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Histopathologically, oral squamous cell carcinoma (OSCC) consists of well-defined interfaces with adjacent non-cancerous epithelium. Previously, we found that SCC tissues expressed higher levels of specific proteins at this interface. Ladinin-1 (LAD1) is one of the specific molecules that has increased expressions in cancer fronts; however, its function in OSCC is unknown. Therefore, this study aimed to elucidate the function of LAD1 in human OSCC cells. LAD1 was localized on the actin arc at the distal periphery of cell clusters in the OSCC cell lines HSC-2, HSC-3, and HSC-4. When LAD1 was knocked down, cellular migration was repressed in wound scratch assays but was reversed in three-dimensional collagen gel invasion assays. Characteristic LAD1 localization along actin arcs forming the leading edge of migrating cells was diminished with loss of filopodia formation and ruffling in knockdown cells, in which the expression levels of cell motility-related genes-p21-activated kinase 1 (PAK1) and caveolin-1 (CAV1)-were upregulated and downregulated, respectively. LAD1 expression was also associated with the downregulation of vimentin and increased histological differentiation of OSCC. These results suggest that LAD1 is involved in actin dynamics during filopodia and lamellipodia formation, and in maintaining the epithelial phenotype of OSCC cells., (© 2024. The Author(s).)

Pemphigus vulgaris (PV) is a rare autoimmune disease characterized by blisters on the skin and mucous membrane. Since it often appears in the oral mucosa first, it may be diagnosed by oral mucosal cytology. Although the cytologic finding is characterized by acantholytic cells, that is, Tzanck cells, it is important to distinguish PV from neoplastic lesions of the oral mucosal epithelium, including differentiation from atypical parabasal/basal cells, which appear in squamous cell carcinoma (SCC). In this study, we examined the cellular findings in two cases of PV and a case of well-differentiated SCC with loss of epithelial cell cohesion. The samples were prepared using liquid-based cytology, which showed small round-shaped and deeply stained atypical, orangeophilic keratinocytes not only in SCC but also in PV, which made differentiation between the two difficult. However, Tzanck cells found in PV differ from the deep atypical parabasal/basal cells of SCC, suggesting that the cell outline is indistinct and small protrusions and brush-like structures are observed. This feature of Tzanck cells may be useful in cytological judgment., (© 2023 Wiley Periodicals LLC.)

Cholesterol plays an important role in cancer progression, as it is utilized in membrane biogenesis and cell signaling. Cholesterol-lowering drugs have exhibited tumor-suppressive effects in oral squamous cell carcinoma (OSCC), suggesting that cholesterol is also essential in OSCC pathogenesis. However, the direct effects of cholesterol on OSCC cells remain unclear. Here, we investigated the role of cholesterol in OSCC with respect to caveolin-1 (CAV1), a cholesterol-binding protein involved in intracellular cholesterol transport. Cholesterol levels in OSCC cell lines were depleted using methyl-β-cyclodextrin and increased using the methyl-β-cyclodextrin-cholesterol complex. Functional analysis was performed using timelapse imaging, and CAV1 expression in cholesterol-manipulated cells was investigated using immunofluorescence and immunoblotting assays. CAV1 immunohistochemistry was performed on surgical OSCC samples. We observed that cholesterol addition induced polarized cell morphology, along with CAV1 localization at the trailing edge, and promoted cell migration. Moreover, CAV1 was upregulated in the lipid rafts and formed aggregates in the plasma membrane in cholesterol-added cells. High membranous CAV1 expression in tissue specimens was associated with OSCC recurrence. Therefore, cholesterol promotes the migration of OSCC cells by regulating cell polarity and CAV1 localization to the lipid raft. Furthermore, membranous CAV1 expression is a potential prognostic marker for OSCC patients.

