Hamzah Babkair

Podoplanin is a novel myoepithelial cell marker in pleomorphic adenoma and other salivary gland tumors with myoepithelial differentiation

The expression of podoplanin, one of the representative immunohistochemical markers for lymphatic endothelium, is upregulated in various kinds of cancers. Based on our previous studies, we have developed a hypothesis that podoplanin plays a role in cell adhesion via its association with extracellular matrix (ECM). Since salivary pleomorphic adenoma is histologically characterized by its ECM-enriched stroma, we firstly wanted to explore the expression modes of podoplanin in pleomorphic adenoma and related salivary tumors by immunohistochemistry. In normal salivary gland, podoplanin was specifically localized in myoepithelial cells, which were also positively labeled by antibodies against P63, of the intercalated duct as well as acini. In pleomorphic adenoma, podoplanin was colocalized with P63 and CD44 in basal cells of glandular structures as well as in stellate/spindle cells in myxochondroid matrices, where perlecan and hyaluronic acid were enriched. The expression of podoplanin was confirmed at both protein and mRNA levels in pleomorphic adenoma cell systems (SM-AP1 and SM-AP4) by using immunofluorescence, western blotting, and reverse transcription polymerase chain reaction. Podoplanin was localized on the cell border as well as in the external periphery of the cells. Moreover, podoplanin expression was also confirmed in tumor cells with myoepithelial differentiation in myoepithelioma and intraductal papilloma. The results indicate that podoplanin can be regarded as a novel myoepithelial marker in salivary gland tumors and suggest that podoplanin’s communication with ECM molecules is essential to phenotypic differentiation to myoepithelial cells.

Extracellular heat shock protein A9 is a novel interaction partner of podoplanin in oral squamous cell carcinoma cells.

In previous studies, we have shown several lines of evidence that podoplanin (PDPN) plays an important role in cell adhesion via its association with extracellular components in neoplastic conditions, though there has been no trial to search for PDPN-interaction molecules in the extracellular milieu. To screen for those molecules, we performed proteomics-based analysis using liquid chromatography-tandem mass spectrometry followed by co-immunoprecipitation for PDPN in ZK-1, an oral squamous cell carcinoma (SCC) cell system whose cell membrane molecules were cross-linked with each other in their extracellular compartments, and we identified heat shock protein (HSP) A9 as one of the extracellular PDPN bound molecules. Effects of transient PDPN knockdown by siRNA in ZK-1 were also comparatively examined for cellular behaviors in terms of HSPA9 expression and secretion. Finally, HSPA9 expression modes were immunohistochemically visualized in oral SCC tissue specimens. HSPA9 was secreted from ZK-1 cells, and the expression and secretion levels of HSPA9 gene and protein were well coordinated with those of PDPN. Immunohistochemically, HSPA9 and PDPN were co-localized in ZK-1 cells and oral SCC foci, especially in the peripheral zone. In conclusion, the results indicate that HSPA9 secreted by oral SCC cells interacts with PDPN on their cell surface in an autocrine manner and regulates their growth and invasiveness.

Hybrid ameloblastoma and adenomatoid odontogenic tumor: report of a case and review of hybrid variations in the literature

Hybrid odontogenic tumors including 2 or more different histologic types have been documented, but their occurrences are not very common. We present a case of hybrid odontogenic tumor composed of ameloblastoma and adenomatoid odontogenic tumor (AOT) arising in the mandibular molar region of a 31-year-old Japanese woman who had a history of familial adenomatous polyposis. The tumor, measuring 10 mm in diameter, was surgically removed from the alveolar bone. Histopathologically, the tumor consisted of both follicular and plexiform types of ameloblastoma in which multiple and smaller foci of AOT were intermingled. There have been 3 reported cases of hybrid ameloblastoma and AOT, all of which presented unicystic types as ameloblastoma components. This, however, is the first report of a hybrid tumor containing an authentic solid-type ameloblastoma compartment and an AOT compartment in a patient with a background of familial adenomatous polyposis.

