Multicolour lineage tracing reveals clonal dynamics of squamous carcinoma evolution from initiation to metastasis.

Tumour cells are subjected to evolutionary selection pressures during progression from initiation to metastasis. We analysed the clonal evolution of squamous skin carcinomas induced by DMBA/TPA treatment using the K5CreER-Confetti mouse and stage-specific lineage tracing. We show that benign tumours are polyclonal, but only one population contains the Hras driver mutation. Thus, benign papillomas are monoclonal in origin but recruit neighbouring epithelial cells during growth. Papillomas that never progress to malignancy retain several distinct clones, whereas progression to carcinoma is associated with a clonal sweep. Newly generated clones within carcinomas demonstrate intratumoural invasion and clonal intermixing, often giving rise to metastases containing two or more distinct clones derived from the matched primary tumour. These data demonstrate that late-stage tumour progression and dissemination are governed by evolutionary selection pressures that operate at a multicellular level and, therefore, differ from the clonal events that drive initiation and the benign-malignant transition

A murine living skin equivalent amenable to live cell imaging: analysis of the roles of connexins in the epidermis

Three-dimensional (3D) organotypic models are increasingly used to study the aspects of epidermal organisation and cutaneous wound-healing events. However, these are largely dependent on laborious histological analysis and immunohistochemical approaches. Despite the large resource of transgenic and knockout mice harboring mutations relevant to skin disorders, few organotypic mouse skin models are available. We have developed a versatile in vitro 3D organotypic mouse skin equivalent that reflects epidermal organisation in vivo. The system is optically transparent and ideally suited to real-time analysis using a variety of integrated in situ imaging techniques. As a paradigm for coordination of cellular events, the epidermal gap junction network was investigated and the model displayed predominant connexin 43 (Cx43) expression in basal proliferating cells and Cx26 and Cx30 expression in differentiated keratinocytes. We show that attenuation of Cx43-mediated communication by a Cx mimetic peptide enhanced wound closure rates in keratinocyte monocultures and in the living skin equivalent system, emphasising the utility of the model to systematically unravel the molecular mechanisms underlying epidermal morphogenesis, assess promising therapeutic strategies, and reduce animal experimentation. Furthermore, we visualise epidermal regeneration following injury in real time, thereby facilitating avenues to explore distinctive modes of wound re-epithelialisation in a non-invasive manner

Multicolour lineage tracing reveals clonal dynamics of squamous carcinoma evolution from initiation to metastasis.

Tumour cells are subjected to evolutionary selection pressures during progression from initiation to metastasis. We analysed the clonal evolution of squamous skin carcinomas induced by DMBA/TPA treatment using the K5CreER-Confetti mouse and stage-specific lineage tracing. We show that benign tumours are polyclonal, but only one population contains the Hras driver mutation. Thus, benign papillomas are monoclonal in origin but recruit neighbouring epithelial cells during growth. Papillomas that never progress to malignancy retain several distinct clones, whereas progression to carcinoma is associated with a clonal sweep. Newly generated clones within carcinomas demonstrate intratumoural invasion and clonal intermixing, often giving rise to metastases containing two or more distinct clones derived from the matched primary tumour. These data demonstrate that late-stage tumour progression and dissemination are governed by evolutionary selection pressures that operate at a multicellular level and, therefore, differ from the clonal events that drive initiation and the benign-malignant transition

Label Retaining Cells (LRCs) with Myoepithelial Characteristic from the Proximal Acinar Region Define Stem Cells in the Sweat Gland