Background: Spindle cell squamous cell carcinoma is an uncommon variant of squamous cell carcinoma; its diagnosis is sometimes challenging because it histopathologically resembles neoplastic or reactive spindle cell lesions of mesenchymal origins. Here, we report a rare case of spindle cell squamous cell carcinoma exhibiting prominent neutrophil phagocytosis., Case Presentation: A 69-year-old Japanese man presented with pain and a polypoid mass on the lower left gingiva. He had received chemoradiotherapy for squamous cell carcinoma of the buccal mucosa 15 years prior to this consultation. In addition, he was treated for mandibular osteonecrosis 6 years after chemoradiotherapy without evidence of cancer recurrence. A biopsy revealed atypical spindle or pleomorphic cells scattered in the edematous and fibrin-rich stroma; however, no malignant squamous components were apparent. These atypical cells frequently contained neutrophils within their cytoplasm that formed cell-in-cell figures. Immunohistochemically, the atypical cells were negative for cytokeratins, epithelial membrane antigen, and E-cadherin, but positive for p63, vimentin, and p53. Although these findings suggested spindle cell squamous cell carcinoma, it was difficult to reach a definitive diagnosis. Based on a clinical diagnosis of a malignant tumor, the patient underwent a hemimandibulectomy. The surgically resected specimen had a typical spindle cell squamous cell carcinoma histology consisting of biphasic spindle cells and conventional squamous cell carcinoma components. Moreover, the surgical specimen also exhibited spindle tumor cells that frequently included neutrophils, around which intense staining for lysosomal-associated membrane protein 1 and cathepsin B was observed. This suggested that the cell-in-cell figures represent active neutrophil phagocytosis by tumor cells, and not emperipolesis., Conclusion: The presence of neutrophil phagocytosis may be a potent indicator of malignancy., (© 2021. The Author(s).)

A 75-year-old woman with liver cirrhosis was admitted for treatment of portal vein thrombosis (PVT). Computed tomography (CT) showed PVT, massive ascites, and multiple abdominal organ embolism. Blood tests revealed a decreased liver function (Child-Pugh grade C). Language impairment followed by progressive left hemi-paralysis was subsequently detected. Magnetic resonance imaging revealed multiple small acute cerebral infarctions and, on CT, a 30-mm bladder tumour; a biopsy specimen examination showed squamous cell carcinoma. Her general condition worsened rapidly, and the best supportive care was chosen. Our findings suggest that, in patients with PVT, Trousseau syndrome should be considered, even in cases of liver cirrhosis.

Apoptotic cell death frequently occurs in human cancer tissues including oral squamous cell carcinoma (SCC), wherein apoptotic tumor cells are phagocytosed not only by macrophages but also by neighboring tumor cells. We previously reported that the engulfment of apoptotic SCC cells by neighboring SCC cells frequently occurs at the invading front. Therefore, we hypothesized that the phagocytosis of these apoptotic cells by tumor cells contributes to disease progression. Herein, using cultured oral SCC cells, we aimed to confirm whether tumor cells actually phagocytose apoptotic cells and to examine whether cellular activities are regulated by the phagocytosis of apoptotic cells. Co-culture experiments showed that living cells could ingest apoptotic cells into phagolysosomes. NSC23766, an inhibitor of Rac1, which is a key regulator of phagocytic cup formation in professional phagocytes, dramatically suppressed the phagocytosis of apoptotic cells by living cells. Additionally, cell migration and the secretion of DKK1, a tumor-promoting protein, were enhanced by co-culture with apoptotic cells, whereas NSC23766 inhibited these effects. These results show that tumor cells can actively phagocytose apoptotic neighbors in a Rac1-dependent manner and that such activity increases their migration. The regulation of apoptotic cell phagocytosis thus represents new directions for therapeutic intervention for oral cancer., Competing Interests: Declaration of competing interest We declare that we have no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Transcription factor SRY‑box 9 (SOX9) is a key regulator of chondrocyte differentiation and sex determination, and it is also involved in the progression of various types of human cancer. However, its putative association with oral squamous cell carcinoma (OSCC) remains elusive. The aim of the present study was to investigate the expression profiles of SOX9 in various oral epithelial lesions, including OSCC. We performed immunohistochemical analysis of SOX9 expression in surgical specimens of OSCC, which simultaneously exhibited different grades of epithelial lesions, and analyzed the correlation between SOX9 expression and several clinicopathological factors. Moreover, we performed immunofluorescent staining, western blot analysis and real‑time reverse transcription‑polymerase chain reaction to assess SOX9 expression in OSCC HSC‑3 (a metastatic cell line) and HSC‑4 (a non‑metastatic cell line) cell lines. In surgical specimens, SOX9 expression was detected in the nuclei of proliferating cells in areas with epithelial dysplasia and carcinoma in situ, but not in areas with normal epithelia. Nuclear SOX9 expression was observed in most SCC cells. Notably, cytoplasmic SOX9 expression was confirmed only in some SCC cells; however, cytoplasmic SOX9 expression was significantly and positively correlated with poor clinical outcomes. Both protein and mRNA expression of SOX9 were significantly higher in the HSC‑3 cell line than that in the HSC‑4 line. Notably, however, only HSC‑3 cells exhibited cytoplasmic localization of SOX9 expression. Our findings indicate that SOX9 may be involved in the tumorigenesis and progression of OSCC. Furthermore, its cytoplasmic expression represents a potential predictive biomarker for tumor aggressiveness and OSCC prognosis.