MFG-E8 expression for progression of oral squamous cell carcinoma and for self-clearance of apoptotic cells

Milk fat globule—epidermal growth factor (EGF)—factor VIII (MFG-E8) is a secreted glycoprotein that promotes clearance of apoptotic cells by bridging phosphatidylserine on apoptotic cells and integrin αvβ3/5 on phagocytes. High expression of MFG-E8 has been reported in various types of cancer in humans. Apoptotic figures are frequently found in the surgical samples of oral squamous cell carcinoma (SCC) and carcinoma in situ, and we have often observed apoptotic carcinoma cells engulfed by macrophages or even by neighboring carcinoma cells. Thus we hypothesized that MFG-E8 might promote engulfment of apoptotic carcinoma cells by living carcinoma cells and that MFG-E8 expressed by carcinoma cells could contribute to tumor progression. The aim of this study was to elucidate the biological role of MFG-E8 in oral SCC. Fifty-three surgical specimens of oral SCC were used for immunohistochemistry for MFG-E8, and the expression profiles were correlated with clinicopathological properties. Also, we examined the MFG-E8 expression patterns and functions using three human oral SCC cell lines. Most of the cases had MFG-E8-positive SCC cells, and the expression of MFG-E8 was correlated with such clinicopathological features as tumor size, pathological stage, locoregional recurrence, scattering invasion pattern, and SCC cell figures engulfing apoptotic SCC cells. The MFG-E8 staining was enhanced in apoptotic SCC cells, some of which were apparently engulfed by the neighboring SCC cells. ZK-1 cells showed high MFG-E8 expression, and its localization was found in the cytoplasm and the cell surface. Transient MFG-E8 knockdown by siRNA in ZK-1 decreased cell proliferation and invasiveness and increased cell death. Thus we have demonstrated that MFG-E8 promotes tumor progression in oral SCC and that it might be involved in the clearance of apoptotic SCC cells by living SCC cells.

Hemophagocytosis-mediated keratinization in oral carcinoma in situ and squamous cell carcinoma: A possible histopathogenesis of keratin pearls

Although the histopathogenetic process of keratin pearls is still poorly understood, acceleration of keratinization in squamous cell carcinoma (SCC) cells may represent one possible therapeutic avenue. Based on our histopathological observations, we have hypothesized that SCC cells are keratinized by phagocytosis of extravasated erythrocytes. To confirm this hypothesis, we firstly examined immature keratin pearls in oral carcinoma in situ (CIS) and mature ones in SCC by immunohistochemistry. Concentric dyskeratotic cells in CIS keratin pearls became positive for keratin (K) 10, K17, heme oxygenase‐1 (HO‐1), or protease activated receptor‐2 (PAR‐2), a candidate regulator for hemophagocytosis. When ZK‐1 cells, an SCC cell system, were incubated with human peripheral blood erythrocytes, or with crude and purified hemoglobins (Hbs), their erythro‐hemophagocytotic activities were confirmed by immunofluorescence. Immunofluorescence signals for K10, K17, and HO‐1 were enhanced due to hemophagocytosis in time‐dependent manners. mRNA expression levels for the three molecules were most enhanced by purified Hb, followed by crude Hb and erythrocytes. K17/K10 mRNA expression levels were more elevated when PAR‐2 was activated in ZK‐1 cells. The results indicated that immature and mature keratin pearls in CIS and SCC were generated by oxidative stresses derived from erythro‐hemophagocytosis, which might mediate HO‐1 expression and be regulated by PAR‐2. Thus, hemorrhage from the rupture of blood vessels can be one of the triggers for keratin pearl formation in oral CIS and SCC. J. Cell. Physiol. 228: 1977–1988, 2013.

