Differences in the Nemosis Response of Normal and Cancer-Associated Fibroblasts from Patients with Oral Squamous Cell Carcinoma

Background

Tumor-stroma reaction is associated with activation of fibroblasts. Nemosis is a novel type of fibroblast activation. It leads to an increased production of growth factors and proinflammatory and proteolytic proteins, while at the same time cytoskeletal proteins are degraded. Here we used paired normal skin fibroblasts and cancer-associated fibroblasts (CAF) and primary and recurrent oral squamous cell carcinoma (SCC) cells to study the nemosis response.

Principal Findings

Fibroblast nemosis was analyzed by protein and gene expression and the paracrine regulation with colony formation assay. One of the normal fibroblast strains, FB-43, upregulated COX-2 in nemosis, but FB-74 cells did not. In contrast, CAF-74 spheroids expressed COX-2 but CAF-43 cells did not. Alpha-SMA protein was expressed in both CAF strains and in FB-74 cells, but not in FB-43 fibroblasts. Its mRNA levels were downregulated in nemosis, but the CAFs started to regain the expression. FSP1 mRNA was downregulated in normal fibroblasts and CAF-74 cells, but not in CAF-43 fibroblasts. Serine protease FAP was upregulated in all fibroblasts, more so in nemotic CAFs. VEGF, HGF/SF and FGF7 mRNA levels were upregulated to variable degree in nemosis. CAFs increased the colony formation of primary tumor cell lines UT-SCC-43A and UT-SCC-74A, but normal fibroblasts inhibited the anchorage-independent growth of recurrent UT-SCC-43B and UT-SCC-74B cells.

Conclusions

Nemosis response, as observed by COX-2 and growth factor induction, and expression of CAF markers α-SMA, FSP1 and FAP, varies between fibroblast populations. The expression of CAF markers differs between normal fibroblasts and CAFs in nemosis. These results emphasize the heterogeneity of fibroblasts and the evolving tumor-promoting properties of CAFs.     

Habitat use and diet of sympatric Arctic charr (<Emphasis Type="Italic">Salvelinus alpinus</Emphasis>) and whitefish (<Emphasis Type="Italic">Coregonus lavaretus</Emphasis>) in five lakes in southern Norway: not only interspecific population dominance?

Stable coexistence of Arctic charr and whitefish does occur in a number of native lake fish communities in Scandinavia. Even so, whitefish introductions into Arctic charr lakes have resulted in serious decline and possibly local extinction of Arctic charr. In this article, we analyze the habitat use and diet of the two species in five Norwegian lakes differing in basin shape and environmental conditions. In two of the lakes, both species are native, and appear to live in a relatively stable coexistence. Here, whitefish mainly occupy the littoral and upper pelagic zone, while Arctic charr live in the deeper habitats. Diets are generally quite different in terms of the zooplankton species eaten. In the three other lakes, either whitefish or both species have been introduced. In the shallowest lake, habitat segregation is similar to that seen in the pristine lakes, although Arctic charr appears to be on the brink of extinction. In the remaining two lakes, however, Arctic charr dominates, and occurs in higher numbers than whitefish in all the habitats. Our observations indicate that coexistence of the two species in oligotrophic and relatively pristine lakes requires an extensive profundal zone to serve as a refugium for Arctic charr. If the littoral zone is rendered inaccessible or unprofitable for whitefish due to dominance of a third competitor or predator, or as a result of lake regulation, then Arctic charr may be the dominant species.     

Rheological and structural characterization of the interactions between cyclodextrin compounds and hydrophobically modified alginate

Interactions in semidilute solutions of a hydrophobically modified alginate (HM-alginate) in the presence of hydroxypropyl-beta-cyclodextrin (HP-beta-CD) monomer or a beta-cyclodextrin polymer (poly(beta-CD)) have been characterized at different temperatures with the aid of rheology and small-angle neutron scattering (SANS). The viscosity results for the HM-alginate/HP-beta-CD system reveal progressive deactivation of the hydrophobic associations as the concentration of HP-beta-CD increases. For the HM-alginate/poly(beta-CD) system, on the other hand, addition of poly(beta-CD) sets up bridges between adjacent polymer chains and thereby strengthens the associative network. A novel shear-thickening effect is observed at fairly high shear rates for the HM-alginate/poly(beta-CD) system, and this feature is influenced by temperature. Elevated temperature induces higher chain mobility and the formation of weaker network associations. Analyses of the SANS data disclosed that the association strength in HM-alginate/poly(beta-CD) mixtures increases strongly with increasing cosolute concentration, whereas no effect or a moderate weakening of the strength can be traced in HM-alginate/HP-beta-CD solutions upon addition of HP-beta-CD. The value of the correlation length xi is virtually not affected by the addition of cosolute for the HM-alginate/poly(beta-CD) system, whereas the decoupling of hydrophobic moieties of the polymer upon addition of HP-beta-CD gives rise to a smaller value of xi, suggesting that the size of the heterogeneity patches is reduced. The SANS results suggest that compact association structures are formed in the HM-alginate/poly(beta-CD) solutions.     

