Keratinocyte Growth Factor Induces Gene Expression Signature Associated with Suppression of Malignant Phenotype of Cutaneous Squamous Carcinoma Cells

Keratinocyte growth factor (KGF, fibroblast growth factor-7) is a fibroblast-derived mitogen, which stimulates proliferation of epithelial cells. The expression of KGF by dermal fibroblasts is induced following injury and it promotes wound repair. However, the role of KGF in cutaneous carcinogenesis and cancer progression is not known. We have examined the role of KGF in progression of squamous cell carcinoma (SCC) of the skin. The expression of KGF receptor (KGFR) mRNA was lower in cutaneous SCCs (n = 6) than in normal skin samples (n = 6). Expression of KGFR mRNA was detected in 6 out of 8 cutaneous SCC cell lines and the levels were downregulated by 24-h treatment with KGF. KGF did not stimulate SCC cell proliferation, but it reduced invasion of SCC cells through collagen. Gene expression profiling of three cutaneous SCC cell lines treated with KGF for 24 h revealed a specific gene expression signature characterized by upregulation of a set of genes specifically downregulated in SCC cells compared to normal epidermal keratinocytes, including genes with tumor suppressing properties (SPRY4, DUSP4, DUSP6, LRIG1, PHLDA1). KGF also induced downregulation of a set of genes specifically upregulated in SCC cells compared to normal keratinocytes, including genes associated with tumor progression (MMP13, MATN2, CXCL10, and IGFBP3). Downregulation of MMP-13 and KGFR expression in SCC cells and HaCaT cells was mediated via ERK1/2. Activation of ERK1/2 in HaCaT cells and tumorigenic Ha-ras-transformed HaCaT cells resulted in downregulation of MMP-13 and KGFR expression. These results provide evidence, that KGF does not promote progression of cutaneous SCC, but rather suppresses the malignant phenotype of cutaneous SCC cells by regulating the expression of several genes differentially expressed in SCC cells, as compared to normal keratinocytes

Factors Affecting Intracellular Delivery and Release of Hydrophilic Versus Hydrophobic Cargo from Mesoporous Silica Nanoparticles on 2D and 3D Cell Cultures

Intracellular drug delivery by mesoporous silica nanoparticles (MSNs) carrying hydrophilic and hydrophobic fluorophores as model drug cargo is demonstrated on 2D cellular and 3D tumor organoid level. Two different MSN designs, chosen on the basis of the characteristics of the loaded cargo, were used: MSNs with a surface-grown poly(ethylene imine), PEI, coating only for hydrophobic cargo and MSNs with lipid bilayers covalently coupled to the PEI layer as a diffusion barrier for hydrophilic cargo. First, the effect of hydrophobicity corresponding to loading degree (hydrophobic cargo) as well as surface charge (hydrophilic cargo) on intracellular drug release was studied on the cellular level. All incorporated agents were able to release to varying degrees from the endosomes into the cytoplasm in a loading degree (hydrophobic) or surface charge (hydrophilic) dependent manner as detected by live cell imaging. When administered to organotypic 3D tumor models, the hydrophilic versus hydrophobic cargo-carrying MSNs showed remarkable differences in labeling efficiency, which in this case also corresponds to drug delivery efficacy in 3D. The obtained results could thus indicate design aspects to be taken into account for the development of efficacious intracellular drug delivery systems, especially in the translation from standard 2D culture to more biologically relevant organotypic 3D cultures

Altered vascular pattern in granulation tissue of <i>Mmp13^−/−</i> mice.

