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Loss-of-function (LOF) mutations in NFKB1, coding for p105, may cause common variable immunodeficiency due to dysregulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κΒ) pathway. Monoallelic LOF variants of NFKB1 can predispose to uncontrolled inflammation including sterile necrotizing fasciitis or pyoderma gangrenosum. In this study, we explored the impact of a heterozygous NFKB1 c.C936T/p.R157X LOF variant on immunity in sterile fasciitis patients and their family members. The p50 or p105 protein levels were reduced in all variant carriers. Interleukin-1β (IL-1β) and interleukin-8 (IL-8) levels were elevated in vitro, potentially contributing to the very high neutrophil counts observed during fasciitis episodes. Phosphorylation of p65/RelA was reduced in p.R157X neutrophils suggesting defective activation of canonical NF-κB. Oxidative burst after NF-κB-independent phorbol 12-myristate 13-acetate (PMA) stimulation was similar in both p.R157X and control neutrophils. Comparable amounts of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex subunits were found in p.R157X and control neutrophils. However, a compromised oxidative burst was observed in p.R157X neutrophils following activation of NF-κB-dependent mechanisms following stimulation of toll-like receptor 2 (TLR2) and Dectin-1. Neutrophil extracellular trap formation was not affected by p.R157X. In summary, the NFKB1 c.C936T/p.R157X LOF variant has an impact on inflammation and neutrophil function and may play a role in the pathogenesis of sterile necrotizing fasciitis.

Matrix metalloproteinases (MMPs) modify bioactive factors via selective processing or degradation resulting in tumour-promoting or tumour-suppressive effects, such as those by MMP8 in various cancers. We mapped the substrates of MMP8 to elucidate its previously shown tumour-protective role in oral tongue squamous cell carcinoma (OTSCC). MMP8 overexpressing (+) HSC-3 cells, previously demonstrated to have reduced migration and invasion, showed enhanced cell-cell adhesion. By analysing the secretomes of MMP8 + and control cells with terminal amine isotopic labelling of substrates (TAILS) coupled with liquid chromatography and tandem mass spectrometry (LC-MS/MS), we identified 36 potential substrates of MMP8, including FXYD domain-containing ion transport regulator 5 (FXYD5). An anti-adhesive glycoprotein FXYD5 has been previously shown to predict poor survival in OTSCC. Cleavage of FXYD5 by MMP8 was confirmed using recombinant proteins. Furthermore, we detected a loss of FXYD5 levels on cell membrane of MMP8 + cells, which was rescued by inhibition of the proteolytic activity of MMP8. Silencing (si) FXYD5 increased the cell-cell adhesion of control but not that of MMP8 + cells. siFXYD5 diminished the viability and motility of HSC-3 cells independent of MMP8 and similar effects were seen in another tongue cancer cell line, SCC-25. FXYD5 is a novel substrate of MMP8 and reducing FXYD5 levels either with siRNA or cleavage by MMP8 increases cell adhesion leading to reduced motility. FXYD5 being a known prognostic factor in OTSCC, our findings strengthen its potential as a therapeutic target.

Lymphatic metastasis represents the main route of tumour cell dissemination in oral squamous cell carcinoma (OSCC). Yet, there are no FDA-approved therapeutics targeting cancer-related lymphangiogenesis to date. The lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE-1), a specific lymphatic marker, is associated with poor survival in OSCC patients. In this study, we present a potential novel mechanism of lymphatic metastasis in OSCC—lymphatic mimicry (LM), a process whereby tumour cells form cytokeratin+/LYVE-1+, but podoplanin-negative, mosaic endothelial-like vessels. LM was detected in one-third (20/57; 35.08%) of randomly selected OSCC patients. The LM-positive patients had shorter overall survival (OS) compared to LM-negative group albeit not statistically significant. Highly-metastatic tumour cells formed distinct LM structures in vitro and in vivo. Importantly, the siRNA-mediated knockdown of LYVE-1 not only impaired tumour cell migration but also blunted their capacity to form LM-vessels in vitro and reduced tumour metastasis in vivo. Together, our findings uncovered, to our knowledge, a previously unknown expression and function of LYVE-1 in OSCC, whereby tumour cells could induce LM formation and promote lymphatic metastasis. Finally, more detailed studies on LM are warranted to better define this phenomenon in the future. These studies could benefit the development of targeted therapeutics for blocking tumour-related lymphangiogenesis.

