Multiple Epithelioid Hemangiomas with Orbital Involvement

Epithelioid hemangioma, also known as angiolymphoid hyperplasia with eosinophilia, is a cutaneous angioproliferative lesion that follows a benign clinical course. It is most frequently localized in the skin of the head and neck region; although it may sometimes arise deeper in soft tissues, orbital involvement is rare. Here we describe a patient who developed multiple epithelioid hemangiomas, including an intraorbital lesion. The histopathological parallels with other reactive and neoplastic lesions as well as therapeutic options are discussed

Cysteine cathepsins B, X and K expression in peri-arteriolar glioblastoma stem cell niches

Glioblastoma (GBM) is the most lethal brain tumor also due to malignant and therapy-resistant GBM stem cells (GSCs) that are localized in protecting hypoxic GSC niches. Some members of the cysteine cathepsin family of proteases have been found to be upregulated in GBM. Cathepsin K gene expression is highly elevated in GBM tissue versus normal brain and it has been suggested to regulate GSC migration out of the niches. Here, we investigated the cellular distribution of cathepsins B, X and K in GBM tissue and whether these cathepsins are co-localized in GSC niches. Therefore, we determined expression of these cathepsins in serial paraffin sections of 14 human GBM samples and serial cryostat sections of two samples using immunohistochemistry and metabolic mapping of cathepsin activity using selective fluorogenic substrates. We detected cathepsins B, X and K in peri-arteriolar GSC niches in 9 out of 16 GBM samples, which were defined by co-expression of the GSC marker CD133, the niche marker stromal-derived factor-1α (SDF-1α) and smooth muscle actin as a marker for arterioles. The expression of cathepsin B and X was detected in stromal cells and cancer cells throughout the GBM sections, whereas cathepsin K expression was more restricted to arteriole-rich regions in the GBM sections. Metabolic mapping showed that cathepsin B, but not cathepsin K is active in GSC niches. On the basis of these findings, it is concluded that cathepsins B, X and K have distinct functions in GBM and that cathepsin K is the most likely GSC niche-related cathepsin of the three cathepsins investigated

TRIM28 and β-actin identified via nanobody-based reverse proteomics approach as possible human glioblastoma biomarkers.

Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry analysis revealed two proteins, TRIM28 and β-actin, that were up-regulated in the GBM stem-like cells compared to the controls

Schematic presentation of the structure of TRIM28.

<p>The conserved RBCC motif is present at the N-terminal domain; the variable C-domain includes the PHD and BROMO domains. Image adapted from Hatakeyama <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113688#pone.0113688-Hatakeyama1" target="_blank">[45]</a>. Author's approval was obtained for the use of this image; original image is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113688#pone.0113688.s002" target="_blank">Figure S2</a>.</p

Schematic presentation of the whole workflow.

<p>1. Material preparation – expanding a GBM cell line, laminin grown and enriched in stem-like cells, 2. Llama immunization with whole GBM cells, 3. Construction of a nanobody library, 4. Phage display cycle – enrichment of antigen specific nanobodies on various biological samples (whole protein extract from GBM tissues, membrane protein-enriched fractions from GBM tissues and GBM stem-like cell lines), 5. ELISA - selection of GBM specific nanobodies, 6. Nanobody production – large scale production and purification of specific nanobodies, 7. Nanobody:antigen pairs – immobilizing the nanobodies and binding to the corresponding antigens, 8. Antigen identification by mass spectrometry, 9. Antigen validation by Western blot. See Materials and methods for additional experimental details.</p

Relative band intensities of antigens validated with Western blot.

<p>The relative band intentisy of the antigens, TRIM28 and β-actin, was calculated as the ratio between arbitrary units of the band of the antigen and the arbitrary units of the band of the internal control, GAPDH [AU(antigen)/AU(GAPDH)]. Samples: GSCc – cytosolic/nuclear protein fraction isolated from GBM stem-like cell lines; GBMc – cytosolic/nuclear protein fraction isolated from GBM tissues; NBTc – cytosolic/nuclear protein fraction isolated from human brain samples.</p

Sequences of the nanobodies that were chosen for the large-scale production.

<p>The amino acid sequence of the CDR3 regions of the nanobodies is given in alphabetic order.</p

Antigen validation.

<p>Western blot membrane after probing with a monoclonal anti-GAPDH antibody (36 kDa band) used as an internal control for amount of protein loaded in each lane, a monoclonal anti-β-actin antibody (48 kDa band) and monoclonal anti-TRIM28 antibody (100 kDa band). Samples: GSCm – membrane protein-enriched extract isolated from GBM stem-like cell lines; GBMm – membrane protein-enriched extract isolated from GBM tissues; NBTm – membrane protein-enriched extract isolated from human brain samples; GSCc – cytosolic/nuclear protein fraction isolated from GBM stem-like cell lines; GBMc – cytosolic/nuclear protein fraction isolated from GBM tissues; NBTc – cytosolic/nuclear protein fraction isolated from human brain samples; GBMw – whole protein extract from GBM tissues; NBTw – whole protein extract from human brain samples.</p