshold the average standard deviation is shown (blue line). (A–C) and (G–I): PFGE purification of BAC clones; (D–F) and (J–L) Qiagen preparation of BAC clones. (A, D, G, J): adapter-linker PCR; (B, E, H, K): DOP-PCR; (C, F, I, L): Phi29 rolling circle amplification. (M) Statistical evaluation of results from different BAC-preparations. Averages from three independent experiments are shown. (Mse: Adaptor-linker-PCR; STDEV: standard deviation).

Figure 3.

Performance of 3 K BAC array. (A) and (B) Hybridization of amplified single cell-DNA from a female healthy donor and a patient with trisomy 21, respectively, to 3 K BAC array versus male reference DNA. The log2-transformed fluorescence intensity ratios are plotted against the chromosomal locations of the BAC clones. Gray color of dots indicates normal clones, green indicates loss and red indicates gain of chromosomal material. Blue indicates high-copy amplification. Outliers are marked by a box around the data point; (data normalization and visualization were performed with CAPweb software). (C) GC-content (%) of BAC clones from chromosomes 3 (light gray) and 19 (dark gray) (n = 30 clones were analyzed for both chromosomes). (D) and (E) Details of chromosomes 3 and 19 from panels A and B after selection of chromosome 19 BAC clones with GC-content < 45% (n = 6).

Figure 4.

Comparison of the 3 K BAC array and the 19 K and 244 K oligonucleotide arrays. (A) Hybridization profiles of chromosome 10 from hybridization with T47D cell line DNA. From left to right: Hybridization profile of 10⁶ T47D cells to a 32 K BAC array taken from Shadeo et al., hybridization of T47D DNA amplified from two individual single cells on the 3 K BAC array, the 19 K and 244 K oligonucleotide arrays. In all profiles, the blue line represents the hybridization profile of 10⁶ T47D cells on the 244 K oligonucleotide array as a reference. The red line represents the single cell profiles on the various platforms. Log2 ratios are plotted against chromosomal location of data points. The vertical lines in the profiles indicate the threshold of significant deviations from normal (±0.2). (B–D) Percentage of correctly detected amplifications, deletions and normal regions in T47D single cell profiles on the three different platforms. Regions between 1 and 20 Mb detected in single cell 1 (B) and single cell 2 (C). Regions between >20 and 200 Mb are provided as an average of single cell 1 and 2 (D).

Figure 5.

Hybridization of two adapter-linker amplified DNA samples from disseminated tumor cells isolated from bone marrow of a breast cancer patient. The metaphase CGH profile of each sample is plotted beneath the array CGH profile. Chromosome ideograms are placed horizontally to position the corresponding genomic regions of metaphase and array CGH next to each other for direct comparison. (A) Single tumor cell 1, (B) single tumor cell 2, (C) gain of resolution by array CGH. Two small inserted deletions on the q-arm of chromosome 11 (7 Mb and 500 kb in size; from centromer to telomer) and the high-copy amplifications on chromosomes 12p (4.4 Mb) and 14q (5 Mb) are present in both single cells. Colour code and axes as in Figure 3. Arrows and asterisks point to primer binding sites used as controls or target regions, respectively, for qPCR. (D) Calculated copy numbers of AKAP3 and 6 in single disseminated tumor cell 1, 2 and 3 as calculated from qPCR results (assuming a diploid genome).