The Ambystoma maxicanum (axolotl) regenerates strikingly from wounds and amputations. Comparing its healing ability to non-regenerative species such as the mouse should help narrow in on mechanisms to improve human wound healing. Here, the tongue and intermandibular soft tissues of both mice (C57BL/6NCrl) and axolotls were wounded with a 2–2.5 mm punch biopsy. The study aimed to compare the differences between these 2 species following surgical resection with regard to the macroscopic and histological characteristics. These include wound closure times, epithelial wound sealing and thickness as well as acute immune marker myeloperoxidase (MPO) response over 30 days. Post surgery, mice visually showed greater haemorrhage; their wounds immediately collapsed while it took 14 days for the axolotls mandibular void to close. The epithelium sealed the axolotls’ wound margins within 24 h with a maximal mean thickness of 0.42 ± 0.13-fold normalized to unwounded skin. In mice, the epithelium separately sealed the ventral and dorsal sides, respectively at 7 and 7–30 days with mean maximal epithelial thicknesses reaching 13 ± 5.6 and 3.0 ± 0.63-fold. Mean MPO-positive cell values peaked in axolotls at 14 ± 1.5-fold between hours 6–12; while in mice, it peaked at 8.7 ± 0.9-fold between hours 24–96. We conclude that axolotls form smaller blood clots, have a faster and thinner epithelial cell migrating front, and a shorter MPO-positive cell response in comparison to mice. These observations may help refine future oral and facial wound-healing research and treatment.

Puumala hantavirus (PUUV) hemorrhagic fever with renal syndrome (HFRS) is common in Northern Europe; this infection is usually self-limited and severe complications are uncommon. PUUV and other hantaviruses, however, can rarely cause encephalitis. The pathogenesis of these rare and severe events is unknown. In this study, we explored the possibility that genetic defects in innate anti-viral immunity, as analogous to Toll-like receptor 3 (TLR3) mutations seen in HSV-1 encephalitis, may explain PUUV encephalitis. We completed exome sequencing of seven adult patients with encephalitis or encephalomyelitis during acute PUUV infection. We found heterozygosity for the TLR3 p.L742F novel variant in two of the seven unrelated patients (29%, p = 0.0195). TLR3-deficient P2.1 fibrosarcoma cell line and SV40-immortalized fibroblasts (SV40-fibroblasts) from patient skin expressing mutant or wild-type TLR3 were tested functionally. The TLR3 p.L742F allele displayed low poly(I:C)-stimulated cytokine induction when expressed in P2.1 cells. SV40-fibroblasts from three healthy controls produced increasing levels of IFN-λ and IL-6 after 24 h of stimulation with increasing concentrations of poly(I:C), whereas the production of the cytokines was impaired in TLR3 L742F/WT patient SV40-fibroblasts. Heterozygous TLR3 mutation may underlie not only HSV-1 encephalitis but also PUUV hantavirus encephalitis. Such possibility should be further explored in encephalitis caused by these and other hantaviruses. Electronic supplementary material The online version of this article (10.1007/s10875-020-00834-2) contains supplementary material, which is available to authorized users.

