Defining the Design Principles of Skin Epidermis Postnatal Growth.

During embryonic and postnatal development, organs and tissues grow steadily to achieve their final size at the end of puberty. However, little is known about the cellular dynamics that mediate postnatal growth. By combining in vivo clonal lineage tracing, proliferation kinetics, single-cell transcriptomics, and in vitro micro-pattern experiments, we resolved the cellular dynamics taking place during postnatal skin epidermis expansion. Our data revealed that harmonious growth is engineered by a single population of developmental progenitors presenting a fixed fate imbalance of self-renewing divisions with an ever-decreasing proliferation rate. Single-cell RNA sequencing revealed that epidermal developmental progenitors form a more uniform population compared with adult stem and progenitor cells. Finally, we found that the spatial pattern of cell division orientation is dictated locally by the underlying collagen fiber orientation. Our results uncover a simple design principle of organ growth where progenitors and differentiated cells expand in harmony with their surrounding tissues.

Mechanisms of stretch-mediated skin expansion at single-cell resolution.

The ability of the skin to grow in response to stretching has been exploited in reconstructive surgery1. Although the response of epidermal cells to stretching has been studied in vitro2,3, it remains unclear how mechanical forces affect their behaviour in vivo. Here we develop a mouse model in which the consequences of stretching on skin epidermis can be studied at single-cell resolution. Using a multidisciplinary approach that combines clonal analysis with quantitative modelling and single-cell RNA sequencing, we show that stretching induces skin expansion by creating a transient bias in the renewal activity of epidermal stem cells, while a second subpopulation of basal progenitors remains committed to differentiation. Transcriptional and chromatin profiling identifies how cell states and gene-regulatory networks are modulated by stretching. Using pharmacological inhibitors and mouse mutants, we define the step-by-step mechanisms that control stretch-mediated tissue expansion at single-cell resolution in vivo.

Distinct contribution of stem and progenitor cells to epidermal maintenance.

The skin interfollicular epidermis (IFE) is the first barrier against the external environment and its maintenance is critical for survival. Two seemingly opposite theories have been proposed to explain IFE homeostasis. One posits that IFE is maintained by long-lived slow-cycling stem cells that give rise to transit-amplifying cell progeny, whereas the other suggests that homeostasis is achieved by a single committed progenitor population that balances stochastic fate. Here we probe the cellular heterogeneity within the IFE using two different inducible Cre recombinase­oestrogen receptor constructs targeting IFE progenitors in mice. Quantitative analysis of clonal fate data and proliferation dynamics demonstrate the existence of two distinct proliferative cell compartments arranged in a hierarchy involving slow-cycling stem cells and committed progenitor cells. After wounding, only stem cells contribute substantially to the repair and long-term regeneration of the tissue, whereas committed progenitor cells make a limited contribution.

Distinct stem cells contribute to mammary gland development and maintenance.

The mammary epithelium is composed of several cell lineages including luminal, alveolar and myoepithelial cells. Transplantation studies have suggested that the mammary epithelium is maintained by the presence of multipotent mammary stem cells. To define the cellular hierarchy of the mammary gland during physiological conditions, we performed genetic lineage-tracing experiments and clonal analysis of the mouse mammary gland during development, adulthood and pregnancy. We found that in postnatal unperturbed mammary gland, both luminal and myoepithelial lineages contain long-lived unipotent stem cells that display extensive renewing capacities, as demonstrated by their ability to clonally expand during morphogenesis and adult life as well as undergo massive expansion during several cycles of pregnancy. The demonstration that the mammary gland contains different types of long-lived stem cells has profound implications for our understanding of mammary gland physiology and will be instrumental in unravelling the cells at the origin of breast cancers.

A vascular niche and a VEGF-Nrp1 loop regulate the initiation and stemness of skin tumours.

