Peter Hyckel

Synthesis and protein distribution of the unspliced large tenascin‐C isoform in oral squamous cell carcinoma

The inclusion or omission of the alternatively spliced region in the tenascin‐C (Tn‐C) mRNA gives rise to the large (Tn‐CL) or small (Tn‐CS) variant, respectively. Tn‐CL is thought to be a typical component of provisional extracellular matrices (ECMs) and is expressed during tumour stroma remodelling. Tn‐CL synthesis has been studied using RNA/RNA in situ hybridization, and Tn‐CL protein distribution, using immunohistochemistry (clone BC‐2), in 18 oral squamous cell carcinomas (OSCCs) of different grades of malignancy. While the Tn‐CL protein was demonstrated within the whole stromal compartment regardless of grade of malignancy, the majority of the Tn‐CL mRNA signal‐bearing cells were carcinoma cells. Only a few stromal myofibroblasts were able to synthesize Tn‐CL, as revealed by α‐smooth muscle actin double staining. In well‐differentiated carcinomas (G1), the Tn‐CL synthesizing carcinoma cells were localized as a single positive cell layer in the tumour stroma interface, particularly in invasive areas. A higher grade of malignancy (G2/G3) is associated with a significantly increased number of Tn‐CL synthesizing carcinoma cells randomly distributed within the invading tumour areas. Double‐staining experiments (Tn‐CL mRNA ISH/BC‐2 immunohistochemistry) indicate that these cells are capable of organizing and depositing a three‐dimensional Tn‐CL matrix. Even though an instructive and/or inductive role of the carcinoma cells in tumour stroma formation cannot be excluded, these results demonstrate that carcinoma cells can directly produce the ECM components of tumour stroma. Copyright

Expression of Snail is associated with myofibroblast phenotype development in oral squamous cell carcinoma

Snail is a regulator of epithelial–mesenchymal transition (EMT) and considered crucial to carcinoma metastasis, myofibroblast transdifferentiation, and fibroblast activation. To investigate the role of Snail in oral squamous cell carcinoma (OSCC), its immunohistochemical expression was analysed in 129 OSCC samples and correlated to nodal metastasis, histological grade, E-cadherin, and alpha smooth-muscle-actin (αSMA). The results were compared to findings in 23 basal cell carcinomas (BCC). Additionally, the influence of TGFβ1 and EGF on Snail, E-cadherin, vimentin, and αSMA expression was analysed in two OSCC cell lines. As a result, Snail-positive cells were mainly found in the stroma of the OSCC invasive front without statistically significant correlation to histological grade or nodal metastasis. Snail was co-localised to αSMA but not to E-cadherin or cytokeratin and showed a significant correlation to the loss of membranous E-cadherin. All BCCs were Snail negative. In OSCC culture, the growth-factor-mediated EMT-like phenomenon was accompanied by αSMA down-regulation. In summary, Snail expression in OSCC is a stromal phenomenon associated with the myofibroblast phenotype and not related to growth-factor-mediated transdifferentiation of the carcinoma cells themselves. Consequently, Snail immunohistochemistry cannot contribute to the prediction of the metastatic potential. Furthermore, stromal Snail expression is suggested to be the result of mutual paracrine interaction of fibro-/myofibroblasts and dedifferentiated carcinoma cells leading to the generation of a special type of carcinoma-associated fibroblasts.

Fibrillary co-deposition of laminin-5 and large unspliced tenascin-C in the invasive front of oral squamous cell carcinoma in vivo and in vitro

Purpose: Invasion of oral squamous cell carcinoma (OSCC) is associated with laminin-5 (Ln-5) synthesis, focal Ln-5 loss from the basement membrane (BM), and Ln-5 depositions in the stroma beneath invading carcinoma cell complexes. Methods: The study is focused on the laminin-5 matrix reorganisation within the stroma of the OSCC invasive front outside the basement membrane region as well as in OSCC-fibroblast co-culture in relation to unspliced tenascin-C (Tn-CL) and ED-B+ fibronectin (ED-B+ fn) using confocal laser scanning microscopy. Results: In vivo, Ln-5 was demonstrated as fibrillary deposition in the invasive front. It was co-localised to Tn-CL. In pure OSCC cultures, Ln-5 was synthesised and deposited as a spot-like matrix. Fibrillary structures were not found. In contrast, in the OSCC-fibroblast co-culture, a fibrillary Ln-5 matrix organisation was revealed within the interface of OSCC cell-fibroblast complexes exclusively in co-distribution with Tn-CL and ED-B+ fn. Conclusion: At least in vitro, a carcinoma cell-stroma fibroblast interaction is indispensable for fibrillary Ln-5/Tn-CL matrix organisation. Behind the parallels to the initial basement membrane formation in organotypic cultures, the fibrillary multiprotein complexes at the OSCC cell-fibroblast interface are suggested as provisional basement membrane fragments with a possible supportive role for invasive tumour behaviour.

