A Modified Bleaching Method for Multiplex Immunofluorescence Staining of FFPE Tissue Sections.

Multiplex immunofluorescence (mIF) staining plays an important role in profiling biomarkers and allows investigation of co-relationships between multiple biomarkers in the same tissue section. The Cell DIVE mIF platform (Leica Microsystems) employs an alkaline solution of hydrogen peroxide as a fluorophore inactivation reagent in the sequential staining, imaging, and bleaching protocol for use on FFPE sections. Suboptimal bleaching efficiency, degradation of tissue structure, and loss of antigen immunogenicity occasionally are encountered with the standard bleaching process.

Targeting tumour-associated macrophages in hodgkin lymphoma using engineered extracellular matrix-mimicking cryogels.

Tumour-associated macrophages are linked with poor prognosis and resistance to therapy in Hodgkin lymphoma; however, there are no suitable preclinical models to identify macrophage-targeting therapeutics. We used primary human tumours to guide the development of a mimetic cryogel, wherein Hodgkin (but not Non-Hodgkin) lymphoma cells promoted primary human macrophage invasion. In an invasion inhibitor screen, we identified five drug hits that significantly reduced tumour-associated macrophage invasion: marimastat, batimastat, AS1517499, ruxolitinib, and PD-169316.

Quantitative single-cell analysis of immunofluorescence protein multiplex images illustrates biomarker spatial heterogeneity within breast cancer subtypes.

Background: The extent of cellular heterogeneity in breast cancer could have potential impact on diagnosis and long-term outcome. However, pathology evaluation is limited to biomarker immunohistochemical staining and morphology of the bulk cancer. Inter-cellular heterogeneity of biomarkers is not usually assessed.

Heterogeneity of Circulating Tumor Cell-Associated Genomic Gains in Breast Cancer and Its Association with the Host Immune Response.

Tumor cells that preferentially enter circulation include the precursors of metastatic cancer. Previously, we characterized circulating tumor cells (CTC) from patients with breast cancer and identified a signature of genomic regions with recurrent copy-number gains. Through FISH, we now show that these CTC-associated regions are detected within the matched untreated primary tumors of these patients (21% to 69%, median 55.

Whole-mount pathology of breast lumpectomy specimens improves detection of tumour margins and focality.

Aims: Technical limitations in conventional pathological evaluation of breast lumpectomy specimens may reduce diagnostic accuracy in the assessment of margin and focality. A novel technique based on whole-mount serial sections enhances sampling while preserving specimen conformation and orientation. The aim of this study was to investigate assessment of focality and margin status by the use of whole-mount serial sections versus simulated conventional sections in lumpectomies.

C4ST-1/CHST11-controlled chondroitin sulfation interferes with oncogenic HRAS signaling in Costello syndrome.

Costello syndrome is a pediatric genetic disorder linked to oncogenic germline mutations in the HRAS gene. The disease is characterized by multiple developmental abnormalities, as well as predisposition to malignancies. Our recent observation that heart tissue from patients with Costello syndrome showed a loss of the glycosaminoglycan chondroitin-4-sulfate (C4S) inspired our present study aimed to explore a functional involvement of the chondroitin sulfate (CS) biosynthesis gene Carbohydrate sulfotransferase 11/Chondroitin-4-sulfotransferase-1 (CHST11/C4ST-1), as well as an impaired chondroitin sulfation balance, as a downstream mediator of oncogenic HRAS in Costello syndrome.

Development and evaluation of a robust algorithm for computer-assisted detection of sentinel lymph node micrometastases.

Aims: Increasing the sectioning rate for breast sentinel lymph nodes can increase the likelihood of detecting micrometastases. To make serial sectioning feasible, we have developed an algorithm for computer-assisted detection (CAD) with digitized lymph node sections.

Methods And Results: K-means clustering assigned image pixels to one of four areas in a colourspace (representing tumour, unstained background, counterstained background and microtomy artefacts).

Heightened aberrant deposition of hard-wearing elastin in conduit arteries of prehypertensive SHR is associated with increased stiffness and inward remodeling.

Elastin is a major component of conduit arteries and a key determinant of vascular viscoelastic properties. Aberrant organization of elastic lamellae has been reported in resistance vessels from spontaneously hypertensive rats (SHR) before the development of hypertension. Hence, we have characterized the content and organization of elastic lamellae in conduit vessels of neonatal SHR in detail, comparing the carotid arteries from 1-wk-old SHR with those from Wistar-Kyoto (WKY) and Sprague Dawley (SD) rats.

Neuraminidase-1, a subunit of the cell surface elastin receptor, desialylates and functionally inactivates adjacent receptors interacting with the mitogenic growth factors PDGF-BB and IGF-2.