Angiogenesis is critical during tumour initiation and malignant progression. Different strategies aimed at blocking vascular endothelial growth factor (VEGF) and its receptors have been developed to inhibit angiogenesis in cancer patients. It has become increasingly clear that in addition to its effect on angiogenesis, other mechanisms including a direct effect of VEGF on tumour cells may account for the efficiency of VEGF-blockade therapies. Cancer stem cells (CSCs) have been described in various cancers including squamous tumours of the skin. Here we use a mouse model of skin tumours to investigate the impact of the vascular niche and VEGF signalling on controlling the stemness (the ability to self renew and differentiate) of squamous skin tumours during the early stages of tumour progression. We show that CSCs of skin papillomas are localized in a perivascular niche, in the immediate vicinity of endothelial cells. Furthermore, blocking VEGFR2 caused tumour regression not only by decreasing the microvascular density, but also by reducing CSC pool size and impairing CSC renewal properties. Conditional deletion of Vegfa in tumour epithelial cells caused tumours to regress, whereas VEGF overexpression by tumour epithelial cells accelerated tumour growth. In addition to its well-known effect on angiogenesis, VEGF affected skin tumour growth by promoting cancer stemness and symmetric CSC division, leading to CSC expansion. Moreover, deletion of neuropilin-1 (Nrp1), a VEGF co-receptor expressed in cutaneous CSCs, blocked VEGF's ability to promote cancer stemness and renewal. Our results identify a dual role for tumour-cell-derived VEGF in promoting cancer stemness: by stimulating angiogenesis in a paracrine manner, VEGF creates a perivascular niche for CSCs, and by directly affecting CSCs through Nrp1 in an autocrine loop, VEGF stimulates cancer stemness and renewal. Finally, deletion of Nrp1 in normal epidermis prevents skin tumour initiation. These results may have important implications for the prevention and treatment of skin cancers.

Skin squamous cell carcinoma propagating cells increase with tumour progression and invasiveness.

Cancer stem cells have been described in various cancers including squamous tumours of the skin by their ability to reform secondary tumours upon transplantation into immunodeficient mice. Here, we used transplantation of limiting dilution of different populations of FACS-isolated tumour cells from four distinct mouse models of squamous skin tumours to investigate the frequency of tumour propagating cells (TPCs) at different stages of tumour progression. We found that benign papillomas, despite growing rapidly in vivo and being clonogenic in vitro, reformed secondary tumours upon transplantation at very low frequency and only when tumour cells were co-transplanted together with tumour-associated fibroblasts or endothelial cells. In two models of skin squamous cell carcinoma (SCC), TPCs increased with tumour invasiveness. Interestingly, the frequency of TPCs increased in CD34(HI) but not in CD34(LO) SCC cells with serial transplantations, while the two populations initially gave rise to secondary tumours with the same frequency. Our results illustrate the progressive increase of squamous skin TPCs with tumour progression and invasiveness and reveal that serial transplantation may be required to define the long-term renewal potential of TPCs.

Stem cell dynamics, migration and plasticity during wound healing.

Tissue repair is critical for animal survival. The skin epidermis is particularly exposed to injuries, which necessitates rapid repair. The coordinated action of distinct epidermal stem cells recruited from various skin regions together with other cell types, including fibroblasts and immune cells, is required to ensure efficient and harmonious wound healing. A complex crosstalk ensures the activation, migration and plasticity of these cells during tissue repair.

Defining stem cell dynamics and migration during wound healing in mouse skin epidermis.

Wound healing is essential to repair the skin after injury. In the epidermis, distinct stem cells (SCs) populations contribute to wound healing. However, how SCs balance proliferation, differentiation and migration to repair a wound remains poorly understood. Here, we show the cellular and molecular mechanisms that regulate wound healing in mouse tail epidermis. Using a combination of proliferation kinetics experiments and molecular profiling, we identify the gene signatures associated with proliferation, differentiation and migration in different regions surrounding the wound. Functional experiments show that SC proliferation, migration and differentiation can be uncoupled during wound healing. Lineage tracing and quantitative clonal analysis reveal that, following wounding, progenitors divide more rapidly, but conserve their homoeostatic mode of division, leading to their rapid depletion, whereas SCs become active, giving rise to new progenitors that expand and repair the wound. These results have important implications for tissue regeneration, acute and chronic wound disorders.

Abolition of peroxiredoxin-5 mitochondrial targeting during canid evolution.