Head and neck cancer cells are efficiently infected by Ad5/35 hybrid virus

BACKGROUND: Clinical gene therapy trials using standard Ad5-based vectors have thus far demonstrated limited efficacy, most likely due to low expression levels of adenoviral receptors on tumor cells. We sought to analyze adenoviral receptor expression levels on primary head and neck squamous cell carcinoma (HNSCC) cells and to determine whether adenoviral retargeting to the CD46 receptor via the Ad5/35 system would increase therapeutic potential for HNSCC. METHODS: We used flow cytometric analyses to determine adenoviral receptor expression levels on nine primary HNSCC cells collected from cancer patients. Adenoviruses Ad5.LacZ and Ad5/35.LacZ were used to analyze the differences in viral transduction both in vitro and in a HNSCC tumor mouse model. RESULTS: Flow cytometric analyses demonstrated uniformly high CD46 expression in all cells studied (85-99%). In contrast, coxsackievirus and adenovirus receptor (CAR) expression was substantially lower and highly variable (1.6-62%). Alpha(v) integrin expression was between 39-98%. In situ stainings for beta-galactosidase gene expression demonstrated that Ad5/35.LacZ was clearly more effective than Ad5.LacZ in transducing primary HNSCC cells. Quantification of beta-galactosidase expression revealed up to 65 times higher transgene expression from Ad5/35.LacZ than Ad5.LacZ. In vivo, beta-galactosidase expression was detected in a substantial area after a single intratumoral injection of Ad5/35.LacZ, whereas injection with Ad5.LacZ resulted in gene expression only in a few cells. CONCLUSIONS: Our results demonstrate that the low and variable CAR expression levels limit the therapeutic efficacy of Ad5-based strategies for HNSCC. In contrast, the effective in vivo transduction capacity of Ad5/35 warrants further development of this vector for the treatment of head and neck cancer.     

Evaluation of a new fiber-optic pressure recording system for cardiovascular measurements in mice

We have tested a new fiber-optic pressure recording system, Samba, with a thin fiber [outer diameter (OD) = 0.25 mm] and a pressure sensor (length and OD = 0.42 mm) attached to the end. The accuracy of the system tested in vitro was good, with a coefficient of variation of 2.54% at 100 mmHg. The drift was <0.45 mmHg/h, and the temperature sensitivity was approximately 0.07 mmHg/1 degrees C between 22 and 37 degrees C. The frequency response characteristics were similar to a 1.4-Fr Millar catheter (0-200 Hz). Introduction of the Samba sensor from the right carotid artery into the left ventricle in six mice caused no drop in mean aortic pressure, whereas introduction of a 1.4-Fr Millar catheter (OD = 0.47 mm; n = 6) caused a pressure drop from 91.6 +/- 9.2 to 65.1 +/- 6.2 mmHg; P < 0.05. Thus the Samba sensor system may represent a new alternative to assess hemodynamic variables in the murine cardiovascular system.     

Snail-dependent and -independent epithelial-mesenchymal transition in oral squamous carcinoma cells.

Disappearance of E-cadherin is a milestone for epithelial-mesenchymal transition (EMT), found both in carcinomas and in some fibrotic diseases. We have studied the mechanisms of EMT in oral squamous cell carcinoma (SCC) cells isolated from primary tumor (43A) and its recurrent tumor (43B). Whereas the cells from primary carcinoma displayed a typical phenotype of squamous epithelial cells including E-cadherin and laminin-332 (laminin-5), cells from recurrent tumor expressed characteristics of dedifferentiated, EMT-experienced tumors. 43B cells expressed E-cadherin repressors ZEB-1/deltaEF1 and especially ZEB-2/SIP1, which therefore appear as candidates for endogenous EMT in these cells. Differences between endogenous and exogenous EMT were assessed by transfecting 43A cells with SNAIL cDNA. SNAIL-transfected cells showed complete EMT phenotype with fibroblastoid appearance, vimentin filaments, E-cadherin/N-cadherin switch, lack of hemidesmosomes and, as a new feature of EMT, lack of laminin-332 synthesis. Upregulation of ZEB-1 and ZEB-2 was evident in these cells, suggesting that SNAIL can regulate these E-cadherin repressors. New monoclonal antibodies against SNAIL showed nuclear immunoreactivity not only in the SNAIL-transfected cells but also in carcinoma cells lacking production of Lm-332 and showing signs of EMT. These results suggest that changes in the epithelial cell differentiation program and EMT in SCC cells can result from the interplay among several E-cadherin repressors; however, SNAIL alone is able to accomplish a complete EMT.     