Background: Tongue cancer is more common in men than in women. Yet the effects of sex steroid hormones on the behaviour of oral tongue squamous cell carcinoma (OTSCC) are not well known. Matrix metalloproteinase 8 (MMP8) is expressed in OTSCC and can degrade estrogen receptors (ERs). Materials and Methods: Western blot was used to examine the levels of ERβ in OTSCC cell lines (HSC-3 and SCC-25). We evaluated the effects of estradiol and dihydrotestosterone (DHT) on HSC-3 and SCC-25 cell migration, invasion and viability. The effect of estradiol on the invasion of MMP8-overexpressing (MMP8+) and empty vector HSC-3 cells was examined using 3D spheroid invasion assay. Results: Both HSC-3 and SCC-25 cells expressed ERβ. In scratch assay, estradiol, but not DHT, reduced the migration and invasion of HSC-3 and SCC-25 cells. MMP8+ HSC-3 cells showed weaker invasion than empty vector cells, in line with previous reports. However, MMP8 overexpression did not alter the effect of estradiol on HSC-3 cell invasion in spheroid assay. Conclusion: Estradiol inhibited the migration and invasion of OTSCC cells, whereas DHT had no effect. Our data suggest that MMP8 does not modulate the effect of estradiol in OTSCC cells. However, the sex difference in OTSCC incidence might partly be due to protective actions of estradiol in epithelial cell carcinogenesis.

Tongue cancer is more common in men than in women. Yet the effects of sex steroid hormones on the behaviour of oral tongue squamous cell carcinoma (OTSCC) are not well known. Matrix metalloproteinase 8 (MMP8) is expressed in OTSCC and can degrade estrogen receptors (ERs).Western blot was used to examine the levels of ERβ in OTSCC cell lines (HSC-3 and SCC-25). We evaluated the effects of estradiol and dihydrotestosterone (DHT) on HSC-3 and SCC-25 cell migration, invasion and viability. The effect of estradiol on the invasion of MMP8-overexpressing (MMP8Both HSC-3 and SCC-25 cells expressed ERβ. In scratch assay, estradiol, but not DHT, reduced the migration and invasion of HSC-3 and SCC-25 cells. MMP8Estradiol inhibited the migration and invasion of OTSCC cells, whereas DHT had no effect. Our data suggest that MMP8 does not modulate the effect of estradiol in OTSCC cells. However, the sex difference in OTSCC incidence might partly be due to protective actions of estradiol in epithelial cell carcinogenesis.

B7-H3 was the only molecule identified with prognostic potential from a recent systematic review of the prognostic value of immune checkpoints in oral cancer. We aimed to validate this finding in a multicenter international cohort. We retrospectively retrieved 323 oral tongue squamous cell carcinoma (OTSCC) samples from three different countries (Brazil, Finland, and Norway) for immunostaining and scoring for B7-H3. We evaluated tumor immunogenicity by analyzing the amount of tumor-infiltrating lymphocytes and divided the tumors into immune hot and cold. To increase the reliability of the results, both digital and manual visual scoring were used. Survival curves were constructed based on the Kaplan-Meier method, and the Cox proportional hazard model was utilized for univariate and multivariate survival analysis. B7-H3 expression was not significantly associated with overall or disease-specific survival in the whole OTSCC cohort. When divided into immune hot and cold tumors, high B7-H3 expression was significantly associated with poor disease-specific and overall survival in the immune hot group, depending on the scoring method and the country of the cohort. This was achieved only in the univariate analysis. In conclusion, B7-H3 was a negative prognosticator for OTSCC patient survival in the subgroup of immune hot tumors, and was not validated as a prognosticator in the full cohort. Our findings suggest that the immune activity of the tumor should be considered when testing immune checkpoints as biomarkers. Electronic supplementary material The online version of this article (doi:10.1007/s12105-020-01222-3) contains supplementary material, which is available to authorized users.

