fully-human-tumor-based-matrix-three-dimensional-spheroid-invasion

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

University of Helsinki

University of Oulu

Oulu University Horspital

Helsinki University Hospital

University of oulu

Incidence rates for oral tongue squamous cell carcinoma (SCC) are steadily rising worldwide.

Improved outcomes with oral tongue squamous cell carcinoma in Finland.

Urokinase plasminogen activator (uPA) receptor (uPAR) is up-regulated at the invasive tumour front of human oral squamous cell carcinoma (OSCC), indicating a role for uPAR in tumour progression. We previously observed elevated expression of uPAR at the tumour-stroma interface in a mouse model for OSCC, which was associated with increased proteolytic activity. The tumour microenvironment regulated uPAR expression, as well as its glycosylation and cleavage. Both full-length- and cleaved uPAR (uPAR (II-III)) are involved in highly regulated processes such as cell signalling, proliferation, migration, stem cell mobilization and invasion. The aim of the current study was to analyse tumour associated factors and their effect on uPAR cleavage, and the potential implications for cell proliferation, migration and invasion.

Cleavage of the urokinase receptor (uPAR) on oral cancer cells: regulation by transforming growth factor - β1 (TGF-β1) and potential effects on migration and invasion.

Oxidized low-density lipoproteins (oxLDL) are formed as a result of lipid peroxidation and are highly immunogenic and proatherogenic. In this study, saliva antibodies binding to oxLDL, Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) were characterized and their cross-reactivity was evaluated.

Cross-reactive saliva IgA antibodies to oxidized LDL and periodontal pathogens in humans.

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  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 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  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 Additionally, Myogel can replace Matrigel 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  cancer assays.This article is part of the discussion meeting issue 'Extracellular vesicles and the tumour microenvironment'.

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

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.

Human Tumor Tissue-Based 3D In Vitro Invasion Assays.

Different regions of oral squamous cell carcinoma (OSCC) have particular histopathological and molecular characteristics limiting the standard tumor-node-metastasis prognosis classification. Therefore, defining biological signatures that allow assessing the prognostic outcomes for OSCC patients would be of great clinical significance. Using histopathology-guided discovery proteomics, we analyze neoplastic islands and stroma from the invasive tumor front (ITF) and inner tumor to identify differentially expressed proteins. Potential signature proteins are prioritized and further investigated by immunohistochemistry (IHC) and targeted proteomics. IHC indicates low expression of cystatin-B in neoplastic islands from the ITF as an independent marker for local recurrence. Targeted proteomics analysis of the prioritized proteins in saliva, combined with machine-learning methods, highlights a peptide-based signature as the most powerful predictor to distinguish patients with and without lymph node metastasis. In summary, we identify a robust signature, which may enhance prognostic decisions in OSCC and better guide treatment to reduce tumor recurrence or lymph node metastasis.

Combining discovery and targeted proteomics reveals a prognostic signature in oral cancer.

Despite the importance of immune checkpoints in immunotherapy, the prognostic value of these molecules remains controversial in oral squamous cell carcinoma (OSCC). We performed a systematic review to investigate the prognostic significance of the immune checkpoints in OSCC.

The prognostic value of immune checkpoints in oral squamous cell carcinoma.

The analysis of the salivary metabolomic profile may offer an early phase approach to assess the changes associated with a wide range of diseases including head and neck cancer. The aim of the present study was to investigate the potential of nuclear magnetic resonance (NMR) spectroscopy for detecting the salivary metabolic changes associated with head and neck squamous cell carcinoma (HNSCC). Unstimulated whole-mouth saliva samples collected from HNSCC patients (primary tumour was located either in the larynx or in the oral cavity) and healthy controls were analysed by H-NMR spectroscopy. Reliably identified salivary metabolites were quantified and the determined concentration values were compared group-wise using a Mann-Whitney U-test. Multivariate discrimination function analysis (DFA) was conducted to identify such a combination of metabolites, when considered together, that gives maximum discrimination between the groups. HNSCC patients exhibited significantly increased concentrations of 1,2-propanediol (P=0.032) and fucose (P=0.003), while proline levels were significantly decreased (P=0.043). In the DFA model, the most powerful discrimination was achieved when fucose, glycine, methanol and proline were considered as combined biomarkers, resulting in a correct classification rate of 92.1%, sensitivity of 87.5% and specificity of 93.3%. To conclude, NMR spectrometric analysis was revealed to be a feasible approach to study the metabolome of saliva that is sensitive to metabolic changes in HNSCC and straightforward to collect in a non-invasive manner. Salivary fucose was of particular interest and therefore, controlled longitudinal studies are required to assess its clinical relevance as a diagnostic biomarker in HNSCC.

Potential role of nuclear magnetic resonance spectroscopy to identify salivary metabolite alterations in patients with head and neck cancer.

The aim was to update information on oral proliferative verrucous leukoplakia (PVL), a disease of verrucous-like lesions with high risk of malignancy, and its biomarkers.

Proliferative verrucous leukoplakia and its tumor markers: Systematic review and meta-analysis.

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.

Article	Year
Improved outcomes with oral tongue squamous cell carcinoma in Finland. Head & neck\| PubMed ID: 28481417	2017
Cleavage of the urokinase receptor (uPAR) on oral cancer cells: regulation by transforming growth factor - β1 (TGF-β1) and potential effects on migration and invasion. BMC cancer\| PubMed ID: 28526008	2017
Cross-reactive saliva IgA antibodies to oxidized LDL and periodontal pathogens in humans. Journal of clinical periodontology\| PubMed ID: 28548243	2017
Organotypic three-dimensional assays based on human leiomyoma-derived matrices. Philosophical transactions of the Royal Society of London. Series B, Biological sciences\| PubMed ID: 29158312	2018
Human Tumor Tissue-Based 3D In Vitro Invasion Assays. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 29318556	2018
Combining discovery and targeted proteomics reveals a prognostic signature in oral cancer. Nature communications\| PubMed ID: 30185791	2018
The prognostic value of immune checkpoints in oral squamous cell carcinoma. Oral diseases\| PubMed ID: 30315712	2018
Potential role of nuclear magnetic resonance spectroscopy to identify salivary metabolite alterations in patients with head and neck cancer. Oncology letters\| PubMed ID: 30344764	2018
Proliferative verrucous leukoplakia and its tumor markers: Systematic review and meta-analysis. Head & neck\| PubMed ID: 30536498	2019
Matrix metalloproteinase 9 inhibits the motility of highly aggressive HSC-3 oral squamous cell carcinoma cells. Experimental cell research\| PubMed ID: 30710501	2019

Tuula Salo

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