Oral squamous cell carcinomas (SCCs) are frequently associated with pre-invasive lesions including carcinoma in-situ (CIS), and CISs further form lateral interfaces against surrounding normal or dysplastic epithelia (ND). At the interface where keratin (K) 17 positive (+) SCC/CIS cells are in contact with K13+ ND cells, "cell competition" must be evoked between two such different cell types. Thus, the aim of this study was to characterize the histopathology of the SCC/CIS-ND interface and to determine protein profiles around the interface by proteomics. A total of 112 lateral interfaces were collected from 55 CIS and 57 SCC foci, and they were investigated by immunohistochemistry and liquid chromatography-tandem mass spectrometry. The interfaces were morphologically classified into three types: vertical, oblique, and convex. There were several cellular changes characteristic to the interface, including apoptosis and hyaline bodies, which were more emphasized in SCC/CIS sides. The results suggested that ND cells were winners of cell competition against SCC/CIS cells. Then, the interfaces were divided into four vertical segments, and each segment was separately laser-microdissected from tissue sections with immunostaining for K13 or K17; the four segments included SCC/CIS away from (#1) or adjacent to (#2) the interface, and ND adjacent to (#3) or away from (#4) the interface. Proteome analyses revealed approximately 4000 proteins from SCC/CIS sides [#1 and #2] and 2800 proteins from ND sides [#3 and #4]. We quantitatively selected the top 25 proteins including ladinin-1 or interleukin-1 receptor antagonist protein, which were most contrastively increased or decreased in SCC/CIS or ND sides, respectively, and their specific immunohistochemical expression modes were confirmed in tissue sections as well as in cultured SCC cells. These molecules should be involved in the cellular crosstalk toward cell competition at the lateral interface of oral SCC/CIS and would be new candidates for histopathological distinction of oral malignancies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The intercellular deposit of perlecan, a basement-membrane type heparan sulfate proteoglycan, is considered to function as a growth factor reservoir and is enhanced in oral epithelial dysplasia and carcinoma in situ (CIS). However, it remains unknown which types of growth factors function in these perlecan-enriched epithelial conditions. The aim of this study was to determine immunohistochemically which growth factors were associated with perlecan in normal oral epithelia and in different epithelial lesions from dysplasia and CIS to squamous cell carcinoma (SCC). Eighty-one surgical tissue specimens of oral SCC containing different precancerous stages, along with ten of normal mucosa, were examined by immunohistochemistry for growth factors. In normal epithelia, perlecan and growth factors were not definitely expressed. In epithelial dysplasia, VEGF, SHH, KGF, Flt-1, and Flk-1were localized in the lower half of rete ridges (in concordance with perlecan, 33-100%), in which Ki-67 positive cells were densely packed. In CIS, perlecan and those growth factors/receptors were more strongly expressed in the cell proliferating zone (63-100%). In SCC, perlecan and KGF disappeared from carcinoma cells but emerged in the stromal space (65-100%), while VEGF, SHH, and VEGF receptors remained positive in SCC cells (0%). Immunofluorescence showed that the four growth factors were shown to be produced by three oral SCC cell lines and that their signals were partially overlapped with perlecan signals. The results indicate that perlecan and its binding growth factors are differentially expressed and function in specific manners before (dysplasia/CIS) and after (SCC) invasion of dysplasia/carcinoma cells., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

