Organotypic three-dimensional assays based on human leiomyoma-derived matrices

Alongside cancer cells, tumours exhibit a complex stroma containing a repertoire of cells, matrix molecules and soluble factors that actively crosstalk between each other. Recognition of this multifaceted concept of the tumour microenvironment (TME) calls for authentic TME mimetics to study cancer in vitro. Traditionally, tumourigenesis has been investigated in non-human, three-dimensional rat type I collagen containing organotypic discs or by means of mouse sarcoma-derived gel, such as Matrigel (R). However, the molecular compositions of these simplified assays do not properly simulate human TME. Here, we review the main properties and benefits of using human leiomyoma discs and their matrix Myogel for in vitro assays. Myoma discs are practical for investigating the invasion of cancer cells, as are cocultures of cancer and stromal cells in a stiff, hypoxic TME mimetic. Myoma discs contain soluble factors and matrix molecules commonly present in neoplastic stroma. In Transwell, IncuCyte, spheroid and sandwich assays, cancer cells move faster and form larger colonies in Myogel than in Matrigel (R). Additionally, Myogel can replace Matrigel (R) in hanging-drop and tube-formation assays. Myogel also suits three-dimensional drug testing and extracellular vesicle interactions. To conclude, we describe the application of our myoma-derived matrices in 3D in vitro cancer assays. This article is part of the discussion meeting issue 'Extracellular vesicles and the tumour microenvironment'.Peer reviewe

Evaluation of the budding and depth of invasion (BD) model in oral tongue cancer biopsies

It is of great clinical importance to identify simple prognostic markers from preoperative biopsies that could guide treatment planning. Here, we compared tumor budding (B), depth of invasion (D), and the combined scores (i.e., budding and depth of invasion (BD) histopathologic model) in preoperative biopsies and the corresponding postoperative specimens of oral tongue squamous cell carcinoma (OTSCC). Tumor budding and depth of invasion were evaluated in the pre- and postoperative samples from 100 patients treated for OTSCC. Sensitivity and specificity statistics were used. Our results showed statistically significant (P <0.001) relationship between pre- and postoperative BD scores. There was an agreement between the pre- and postoperative BD model scores in 83 cases (83%) with 57.1% sensitivity (95% CI 39.4 to 73.7%) and 96.9% specificity (95% CI 89.3 to 99.6%). Our findings suggest that the BD model, analyzed from representative biopsies, could be used for the treatment planning of OTSCC.Peer reviewe

Tenascin-C and fibronectin expression divide early stage tongue cancer into low- and high-risk groups

Background:Oral tongue squamous cell carcinoma (OTSCC) metastasises early, especially to regional lymph nodes. There is an ongoing debate on which early stage (T1-T2N0) patients should be treated with elective neck dissection. We need prognosticators for early stage tongue cancer. Methods: Mice immunisation with human mesenchymal stromal cells resulted in production of antibodies against tenascin-C (TNC) and fibronectin (FN), which were used to stain 178 (98 early stage), oral tongue squamous cell carcinoma samples. TenascinC and FN expression in the stroma (negative, moderate or abundant) and tumour cells (negative or positive) were assessed. Similar staining was obtained using corresponding commercial antibodies. Results: Expression of TNC and FN in the stroma, but not in the tumour cells, proved to be excellent prognosticators both in all stages and in early stage cases. Among early stages, when stromal TNC was negative, the 5-year survival rate was 88%. Correspondingly, when FN was negative, no cancer deaths were observed. Five-year survival rates for abundant expression of TNC and FN were 43% and 25%, respectively. Conclusions: Stromal TNC and, especially, FN expressions differentiate patients into low-and high-risk groups. Surgery alone of early stage primary tumours might be adequate when stromal FN is negative. Aggressive treatments should be considered when both TNC and FN are abundant.Peer reviewe

FRAXE-associated mental retardation protein (FMR2) is an RNA-binding protein with high affinity for G-quartet RNA forming structure