We recently established that the elastin-binding protein, which is identical to the spliced variant of beta-galactosidase, forms a cell surface-targeted complex with two proteins considered "classic lysosomal enzymes": protective protein/cathepsin A and neuraminidase-1 (Neu1). We also found that cell surface-residing Neu1 can desialylate neighboring microfibrillar glycoproteins and facilitate the deposition of insoluble elastin, which contributes to the maintenance of cellular quiescence. Here we provide evidence that cell surface-residing Neu1 contributes to a novel mechanism that limits cellular proliferation by desialylating cell membrane-residing sialoglycoproteins that directly propagate mitogenic signals.

Aldosterone induces elastin production in cardiac fibroblasts through activation of insulin-like growth factor-I receptors in a mineralocorticoid receptor-independent manner.

Aldosterone is known to regulate electrolyte homeostasis, but it may also contribute to other processes, including the maladaptive remodeling of postinfarct hearts. Because aldosterone has been implicated in the stimulation of collagen production in the heart, we investigated whether it would also affect elastin deposition in cultures of human cardiac fibroblasts. We first demonstrated that treatment with 1 to 50 nmol/L aldosterone leads to a significant increase in collagen type I mRNA levels and in subsequent collagen fiber deposition.

Ellagic and tannic acids protect newly synthesized elastic fibers from premature enzymatic degradation in dermal fibroblast cultures.

Progressive proteolytic degradation of cutaneous elastic fibers, that cannot be adequately replaced or repaired by adult dermal fibroblasts, constitutes a major feature of aging skin. Our present investigations, employing monolayer cultures of human dermal fibroblasts and organ cultures of skin biopsies, were aimed at testing whether the hydrophilic tannic acid (TA) and lipophilic ellagic acid (EA) would protect dermal elastin from exogenous and endogenous enzymatic degradation. Results from both culture systems indicated that dermal fibroblasts, maintained with TA or EA, deposit significantly more elastic fibers than untreated control cultures despite the fact that neither polyphenol enhanced transcription of elastin mRNA or cellular proliferation.

Proteolytic digest derived from bovine Ligamentum Nuchae stimulates deposition of new elastin-enriched matrix in cultures and transplants of human dermal fibroblasts.

Background: Diverse topical products and injectable fillers used for correcting facial wrinkles induce rather short-lived effects because they target replacement of dermal collagen and hyaluronan, matrix components of limited biologic durability.

Objective: Present studies were aimed at stimulation of fully differentiated human dermal fibroblasts to resume deposition of new extracellular matrix rich of elastin, the most durable and metabolically inert component of dermal ECM.

Methods: We have created a novel proteolytic digest of bovine ligamentum nuchae (ProK-60), and tested its potential biological effect on dermal fibroblasts derived from females of different ages.

Retrovirally mediated overexpression of versican v3 reverses impaired elastogenesis and heightened proliferation exhibited by fibroblasts from Costello syndrome and Hurler disease patients.

The phenotypic resemblance of patients with Costello syndrome and Hurler disease has been linked to impaired formation of elastic fibers that coincides with elevated cellular proliferation. Impaired elastogenesis in these diseases associates with respective abnormal accumulation of chondroitin sulfate and dermatan sulfate proteoglycans that induce cell surface shedding of elastin-binding protein (EBP) normally required for intracellular chaperoning of tropoelastin and its assembly into elastic fibers. A variant of the chondroitin sulfate proteoglycan versican, V3, which lacks chondroitin sulfate, has recently been shown to stimulate elastic fiber assembly and decrease proliferation when expressed by retroviral transduction in arterial smooth muscle cells.

Concomitant loss of mitochondria and the DNA repair protein hOGG1 in clear cell carcinoma of the kidney.

The kidney is subjected to DNA oxidative damage from reactive oxygen species generated by free radicals and toxic metabolites, leading to formation of DNA base lesions. One such DNA lesion is 8-oxoguanine, which, if not sufficiently removed, is potentially mutagenic because it can cause G:C to T:A transversion in subsequent DNA replication. The human 8-oxoguanine DNA glycosylase 1 (hOGG1) gene on chromosome 3, a region (3p25-26) that shows frequent loss of heterozygosity in clear cell renal cell carcinoma (CC-RCC), encodes for a DNA repair enzyme capable of excision repair of 8-oxoguanine.

Induced expression of syndecan-1 in the stroma of head and neck squamous cell carcinoma.

Syndecan-1 (CD138), a cell-surface heparan sulfate proteoglycan, is involved in cell-cell, cell-matrix interaction and growth factor binding. Loss of expression of syndecan-1 in tumor cells leads to decreased intercellular cohesion, increased potential for tumor invasiveness, and metastatic spread. Furthermore, induction of syndecan-1 expression in the tumor stroma has been postulated to promote tumor angiogenesis via its binding to growth factors such as basic fibroblast growth factor.

Promoter hypermethylation and inactivation of hMLH1, a DNA mismatch repair gene, in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a multistage process during which adverse genetic alterations accumulate resulting in loss of cell cycle control, selective cell overgrowth, and ultimately formation of malignancy. Among various genetic alterations in HNSCC is increased microsatellite instability (MSI). hMLH1 is one of the major mismatch DNA repair genes, the inactivation of which caused increased MSI in a variety of human cancers including HNSCC.