A Comparative Quantitative Analysis of Laminin-5 in the Basement Membrane of Normal, Hyperplastic, and Malignant Oral Mucosa by Confocal Immunofluorescence Imaging

Laminin-5 (Ln-5) is a heterotrimeric basement membrane (BM) molecule (α3β3γ2). It is a principal protein constituent of the anchoring filaments, which connect the BM with the hemidesmosomes of the basal keratinocytes and possess a crucial function in keratinocyte adhesion. Confocal immunofluorescence imaging is introduced for a quantitative evaluation of the Ln-5 content in the BM of oral squamous epithelium. The BM of normal oral mucosa was used as a reference (100%) for comparative analysis and showed a nearly uniform Ln-5 immunofluorescence intensity (99–100%). In all hyperplastic lesions of oral mucosa, the Ln-5 immunofluorescence intensity was increased (107–141%). The increased Ln-5 content in the BM of hyperplastic lesions suggests an increased keratinocyte-BM adhesion, possibly resulting in a higher stability of the oral mucosa. In contrast, in the oral squamous cell carcinoma (OSCC) invasive front, the remaining BM segments were characterized by a decrease in Ln-5 immunofluorescence intensity (35–74%). A stronger decrease of Ln-5-linked kerationocyte-BM adhesion correlates with a higher tumor grade. Because in central areas of carcinoma BM segments with a normal Ln-5 content could be demonstrated, the fundamental Ln-5 diminution in BM segments of the invasive front should be considered as an invasion-associated phenomenon.

Small oral squamous cell carcinomas with nodal lymphogenic metastasis show increased infiltration of M2 polarized macrophages – An immunohistochemical analysis

BACKGROUND In solid malignancies the influence of immunological parameters - especially of macrophages - on invasiveness, metastatic potential and prognosis has been shown. There are no studies quantitatively analysing the macrophage polarization in oral squamous cell carcinoma (oscc). The aim of this study was to correlate macrophage polarization in the epithelial and stromal compartment of oscc with histopathologic parameters. METHODS T1 and T2 oscc samples (n = 34) were used. Automated immunohistochemical staining detected CD68, CD11c, CD163 and MRC1 positive cells. All samples were completely digitalized using whole slide imaging and the number of stained cells per area was assessed quantitatively. RESULTS Primary tumours with lymphogenic metastasis (N+) showed a significantly (p < 0.05) increased count of CD68, CD11c, CD163 and MRC1 positive cells in the epithelial fraction compared to N0 tumours. The ratio of CD163 positive cells (M2 macrophages) to CD68 positive cells (M1 and M2 macrophages) was significantly (p < 0.05) increased in N+ tumours. CONCLUSION An increased macrophage infiltration and an increased M2 polarization in primary oral squamous cell carcinomas with lymphogenic metastasis was shown. Macrophages that migrated into the epithelial tumour fraction seem to be of special biological importance. The results indicate a central role of macrophages in the progression of oscc.

Increased malignancy of oral squamous cell carcinomas (oscc) is associated with macrophage polarization in regional lymph nodes – an immunohistochemical study

