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

A recurrent aphthous ulcer (RAU) is a common inflammatory ulcerative lesion affecting oral mucosa. We studied the eventual apoptosis of epithelial cells from the point of view of ulcer and inflammation. RAU lesions and healthy mucosa samples were immunostained for caspase-3 and high-mobility group box 1 (HMGB1). DNA nicks were identified using TUNEL staining. We studied the effects of tumor necrosis factor α (TNFα) and interferon γ (IFNγ) on the toll-like receptor 2 and 4 (TLR2 and TLR4) expression of human oral SCC-25 keratinocytes. We also studied the effects of self-DNA, all-thiol-HMGB1, and disulfide-HMGB1 on epithelial cells, with or without IFNγ. At the edge of RAU lesions, all epithelial cell layers were caspase-3(+), TUNEL(+), and HMGB-1(+) and had widened intercellular spaces. In contrast, healthy epithelial cells were negative for caspase-3 and TUNEL staining. HMGB1 was seen in only the basal cell layers, and the cells retained close cell-to-cell contacts. Self-DNA increased TNF-α mRNA (P = 0.02) in SCC-25 cells. Both TNFα and IFNγ (P = 0.01) increased TLR2. Upon TNFα stimulation, SCC-25 cells lost their nuclear HMGB1 staining. HMGB1 did not increase IL-8, IL-6, or TNF-α mRNA in SCC-25 cells, which was unaffected by the presence of IFNγ. We conclude that in healthy epithelium, the most superficial cells at the end of their life cycle are simply desquamated. In contrast, RAU is characterized by top-to-bottom apoptosis such that dead cells may slough off, leading to an ulcer. Because of a lack of scavenging anti-inflammatory macrophages, apoptotic cells probably undergo secondary necrosis releasing proinflammatory danger signals, which may contribute to the peripheral inflammatory halo. This is supported by self-DNA-induced TNFα synthesis. In contrast to TLR4- and TLR2-binding lipopolysaccharide used as a positive control, disulfide-HMGB1 did not stimulate proinflammatory cytokines. 

Epithelial Cell Apoptosis in Recurrent Aphthous Ulcers.

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.

Oral tongue squamous cell carcinoma (SCC) is characterized by early metastasis and poor prognosis. Interleukin-17F (IL-17F) plays a protective role in many tumors. However, IL-17F expression in oral tongue SCC tissue has not been investigated.

Extracellular interleukin-17F has a protective effect in oral tongue squamous cell carcinoma.

Oral mucosal dysplasia is a histologic feature of potentially malignant disorders that is associated with the risk of transformation to carcinoma. Dysplastic cells use many strategies during their transformation to cancer, including escape from the immune mediated destruction. We hypothesized that adaptive immunity is inhibited by activation of distinct immune checkpoint molecules, such as indoleamine 2,3-dioxygenase 1 (IDO1) and programmed death-ligand 1 (PD-L1).

Immune checkpoints indoleamine 2,3-dioxygenase 1 and programmed death-ligand 1 in oral mucosal dysplasia.

Oral tongue squamous cell carcinoma (OTSCC) cells are highly proliferative and invasive. Lingonberry contains several polyphenolic compounds similar to curcumin. We hypothesize that fermented lingonberry juice (FLJ) has an anti-invasive and anti-proliferative effect on OTSCC cells similarly to curcumin, which is known to be anti-carcinogenic.

Fermented Lingonberry Juice Inhibits Oral Tongue Squamous Cell Carcinoma Invasion  Similarly to Curcumin.

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.

Article	Year
Epithelial Cell Apoptosis in Recurrent Aphthous Ulcers. Journal of dental research\| PubMed ID: 25861801	2015
Human Tumor Tissue-Based 3D In Vitro Invasion Assays. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 29318556	2018
Extracellular interleukin-17F has a protective effect in oral tongue squamous cell carcinoma. Head & neck\| PubMed ID: 29761594	2018
Immune checkpoints indoleamine 2,3-dioxygenase 1 and programmed death-ligand 1 in oral mucosal dysplasia. Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology\| PubMed ID: 29851145	2018
Fermented Lingonberry Juice Inhibits Oral Tongue Squamous Cell Carcinoma Invasion Similarly to Curcumin. In vivo (Athens, Greece)\| PubMed ID: 30150430	2018
The prognostic value of immune checkpoints in oral squamous cell carcinoma. Oral diseases\| PubMed ID: 30315712	2018

Ahmed Al-Samadi

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