Tetranectin Is a Novel Marker for Myogenesis during Embryonic Development, Muscle Regeneration, and Muscle Cell Differentiationin Vitro

Tetranectin, a plasminogen-binding protein with a C-type lectin domain, is found in both serum and the extracellular matrix. In the present study we report that tetranectin is closely associated with myogenesis during embryonic development, skeletal muscle regeneration, and muscle cell differentiationin vitro.We find that tetranectin expression coincides with muscle differentiation and maturation in the second half of gestation and further that tetranectin is enriched at the myotendinous and myofascial junctions. The tetranectin immunostaining declines after birth and no immunostaining is observed in normal adult muscle. However, during skeletal muscle regeneration induced by the intramuscular injection of the myotoxic anesthetic Marcaine, myoblasts, myotubes, and the stumps of damaged myofibers exhibit intense tetranectin immunostaining. Tetranectin is also present in regenerating muscle cells in dystrophicmdxmice. Murine C2C12 myogenic cells and pluripotent embryonic stem cells can undergo muscle cell differentiationin vitro.Tetranectin is not expressed in the undifferentiated myogenic cells, but during the progression of muscle differentiation, tetranectin mRNA is induced, and both cytoplasmic and cell surface tetranectin immunostaining become apparent. Finally, we demonstrate that while tetranectin mRNA is translated to a similar degree in developing limbs and lung, the protein does not seem to be tissue associated in the lung as it is in the limbs. This indicates that in some tissues, such as the limbs, tetranectin may function locally, whereas in other tissues, such as the lung, tetranectin production may be destined for body fluids. In summary, these results suggest that tetranectin is a matricellular protein and plays a role in myogenesis.     

Leptomeric fibrils and T-tubule desmosomes in the Z-band region of the mouse heart papillary muscle

Summary The papillary muscle of the heart of adult white mice is investigated. Intrafibrillarly located leptomeric fibrils, frequently encountered in the Z-band region of the myofibrils. The leptomeric fibrils are always running in a transverse direction and often in close proximity to the transverse tubules (which are also located at this level). There seems to be a close connection between the dense striae of the leptofibrils and the Z-bands of ordinary myofibrils. The leptomeric fibrils are spindle-shaped and have a length varying between 0.6 and 1.2 m. The banding periodicity of the fibrils is approximately 0.16 m.Occasionally desmosomes are observed in the T-tubule system.     

The membrane systems of the cardiac muscle cell of Meganychtiphanes norvegica (M. Sars), euphauciacea,crustacea

Summary The membrane systems of cardiac muscle cells of the euphausiacean Meganychtiphanes norvegica are described. Transverse tubules are found both at the Z-band level (T_z-tubules) and at the H-band level (T_h-tubules). Within the sarcomere narrow longitudinal tubules branch off from the T_z-tubules. At the H-band level these tubules expand forming flattened cisternae in dyadic and triadic couplings with the sarcoplasmic reticulum (SR). Adjacent myofibrils are separated by a well developed SR. Modifications of the SR are seen at the H-band level where junctional cisternae are formed.     

Effects of isoproterenol on the dense core and perigranular membrane of atrial specific granules

Summary Following subcutaneous injections of isoproterenol hydrochloride (ISO), atrial cells present a large number of partly degranulated or completely clear specific granules enclosed by an intact membrane. Such profiles were never encountered in normal controls and might suggest ISO-induced release of a secretory product. Permeability of perigranular membrane was tested using the extracellular macromolecular tracer horseradish peroxidase (HRP). Reaction product was entirely absent within granules of atrial cells in which the sarcolemma was made permeable to HRP molecules by the ISO injections. This seemed to be the case even in heavily labelled cells in which the peroxidase had penetrated the mitochondrial membranes. In atrial cells impermeable to the tracer, the specific granules closely apposed to the sarcolemma were always HRP-negative. The release mechanism of a possible secretory substance from the specific granules is discussed.