<p>(<b>A</b>) Sections of experimental granulation tissue of wild-type (WT) and MMP-13 knockout (<i>Mmp13^−/−</i>) mice harvested at indicated time points were immunostained for blood vessels using CD34 as a marker. The <i>arrowheads</i> indicate microvessels and medium sized vessels (diameter<40 µm) and <i>arrows</i> indicate large vessel structures (diameter>40 µm). (s, implant surface; scale bar = 200 µm. (<b>B</b>) The number and the diameter of CD34-positive blood vessels were determined in defined areas of cellular granulation tissues with digital image analysis. *Statistically significant difference in the density of microvessels (<10 µm) at 14 d and of the large vessels (>40 µm) at 21 d (<i>P</i><0.05, MannWhitney U test, n = 5–6).</p

Delayed growth of experimental granulation tissue in <i>Mmp13^−/−</i> mice.

<p>Subcutaneous viscose cellulose sponges (VCS) implanted in wild type (WT) and MMP-13 knockout (<i>Mmp13^−/−</i>) mice were harvested at different time points, as indicated. (<b>A</b>) Hematoxylin-eosin staining of representative sections demonstrating reduced growth of granulation tissue in <i>Mmp13^−/−</i> mice at 21 d. The border of cellular granulation tissue is marked with dashed line. The area enclosed by a square is shown in (C) with higher magnification. (Scale bar = 1 mm). (<b>B</b>) The growth of granulation tissue inside VCS was quantified blinded by determining the portion of cellular tissue relative to the implant area in a tissue section. The border of granulation tissue was determined as exemplified with dashed lines in (A). (*<i>P</i><0.05, Independent samples T-test, n = 5–6). (<b>C</b>) Higher resolution images from the tissue sections presented in (A) showing the border region at the endpoint of the granulation tissue (the area enclosed by a square in A). (s, implant surface; scale bar = 200 µm).</p

Summary of statistically significant biofunctions associated with the molecules that are differently regulated in <i>Mmp13^−/−</i> granulation tissues at day 14 compared to the corresponding WT samples (IPA Functional Analysis).¹

1<p>The threshold with FC>0.5 and p<0.05 was used to determine differentially expressed molecules.</p>2<p>Category of related biofunctions.</p>3<p>The probability that the association between a set of genes in the dataset and a related function is due to random association.</p>4<p>The z-score predicts the direction of change for the function. A positive z-score ≥2 indicates increased function and negative z-score indicates reduced function. An absolute z-score of ≥2 is considered significant.</p>5<p>Others includes categories: Cancer, Tissue Development, Cellular Development, Connective Tissue Disorders, Cardiovascular Disease, Organismal Injury and Abnormalities and Protein Synthesis.</p

Delayed maturation of myofibroblasts in granulation tissue of <i>Mmp13^−/−</i> mice.

<p>Sections of experimental granulation tissue of wild type (WT) and MMP-13 knockout (<i>Mmp13^−/−</i>) mice were stained with α-smooth muscle actin (α-SMA) antibody. (<b>A</b>) The panel shows three representative image pairs from comparable locations of WT and <i>Mmp13^−/−</i> granulation tissue at 7 d. α-SMA-positive myofibroblasts were detected close to implant surface (s). The staining pattern was denser and followed parallel orientation more strictly in WT mice compared to <i>Mmp13^−/−</i> granulation tissue. (<b>B</b>) (Upper panels) at 14 d, α-SMA-staining pattern was strong and comparable in WT and <i>Mmp13^−/−</i>. (Lower panels) representative image pair of WT and <i>Mmp13^−/−</i> granulation tissues at 21 d immunostained for α-SMA. The expression of α-SMA was evident in the inner parts of implants in WT mouse granulation tissue, whereas in the <i>Mmp13^−/−</i> granulation tissue α-SMA-positive cells were mainly abundant close to implant surface. (s, implant surface; scale bar = 100 µm).</p

The expression of <i>Mmp2, Mmp3, Mmp9, Adamts4, and Npy</i> mRNA in <i>Mmp13^−/−</i> and WT mouse granulation tissue.