BackgroundIt is largely accepted that specific immunological parameters in solid malignancies are associated with patient’s prognosis. Recently a correlation of macrophage polarization with histomorphological parameters could also be shown in oral squamous cell carcinoma (oscc). The observed tumor derived peripheral immune tolerance could be associated with the macrophage polarization in regional tumor draining lymph nodes.So far there are no studies analyzing the macrophage polarization in cervical lymph nodes of oscc patients. In the present study we aimed to correlate macrophage polarization in different anatomical lymph node compartments of patients diagnosed with oscc with histopathologic parameters of the primary tumor (T-, N-, L-, V-, Pn-status, grading).MethodsTumor free (n = 37) and metastatic (n = 17) lymph nodes of T1 and T2 oscc patients were processed for immunohistochemistry to detect CD68, CD11c, CD163 and MRC1 positive cells. Samples were digitized using whole slide imaging and the number of cells expressing the aforementioned markers in the region of interest quantitatively analyzed.ResultsThe malignancy of the primary tumor (defined by T-, L-, Pn-status, grading) correlated with the lymph node macrophage polarization. L1 and Pn1 tumor cases displayed a significantly (p < 0.05) decreased M1 and increased M2 polarization in the sinus of the lymph nodes. G3 cases presented a significantly (p < 0.05) increased M2 polarization in the sinus compared to G2 cases. T2 tumors had significantly (p < 0.05) increased M2 polarization in the interfollicular zone of regional lymph nodes compared to T1 tumors. Metastatic and non-metastatic lymph nodes did not differ regarding their macrophage polarization.ConclusionsThe current study revealed for the first time an influence of oscc on the macrophage polarization in regional lymph nodes. Markers of malignant behavior in the primary tumor were associated with a shift of macrophage polarization in lymph nodes from the anti-tumoral M1 type to the tumor-promoting M2 type. As tumor free and metastatic lymph nodes did not differ in terms of their macrophage polarization pattern, there must be other factors influencing the location for lymph node metastasis formation.

Msx-1 is suppressed in bisphosphonate-exposed jaw bone analysis of bone turnover-related cell signalling after bisphosphonate treatment

OBJECTIVES Bone-destructive disease treatments include bisphosphonates and antibodies against receptor activator for nuclear factor κB ligand (aRANKL). Osteonecrosis of the jaw (ONJ) is a side-effect. Aetiopathology models failed to explain their restriction to the jaw. The osteoproliferative transcription factor Msx-1 is expressed constitutively only in mature jaw bone. Msx-1 expression might be impaired in bisphosphonate-related ONJ. This study compared the expression of Msx-1, Bone Morphogenetic Protein (BMP)-2 and RANKL, in ONJ-affected and healthy jaw bone. MATERIAL AND METHODS An automated immunohistochemistry-based alkaline phosphatase-anti-alkaline phosphatase method was used on ONJ-affected and healthy jaw bone samples (n = 20 each): cell-number ratio (labelling index, Bonferroni adjustment). Real-time RT-PCR was performed to quantitatively compare Msx-1, BMP-2, RANKL and GAPDH mRNA levels. RESULTS Labelling indices were significantly lower for Msx-1 (P < 0.03) and RANKL (P < 0.003) and significantly higher (P < 0.02) for BMP-2 in ONJ compared with healthy bone. Expression was sevenfold lower (P < 0.03) for Msx-1, 22-fold lower (P < 0.001) for RANKL and eightfold higher (P < 0.02) for BMP-2 in ONJ bone. CONCLUSIONS Msx-1, RANKL suppression and BMP-2 induction were consistent with the bisphosphonate-associated osteopetrosis and impaired bone remodelling in BP- and aRANKL-induced ONJ. Msx-1 suppression suggested a possible explanation of the exclusivity of ONJ in jaw bone. Functional analyses of Msx-1- RANKL interaction during bone remodelling should be performed in the future.

Msx-1 is suppressed in Bisphosphonate exposed jaw bone- analysis of bone turnover related cell signalling

OBJECTIVES Bone-destructive disease treatments include bisphosphonates and antibodies against receptor activator for nuclear factor κB ligand (aRANKL). Osteonecrosis of the jaw (ONJ) is a side-effect. Aetiopathology models failed to explain their restriction to the jaw. The osteoproliferative transcription factor Msx-1 is expressed constitutively only in mature jaw bone. Msx-1 expression might be impaired in bisphosphonate-related ONJ. This study compared the expression of Msx-1, Bone Morphogenetic Protein (BMP)-2 and RANKL, in ONJ-affected and healthy jaw bone. MATERIAL AND METHODS An automated immunohistochemistry-based alkaline phosphatase-anti-alkaline phosphatase method was used on ONJ-affected and healthy jaw bone samples (n = 20 each): cell-number ratio (labelling index, Bonferroni adjustment). Real-time RT-PCR was performed to quantitatively compare Msx-1, BMP-2, RANKL and GAPDH mRNA levels. RESULTS Labelling indices were significantly lower for Msx-1 (P < 0.03) and RANKL (P < 0.003) and significantly higher (P < 0.02) for BMP-2 in ONJ compared with healthy bone. Expression was sevenfold lower (P < 0.03) for Msx-1, 22-fold lower (P < 0.001) for RANKL and eightfold higher (P < 0.02) for BMP-2 in ONJ bone. CONCLUSIONS Msx-1, RANKL suppression and BMP-2 induction were consistent with the bisphosphonate-associated osteopetrosis and impaired bone remodelling in BP- and aRANKL-induced ONJ. Msx-1 suppression suggested a possible explanation of the exclusivity of ONJ in jaw bone. Functional analyses of Msx-1- RANKL interaction during bone remodelling should be performed in the future.