<div><p>Slow cycling is a common feature shared among several stem cells (SCs) identified in adult tissues including hair follicle and cornea. Recently, existence of unipotent SCs in basal and lumenal layers of sweat gland (SG) has been described and label retaining cells (LRCs) have also been localized in SGs; however, whether these LRCs possess SCs characteristic has not been investigated further. Here, we used a H2BGFP LRCs system for <i>in vivo</i> detection of infrequently dividing cells. This system allowed us to specifically localize and isolate SCs with label-retention and myoepithelial characteristics restricted to the SG proximal acinar region. Using an alternative genetic approach, we demonstrated that SG LRCs expressed keratin 15 (K15) in the acinar region and lineage tracing determined that K15 labeled cells contributed long term to the SG structure but not to epidermal homeostasis. Surprisingly, wound healing experiments did not activate proximal acinar SG cells to participate in epidermal healing. Instead, predominantly non-LRCs in the SG duct actively divided, whereas the majority of SG LRCs remained quiescent. However, when we further challenged the system under more favorable isolated wound healing conditions, we were able to trigger normally quiescent acinar LRCs to trans-differentiate into the epidermis and adopt its long term fate. In addition, dissociated SG cells were able to regenerate SGs and, surprisingly, hair follicles demonstrating their <i>in vivo</i> plasticity. By determining the gene expression profile of isolated SG LRCs and non-LRCs <i>in vivo</i>, we identified several Bone Morphogenetic Protein (BMP) pathway genes to be up-regulated and confirmed a functional requirement for BMP receptor 1A (BMPR1A)-mediated signaling in SG formation. Our data highlight the existence of SG stem cells (SGSCs) and their primary importance in SG homeostasis. It also emphasizes SGSCs as an alternative source of cells in wound healing and their plasticity for regenerating different skin appendages.</p></div

Acinar sweat gland cells do not contribute to the epidermis during typical wound healing.

<p>(A) K15CrePR/R26LacZ^RU marked sweat gland cells do not contribute to the epidermis at 24 h, (B) 48 h, and (C) 72 h after wounding. (D) BrdU pulse shows that a few SG cells are activated upon injury (inset, arrows) while most SG LRCs remain quiescent. (E) Ki67 staining confirms that the acinar sweat gland region is quiescent while the SG duct and epidermal basal layer is proliferative at 24 h and (F) 48 h. (G) Under normal homeostasis, cells of the SG duct and epidermal basal layer are active in the cell cycle. (H) Corresponding single Ki67 (red) channel. Abbreviations: du, sweat ducts.</p

Molecular characteristics of sweat gland LRCs define BMP signaling as a requirement for SG formation.

<p>(A) GFP+/α6+ sweat gland LRCs and GFP−/α6+ sweat gland non-LRCs basal cells were compared to the basal layer of the sole’s epidermis. The resulting gene expression profiles of these sweat gland LRCs and basal cells were then compared to each other. (B) Gene expression profiles consistently found in two independent microarray analyses from independent biological samples were categorized into ion and protein transport, signaling, transcription, extracellular matrix (ECM) and cell adhesion based on function. (C) Sodium Potassium ATPases were confirmed to be expressed in the sweat glands. (D) Gja1 is confirmed to be expressed in SG LRCs (arrows) as well as non-LRCs. (E) Phospho-Smad2 co-localizes with SG LRCs, arrows. (F) Corresponding phospho-Smad2 and K8 channels. Arrows indicate LRCs marked in panel “E”. (G) Positive phospho-smad1/5/8 staining indicates active BMP signaling in sweat glands. (H) Corresponding phospho-Smad1/5/8 and K8 channels with arrows indicating co-localization with some LRCs. (I) Downgrowth of sweat glands is observed in P1 control paws but is absent (J) in Bmpr1a/K14Cre/K14-H2BGFP KO paws. (K) Similarly, more developed sweat glands are observed in P8 control paws but are still absent (L) in KO mice. (M) The basal layer of the epidermis as well as the sweat glands is proliferative at P1. (N) Although sweat glands are absent in Bmpr1a/K14Cre/K14-H2BGFP KO paws, the epidermal basal layer is still capable of division.</p

Sweat gland LRCs are attached to the basement membrane and possess myoepithelial characteristics.

<p>(A) Sweat gland LRCs are attached to the basement membrane positive for β4 integrin (red) and are (B) found in the basal layer co-localizing with K14. (C,D) Sweat gland LRCs do not express lumenal layer markers K8 and K18, respectively. (E) Within the basal layer, LRCs co-localize with myoepithelial cell marker p63, inset denotes p63 single channel, arrows and (F) with myoepithelial cell marker smooth muscle actin (SMA), arrows. (G,H) Whole mount staining of SGs with laminin with and without DAPI. Arrows denote co-localization of markers with H2BGFP marked LRCs. Abbreviations: LRCs, label-retaining cells; H2BGFP, histone 2B conjugated with green fluorescent protein; SMA, smooth muscle actin; K, keratin.</p