Background Rare tonsillar granulomas may be caused for example by infections, malignancies or sarcoidosis. Granulomas also occur in inborn errors of immunity (IEI) such as common variable immunodeficiency (CVID) with B cell maturation defects and hypogammaglobulinemia. CVID shares various features with sarcoidosis and drug-induced secondary hypogammaglobulinemia; careful consideration of differential diagnosis between these conditions is warranted. Case presentation A 29-year-old female with epilepsy developed dysphagia, dyspnea and impaired exercise tolerance. Obstruction caused by swollen lingual tonsil and edema in the epiglottis and arytenoid mucosa were found. Lingual tonsil and epiglottis biopsies displayed non-necrotizing granulomas. There was no evidence of viral, bacterial, mycobacterial or fungal infections. Chest X-ray, computerized tomography of chest and ultrasound of neck and abdomen remained unremarkable. Positron emission tomography/computed tomography (PET/CT) showed laryngeal enhancement. Empiric antimicrobials combined with prednisolone were insufficient to control her disease. In immunological evaluation, the patient had normal counts of B and T cells. Proportions of CD27+ memory B cells (30.3%) and IgD−IgM−CD27+ switched memory B cells (7.2%; normal range 6.5–29.2%) were normal. Percentage of activated CD21low B cells was high (6.6%; normal range 0.6–3.5%). IgG (3.5 g/L; normal range 6.77–15.0 g/l) and all IgG subclass concentrations were low. Anti-polysaccharide responses were impaired, with 3/10 serotypes reaching a level of 0.35 µg/ml after immunization with Pneumovax®. The findings were consistent with hypogammaglobulinemia resembling CVID, possibly secondary to antiepileptic medication. Her dyspnea and dysphagia responded favorably to subcutaneous IgG and rituximab. Conclusions Tonsillar granulomas can be the presenting and only clinical feature of B cell deficiency, highlighting the diversity of symptoms and findings in primary or secondary immunodeficiencies.

Susceptibility to herpes simplex virus type 1 (HSV-1) encephalitis (HSE-1) in otherwise healthy individuals, in the course of primary infection, can be caused by single-gene inborn errors of Toll-like receptor 3 (TLR3) dependent, interferon (IFN)-alpha/beta-mediated immunity,(1,2) or by single-gene inborn errors of snoRNA31.(3) These variations underlie infections of the forebrain, whereas mutations of DBR1 underlie infections of the brainstem.(3) HSV-2 encephalitis (HSE-2) is typically observed in neonates, albeit also rarely in older children and adults.(4) Its manifestations include altered level of consciousness, cranial neuropathies or more extensive brainstem encephalitis, hemiparesis, hemisensory loss, and permanent neurologic deficit.(4) MRI in HSE-2 may show normal findings, nonspecific white matter, orbitofrontal, mesial temporal lobe, or brainstem lesions. Inborn errors of immunity underlying HSE-2 have not been described. Non

In vitro cancer drug testing carries a low predictive value. We developed the human leiomyoma&ndash, derived matrix &ldquo, Myogel&rdquo, to better mimic the human tumor microenvironment (TME). We hypothesized that Myogel could provide an appropriate microenvironment for cancer cells, thereby allowing more in vivo&ndash, relevant drug testing. We screened 19 anticancer compounds, targeting the epidermal growth factor receptor (EGFR), MEK, and PI3K/mTOR on 12 head and neck squamous cell carcinoma (HNSCC) cell lines cultured on plastic, mouse sarcoma&ndash, derived Matrigel (MSDM), and Myogel. We applied a high-throughput drug screening assay under five different culturing conditions: cells in two-dimensional (2D) plastic wells and on top or embedded in Matrigel or Myogel. We then compared the efficacy of the anticancer compounds to the response rates of 19 HNSCC monotherapy clinical trials. Cancer cells on top of Myogel responded less to EGFR and MEK inhibitors compared to cells cultured on plastic or Matrigel. However, we found a similar response to the PI3K/mTOR inhibitors under all culturing conditions. Cells grown on Myogel more closely resembled the response rates reported in EGFR-inhibitor monotherapy clinical trials. Our findings suggest that a human tumor matrix improves the predictability of in vitro anticancer drug testing compared to current 2D and MSDM methods.