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., (Copyright © 2016 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Based on our previous finding that protease-activated receptor 2 (PAR-2) regulates hemophagocytosis of oral squamous cell carcinoma (SCC) cells, which induces their heme oxygenase 1-dependent keratinization, we have formulated a hypothesis that PAR-2 functions in wider activities of SCC cells. To confirm this hypothesis, we investigated immunohistochemical profiles of PAR-2 in oral SCC tissues and its functional roles in cell proliferation and invasion in SCC cells in culture. The PAR-2 expression modes were determined in 48 surgical tissue specimens of oral SCC. Using oral SCC-derived cell systems, we determined both gene and protein expression levels of PAR-2. SCC cell proliferation and invasive properties were also examined in conditions in which PAR-2 was activated by the synthetic peptide SLIGRL. PAR-2 was immunolocalized in oral SCC and carcinoma in situ cells, especially in those on the periphery of carcinoma cell foci (100% of cases), but not in normal oral epithelia. Its expression at both gene and protein levels was confirmed in 3 oral SCC cell lines including ZK-1. Activation of PAR-2 induced ZK-1 cell proliferation in a dose-dependent manner. PAR-2-activated ZK-1 cells invaded faster than nonactivated ones. The expression of PAR-2 is specific to oral malignancies, and PAR-2 regulates the growth and invasion of oral SCC cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Expression of keratin (K) 13 is replaced with that of K17 when squamous cells of the oral mucosa transform from normal and dysplastic epithelia to carcinoma in situ (CIS) and squamous cell carcinoma (SCC). Since 14-3-3 sigma is functionally associated with K17, we examined possible relationships between expression of K17 and 14-3-3 sigma in oral CIS and SCC tissues by immunohistochemistry. We furthermore examined whether or not K17 expression or knockdown by small interfering RNA (siRNA) modulates the behavior of SCC cells in culture in terms of cell proliferation and migration. In tissue specimens of oral SCC and CIS, the pattern of cytoplasmic expression of 14-3-3 sigma and K17 was similar but neither was expressed in normal or dysplastic epithelia. Both proteins were demonstrated in the cytoplasm of control oral SCC ZK-1 cells, but expression of 14-3-3 sigma changed from cytoplasmic to nuclear upon knockdown of K17. In carcinoma cells, therefore, cytoplasmic localization of 14-3-3 sigma seems to accompany expression of K17. In K17-knockdown cells, proliferation was significantly suppressed at 4 days after seeding. In addition, the cell size of K17-knockdown cells was significantly smaller than that of control cells; as a result of which in the migration experiments, we found delayed closure of scratch wounds but migration as such was not affected. We conclude that K17 expression promotes SCC cell growth and cell size but does not affect cell migration. K17 expression is accompanied by cytoplasmic expression of 14-3-3 sigma, indicative of their functional relationship.

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., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

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., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Local recurrence remains a challenging clinical issue for the treatment of oral squamous cell carcinoma (SCC). We analyzed retrospectively how effective the frozen section technique (FS) was against recurrences of oral SCC., Methods: We screened 343 surgical samples from 236 patients who had oral SCC, carcinoma in situ (CIS), or epithelial dysplasia, and we followed up their clinical outcomes for at least 5 years. Histopathological states of surgical margins were compared between FS and surgical materials in relapse and relapse-free groups, respectively., Results: Among the 236 patients, 191 were classified into the relapse-free group, and 45 into the relapse group. FS was more frequently performed in the relapse-free group (128/191) than in the relapse group (83/152). Histopathologically, moderate dysplasia or CIS (borderline malignancies) and SCC were recognized in 55 samples of the relapse-free group and in 57 of the relapse group. For those surgical margins with borderline malignancies, additional incisions were performed in 38 of the 55 relapse-free cases, which reduced to 20 from the 38 margins with borderline malignancies (47.4% reduction), and in 39 of the 57 relapse cases, which reduced to only 3 of 39 (7.7% reduction)., Conclusions: The intraoperative assessment of surgical margins by FS is essential in preventing recurrences of oral mucosal malignancies.

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., (Copyright © 2013 Wiley Periodicals, Inc.)

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

It is poorly understood which cell type, tumor cells, or stromal cells are responsible for the production of extracellular matrix molecules in the neoplastic stroma. We studied the expression of 4 extracellular matrix molecules at the protein and messenger RNA levels in monocellular and 2 kinds of coculture systems between human squamous cell carcinoma (ZK-1) and fibroblast (OF-1) cell lines, which may correspond to carcinoma in situ and squamous cell carcinoma, respectively. Squamous cell carcinoma and carcinoma in situ tissue sections were also investigated by immunohistochemistry and in situ hybridization for extracellular matrix. Immunohistochemically, perlecan and tenascin C were localized in carcinoma cells in carcinoma in situ, whereas they were in the stromal space in squamous cell carcinoma. In monocellular culture conditions, expression levels for perlecan, tenascin C, and laminin were more predominant in ZK-1 than in OF-1, although those for fibronectin were more enhanced in OF-1. However, these extracellular matrix expression levels of OF-1 were elevated, whereas those of ZK-1 dropped when they were in coculture conditions. The differences between ZK-1 and OF-1 were significantly more evident in direct contact (ZK-1/OF-1, 56%-22%) than in indirect contact (63%-39%). These results indicate that oral squamous cell carcinoma cells produce extracellular matrix in the absence of stromal fibroblasts (or in carcinoma in situ) and that they stop producing extracellular matrix in the presence of fibroblasts (or in squamous cell carcinoma). It is hence suggested that stromal fibroblasts after direct contact with invading squamous cell carcinoma cells are more responsible than squamous cell carcinoma cells for the formation of neoplastic stroma, whereas carcinoma in situ cells have to produce and deposit extracellular matrix by themselves to form intraepithelial microstromal spaces., (Copyright © 2012 Elsevier Inc. All rights reserved.)