Cell-surface phenotyping identifies CD36 and CD97 as novel markers of fibroblast quiescence in lung fibrosis.

Fibroblasts play an important role in lung homeostasis and disease. In lung fibrosis, fibroblasts adopt a proliferative and migratory phenotype, with increased expression of α-smooth muscle actin (αSMA) and enhanced secretion of extracellular matrix components. Comprehensive profiling of fibroblast heterogeneity is limited because of a lack of specific cell-surface markers. We have previously profiled the surface proteome of primary human lung fibroblasts. Here, we sought to define and quantify a panel of cluster of differentiation (CD) markers in primary human lung fibroblasts and idiopathic pulmonary fibrosis (IPF) lung tissue, using immunofluorescence and FACS analysis. Fibroblast function was assessed by analysis of replicative senescence. We observed the presence of distinct fibroblast phenotypes in vivo, characterized by various combinations of Desmin, αSMA, CD36, or CD97 expression. Most markers demonstrated stable expression over passages in vitro, but significant changes were observed for CD36, CD54, CD82, CD106, and CD140a. Replicative senescence of fibroblasts was observed from passage 10 onward. CD36- and CD97-positive but αSMA-negative cells were present in remodeled areas of IPF lungs. Transforming growth factor (TGF)-ß treatment induced αSMA and collagen I expression but repressed CD36 and CD97 expression. We identified a panel of stable surface markers in human lung fibroblasts, applicable for positive-cell isolation directly from lung tissue. TGF-ß exposure represses CD36 and CD97 expression, despite increasing αSMA expression; we therefore identified complex surface protein changes during fibroblast-myofibroblast activation. Coexistence of quiescence and activated fibroblast subtypes in the IPF lung suggests dynamic remodeling of fibroblast activation upon subtle changes to growth factor exposure in local microenvironmental niches.

Immunohistochemical analysis of vimentin expression in myocardial tissue from autopsy cases of ischemic heart disease.

Vimentin is a type III intermediate filament cytoskeletal protein that is expressed mainly in cells of mesenchymal origin and is involved in a plethora of cellular functions. In this study, myocardial tissues from patients with ischemic heart disease and a mouse model of acute myocardial infarction were subjected to immunohistochemistry for vimentin. We first examined 26 neutral formalin-fixed, paraffin-embedded myocardial tissue samples from autopsies of patients that were diagnosed with ischemic heart disease within 48 h postmortem. Myocardial cells were negative for vimentin, whereas non-myocardial cells, including vascular endothelium, vascular smooth muscle, fibroblasts, nerve fibers, adipocytes and mesothelial cells, showed positivity. Elevated vimentin expression was observed around myocardial cells undergoing remodeling, suggesting fibroblastic and endothelial proliferation in these locations. By contrast, myocardial foci that were completely fibrotic did not show upregulated vimentin expression. Inflammatory foci including macrophages and neutrophils were clearly visualized with vimentin immunostaining. The same vimentin expression phenomena as those found in human samples were observed in the mouse model. Our study indicates that immunostaining of vimentin as a marker for myocardial remodeling and the dynamics of all non-myocardial cell types may be useful for supplementing conventional staining techniques currently used in the diagnosis of ischemic heart disease.

Similar Characteristics of Endometrial and Endometriotic Epithelial Cells.

Epithelial-mesenchymal transition (EMT) is characterized by the loss of epithelial and acquisition of mesenchymal cell characteristics. Our aim was to assess the epithelial phenotype in the pathogenesis of endometriosis with epithelial and mesenchymal markers. We used 2 structural (keratin-18, -19 [K18, K19]), 1 membrane-associated (mucin-1 [MUC1]), and 2 mesenchymal proteins (vimentin; zinc finger E-box-binding homeobox 1, [ZEB1]) to compare epithelial and mesenchymal characteristics in eutopic endometrium with the 3 endometriotic entities, peritoneal, ovarian, and deep infiltrating endometriosis (DIE). Quantitation showed no differences for K18, K19, and MUC1 between endometrium with and without endometriosis. Also, K18 was not different between endometrium and endometriotic lesions. In contrast, K19 and MUC1 were modestly but significantly decreased in the endometriotic lesions compared to endometrium. However, the maintained expression of epithelial markers in all investigated tissues, regardless of the pathological condition, clearly indicates no loss of the epithelial phenotype. This is further supported by the reduced presence of epithelial vimentin in endometriotic lesions which is in contrast to an increase in stromal vimentin in ectopic endometrium, especially in ovarian endometriosis. The ZEB1 increase in endometriotic lesions, especially in DIE, on the other hand suggests a role of partial EMT in the development of endometriotic lesions, possibly connected with the gain of invasive capabilities or stemness. Taken together, although we found some hints for at least a partial EMT, we did not observe a severe loss of the epithelial cell phenotype. Thus, we propose that EMT is not a main factor in the pathogenesis of endometriosis.