Aim: Sorafenib (Nexavar) is a multitargeted tyrosine kinase inhibitor that is used for treatment of hepatocellular carcinoma and renal cell carcinoma. Cancer treatment with sorafenib has been reported to be associated with cardiotoxic side effects such as hypertension, myocardial ischemia, thrombosis and pulmonary embolism, whereas incidence of LV dysfunction is rare. Aim of the current study was to identify the mechanism of sorafenib cardiotoxicity. Results: Healthy mice were treated with sorafenib (50 mg/kg/d) for 4 weeks. Echocardiography analysis revealed no difference in LV systolic function, but LV posterior wall thickness and relative wall thickness were significantly increased, and LV volume decreased in sorafenib treated mice. Hearts of sorafenib treated mice showed a 10-fold increase in expression of heart failure marker gene ANP and a 3-fold increase in the number of apoptotic cells. Electron microscopy analysis indicated damage to cardiac endothelial cells with no evidence of cardiomyocyte loss and co-staining of cardiac sections with endothelial marker CD31 revealed that ~80% of apoptotic cells in sorafenib treated hearts were endothelial cells (ECs). To assess for the time course for EC damage, we treated mice with sorafenib for 8h, 24h, 48h and 72h. The increase in EC death was already detected after 8h treatment and peaking at 24h-48h. RNA profiling analysis showed that expression of angiopoietin-1 (Angpt1) was reduced, whereas expression of Angpt2 was induced in hearts of mice treated with sorafenib for 4 weeks and in EC fractions from hearts of mice treated with sorafenib for 72h. Angpt-1 overexpression induced protective signaling pathways in hearts of mice treated with sorafenib for 48h, but did not rescue from acute EC injury. Treatment of Angpt1 overexpressing mice with sorafenib for 4 weeks resulted in capillary rarefaction and heart failure, evidenced by a decrease in LV systolic function and a 40-fold increase in ANP expression. Conclusion: our data shows that sorafenib cardiotoxicity is due to endothelial cell damage in the heart and results in heart failure with preserved ejection fraction. Furthermore, antagonizing the function of Angpt2 by overexpression of Angpt1 is detrimental during sorafenib treatment.

Backround Obesity has been recognized as a state of subclinical inflammation resulting in a loss of insulin receptors and decreased insulin sensitivity. We here studied in vivo the role of circulating matrix metaloproteinase-8 (MMP-8) among young healthy twin adults. Also in vitro analysis of the cleavage of human insulin receptor (INSR) by MMP-8 was investigated as well its inhibition by doxycycline and other MMP-8 inhibitor, Ilomastat/GM6001, which are broadspectrum MMP-inhibitors. Materials and methods We analyzed serum MMP-8 levels by a time-resolved immunofluorometric assay in obese (n=34), overweight (n=76) and normal weight (n=130) twin individuals. The effect of MMP-8 on INSR and the effects of synthetic MMP-8 inhibitors, doxycycline and Ilomastat/GM6001, were studied by SDS-PAGE. Results We found that in obese individuals relative to normal weight individuals the serum MMP-8 levels and MMP-8/TIMP-1 ratio were significantly increased (P=0.0031 and P=0.031, respectively). Among normal weight and obese individuals also smoking significantly increases serum MMP-8 and MMP-8/TIMP-1 ratio. In vitro we found that INSR was degraded by MMP-8 and this was inhibited by doxycycline and Ilomastat/GM6001. Conclusions Obesity associated with elevated circulating MMP-8 found among young adults may contribute to progression of insulin resistance by cleaving INSR. This INSR cleavage by MMP-8 can be inhibited by synthetic MMP-8 inhibitors such as doxycycline. In addition to obesity, also smoking independently explained increased MMP-8 levels. Our results suggest that MMP-8 is an essential mediator in systemic subclinical inflammatory response in obesity, and a potential drug target. This article is protected by copyright. All rights reserved.

As one of the most abundant constituents of the tumour microenvironment (TME), cancer-associated fibroblasts (CAF) display critical roles during tumour progression and metastasis. Multiple classes of molecules including growth factors, cytokines, proteases and extracellular matrix proteins, are produced by CAF to act as mediators of the stroma-tumour interactions. One of the main channels for this communication is associated with extracellular vesicles (EV), which are secreted particles loaded with protein and genetic information. In this study, we evaluated the effects of EV derived from CAF primary human cell lines (n = 5) on proliferation, survival, migration, and invasion of oral squamous cell carcinoma (OSCC) cells. As controls, EV from human primary-established normal oral fibroblasts (NOF, n = 5) were used. Our in vitro assays showed that CAF-EV significantly induces migration and invasion of OSCC cells and promote a disseminated pattern of HSC-3 cell invasion in the 3D organotypic assay. Furthermore, gene expression analysis of EV-treated cancer cells revealed changes in the pathways associated with tumour metabolism and up-regulation of tumour invasion genes. Our findings suggest a significant role of CAF-EV in promoting the migration and invasion of OSCC cells, which are related to the activation of cancer-related pathways.