FRAXE is a form of mild to moderate mental retardation due to the silencing of the FMR2 gene. The cellular function of FMR2 protein is presently unknown. By analogy with its homologue AF4, FMR2 was supposed to have a role in transcriptional regulation, but robust evidences supporting this hypothesis are lacking. We observed that FMR2 co-localizes with the splicing factor SC35 in nuclear speckles, the nuclear regions where splicing factors are concentrated, assembled and modified. Similarly to what was reported for splicing factors, blocking splicing or transcription leads to the accumulation of FMR2 in enlarged, rounded speckles. FMR2 is also localized in the nucleolus when splicing is blocked. We show here that FMR2 is able to specifically bind the G-quartet-forming RNA structure with high affinity. Remarkably, in vivo, in the presence of FMR2, the ESE action of the G-quartet situated in mRNA of an alternatively spliced exon of a minigene or of the putative target FMR1 appears reduced. Interestingly, FMR1 is silenced in the fragile X syndrome, another form of mental retardation. All together, our findings strongly suggest that FMR2 is an RNA-binding protein, which might be involved in alternative splicing regulation through an interaction with G-quartet RNA structure

A novel human leiomyoma tissue derived matrix for cell culture studies

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 (R), are the most commonly used tumor microenvironment (TME) mimicking matrices for experimental studies. However, since Matrigel (R) 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 (R). Myogel was tested and compared to Matrigel (R) 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 (R). All test results showed that Myogel was comparable with Matrigel (R), and when mixed with low-melting agarose (Myogel-LMA) it was superior to Matrigel (R) in in vitro Transwell (R) 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.Peer reviewe

Cardiovascular disease, chronic kidney disease, and diabetes mortality burden of cardiometabolic risk factors from 1980 to 2010: A comparative risk assessment

Background: High blood pressure, blood glucose, serum cholesterol, and BMI are risk factors for cardiovascular diseases and some of these factors also increase the risk of chronic kidney disease and diabetes. We estimated mortality from cardiovascular diseases, chronic kidney disease, and diabetes that was attributable to these four cardiometabolic risk factors for all countries and regions from 1980 to 2010. Methods: We used data for exposure to risk factors by country, age group, and sex from pooled analyses of population-based health surveys. We obtained relative risks for the effects of risk factors on cause-specific mortality from meta-analyses of large prospective studies. We calculated the population attributable fractions for each risk factor alone, and for the combination of all risk factors, accounting for multicausality and for mediation of the effects of BMI by the other three risks. We calculated attributable deaths by multiplying the cause-specific population attributable fractions by the number of disease-specific deaths. We obtained cause-specific mortality from the Global Burden of Diseases, Injuries, and Risk Factors 2010 Study. We propagated the uncertainties of all the inputs to the final estimates. Findings: In 2010, high blood pressure was the leading risk factor for deaths due to cardiovascular diseases, chronic kidney disease, and diabetes in every region, causing more than 40% of worldwide deaths from these diseases; high BMI and glucose were each responsible for about 15% of deaths, and high cholesterol for more than 10%. After accounting for multicausality, 63% (10·8 million deaths, 95% CI 10·1-11·5) of deaths from these diseases in 2010 were attributable to the combined effect of these four metabolic risk factors, compared with 67% (7·1 million deaths, 6·6-7·6) in 1980. The mortality burden of high BMI and glucose nearly doubled from 1980 to 2010. At the country level, age-standardised death rates from these diseases attributable to the combined effects of these four risk factors surpassed 925 deaths per 100 000 for men in Belarus, Kazakhstan, and Mongolia, but were less than 130 deaths per 100 000 for women and less than 200 for men in some high-income countries including Australia, Canada, France, Japan, the Netherlands, Singapore, South Korea, and Spain. Interpretation: The salient features of the cardiometabolic disease and risk factor epidemic at the beginning of the 21st century are high blood pressure and an increasing effect of obesity and diabetes. The mortality burden of cardiometabolic risk factors has shifted from high-income to low-income and middle-income countries. Lowering cardiometabolic risks through dietary, behavioural, and pharmacological interventions should be a part of the global response to non-communicable diseases. Funding: UK Medical Research Council, US National Institutes of Health. © 2014 Elsevier Ltd