In human, the subcellular targeting of peroxiredoxin-5 (PRDX5), a thioredoxin peroxidase, is dependent on the use of multiple alternative transcription start sites and two alternative in-frame translation initiation sites, which determine whether or not the region encoding a mitochondrial targeting sequence (MTS) is translated. In the present study, the abolition of PRDX5 mitochondrial targeting in dog is highlighted and the molecular mechanism underlying the loss of mitochondrial PRDX5 during evolution is examined. Here, we show that the absence of mitochondrial PRDX5 is generalized among the extant canids and that the first events leading to PRDX5 MTS abolition in canids involve a mutation in the more 5' translation initiation codon as well as the appearance of a STOP codon. Furthermore, we found that PRDX5 MTS functionality is maintained in giant panda and northern elephant seal, which are phylogenetically closely related to canids. Also, the functional consequences of the restoration of mitochondrial PRDX5 in dog Madin-Darby canine kidney (MDCK) cells were investigated. The restoration of PRDX5 mitochondrial targeting in MDCK cells, instead of protecting, provokes deleterious effects following peroxide exposure independently of its peroxidase activity, indicating that mitochondrial PRDX5 gains cytotoxic properties under acute oxidative stress in MDCK cells. Altogether our results show that, although mitochondrial PRDX5 cytoprotective function against oxidative stress has been clearly demonstrated in human and rodents, PRDX5 targeting to mitochondria has been evolutionary lost in canids. Moreover, restoration of mitochondrial PRDX5 in dog MDCK cells, instead of conferring protection against peroxide exposure, makes them more vulnerable.

Adult interfollicular tumour-initiating cells are reprogrammed into an embryonic hair follicle progenitor-like fate during basal cell carcinoma initiation.

Basal cell carcinoma, the most frequent human skin cancer, arises from activating hedgehog (HH) pathway mutations; however, little is known about the temporal changes that occur in tumour-initiating cells from the first oncogenic hit to the development of invasive cancer. Using an inducible mouse model enabling the expression of a constitutively active Smoothened mutant (SmoM2) in the adult epidermis, we carried out transcriptional profiling of SmoM2-expressing cells at different times during cancer initiation. We found that tumour-initiating cells are massively reprogrammed into a fate resembling that of embryonic hair follicle progenitors (EHFPs). Wnt/ ß-catenin signalling was very rapidly activated following SmoM2 expression in adult epidermis and coincided with the expression of EHFP markers. Deletion of ß-catenin in adult SmoM2-expressing cells prevents EHFP reprogramming and tumour initiation. Finally, human basal cell carcinomas also express genes of the Wnt signalling and EHFP signatures.

Characterization and clinical evaluation of CD10+ stroma cells in the breast cancer microenvironment.

PURPOSE: There is growing evidence that interaction between stromal and tumor cells is pivotal in breast cancer progression and response to therapy. Based on earlier research suggesting that during breast cancer progression, striking changes occur in CD10(+) stromal cells, we aimed to better characterize this cell population and its clinical relevance. EXPERIMENTAL DESIGN: We developed a CD10(+) stroma gene expression signature (using HG U133 Plus 2.0) on the basis of the comparison of CD10 cells isolated from tumoral (n = 28) and normal (n = 3) breast tissue. We further characterized the CD10(+) cells by coculture experiments of representative breast cancer cell lines with the different CD10(+) stromal cell types (fibroblasts, myoepithelial, and mesenchymal stem cells). We then evaluated its clinical relevance in terms of in situ to invasive progression, invasive breast cancer prognosis, and prediction of efficacy of chemotherapy using publicly available data sets. RESULTS: This 12-gene CD10(+) stroma signature includes, among others, genes involved in matrix remodeling (MMP11, MMP13, and COL10A1) and genes related to osteoblast differentiation (periostin). The coculture experiments showed that all 3 CD10(+) cell types contribute to the CD10(+) stroma signature, although mesenchymal stem cells have the highest CD10(+) stroma signature score. Of interest, this signature showed an important role in differentiating in situ from invasive breast cancer, in prognosis of the HER2(+) subpopulation of breast cancer only, and potentially in nonresponse to chemotherapy for those patients. CONCLUSIONS: Our results highlight the importance of CD10(+) cells in breast cancer prognosis and efficacy of chemotherapy, particularly within the HER2(+) breast cancer disease.