Pro-tumorigenic activities of matrix metalloproteinase (MMP) 9 have been linked to many cancers, but recently the tumour-suppressing role of MMP9 has also been elucidated. The multifaceted evidence on this subject prompted us to examine the role of MMP9 in the behaviour of oral tongue squamous cell carcinoma (OTSCC) cells. We used gelatinase-specific inhibitor, CTT2, and short hairpin (sh) RNA gene silencing to study the effects of MMP9 on proliferation, motility and invasion of an aggressive OTSCC cell line, HSC-3. We found that the migration and invasion of HSC-3 cells were increased by CTT2 and shRNA silencing of MMP9. Proliferation, in turn, was decreased by MMP9 inhibition. Furthermore, arresten-overexpressing HSC-3 cells expressed increased levels of MMP9, but exhibited decreased motility compared with controls. Interestingly, these cells restored their migratory capabilities by CTT2 inhibition of MMP9. Hence, although higher MMP9 expression could give rise to an increased tumour growth in vivo due to increased proliferation, in some circumstances, it may participate in yet unidentified molecular mechanisms that reduce the cell movement in OTSCC.

Here we describe a protocol to utilize human benign leiomyoma tissue in in vitro 3D model that enables an assessment of cell invasion. The chapter also describes detailed instructions for image analysis to quantify the results. Leiomyoma is a benign tumor of the uterus which mimics authentic components of the tumor microenvironment including fibroblasts, vessels, collagen fibers, and extracellular protein composition. The leiomyoma invasion model represents a superior 3D model for cell invasion studies compared to the other non-human organotypic models.

Here we describe a protocol to utilize human benign leiomyoma tissue in in vitro 3D model that enables an assessment of cell invasion. The chapter also describes detailed instructions for image analysis to quantify the results. Leiomyoma is a benign tumor of the uterus which mimics authentic components of the tumor microenvironment including fibroblasts, vessels, collagen fibers, and extracellular protein composition. The leiomyoma invasion model represents a superior 3D model for cell invasion studies compared to the other non-human organotypic models.

Background: Matrix metalloproteinase-8 (MMP-8) has oncosuppressive properties in various cancers. We attempted to assess MMP-8 function in oral tongue squamous cell carcinoma (OTSCC). Methods: MMP-8 overexpressing OTSCC cells were used to study the effect of MMP-8 on proliferation, apoptosis, migration, invasion and gene and protein expression. Moreover, MMP-8 functions were assessed in the orthotopic mouse tongue cancer model and by immunohistochemistry in patient samples. Results: MMP-8 reduced the invasion and migration of OTSCC cells and decreased the expression of MMP-1, cathepsin-K and vascular endothelial growth factor-C (VEGF-C). VEGF-C was induced by transforming growth factor-beta 1 (TGF-beta 1) in control cells, but not in MMP-8 overexpressing cells. In human OTSCC samples, low MMP-8 in combination with high VEGF-C was an independent predictor of poor cancer-specific survival. TGF-beta 1 treatment also restored the migration of MMP-8 overexpressing cells to the level of control cells. In mouse tongue cancer, MMP-8 did not inhibit metastasis, possibly because it was eliminated in the peripheral carcinoma cells. Conclusions: The suppressive effects of MMP-8 in OTSCC may be mediated through interference of TGF-beta 1 and VEGF-C function and altered proteinase expression. Together, low MMP-8 and high VEGF-C expression have strong independent prognostic value in OTSCC.