The role of tumor microenvironment on oral tongue cancer invasion and prognosis

Abstract Oral tongue squamous cell carcinoma (OTSCC) is the most common cancer of the oral cavity. The 5-year mortality of OTSCC remains at about 50%. The tumor microenvironment (TME) is now recognized as an important factor in cancer progression and metastasis, as well as a tool for prognostication. The aim of this study was to elucidate the roles of TME hypoxia and soluble factors on cancer cell migration and invasion, and the prognostic value of two extracellular matrix (ECM) molecules: tenascin-C (TNC) and fibronectin (FN). Hypoxia was studied using oral squamous cell carcinoma cells in migration and invasion assays. Invasion assays were carried out using a 3D-myoma invasion method. Similarly, the effect of soluble factors as well as ECM alterations were studied using the myoma model: the effect of soluble factors was studied by rinsing the myoma discs prior to experiments, and ECM alterations by lyophilizing and rehydrating. ECM was further studied by analyzing the prognostic value of TNC and FN from OTSCC samples. The effect of hypoxia was shown to be OTSCC cell line dependent: the effect of hypoxia on migration and invasion was increased in aggressive cell lines. Additionally, the response to hypoxia was altered in rinsed tissue. Tissue rinsing media were analyzed and factors affecting cell motility were found. The TME was found to be pivotal for cancer invasion: invasion was impaired in non-neoplastic tissue. Furthermore, changes in the ECM by lyophilization and rehydration led to a change in the invasion mechanism. High expression of stromal TNC and FN were excellent prognosticators in early-stage OTSCC. In conclusion, the present study highlighted the role of various TME components in cancer cell invasion as well as prognostication in OTSCC. Additionally, this study provided feasible tools for more precise diagnosis of early-stage OTSCC.Tiivistelmä Liikkuvan kielen levyepiteelikarsinooma (OTSCC) on suuontelon yleisin syöpä. Viiden vuoden kuolleisuus OTSCC:an on edelleen noin 50 %. Kasvaimen mikroympäristön (TME) tiedetään nykyään olevan tärkeässä roolissa syövän kehityksessä ja etäpesäkkeiden muodostuksessa, sekä tarjoavan työkaluja ennusteiden laadintaan. Tämän tutkimuksen tarkoituksena oli selvittää TME:n hypoksian ja liukoisten tekijöiden vaikutusta syöpäsolujen liikkumiseen ja invaasioon ympäröivään kudokseen, sekä tutkia kahden solunulkoisen matriksin (ECM) proteiinin, tenaskiini-C:n (TNC) ja fibronektiinin (FN), vaikutusta OTSCC:n ennusteeseen. Hypoksian vaikutusta tutkittiin käyttäen suun levyepiteelikarsinoomasoluja liikkuvuus- ja invaasiokokeissa. Invaasiokokeissa hyödynnettiin kolmiulotteista ihmisen myoomaan perustuvaa invaasiomallia. Myös liukoisten tekijöiden ja ECM:n muutosten vaikutusten tutkimisessa käytettiin myoomamallia: liukoisten tekijöiden vaikutusta tutkittiin huuhtomalla myoomakiekot ennen niiden käyttämistä, ja ECM:n muutosten vaikutusta kylmäkuivaamalla ja uudelleen nesteyttämällä myoomakiekot. ECM:ia tutkittiin myös analysoimalla TNC:n ja FN:n värjäytyvyyden merkitystä OTSCC:n ennusteessa. Hypoksian vaikutus osoittautui solulinjariippuvaiseksi: hypoksia lisäsi kielisyöpäsolujen liikkuvuutta ja invaasiota eniten aggressiivisimmilla solulinjoilla. Lisäksi solujen vaste hypoksialle oli erilainen huuhdotussa kudoksessa. Huuhteluliuos analysoitiin ja siitä löydettiin solujen liikkumiseen vaikuttavia tekijöitä. TME:n havaittiin olevan ratkaisevassa roolissa syöpäsolujen invaasiossa: syöpäsolut eivät kyenneet invasoitumaan lainkaan ei-neoplastiseen kudokseen. Lisäksi muutosten ECM:ssä havaittiin johtavan muutoksiin solujen käyttämässä invaasion mekanismissa. Strooman TNC:n ja FN:n värjäytyvyyden todettiin olevan erinomaisia ennustekijöitä aikaisen vaiheen OTSCC:ssa. Tiivistettynä voidaan todeta, että tämä tutkimus alleviivasi useiden TME:n komponenttien vaikutusta syövän invaasiolle ja ennusteelle OTSCC:ssä. Lisäksi se tarjoaa käyttökelpoiset työkalut (TNC ja FN) tarkemmalle diagnostiikalle aikaisen vaiheen OTSCC:ssä

