Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device

Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cancer derived cell lines (LS174T, LS180 and RKO) and fresh colorectal tumors have been individually trapped, their genomes extracted and prepared for sequencing using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 ± 0.06%) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as the lysis is in sub-nanoliter volumes. Our data thus demonstrates that high quality whole genome sequencing of single cells can be achieved using a relatively simple, inexpensive and scalable device. Detection of genetic heterogeneity at the single cell level, as we have demonstrated for freshly obtained single cancer cells, could soon become available as a clinical tool to precisely match treatment with the properties of a patient's own tumor

Single-molecule DNA-mapping and whole-genome sequencing of individual cells

To elucidate cellular diversity and clonal evolution in tissues and tumors, one must resolve genomic heterogeneity in single cells. To this end, we have developed low-cost, mass-producible micro-/nanofluidic chips for DNA extraction from individual cells. These chips have modules that collect genomic DNA for sequencing or map genomic structure directly, on-chip, with denaturation-renaturation (D-R) optical mapping [Marie R, et al. (2013) Proc Natl Acad Sci USA 110:4893-4898]. Processing of single cells from the LS174T colorectal cancer cell line showed that D-R mapping of single molecules can reveal structural variation (SV) in the genome of single cells. In one experiment, we processed 17 fragments covering 19.8 Mb of the cell's genome. One megabase-large fragment aligned well to chromosome 19 with half its length, while the other half showed variable alignment. Paired-end single-cell sequencing supported this finding, revealing a region of complexity and a 50-kb deletion. Sequencing struggled, however, to detect a 20-kb gap that D-R mapping showed clearly in a megabase fragment that otherwise mapped well to the reference at the pericentromeric region of chromosome 4. Pericentromeric regions are complex and show substantial sequence homology between different chromosomes, making mapping of sequence reads ambiguous. Thus, D-R mapping directly, from a single molecule, revealed characteristics of the single-cell genome that were challenging for short-read sequencing

N-Glycomic and Transcriptomic Changes Associated with CDX1 mRNA Expression in Colorectal Cancer Cell Lines

The caudal-related homeobox protein 1 (CDX1) is a transcription factor, which is important in the development, differentiation, and homeostasis of the gut. Although the involvement of CDX genes in the regulation of the expression levels of a few glycosyltransferases has been shown, associations between glycosylation phenotypes and CDX1 mRNA expression have hitherto not been well studied. Triggered by our previous study, we here characterized the N-glycomic phenotype of 16 colon cancer cell lines, selected for their differential CDX1 mRNA expression levels. We found that high CDX1 mRNA expression associated with a higher degree of multi-fucosylation on N-glycans, which is in line with our previous results and was supported by up-regulated gene expression of fucosyltransferases involved in antenna fucosylation. Interestingly, hepatocyte nuclear factors (HNF)4A and HNF1A were, among others, positively associated with high CDX1 mRNA expression and have been previously proven to regulate antenna fucosylation. Besides fucosylation, we found that high CDX1 mRNA expression in cancer cell lines also associated with low levels of sialylation and galactosylation and high levels of bisection on N-glycans. Altogether, our data highlight a possible role of CDX1 in altering the N-glycosylation of colorectal cancer cells, which is a hallmark of tumor development

Natural Compounds' Activity against Cancer Stem-Like or Fast-Cycling Melanoma Cells

<div><p>Background</p><p>Accumulating evidence supports the concept that melanoma is highly heterogeneous and sustained by a small subpopulation of melanoma stem-like cells. Those cells are considered as responsible for tumor resistance to therapies. Moreover, melanoma cells are characterized by their high phenotypic plasticity. Consequently, both melanoma stem-like cells and their more differentiated progeny must be eradicated to achieve durable cure. By reevaluating compounds in heterogeneous melanoma populations, it might be possible to select compounds with activity not only against fast-cycling cells but also against cancer stem-like cells. Natural compounds were the focus of the present study.</p><p>Methods</p><p>We analyzed 120 compounds from The Natural Products Set II to identify compounds active against melanoma populations grown in an anchorage-independent manner and enriched with cells exerting self-renewing capacity. Cell viability, cell cycle arrest, apoptosis, gene expression, clonogenic survival and label-retention were analyzed.</p><p>Findings</p><p>Several compounds efficiently eradicated cells with clonogenic capacity and nanaomycin A, streptonigrin and toyocamycin were effective at 0.1 µM. Other anti-clonogenic but not highly cytotoxic compounds such as bryostatin 1, siomycin A, illudin M, michellamine B and pentoxifylline markedly reduced the frequency of ABCB5 (ATP-binding cassette, sub-family B, member 5)-positive cells. On the contrary, treatment with maytansine and colchicine selected for cells expressing this transporter. Maytansine, streptonigrin, toyocamycin and colchicine, even if highly cytotoxic, left a small subpopulation of slow-dividing cells unaffected. Compounds selected in the present study differentially altered the expression of melanocyte/melanoma specific microphthalmia-associated transcription factor (MITF) and proto-oncogene c-MYC.</p><p>Conclusion</p><p>Selected anti-clonogenic compounds might be further investigated as potential adjuvants targeting melanoma stem-like cells in the combined anti-melanoma therapy, whereas selected cytotoxic but not anti-clonogenic compounds, which increased the frequency of ABCB5-positive cells and remained slow-cycling cells unaffected, might be considered as a tool to enrich cultures with cells exhibiting melanoma stem cell characteristics.</p></div

The summary of natural compound activities at 5 µM.

<p>After initial screening, compounds were grouped based on their activities. A compound was defined as anti-clonogenic when it reduced the percentage of clones formed in soft agar to less than 20% of control treated with vehicle (0.05% DMSO). A compound was named cytostatic when it reduced the viable cell number to less than 50% of control using viable cell number counting, and cytotoxic using the flow cytometry after PI-staining. Numbers corresponding to compounds that accumulated melanoma cells in subG₁ are underlined. Compounds that caused cell cycle arrest are marked in green for G₀/G₁ phase, blue for S phase and red for G₂/M phase. Several compounds (46) were not active in any assay. Few compounds were cytotoxic but not markedly influenced the numbers of colonies formed in agar. Few other compounds exerted their effects only on clonogenic cells or reduced clonogenicity below 20% of control, caused cytostatic/cytostatic effect without inducing substantial cell death. Several compounds were cytostatic and cytotoxic and in addition they efficiently eradicated cells with clonogenic potential. They were in the focus of the further study. See Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090783#pone.0090783.s001" target="_blank">File SI</a> for the names of the compounds corresponding to numbers shown in the Figure.</p

Several natural compounds at 5 µM influenced the clonogenic growth of melanoma cells.

<p>Cells were incubated in compound-containing medium for 4 h and then they were grown on soft agar for 14 days in the presence of drug-free medium. Cell colonies were stained with crystal violet and counted. Compounds were grouped based on their anti-clonogenic activity expressed as percentage of control treated with vehicle (0.05% DMSO). At least two independent experiments were performed in duplicates. See Figure S4 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090783#pone.0090783.s001" target="_blank">File SI</a> for quantitative data.</p

Selected natural compounds induced diverse effects on the expression of <i>MITF</i> and <i>c-MYC</i>.

<p>(A) Cell viability was measured for highly cytotoxic compounds to choose a concentration not reducing cell viability below 40% of control after 24 h of treatment. (B) qRT-PCR was used to assess the fold change in the expression of <i>MITF</i> and <i>c-MYC</i> after treatment with selected compounds at indicated concentrations and time of exposure. (*P<0.05; **P<0.01; ***P<0.001).</p