Olav Schreurs

Liver Metastasis of Cancer Facilitated by Chemokine Receptor CCR6

When injected subcutaneously, mouse plasmacytoma (MOPC315) grew rapidly in situ, and metastatic cells became detectable first in the lymph nodes (LNs) and bone marrow, and later in the liver and lungs. We studied MOPC315 cell migration by tracking metastatic cells labelled with green fluorescent protein (GFP). We measured the levels of their chemokine receptor mRNA (by semiquantitative and real‐time quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR), because chemokines can regulate organ predilection of metastasis. Freshly sorted metastatic cells and tumour cell lines derived from the liver of BALB/c mice overexpressed functional CCR6 and CCR7 molecules compared with primary tumour. Preincubation with the CCR6 ligand (CCL20) induced liver‐sorted tumour cells to preferentially colonize the liver, demonstrating an association between liver metastasis and CCR6 expression in the mouse. Because the liver is a common site for metastasis, second only to draining LNs, we wished to ascertain whether this finding could be generalized, i.e. whether other cancers can use the similar mechanism of metastasis to the liver, and whether it holds true for humans. We found that CCR6 is overexpressed in small liver metastases of colon, thyroid and ovarian carcinomas compared with normal liver. Because human liver constitutively expresses CCL20, it could attract and select CCR6+ cancer cells. We suggest that chemotaxis via CCR6 might be a common mechanism by which malignant cancers metastasize to the liver. As metastasis in patients with cancer poses the biggest peril for survival, inhibition of CCR6 signalling, either during or after medical or surgical treatment, might be useful in preventing liver metastasis.

Aquaporin 11 in the developing mouse submandibular gland

Several aquaporins (AQPs) have been detected in mature and embryonic mammalian salivary glands (AQP1 and AQP3-AQP8). However, AQP11 has, to our knowledge, never before been described in salivary glands, but is known to be important in, for example, kidney development in mice. We therefore thought it relevant to investigate if AQP11 was present during salivary organogenesis. The submandibular salivary gland (SMG) from CD1 mice was studied during prenatal development and early postnatal development, and also in young adult male and female mice. The expression trend of the AQP11 transcript was detected using the reverse transcription-polymerase chain reaction (RT-PCR), and the temporal-spatial pattern was observed using in situ hybridization. The AQP11 transcript was first detected at embryonic day 13.5 and showed a more or less constitutive expression trend during the prenatal and early postnatal SMG development. Spatial studies demonstrated that the AQP11 transcript was present in the developing and mature duct structures at all stages studied. In the end pieces, the AQP11 transcript was reduced during glandular development. Our results point to an important role for AQP11 during salivary gland development.

Trefoil factor family 3 expression in the oral cavity.

This study examined the expression, in oral keratinocytes and in the major and minor salivary glands, of Trefoil factor family 3 (TFF3) peptide. Trefoil factor family 3 messenger RNA (mRNA) and peptide were detected in cultures of normal oral keratinocytes by quantitative real-time polymerase chain reaction (PCR) and western blotting, respectively. Trefoil factor family 3 was found, by immunohistochemical analyses, to be expressed in the basal layers of the oral mucosal epithelium. In salivary glands, immunohistochemical staining showed that TFF3 peptide expression was strongest in the mucous acini of the submandibular and the small salivary glands. Serous cells in the same glands showed weak staining. In the parotid gland, many serous acini showed weak positive staining, while other areas did not. In all glands examined, the intercalated, striated, and collecting ducts were moderately TFF3-positive. Double immunostaining confirmed that mucous (MUC5B positive) cells were moderately or strongly positive for TFF3 and that some serous (MUC7 positive) cells showed restricted TFF3 expression, mostly in a granular pattern. The prevalence of the TFF3 peptide in the salivary secretions of healthy volunteers was detected by western blotting of saliva from minor salivary glands (four of five) and the parotid gland (one of five) and of mixed submandibular/sublingual saliva (five of five). In conclusion, the submandibular and small salivary glands appear to be the major producers of oral TFF3, but duct cells of all glands and keratinocytes of the oral mucosa may also contribute as sources of TFF3 in the oral cavity.

