Metabolism of oxygen, while central to life, also produces reactive oxygen species (ROS) that have been implicated in processes as diverse as cancer, cardiovascular disease, and aging. It has recently been shown that central nervous system stem cells 1, 2 and hematopoietic stem cells and early progenitors [3][4][5][6] contain lower levels of ROS than their more mature progeny and that these differences appear to be critical for maintaining stem cell function. We hypothesized that epithelial tissue stem cells and their cancer stem cell (CSC) counterparts may also share this property. Here we show that normal mammary epithelial stem cells contain lower concentrations of ROS than their more mature # To whom correspondence should be addressed. mfclarke@stanford.edu. * Contributed equally.Author Contributions M.D. and R.W.C. contributed equally to this work. M.D, R.W.C., N.L., T.K., M.J.D., A.K., D.Q., J.S.L., L.A., and M.W. performed the experiments. B.J., M.J.K, I.W., F.W., G.S., C.G., B.P., J.S., and S.K.L. aided in human tumor tissue acquisition. G.S. designed a pre-operative protocol allowing for tissue acquisition. M.D., R.W.C., and M.F.C. designed the experiments and wrote the manuscript. S.R.Q., J.M.B., and I.L.W. provided intellectual input and aided in experimental design.Author Information Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. Given the conservation of low ROS levels in several types of normal tissue stem cells, we hypothesized that CSCs in some tumors may also contain lower concentrations of ROS than their non-tumorigenic progeny. In order to investigate ROS biology in human CSCs, we began by examining the expression of genes involved in ROS metabolism in primary human breast CSCs and NTCs. Using microarray data from human breast CSC-enriched populations and NTCs 13 and a curated list of genes involved in ROS metabolism 5 (see methods), Gene Set Enrichment Analysis (GSEA) 14 revealed that the expression of ROS genes was highly overrepresented in the CD44 + CD24 -/low Lin -breast CSC-enriched population compared to NTCs (p<0.001; Supplementary Fig. S2). The ROS genes identified as the core enriched genes by GSEA included a number of important antioxidant genes (Supplementary Table 2). Thus, gene expression profiles of human breast CSC-containing populations suggest that they contain higher levels of antioxidant defense systems than NTCs. NIH Public AccessNext, we directly assessed ROS levels in human tumor subpopulations. To do this the CD44 + CD24 -/low Lin -breast CSC-enriched population and the corresponding "Not CD44 + CD24 -/low " Lin -NTC population were purified from surgically resected breast tumors ( Supplementary Fig. S3). DCF-DA staining revealed that the CSC-enriched population in the human breast tumors we examined contained significantly lower levels of prooxidants than the NTC population. In some breast tumors, the vast majority of cells in the CSC-containing fraction displayed a low ROS phenotype compared to NTCs (Fig. 1e) while ...

Cancer is often viewed as a caricature of normal developmental processes, but the extent by which its cellular heterogeneity truly recapitulates multi-lineage differentiation processes of normal tissues remains unknown. Here, we implement “single-cell PCR gene-expression analysis” (SINCE-PCR) to dissect the cellular composition of primary human normal colon and colon cancer epithelia. We show that human colon cancer tissues contain distinct cell populations whose transcriptional identities mirror those of the different cellular lineages of normal colon. By creating monoclonal tumor xenografts from injection of a single-cell (n = 1), we show that transcriptional diversity of cancer tissues is largely explained by in vivo multi-lineage differentiation, not only by clonal genetic heterogeneity. Finally, we show that perturbations in gene-expression programs linked to multi-lineage differentiation strongly associate with patient survival. Guided by SINCE-PCR data, we develop two-gene classifier systems (KRT20 vs CA1, MS4A12, CD177, SLC26A3) that predict clinical outcomes with hazard-ratios superior to pathological grade and comparable to microarray-derived multi-gene expression signatures.

Interest in single-cell whole-transcriptome analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. We compared commercially available single-cell RNA amplification methods with both microliter and nanoliter volumes, using sequence from bulk total RNA and multiplexed quantitative PCR as benchmarks to systematically evaluate the sensitivity and accuracy of various single-cell RNA-seq approaches. We show that single-cell RNA-seq can be used to perform accurate quantitative transcriptome measurement in individual cells with a relatively small number of sequencing reads and that sequencing large numbers of single cells can recapitulate bulk transcriptome complexity.