SummaryAccurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.

A key cellular process associated with the invasive or metastatic program in many cancers is the transformation of epithelial cells toward a mesenchymal state, a process called epithelial to mesenchymal transition or EMT. Actin-dependent protrusion of cell pseudopodia is a critical element of mesenchymal cell migration and therefore of cancer metastasis. However, whether EMT occurs in human cancers and, in particular, whether it is a prerequisite for tumor cell invasion and metastasis, remains a subject of debate. Microarray and proteomic analysis of actin-rich pseudopodia from six metastatic human tumor cell lines identified 384 mRNAs and 64 proteins common to the pseudopodia of six metastatic human tumor cell lines of various cancer origins leading to the characterization of 19 common pseudopod-specific proteins. Four of these (AHNAK, septin-9, eIF4E, and S100A11) are shown to be essential for pseudopod protrusion and tumor cell migration and invasion. Knockdown of each of these proteins in metastatic cells resulted in reduced actin cytoskeleton dynamics and induction of mesenchymal-epithelial transition (MET) that could be prevented by the stabilization of the actin cytoskeleton. Actin-dependent pseudopodial protrusion and tumor cell migration are therefore determinants of EMT. Protein regulators of pseudopodial actin dynamics may represent unique molecular targets to induce MET and thereby inhibit the metastatic potential of tumor cells.Cancer Res; 70(9); 3780-90. ©2010 AACR.

Highlights d Hic1 marks multiple quiescent mesenchymal progenitor (MP) subsets within skeletal muscle d Conditional deletion of Hic1 leads to MP hyperplasia and an activated MP phenotype d Hic1 + MPs generate transit-amplifying progeny post-injury that support regeneration d Following injury, select Hic1 + progeny persist and regenerate the myotendinous junction

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OBJECTIVE-Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of diabetes, but the roles of specific ER Ca 2ϩ release channels in the ER stress-associated apoptosis pathway remain unknown. Here, we examined the effects of stimulating or inhibiting the ER-resident inositol trisphosphate receptors (IP 3 Rs) and the ryanodine receptors (RyRs) on the induction of ␤-cell ER stress and apoptosis.RESEARCH DESIGN AND METHODS-Kinetics of ␤-cell death were tracked by imaging propidium iodide incorporation and caspase-3 activity in real time. ER stress and apoptosis were assessed by Western blot. Mitochondrial membrane potential was monitored by flow cytometry. Cytosolic Ca 2ϩ was imaged using fura-2, and genetically encoded fluorescence resonance energy transfer (FRET)-based probes were used to measure Ca 2ϩ in ER and mitochondria. RESULTS-NeitherRyR nor IP 3 R inhibition, alone or in combination, caused robust death within 24 h. In contrast, blocking sarco/endoplasmic reticulum ATPase (SERCA) pumps depleted ER Ca 2ϩ and induced marked phosphorylation of PKR-like ER kinase (PERK) and eukaryotic initiation factor-2␣ (eIF2␣), C/EBP homologous protein (CHOP)-associated ER stress, caspase-3 activation, and death. Notably, ER stress following SERCA inhibition was attenuated by blocking IP 3 Rs and RyRs. Conversely, stimulation of ER Ca 2ϩ release channels accelerated thapsigargin-induced ER depletion and apoptosis. SERCA block also activated caspase-9 and induced perturbations of the mitochondrial membrane potential, resulting eventually in the loss of mitochondrial polarization.CONCLUSIONS-This study demonstrates that the activity of ER Ca 2ϩ channels regulates the susceptibility of ␤-cells to ER stress resulting from impaired SERCA function. Our results also suggest the involvement of mitochondria in ␤-cell apoptosis associated with dysfunctional ␤-cell ER Ca 2ϩ homeostasis and ER stress. Diabetes 58:422-432, 2009