Matrix metalloproteinases (MMPs) have traditionally been considered as tumor promoting enzymes as they degrade extracellular matrix components, thus increasing the invasion of cancer cells. It has become evident, however, that MMPs can also cleave and alter the function of various non-matrix bioactive molecules, leading to both tumor promoting and suppressive effects. We applied systematic review guidelines to study MMP8 in cancer including the use of MMP8 as a prognostic factor or as a target/anti-target in cancer treatment, and its molecular mechanisms. A total of 171 articles met the inclusion criteria. The collective evidence reveals that in breast, skin and oral tongue cancer, MMP8 inhibits cancer cell invasion and proliferation, and protects patients from metastasis via cleavage of non-structural substrates. Conversely, in liver and gastric cancers, high levels of MMP8 worsen the prognosis. Expression and genetic alterations of MMP8 can be used as a prognostic factor by examination of the tumor and serum/plasma. We conclude, that MMP8 has differing effects on cancers depending on their tissue of origin. The use of MMP8 as a prognostic factor alone, or with other factors, seems to have potential. The molecular mechanisms of MMP8 in cancer further emphasize its role as an important regulator of bioactive molecules.

Objectives Oral tongue squamous cell carcinoma (OTSCC)is an aggressive cancer with poor survival and increasing incidence. Matrix metalloproteinase-8 (MMP-8), unlike most MMPs, provides protective effects in various human cancers including OTSCC. Yet, the mechanism behind these effects remain unclear. Knowledge of the molecular basis of disease progression is crucial for developing novel treatments. Thus our study aimed to identify novel candidate substrates of MMP-8 to examine the mechanisms behind its tumor-suppressive actions in OTSCC. Findings We have previously generated stably MMP-8 overexpressing oral squamous cell carcinoma cell line (HSC-3 MMP-8+ cells) and showed that overexpression of MMP-8 significantly decreased the cell migration and invasion. However, the molecular mechanisms hampering the motility of these cells remained unrevealed. The current study aimed to unravel these mechanisms by subjecting the secretomes of MMP-8+ and control HSC-3 cells to Terminal Amine Isotopic Labeling of Substrates (TAILS) analysis to find novel candidate substrates for MMP-8. The analysis revealed cleaved proteins, including dysadherin, 60S ribosomal protein L13, kallikrein-5, lipolysis-stimulated lipoprotein receptor, matrix-remodeling-associated protein 7, POTE ankyrin domain family member E, stathmin 1 and tubulin alpha-1C chain, which were enriched in MMP-8+ secretomes. Dysadherin is known to promote metastasis of various cancers and decrease cell adhesion. MMP-8+ cells showed decreased levels of dysadherin, suggesting a cleavage by MMP-8 from the cell membrane. Moreover, the adhesion of MMP-8+ cells was enhanced which might affect the migration. Conclusions Several novel candidate substrates of MMP-8 were revealed by TAILS analysis. The potential substrates, including dysadherin, may play crucial role in the changed behavior of MMP-8+ cells and needs to be further explored for their potential role in OTSCC. The cleavage of tumor-promoting dysadherin from the cell membrane might be one of the mechanisms by which MMP-8 increases tumor cell adhesion and thereby suppresses migration.

Squamous cell carcinoma (SCC) of the tongue is the most common cancer in the oral cavity and has a high mortality rate. A total of 90 mobile tongue SCC samples were analysed for Bryne's malignancy scores, microvascular density, and thickness of the SCC sections. In addition, the staining pattern of cyclooxygenase-2, alphavbeta6 integrin, the laminin-5 gamma2-chain, and matrix metalloproteinases (MMPs) -2, -7, -8, -9, -20, and -28 were analysed. The expression of MMP-8 (collagenase-2) was positively associated with improved survival of the patients and the tendency was particularly prominent in females. No sufficient evidence for a correlation with the clinical outcome was found for any other immunohistological marker. To test the protective role of MMP-8 in tongue carcinogenesis, MMP-8 knockout mice were used. MMP-8 deficient female mice developed tongue SCCs at a significantly higher incidence than wild-type mice exposed to carcinogen 4-Nitroquinoline-N-oxide. Consistently, oestrogen-induced MMP-8 expression in cultured HSC-3 tongue carcinoma cells, and MMP-8 cleaved oestrogen receptor (ER) alpha and beta. According to these data, we propose that, contrary to the role of most proteases produced by human carcinomas, MMP-8 has a protective, probably oestrogen-related role in the growth of mobile tongue SCCs.

