Rare circulating tumor cells (CTCs) are present in the blood of patients with metastatic epithelial cancers but have been difficult to measure routinely. We report a quantitative automated imaging system for analysis of prostate CTCs, taking advantage of prostate-specific antigen (PSA), a unique prostate tumor–associated marker. The specificity of PSA staining enabled optimization of criteria for baseline image intensity, morphometric measurements, and integration of multiple signals in a three-dimensional microfluidic device. In a pilot analysis, we detected CTCs in prostate cancer patients with localized disease, before surgical tumor removal in 8 of 19 (42%) patients (range, 38 to 222 CTCs per milliliter). For 6 of the 8 patients with preoperative CTCs, a precipitous postoperative decline (<24 hours) suggests a short half-life for CTCs in the blood circulation. Other patients had persistent CTCs for up to 3 months after prostate removal, suggesting early but transient disseminated tumor deposits. In patients with metastatic prostate cancer, CTCs were detected in 23 of 36 (64%) cases (range, 14 to 5000 CTCs per milliliter). In previously untreated patients followed longitudinally, the numbers of CTCs declined after the initiation of effective therapy. The prostate cancer–specific TMPRSS2-ERG fusion was detectable in RNA extracted from CTCs from 9 of 20 (45%) patients with metastatic disease, and dual staining of captured CTCs for PSA and the cell division marker Ki67 indicated a broad range for the proportion of proliferating cells among CTCs. This method for analysis of CTCs will facilitate the application of noninvasive tumor sampling to direct targeted therapies in advanced prostate cancer and warrants the initiation of long-term clinical studies to test the importance of CTCs in invasive localized disease.

Skin scarring, the end result of adult wound healing, is detrimental to tissue form and function. Engrailed-1 lineage–positive fibroblasts (EPFs) are known to function in scarring, but Engrailed-1 lineage–negative fibroblasts (ENFs) remain poorly characterized. Using cell transplantation and transgenic mouse models, we identified a dermal ENF subpopulation that gives rise to postnatally derived EPFs by activating Engrailed-1 expression during adult wound healing. By studying ENF responses to substrate mechanics, we found that mechanical tension drives Engrailed-1 activation via canonical mechanotransduction signaling. Finally, we showed that blocking mechanotransduction signaling with either verteporfin, an inhibitor of Yes-associated protein (YAP), or fibroblast-specific transgenic YAP knockout prevents Engrailed-1 activation and promotes wound regeneration by ENFs, with recovery of skin appendages, ultrastructure, and mechanical strength. This finding suggests that there are two possible outcomes to postnatal wound healing: a fibrotic response (EPF-mediated) and a regenerative response (ENF-mediated).

Author contributions L.X. and A.M contributed equally to this study. S.M., E.M. and A.B. planned the study, with input from M.P.S. and J.W. S.M. and E.M. performed the reprogramming experiments, and analysed and interpreted data. S.M., E.M. and L.X. wrote the manuscript with the help of A.B. S.M. generated, processed and analysed bulk and single-cell RNA-seq datasets, analysed the metabolomics data, and performed most THY1-related and conditioned medium experiments. E.M. generated and propagated transgene-free iPS cell lines. All other studies were done by both E.M and S.M., unless otherwise noted. A.M. and S.M. performed wound healing experiments under the supervision of M.T.L. L.X. helped with reprogramming, FACS, and immunofluorescence experiments. F.J. generated the in vitro single-cell RNA-seq data under the supervision of M.P.S. R.S. generated the ChIP-seq libraries under the supervision of J.W. K.H. helped with statistics and PAGODA analysis. X.L. performed metabolomics experiments and helped with metabolomics data analysis and validation under the supervision of M.P.S. K.D. helped with reprogramming and western blotting experiments. L.P. helped with reprogramming and RT-qPCR experiments. C.E.A. and Y.S. performed the induced neuron reprogramming experiment under the supervision of M.W. B.A.B. helped with analysis of the epigenomic data. A.L.S.C. identified and collected the human samples. All authors discussed the results and commented on the manuscript. Data availabilityAll raw sequencing reads for population RNA-seq, ChIP-seq and single-cell RNA-seq data can be found under BioProject PRJNA316110. The command and configuration files, in addition to a list of all versioned dependencies present in the running environment, are available on the Github repository for this paper (https://github.com/brunetlab/Mahmoudi_et_al_2018) (except for the code for the processing of metabolomics data, which is available upon request).