Expression of Msx-1 is suppressed in bisphosphonate associated osteonecrosis related jaw tissue-etiopathology considerations respecting jaw developmental biology-related unique features

BackgroundBone-destructive disease treatments include bisphosphonates and antibodies against the osteoclast differentiator, RANKL (aRANKL); however, osteonecrosis of the jaw (ONJ) is a frequent side-effect. Current models fail to explain the restriction of bisphosphonate (BP)-related and denosumab (anti-RANKL antibody)-related ONJ to jaws. Msx-1 is exclusively expressed in craniofacial structures and pivotal to cranial neural crest (CNC)-derived periodontal tissue remodeling. We hypothesised that Msx-1 expression might be impaired in bisphosphonate-related ONJ. The study aim was to elucidate Msx-1 and RANKL-associated signal transduction (BMP-2/4, RANKL) in ONJ-altered and healthy periodontal tissue.MethodsTwenty ONJ and twenty non-BP exposed periodontal samples were processed for RT-PCR and immunohistochemistry. An automated staining-based alkaline phosphatase-anti-alkaline phosphatase method was used to measure the stained cells:total cell-number ratio (labelling index, Bonferroni adjustment). Real-time RT-PCR was performed on ONJ-affected and healthy jaw periodontal samples (n = 20 each) to quantitatively compare Msx-1, BMP-2, RANKL, and GAPDH mRNA levels.ResultsSemi-quantitative assessment of the ratio of stained cells showed decreased Msx-1 and RANKL and increased BMP-2/4 (all p < 0.05) expression in ONJ-adjacent periodontal tissue. ONJ tissue also exhibited decreased relative gene expression for Msx-1 (p < 0.03) and RANKL (p < 0.03) and increased BMP-2/4 expression (p < 0.02) compared to control.ConclusionsThese results explain the sclerotic and osteopetrotic changes of periodontal tissue following BP application and substantiate clinical findings of BP-related impaired remodeling specific to periodontal tissue. RANKL suppression substantiated the clinical finding of impaired bone remodelling in BP- and aRANKL-induced ONJ-affected bone structures. Msx-1 suppression in ONJ-adjacent periodontal tissue suggested a bisphosphonate-related impairment in cellular differentiation that occurred exclusively jaw remodelling. Further research on developmental biology-related unique features of jaw bone structures will help to elucidate pathologies restricted to maxillofacial tissue.

Stromal laminin chain distribution in normal, hyperplastic and malignant oral mucosa: relation to myofibroblast occurrence and vessel formation.

BACKGROUND The contribution of stromal laminin chain expression to malignant potential, tumour stroma reorganization and vessel formation in oral squamous cell carcinoma (OSCC) is not fully understood. Therefore, the expression of the laminin chains alpha2, alpha3, alpha4, alpha5 and gamma2 in the stromal compartment/vascular structures in OSCC was analysed. METHODS Frozen tissue of OSCC (9x G1, 24x G2, 8x G3) and normal (2x)/hyperplastic (11x) oral mucosa was subjected to laminin chain and alpha-smooth muscle actin (ASMA) immunohistochemistry. Results were correlated to tumour grade. The relation of laminin chain positive vessels to total vessel number was assessed by immunofluorescence double labelling with CD31. RESULTS Stromal laminin alpha2 chain significantly decreases and alpha3, alpha4, alpha5 and gamma2 chains and also ASMA significantly increase with rising grade. The amount of stromal alpha3, alpha4 and gamma2 chains significantly increased with rising ASMA positivity. There is a significant decrease in alpha3 chain positive vessels with neoplastic transformation. CONCLUSIONS Mediated by myofibroblasts, OSCC development is associated with a stromal up-regulation of laminin isoforms possibly contributing to a migration promoting microenvironment. A vascular basement membrane reorganization concerning alpha3 and gamma2 chain laminins during tumour angioneogenesis is suggested.