Aberrant expression of the tight junction molecules claudin-1 and zonula occludens-1 mediates cell growth and invasion in oral squamous cell carcinoma☆

We reported that altered cell contact mediated by E-cadherin is an initial event in the pathogenesis of oral epithelial malignancies. To assess other effects of cell adhesion, we examined the expression levels of tight junction (TJ) molecules in oral carcinoma in situ (CIS) and squamous cell carcinoma (SCC). To identify changes in the expression of TJ molecules, we conducted an analysis of the immunohistochemical profiles of claudin-1 (CLDN-1) and zonula occludens-1 (ZO-1) in surgical specimens acquired from patients with oral SCC containing foci of epithelial dysplasia or from patients with CIS. We used immunofluorescence, Western blotting, reverse-transcription polymerase chain reaction, and RNA interference to evaluate the functions of CLDN-1 and ZO-1 in cultured oral SCC cells. TJ molecules were not detected in normal oral epithelial tissues but were expressed in SCC/CIS cells. ZO-1 was localized within the nucleus of proliferating cells. When CLDN-1 expression was inhibited by transfecting cells with specific small interference RNAs, SCC cells dissociated, and their ability to proliferate and invade Matrigel was inhibited. In contrast, although RNA interference-mediated inhibition of ZO-1 expression did not affect cell morphology, it inhibited cell proliferation and invasiveness. Our findings indicated that the detection of TJ molecules in the oral epithelia may serve as a marker for the malignant phenotype of cells in which CLDN-1 regulates proliferation and invasion.

Simultaneous immunolocalization of desmoglein 3 and IgG4 in oral pemphigus vulgaris: IgG4 predominant autoantibodies in its pathogenesis.

BACKGROUND Oral pemphigus vulgaris (PV), an autoimmune blistering disease, is mainly mediated by autoantibodies against desmoglein (Dsg) 3. However, no attention has been paid to IgG subclasses of the autoantibodies against Dsg3 in the diagnostic procedure for PV. Thus, our aim in this study was to investigate whether Dsg3 and any of IgG subclasses are immunohistochemically colocalized in tissue sections of PV oral mucosa. MATERIALS AND METHODS Serial sections cut from formalin-fixed paraffin blocks of biopsy specimens of 9 PV cases and those of normal buccal mucosa surgically removed for fibro-epithelial polyps were comparatively examined for immunohistochemical localizations for Dsg3, IgG4, and IgG. RESULTS Dsg3 was demonstrated in a dot-like pattern on the cell border and in the cytoplasm of the whole epithelial layer in both normal and PV specimens, while its staining was irregular among floating epithelial sheets of PV. IgG4 was also demonstrated in a punctuated fashion on the cell border among floating epithelial sheets, which was nearly identical to the immunohistochemical profile of Dsg3. In addition to being detected in the epithelial part, IgG4 signals were prominently localized in plasma cells scattered in the granulation tissue, where ratios of IgG4-positive (+) plasma cells to IgG+ cells were extraordinarily higher (mean 28%) than those in normal mucosa. DISCUSSION These findings confirmed for the first time that autoantibodies against Dsg3 are mainly composed of IgG4 in oral PV and that the combined immunohistochemistry for Dsg3 and IgG4 can be a valuable aid in confirming a histopathological diagnosis of PV.

Three-dimensional visualization of perlecan-rich neoplastic stroma induced concurrently with the invasion of oral squamous cell carcinoma

BACKGROUND We have demonstrated the induction of perlecan-rich stroma of oral squamous cell carcinoma (SCC) on and after its start of invasion. However, it remains unknown how such a neoplastic stroma is actually arranged in tumor tissues. METHODS To this end, tissue microarray samples, in which keratin and perlecan were contrastively labeled by immunohistochemistry, were three-dimensionally analyzed using digital images and image analysis software to demonstrate the relationship between SCC foci and the perlecan-positive stromal space or that between carcinoma in situ (CIS) and invasive SCC foci. RESULTS The three-dimensional (3D) reconstruction demonstrated three kinds of perlecan profiles for inside (I) and outside (O) areas of the carcinoma cell focus: mode 1, I(+)/O(-) ; mode 2, I(+)/O(+) ; and mode 3, I(-)/O(+). Mode 1 was seen in CIS as well as SCC tumor massifs in the surface part. Mode 2 was seen in small SCC foci, which seemed isolated in 2D sections but were mostly continuous with the tumor massif in 3D reconstructions. Mode 3 was limited to small SCC foci, which were truly segregated from the tumor massif. CONCLUSIONS The results indicated that the 2D SCC focus isolation could not be regarded as invasion but that the SCC foci surrounded by perlecan-positive stroma (modes 2 and 3) could be regarded as a more objective measure for invasion of SCC. This is the first 3D tissue-level demonstration of the neoplastic stroma space induced with oral SCC invasion, the presence of which we have predicted based on our previous 2D and tissue culture evidence.