Induction of invasion in an organotypic oral cancer model by CoCl2, a hypoxia mimetic

Invasion is a hallmark of malignancy. The aim of this study was to develop an in vitro model that can be used for experimental studies of cancer cell invasion. The organotypic oral cancer model was constructed by growing oral squamous cell carcinoma (OSCC) cells on a collagen matrix in which normal human fibroblasts were incorporated. Immunohistochemical staining of the model showed that the expression of invasion-related molecules such as phosphorylated extracellular signal-regulated kinases 1 and 2 (p-ERK1/2), cyclooxygenase-2 (COX-2), p75(NTR), and hepatocyte growth factor receptor (Met) was similar to that seen in OSCC. Treatment of the model with cobalt chloride (CoCl(2)) to mimic hypoxic conditions increased cancer cell invasion, defined as the appearance of cancer cell islands protruding into the matrix. Models treated with CoCl(2) showed increased expression of p75(NTR) and laminin-5 in the cancer cells, and a more pronounced fragmentation of collagen IV in the basal membrane area, in contrast to models that were left untreated. The results indicate that the present model is well suited for studies on cancer cell invasion in the matrix and that the addition of CoCl(2) on day 3 of the experiment is indicated because it markedly increases the invasion and improves the model.

Distribution of nerve growth factor, pro‐nerve growth factor, and their receptors in human salivary glands

Nerve growth factor (NGF) is a pluripotent mediator that is present in a range of human tissues. Nerve growth factor was originally considered important only in neuronal homeostasis and pathophysiology, but later it was also implicated in the pathophysiology of inflammation, epithelial differentiation, and wound healing. In this study, the distribution of nerve growth factor beta (NGF-β) and pro-NGF, and their receptors - tyrosine kinase A (TrkA) and p75(NTR) - was examined in human parotid, submandibular, sublingual, and labial salivary glands by immunohistochemistry. Intercalated, striated, and collecting-ducts in all gland types showed strong staining for pro-NGF but only weak cytoplasmic or sparse nuclear staining for NGF-β. Tyrosine kinase A was strongly expressed in the ducts of all gland types, whereas p75(NTR) expression was mainly confined to collecting ducts. In acini, no or only weak cytoplasmic staining was found for all markers, and some nuclei stained positive for NGF-β, pro-NGF, and TrkA. Western blotting of saliva showed secretion of several forms of pro-NGF, while no mature NGF-β was detected. Salivary pro-NGF may play a role in oral wound healing.

NGF and its receptors TrkA and p75NTR in the epithelium of oral lichen

BACKGROUND Nerve growth factor (NGF) can through its receptors TrkA and p75(NTR) convey signals for cell survival, differentiation and death. The aim of this study was to examine whether NGF can play a role in the pathology of oral lichen (OL). METHODS Sections from biopsies taken from patients with erythematous (ERY) OL and from volunteers with normal oral mucosa (NOM) were immunostained with antibodies against NGF, proNGF, TrkA, phosphorylated Trk, p75(NTR) and phosphorylated Akt (pAkt) and expression of RNA coding for proNGF/NGF was investigated by in situ hybridization. RESULTS Both in ERY OL and NOM, cytoplasmic staining for NGF was seen in granular and upper spinous cell layers of the epithelium, whereas proNGF staining was seen in all epithelial cell layers. In situ hybridization showed that the proNGF protein was produced in the same cell layers. In OL, strong cytoplasmic stainings for TrkA and activated Trk (pTrk) were observed in all epithelial cell layers while these stainings were only weak in NOM. Basal keratinocytes in OL showed no or only weak cytoplasmic staining for p75(NTR), but in NOM there was a clear cell membrane staining. In OL, strong cytoplasmic and intermittent nuclear staining for pAkt was observed in spinous, granular and superficial layers, while basal and parabasal keratinocytes were negative. This staining was weak or absent in the entire epithelium of NOM. CONCLUSIONS TrkA upregulation and activation in OL is one of the pathways that can activate pAkt and thereby rescue epithelial cells from untimely cell death.