Background The composition of the matrix molecules is important in in vitro cell culture experiments of e.g. human cancer invasion and vessel formation. Currently, the mouse Engelbreth-Holm-Swarm (EHS) sarcoma -derived products, such as Matrigel®, are the most commonly used tumor microenvironment (TME) mimicking matrices for experimental studies. However, since Matrigel® is non-human in origin, its molecular composition does not accurately simulate human TME. We have previously described a solid 3D organotypic myoma disc invasion assay, which is derived from human uterus benign leiomyoma tumor. Here, we describe the preparation and analyses of a processed, gelatinous leiomyoma matrix, named Myogel. Methods A total protein extract, Myogel, was formulated from myoma. The protein contents of Myogel were characterized and its composition and properties compared with a commercial mouse Matrigel®. Myogel was tested and compared to Matrigel® in human cell adhesion, migration, invasion, colony formation, spheroid culture and vessel formation experiments, as well as in a 3D hanging drop video image analysis. Results We demonstrated that only 34 % of Myogel’s molecular content was similar to Matrigel®. All test results showed that Myogel was comparable with Matrigel®, and when mixed with low-melting agarose (Myogel-LMA) it was superior to Matrigel® in in vitro Transwell® invasion and capillary formation assays. Conclusions In conclusion, we have developed a novel Myogel TME matrix, which is recommended for in vitro human cell culture experiments since it closely mimics the human tumor microenvironment of solid cancers. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1944-z) contains supplementary material, which is available to authorized users.

Matrix metalloproteinase-8 (MMP-8)-deficient mice (Mmp8-/-) exhibit delayed dermal wound healing, but also partly contradicting results have been reported. Using the Mmp8-/- mice we investigated the role of MMP-8 in acute wound healing of the mobile tongue, and analyzed the function of tongue fibroblasts in vitro. Interestingly, in the early phase the tongue wounds of Mmp8-/- mice healed faster than those of wild type (wt) mice resulting in significant difference in wound widths (P=0.001, 6–24 h). The Mmp8-/- wounds showed no change in myeloperoxidase positive myeloid cell count, but the level of transforming growth factor (TGF)-β1 was significantly increased (P=0.007) compared to the wt tongues. Fibroblasts cultured from wt tongues expressed MMP-8 and TGF-β1. However, higher TGF-β1 levels were detected in Mmp8-/- fibroblasts, and MMP-8 treatment decreased phosphorylated Smad-2 levels and α-smooth muscle actin expression in these fibroblasts suggesting reduced TGF-β1 signaling. Consistently, a degradation of recombinant TGF-β1 by MMP-8 decreased its ability to activate the signaling cascade in fibroblasts. Moreover, collagen gels with Mmp8-/- fibroblasts reduced more in size. We conclude that MMP-8 regulates tongue wound contraction rate and TGF-β1 levels. In vitro analyses suggest that MMP-8 may also play a role in regulating TGF-β1 signaling of stromal fibroblasts.

Matrix metalloproteinase-8 (MMP-8) participates in skin wound healing and inflammation. We hypothesized that MMP-8 plays a role in wound healing after tooth extraction and in periapical inflammation. Bone formation, collagen metabolism, and inflammation in tooth extraction socket and in periapical lesions were analyzed in wild-type mice and in MMP-8-deficient (MMP-8(-/-)) mice. New trabecular bone area in the extraction sockets and in periapical lesions were similar in both groups. In extraction sockets significantly more type III procollagen was synthesized, and the neutrophil and MMP-9 levels were lower in MMP-8(-/-) mice. The amount of Fas ligand, identified as a substrate for MMP-8, was lower in alveolar mucosa but higher in alveolar bone of MMP-8(-/-) mice. These results indicate that MMP-8 can modulate inflammation and collagen metabolism of alveolar bone and mucosa.