Positive regulatory interactions between YAP and Hedgehog signalling in skin homeostasis and BCC development in mouse skin in vivo

Skin is a highly plastic tissue that undergoes tissue turnover throughout life, but also in response to injury. YAP and Hedgehog signalling play a central role in the control of epidermal stem/progenitor cells in the skin during embryonic development, in postnatal tissue homeostasis and in skin carcinogenesis. However, the genetic contexts in which they act to control tissue homeostasis remain mostly unresolved. We provide compelling evidence that epidermal YAP and Hedgehog/GLI2 signalling undergo positive regulatory interactions in the control of normal epidermal homeostasis and in basal cell carcinoma (BCC) development, which in the large majority of cases is caused by aberrant Hedgehog signalling activity. We report increased nuclear YAP and GLI2 activity in the epidermis and BCCs of K14-CreER/Rosa-SmoM2 transgenic mouse skin, accompanied with increased ROCK signalling and ECM remodelling. Furthermore, we found that epidermal YAP activity drives GLI2 nuclear accumulation in the skin of YAP2-5SA-ΔC mice, which depends on epidermal β-catenin activation. Lastly, we found prominent nuclear activity of GLI2, YAP and β-catenin, concomitant with increased ROCK signalling and stromal fibrosis in human BCC. Our work provides novel insights into the molecular mechanisms underlying the interplay between cell signalling events and mechanical force in normal tissue homeostasis in vivo, that could potentially be perturbed in BCC development

snPATHO-seq: unlocking the FFPE archives for single nucleus RNA profiling

FFPE (formalin-fixed, paraffin-embedded) tissue archives are the largest repository of clinically annotated human specimens. Despite numerous advances in technology, current methods for sequencing of FFPE-fixed single-cells are slow, labour intensive, insufficiently sensitive and have a low resolution, making it difficult to fully exploit their enormous research and clinical potential. Here we introduce single nuclei pathology sequencing (snPATHO-Seq), a sensitive and efficient high-throughput platform to profile the transcriptome of single nuclei extracted from formalin-fixed paraffin-embedded (FFPE) samples. snPATHO-Seq combines an optimised nuclei extraction protocol from archival samples with 10x Genomics probe-based technology targeting the whole transcriptome. We performed direct comparison of the Fixed RNA Profiling (FRP) and established 3’ single cell RNA-Sequencing (scRNA-Seq) workflows through a comprehensive bioinformatics analysis of matched fresh and fixed samples derived from the LNCaP prostate cancer cell line. FRP detected 2.1 times more transcripts in the fixed sample than the 3’ kit did in the fresh sample. Low mitochondrial genes detection using the FRP was translated into 99.9 percent of cells passing the QC filters, compared to 81.6 percent of cells using the v3.1 chemistry. We then optimized snPATHO-Seq and applied it to a human breast cancer metastasis to the liver collected at autopsy and preserved in FFPE, a particularly challenging sample type. Remarkably, at 28,000 reads/cell snPATHO-Seq was able to detect a median of 1850 genes/cell and 3,216 UMI counts/cell. Comparison of snPATHO-Seq with spatial transcriptomics data (10x Genomics Visium FFPE v1) derived from an adjacent section of the same sample revealed a strong correlation, validating the accuracy of the snPATHO-Seq data. Gene expression data from snPATHO-Seq was used to predict cell type composition within each spatial transcriptomic location via deconvolution. Overall, snPATHO-Seq enables high quality and sensitivity snRNA-Seq from preserved tissue samples, unlocking the vast archives of FFPE tissues and thereby allowing extensive retrospective clinical genomic studies

Human BCCs exhibit nuclear YAP and β-catenin in association with ROCK signalling activation and increased ECM collagen deposition.

<p>Representative images of immunohistochemical staining (brown) of Gli2 (A), YAP (B), Thr696-phosphorylated MYPT (C) and β-catenin (D) in normal and human BCCs skin samples. (E) Masson’s trichrome histological staining. IHC, Immunohistochemistry. Scale bars = 20 <b>μ</b>m.</p

Activated ROCK-signalling, increased dermal fibroblast numbers, and dermal fibrosis in the skin of K14-CreER/Rosa-SmoM2 transgenic mice.