Analysis of circulating tumor cells from lung cancer patients with multiple biomarkers using high-performance size-based microfluidic chip.

Circulating tumor cells (CTCs) have attracted pretty much attention from scientists because of their important relationship with the process of metastasis. Here, we developed a size-based microfluidic chip containing triangular pillar array and filter channel array for detecting single CTCs and CTC clusters independent of tumor-specific markers. The cell populations in chip were characterized by immune-fluorescent staining combining an epithelial marker and a mesenchymal marker. We largely decreased the whole time of detection process to nearly 1.5h with this microfluidic device. The CTCs were subsequently measured in 77 patients with lung cancer and 39 healthy persons. The microfluidic device allowed for the detection of CTCs with apparent high sensitivity and specificity (82.7% sensitivity and 100% specificity). Furthermore, the total CTC counts were found to be elevated in advanced patients with metastases when compared with those without (20.89±14.57 vs 8.428±5.858 cells/mL blood; P<0.01). Combined epithelial marker and mesenchymal marker analysis of CTCs could provide more information about metastasis in patients than only usage of epithelial marker. In conclusion, the development of the size-based microfluidic device for efficient capture of CTCs will enable detailed characterization of their biological properties and values in cancer diagnosis.

Heparanase promotes myeloma progression by inducing mesenchymal features and motility of myeloma cells.

Bone dissemination and bone disease occur in approximately 80% of patients with multiple myeloma (MM) and are a major cause of patient mortality. We previously demonstrated that MM cell-derived heparanase (HPSE) is a major driver of MM dissemination to and progression in new bone sites. However the mechanism(s) by which HPSE promotes MM progression remains unclear. In the present study, we investigated the involvement of mesenchymal features in HPSE-promoted MM progression in bone. Using a combination of molecular, biochemical, cellular, and in vivo approaches, we demonstrated that (1) HPSE enhanced the expression of mesenchymal markers in both MM and vascular endothelial cells; (2) HPSE expression in patient myeloma cells positively correlated with the expression of the mesenchymal markers vimentin and fibronectin. Additional mechanistic studies revealed that the enhanced mesenchymal-like phenotype induced by HPSE in MM cells is due, at least in part, to the stimulation of the ERK signaling pathway. Finally, knockdown of vimentin in HPSE expressing MM cells resulted in significantly attenuated MM cell dissemination and tumor growth in vivo. Collectively, these data demonstrate that the mesenchymal features induced by HPSE in MM cells contribute to enhanced tumor cell motility and bone-dissemination.

A Case of Cutaneous Spindle Cell Squamous Cell Carcinoma / 대한피부과학회지

Spindle cell squamous cell carcinoma (SCSCC) is rare. As in many rare cases, the dignity is difficult to determine. A comprehensive immunohistochemical analysis was conducted, and histopathological findings were also assessed prior to diagnosis. A 77-year-old female patient presented with a dome-shaped ulcerative nodule on her left cheek which was 1.5 cm in diameter and expanding slowly. After resection, conventional histopathological investigation showed spindle-shaped cells with hyperchromatic nuclei infiltrating the dermis in a whorl-like pattern. Furthermore, immunohistochemical investigation revealed SCSCC was positive for cytokeratins (cam 5.2, 34betaE12), and mesenchymal marker (vimentin), but was negative for pancytokeratin (AE1/3). The SCSCC was surgically removed 12 months ago and no relapse has been observed since the removal. Comprehensive immunohistochemical analysis is essential, along with assessment of morphological findings prior to diagnosis. In addition, more case reports on SCSCC should be cumulated to determine a precise prognosis.