Expression of the T‐cell‐specific adapter protein in oral epithelium

The multifunctional T-cell-specific adapter protein (TSAd) was originally described in T cells but is also expressed in epithelial cells from the respiratory tract and in endothelium. In this study, we found expression of TSAd messenger RNA (mRNA) and protein in both human and murine oral mucosal epithelium as well as in human primary oral keratinocyte cell cultures. In TSAd(-/-) mice, the mucosa and skin appeared macroscopically normal, but severe disturbances were observed in the fine structures of the basal membrane and intercellular epithelial spaces upon analysis using transmission electron microscopy. Oral epithelial cells from TSAd(-/-) mice displayed decreased migration compared with cells from wild-type mice, whereas overexpression of TSAd in a human epithelial cell line resulted in impaired proliferation. This study is the first to show that TSAd is expressed in normal oral mucosa, that it is important for the normal ultrastructural morphology of the epithelium and the basal membrane, and that it is involved in the migration and proliferation of oral keratinocytes.

Phenotypically non‐suppressive cells predominate among FoxP3‐positive cells in oral lichen planus

BACKGROUND Oral lichen planus (OLP) is a common T-cell-dominated oral chronic inflammatory disease occurring in periods of remission, quiescence, activity with pronounced inflammation, and acute ulceration. Cell infiltrates in OLP contain varying numbers of CD4+ T cells expressing the transcription factor FoxP3. FoxP3+ CD4+ T cells are, however, a heterogeneous cell population containing suppressive and non-suppressive cells, and their distribution in infiltrates from OLP is unknown. METHODS Biopsies were taken from normal oral mucosa (n = 8) and OLP lesions (n = 19), and a set of in situ methods for the determination of the functional phenotype of FoxP3+ CD4+ T cells was applied. RESULTS Numbers of FoxP3+ CD4+ T cells were highest in the atrophic form of the disease, yet low in the ulcerative form. The main FoxP3+ CD4+ T-cell population observed was FoxP3+ CD45RA- CD25+ CD45RO+ and CD15s- , a phenotype delineating a non-suppressive subset. Numbers of cells with an actively suppressing phenotype (FoxP3+ CD45RA- CD25+ CD45RO+ and CD15s+ ) were, however, about twice as high in reticular lesions as compared with the atrophic form. Many FoxP3+ CD4+ T cells expressed T-bet, the hallmark transcription factor for IFN-γ-producing T cells, indicating that they may enhance immune and inflammatory responses rather than suppress them. CONCLUSIONS The absence of actively suppressing FoxP3+ CD4+ T cells may in part explain why OLP is a remarkably persisting condition, in spite of the presence of substantially high numbers of FoxP3+ CD4+ T cells. The findings emphasize that it is crucial to examine not only numbers but also functional phenotype of FoxP3+ CD4+ T cells in human tissues.

Lysophosphatidic acid regulates adhesion molecules and enhances migration of human oral keratinocytes

Oral keratinocytes are connected via cell-to-cell adhesions to protect underlying tissues from physical and bacterial damage. Lysophosphatidic acids (LPAs) are a family of phospholipid mediators that have the ability to regulate gene expression, cytoskeletal rearrangement, and cytokine/chemokine secretion, which mediate proliferation, migration, and differentiation. Several forms of LPA are found in saliva and gingival crevicular fluid, but it is unknown how they affect human oral keratinocytes (HOK). The aim of the present study was therefore to examine how different LPA forms affect the expression of adhesion molecules and the migration and proliferation of HOK. Keratinocytes were isolated from gingival biopsies obtained from healthy donors and challenged with different forms of LPA. Quantitative real-time RT-PCR, immunocytochemistry, and flow cytometry were used to analyze the expression of adhesion molecules. Migration and proliferation assays were performed. Lysophosphatidic acids strongly promoted expression of E-cadherin and occludin mRNAs and translocation of E-cadherin protein from the cytoplasm to the membrane. Occludin and claudin-1 proteins were up-regulated by LPA. Migration of HOK in culture was increased, but proliferation was reduced, by the addition of LPA. This indicates that LPA can have a role in the regulation of the oral epithelial barrier by increasing the expression of adhesion molecules of HOK, by promotion of migration and by inhibition of proliferation.