Expression of FHOD1 in various tissues and cell types.

*<p>0 =  no staining, + =  weak staining, ++ =  moderate staining, +++ =  strong staining.</p

Transcriptomic analysis of the UT-SCC-43A, UT-SCC-43B and 43A-SNA oral SCC cell lines.

<p>A) Microarray transcriptomic profiling of UT-SCC-43A, UT-SCC-43B and 43A-SNA cells. Transcripts significantly altered in UT-SCC-43B cells in comparison with UT-SCC-43A cells are indicated by the blue circle and transcripts altered in 43A-SNA cells in comparison with UT-SCC-43A are indicated by the green circle. The number of genes altered in both comparisons is shown in the overlapping area. The number of up-regulated genes is shown on the top and down-regulated genes on the bottom. B) mRNA level alterations for selected epithelial (top) and mesenchymal (bottom) genes. Depicted genes encode following proteins: CDH1 =  E-cadherin, CLDN3 =  Claudin 3, CLDN 7 =  Claudin 7, KRT5 =  Keratin 5, KRT6B  =  Keratin 6b, CDH2 =  N-Cadherin, VIM  =  Vimentin. C) mRNA levels for formins with significant alterations in both UT-SCC-43B and 43A-SNA cells that were subsequently confirmed by RT-PCR. D) mRNA levels for formins confirmed by RT-PCR.</p

FHOD1 siRNA knockdown changes the morphology of UT-SCC-43B cells and reduces the number of stress fibres.

<p>A) Western blotting shows that FHOD1 siRNA treatment significantly reduces FHOD1 expression. The cells are still E-cadherin negative and the expression of N-cadherin is unaltered. B) Cells treated with non-targeting siRNA (upper panel) express FHOD1 and have a mesenchymal phenotype. Stress fibres are abundant. FHOD1 siRNA abolishes FHOD1 staining (lower panel). siRNA treated cells have less actin stress fibres and are morphologically rounder and flatter. Nuclei are stained with DAPI (blue). C) F-actin staining is significantly reduced in FHOD1 siRNA treated cells. Phalloidin staining intensity is reduced by 49%. Bars indicate standard error of mean. *** p<0.0001. AU  =  arbitrary units.</p

FHOD1 is upregulated in clinical oral SCC, as well as <i>in vitro</i> in SCC cells with EMT features.

<p>A) In normal or non-neoplastic stratified squamous epithelium, no FHOD1 can be detected (top). In invasive squamous cell carcinomas, moderate to strong FHOD1 immunoreactivity is seen in spindle-shaped cells at the invasive front that on morphological grounds have undergone EMT (bottom; details in inset). In the tumour bulk, which consists of cells with an epithelial morphology, only weak immunoreactivity is present. Scale bar: 200 μm. B) Western blot analysis of cell lines show that the epithelial SCC cell line UT-SCC-43A does not express detectable FHOD1, while both the spontaneous EMT cell line UT-SCC-43B and the Snail-induced EMT cell line 43A-SNA express FHOD1. UT-SCC-43A expresses the epithelial marker E-cadherin but not N-cadherin, whereas UT-SCC43-B and 43A-SNA express N-cadherin but not E-cadherin. C) F-actin organization of UT-SCC-43A (upper panel) is typically epithelial, with distinct cell submembraneous filaments and scant stress fibres. UT-SCC-43B shows features of mesenchymal organization (middle panel). Cell-cell contacts are few, the cells are elongated and contain lamellopodia, filopodia and stress fibres. The insert (bottom panel) shows that a fraction of FHOD1 co-localizes with stress fibres in UT-SCC-43B cells (arrowheads). Nuclei are stained with DAPI (blue). D) FHOD1 upregulation in UT-SCC-43B cells is dependent of PI3K signalling. Treatment with MEK 1/2 inhibitor U0126 reduces phosphorylation of ERK 1/2 but does not influence FHOD1 expression. In contrast, PI3K inhibition by LY294992 markedly reduces FHOD1 expression. The reduction of p-Akt indicates that the pathway is efficiently inhibited.</p

FHOD1 silencing inhibits proteolytic activity and invadopodia formation.

<p>A) Images from a zymography assay performed with untreated, nt siRNA and FHOD1 siRNA treated SCC-43B cells. Degradation of Cy 3 labelled gelatin is reduced in FHOD1 siRNA treated cells. B) Quantification of the degraded area/cell. C) Invadopodia formation is visualized by phalloidin staining, which reveals the comet- or ring-shaped actin structures on the ventral cell surface (arrowheads). Invadopodia are present in a larger proportion of untreated and control cells than in FHOD1 siRNA treated cells. D) Quantification of percentage of cells with invadopodia. (* P<0.05). N.S.  =  not significant.</p

Feasibility of experimental BT4C glioma models for somatostatin receptor 2-targeted therapies

<div><p></p><p>Somatostatin receptor subtype 2 (sstr₂) is regarded as a potential target in malignant gliomas for new therapeutic approaches. Therefore, visualizing and quantifying tumor sstr₂ expression in vivo would be highly relevant for the future development of sstr₂-targeted therapies. The purpose of this study was to evaluate sstr₂ status in experimental BT4C malignant gliomas.</p><p><b>Methods.</b> Rat BT4C malignant glioma cells were injected into BDIX rat brain or subcutaneously into nude mice. Tumor uptake of [⁶⁸Ga]DOTA-(Tyr³)-Octreotide ([⁶⁸Ga]DOTATOC), a somatostatin analog binding to sstr₂, was studied by positron emission tomography/computed tomography (PET/CT). Additionally, subcutaneous tumor-bearing mice underwent PET imaging with 5-deoxy-5-[¹⁸F]fluororibose-NOC ([¹⁸F]FDR-NOC), a novel glycosylated peptide tracer also targeting sstr₂. Ex vivo tissue radioactivity measurements, autoradiography and immunohistochemistry were performed to study sstr₂ expression.</p><p><b>Results.</b> Increased tumor uptake of [⁶⁸Ga]DOTATOC was detected at autoradiography with mean tumor-to-brain ratio of 68 ± 30 and tumor-to-muscle ratio of 9.2 ± 3.8 for rat glioma. High tumor-to-muscle ratios were also observed in subcutaneous tumor-bearing mice after injection with [⁶⁸Ga]DOTATOC and [¹⁸F]FDR-NOC with both autoradiography (6.7 ± 1.5 and 4.3 ± 0.8, respectively) and tissue radioactivity measurements (6.5 ± 0.8 and 4.8 ± 0.6, respectively). Furthermore, sstr₂ immunohistochemistry showed positive staining in both tumor models. However, surprisingly low tumor signal compromised PET imaging. Mean SUV_max for rat gliomas was 0.64 ± 0.28 from 30 to 60 min after [⁶⁸Ga]DOTATOC injection. The majority of subcutaneous tumors were not visualized by [⁶⁸Ga]DOTATOC or [¹⁸F]FDR-NOC PET.</p><p><b>Conclusions.</b> Experimental BT4C gliomas show high expression of sstr₂. Weak signal in PET imaging, however, suggests only limited benefit of [⁶⁸Ga]DOTATOC or [¹⁸F]FDR-NOC PET/CT in this tumor model for in vivo imaging of sstr₂ status.</p></div