<p>(A-G) Immunofluorescence staining and area coverage analysis of dorsal skin tamoxifen- and vehicle-treated K14-CreER/Rosa-SmoM2 transgenic littermate mice detecting Fsp1 (A & B) and Vimentin (C), Phalloidin (G), DIAPH3 (H), Thr696-phosphorylated MYPT1 (I & J), Thr18/Ser19-phosphorylated MLC2 (K & L). (D) Masson’s trichrome histological staining of sections through the dorsal neck skin of tamoxifen- and vehicle-treated K14-CreER/Rosa-SmoM2 mice. (E & J) Dual two-photon SHG and monochromatic transmission (Trans; grayscale in merge) images showing collagen (white in single channel, magenta in merged) in tamoxifen- and vehicle-treated K14-CreER/Rosa-SmoM2 skin sections. Area coverage analysis (5 fields/sample from three mice per genotype) of SHG is quantified. Basement membranes and hair follicles are demarcated with dashed lines. DAPI, 4, 6-diamidino-2-phenylindole. Scale bars = 20 μm.</p

A model outlining the cross-regulatory interactions between epidermal YAP, ROCK, β-catenin and Hh signalling.

<p>(A) Epidermal SmoM2 activates YAP, ROCK signalling and dermal fibroblasts in the dorsal skin of K14-CreER/Rosa-SmoM2 transgenic mice (based on Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.g002" target="_blank">2</a>). (B) Epidermal YAP activates GLI2 mediated by β-catenin activation in the dorsal skin of YAP2-5SA-ΔC transgenic mice (based on Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.g004" target="_blank">4</a>). (C) A model outlining the proposed regulatory interactions between epidermal YAP, Hedgehog and ROCK-dependent mechanosignalling to balance skin regeneration based on our findings (red arrows) and on cited studies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref025" target="_blank">25</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref027" target="_blank">27</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref029" target="_blank">29</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref034" target="_blank">34</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref039" target="_blank">39</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref040" target="_blank">40</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref045" target="_blank">45</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref047" target="_blank">47</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref048" target="_blank">48</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref063" target="_blank">63</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref064" target="_blank">64</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref065" target="_blank">65</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref066" target="_blank">66</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref069" target="_blank">69</a>].</p

YAP activation in the skin of K14-CreER/Rosa-SmoM2 transgenic mice.

<p>H&E histological staining (A) and Immunofluorescence (B) staining of dorsal skin sections of tamoxifen- and vehicle-treated K14-CreER/Rosa-SmoM2 transgenic mice detecting GLI2 and YAP. Quantification of % GLI2-YAP co-positive (C), % GLI2 positive (D) and % YAP positive nuclei (E). (F) qPCR quantification of mRNA levels of <i>Thbs</i>, <i>Ctgf</i>, <i>Inhba</i> and <i>Gli2</i> genes relative to <i>18S</i> in lysates extracted from the dorsal skin of tamoxifen (control) and vehicle-treated K14-CreER/Rosa-SmoM2 transgenic mice. Basement membranes are demarcated with dashed lines. DAPI, 4, 6-diamidino-2-phenylindole. Scale bars = 20 μm.</p

GLI2 activation in the skin of YAP2-5SA-ΔC transgenic mice.

<p>(A) Immunofluorescence staining of dorsal skin sections of YAP2-5SA-ΔC transgenic and wildtype mice detecting GLI2 (green) and YAP (red). Quantification of % YAP-GLI2 co-positive (arrowheads—B), % GLI2 positive (C) and % YAP (D) positive nuclei in the skin sections of YAP2-5SA-ΔC transgenic and wildtype mice. Basement membranes are demarcated with dashed lines. DAPI, 4, 6-diamidino-2-phenylindole. Scale bars = 20 μm.</p

A single-cell and spatially resolved atlas of human breast cancers

Breast cancers are complex cellular ecosystems where heterotypic interactions play central roles in disease progression and response to therapy. However, our knowledge of their cellular composition and organization is limited. Here we present a single-cell and spatially resolved transcriptomics analysis of human breast cancers. We developed a single-cell method of intrinsic subtype classification (SCSubtype) to reveal recurrent neoplastic cell heterogeneity. Immunophenotyping using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) provides high-resolution immune profiles, including new PD-L1/PD-L2+ macrophage populations associated with clinical outcome. Mesenchymal cells displayed diverse functions and cell-surface protein expression through differentiation within three major lineages. Stromal-immune niches were spatially organized in tumors, offering insights into antitumor immune regulation. Using single-cell signatures, we deconvoluted large breast cancer cohorts to stratify them into nine clusters, termed ‘ecotypes’, with unique cellular compositions and clinical outcomes. This study provides a comprehensive transcriptional atlas of the cellular architecture of breast cancer