Fluctuating role of antimicrobial peptide hCAP18/LL-37 in oral tongue dysplasia and carcinoma

Oral tongue squamous cell carcinoma (OTSCC), the most common cancer in the oral cavity, is aggressive and its incidence is increasing globally. Human host defense cationic antimicrobial peptide-18/antimicrobial peptide leucine-leucine-37 (hCAP18/LL-37) plays a complex role in various types of cancers. In the present study, we characterized the effects of exogenous LL-37 on three OTSCC cell lines and determined the expression of hCAP18/LL-37 in oral dysplastic and OTSCC patient samples. Our data revealed that LL-37, especially in high doses, mostly reduced the proliferation of OTSCC cells, but the effect was fluctuating. However, LL-37 stimulated the migration and invasion of OTSCC cells. The high dose of LL-37 also increased the amount of total epidermal growth factor receptor (EGFR) probably due to stabilization of the receptor to the plasma membrane. However, activation of EGFR downstream pathways was mostly decreased. Our immunohistochemical analysis showed that the hCAP18/LL-37 expression was higher in normal/mild dysplasia than in moderate/severe dysplasia and OTSCC. The hCAP18/LL-37 expression did not correlate with clinicopathological features or outcome of OTSCC patients. Our data suggest that LL-37 has a fluctuating effect on proliferation, migration and invasion of OTSCC cells, but it does not seem to play a role in the progression of OTSCC.Peer reviewe

Neoplastic extracellular matrix environment promotes cancer invasion in vitro

The invasion of carcinoma cells is a crucial feature in carcinogenesis. The penetration efficiency not only depends on the cancer cells, but also on the composition of the tumor microenvironment. Our group has developed a 3D invasion assay based on human uterine leiomyoma tissue. Here we tested whether human, porcine, mouse or rat hearts as well as porcine tongue tissues could be similarly used to study carcinoma cell invasion in vitro. Three invasive human oral tongue squamous cell carcinoma (HSC-3, SCC-25 and SCC-15), melanoma (G-361) and ductal breast adenocarcinoma (MDA-MB-231) cell lines, and co-cultures of HSC-3 and carcinoma-associated or normal oral fibroblasts were assayed. Myoma tissue, both native and lyophilized, promoted invasion and growth of the cancer cells. However, the healthy heart or tongue matrices were unable to induce the invasion of any type of cancer cells tested. Moreover, when studied in more detail, small molecular weight fragments derived from heart tissue rinsing media inhibited HSC-3 horizontal migration. Proteome analysis of myoma rinsing media, on the other hand, revealed migration enhancing factors. These results highlight the important role of matrix composition for cancer invasion studies in vitro and further demonstrate the unique properties of human myoma organotypic model. (C) 2016 Elsevier Inc. All rights reserved.Peer reviewe