<p>(<b>A</b>) Microarray data of MMP-13 knockout (<i>Mmp13^−/−</i>) and wild type (WT) mouse granulation tissue at 7, 14 and 21 d were analyzed for MMP gene expression, and the signal intensities are illustrated as a heatmap. (<b>B,C</b>) Total RNA harvested from WT and <i>Mmp13^−/−</i> granulation tissues at the indicated time points was analyzed for <i>Mmp2, Mmp3, Mmp9, Adamts4, and Npy</i> mRNA levels by real-time qRT-PCR. A dot represents a mean of triplicate analysis of a sample with SD≤2% of the mean and the black horizontal bar represents the mean of the experimental replicates. The amplification result of a given mRNA was normalized for β-actin mRNA level in each sample. (*<i>P</i><0.05, **<i>P</i><0.001, ***<i>P</i><0.0001, independent samples T-test, n = 4–6).</p

Comparison of gene expression profiles in granulation tissue of <i>Mmp13^−/−</i> and WT mice.

<p>(<b>A</b>) Microarray data of MMP-13 knockout (<i>Mmp13^−/−</i>) and wild type (WT) mouse granulation tissue at 7, 14 and 21 d were analyzed for differential gene expression by comparing <i>Mmp13^−/−</i> granulation tissue samples to WT. The genes, which showed significant difference (<i>P</i><0.05) and FC>0.75 in the expression are illustrated as heatmap. *Genes with FC>1 and <i>P</i><0.001. (<b>B</b>) Differentially expressed genes at indicated time points were categorized based on molecular function according to Ingenuity Pathway Analysis® (IPA) software.</p

Reduced collagen gel contraction by <i>Mmp13^−/−</i> mouse skin fibroblasts.

<p>(<b>A</b>) Skin fibroblasts (MSF) established from wild type (WT) and MMP-13 knockout (<i>Mmp13^−/−</i>) mice were cultured in mechanically unloaded (floating) 3D collagen gel at density 2×10⁵/ml for 24 h in the presence of 0.5% FCS, 10% FCS or 0.5% FCS+TGF-β (5 ng/ml), as indicated. The cells were fixed, stained with fluorescently labeled phalloidin and Hoechst, and photographed with 20× magnification to observe morphological appearance. In contrast to <i>Mmp13^−/−</i> MSF, WT fibroblasts displayed stellate morphology with numerous thick cell extensions in response to TGF-β or 10% FCS (Scale bar = 10 µm). (<b>B</b>) WT and <i>Mmp13^−/−</i> MSF were cultured in mechanically unloaded 3D collagen gel at density 5×10⁵/ml for 24 and 48 h in the presence of 10% FCS. Contraction of collagen gels was measured from digital images of the gels and is shown as relative to the original gel size. (*<i>P</i><0.005 compared to control, Independent samples T-test, n = 4) (<b>C</b>) WT and <i>Mmp13^−/−</i> MSF were cultured in attached 3D collagen gel at density 5×10⁵/ml for 72 h in the presence of 10% FCS. Subsequently the gels were detached from the well walls and contraction was quantified after 24 h. (*<i>P</i><0.005 compared to control. Independent samples T-test, n = 3). (<b>D</b>) MSF were cultured for 72 h in 3D collagen gel in the presence 10% FCS. Equal aliquots of conditioned media were analyzed in gelatinase zymography.</p

Evaluation of myofibroblast orientation in WT and <i>Mmp13^−/−</i> mouse granulation tissue.

<p>Sections of experimental granulation tissue of wild-type (WT) and MMP-13 knockout (<i>Mmp13^−/−</i>) mice were stained for myofibroblasts with α-SMA antibody and analyzed for myofibroblast orientation. Scoring: +, weak; ++, moderate; +++, strong. Scoring is based on the parallel orientation of myofibroblasts to the implant surface where <i>weak</i> implies negligible orientation, <i>moderate</i> implies lining of occasional myofibroblasts in certain areas, and <i>strong</i> indicates intensive parallel lining of myofibroblast masses. Statistical significance (<i>p</i>) was determined by Pearson's χ²-test. n.s., not significant.</p