Tumour-associated macrophages are recruited and differentiated in the neoplastic stroma of oral squamous cell carcinoma

To confirm our hypothesis that macrophages recruited to fight against oral squamous cell carcinoma (SCC) invasion are functionally differentiated within neoplastic stromata, we analysed arrangements of macrophage subtypes and cancer-associated fibroblasts (CAFs) in their association with blood vasculatures in the neoplastic stroma. Surgical specimens of oral SCC were immunohistochemically examined for macrophage phenotypes (CD68, CD163, and CD204) and stromal environments (perlecan, connexin 43, and CD31). Human monocytes were co-cultured with ZK-1 cells of oral SCC origin in different culture conditions. SCC stromata were divided into two types: fascicular (fibroblast-rich) and reticular (perlecan-rich). Regardless of stromal types, CD68 positive (+)/CD163+/CD204+ macrophages were recruited when blood vessels were abundant. Connexin 43+ fibroblasts were enriched in the fascicular stroma, where blood vessels were depleted. In co-culture experiments, monocytes, in the presence of ZK-1 cells, showed TNF-α(low)/IL-12(low) and IL-10(high)/VEGF(high)/MMP-9(high) with increased expression levels for fibronectin and perlecan. With direct contact with monocytes, SCC cells also expressed CD68 and CD163. SCC stromata were characterised by CD163+/CD204+ tumour-associated macrophages (TAMs) and connexin 43+ CAFs. TAMs are differentiated from monocytes by the physical contact with oral SCC cells in the perlecan-rich neoplastic stroma, which is also induced by the cross-talk between SCC cells and stromal cells including TAMs.

Keratin pearl degradation in oral squamous cell carcinoma: reciprocal roles of neutrophils and macrophages

BACKGROUND We have reported that neutrophilic infiltration was associated with round-shaped dyskeratosis foci, a kind of keratin pearl, of oral carcinoma in situ and that those inflammatory cells are recruited from intra-epithelially entrapped blood vessels. Based on these lines of evidence, we have formulated a hypothesis that keratin pearls are terminally degraded by neutrophils. To confirm this hypothesis, we investigated immunohistochemically stepwise degradation of keratin pearls in oral squamous cell carcinoma (SCC) to clarify any other type scavenger cells in addition to neutrophils are involved in this particular degradation process. METHODS Neutrophils (neutrophil elastase) and macrophage subpopulations (CD68, CD163 and CD204) were immunohistochemically localized in 30 cases of oral SCC with typical round-shaped keratin pearls. SCC cells were revealed by immunohistochemistry for keratin (K) 17, and blood vessels were demonstrated by CD31. RESULTS Keratin pearl degradation process was divided into four steps: (i) intact stage: no macrophage infiltration but minimal neutrophils were found in keratin pearls; (ii) neutrophil recruit stage: no macrophage infiltration but focal neutrophilic infiltration within the pearls; (iii) neutrophil predominant stage: dense neutrophil infiltration with minimal macrophages and segregated keratinized cancer cells strongly positive for K17; and (iv) macrophage predominant stage: dense infiltration of CD68-, CD163 (mononuclear)- and CD204 (multinucleated)-positive macrophages engulfing detached keratinized SCC cells. CONCLUSION Keratin pearl degradation in oral SCC is strictly regulated by two types of scavenger cells: neutrophils, which perform initial tasks, and macrophages, which reciprocally take over from neutrophils the role to finalize the degradation processes.