Biopsy-derived oral keratinocytes – A model to potentially test for oral mucosa radiation sensitivity

Thomsen, Andreas; Aldrian, Christine; Luka, Benedikt; Hornhardt, Sabine; Gomolka, Maria; Moertl, Simone; Hess, Joseph; Zitzelsberger, Horst; Heider, T.; Schlueter, Nadine; Rau, Shane W.; Ordonez, B. Monroy; Schäfer, Henning; Rücker, Gerta; Henke, M.

doi:10.1016/j.ctro.2022.03.007

Cited by 3 publications

(10 citation statements)

References 33 publications

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“…When the basal stratum was analyzed, we found a positive immunostaining signal for cytokeratins AE5/AE3, CK5 and CK14, and a negative signal for other types of cytokeratins, with no differences between POM and OOM. Apart from AE1/AE3, which is an unspecific cocktail of cytokeratins, CK5 and CK14 are typically expressed by proliferating epithelia, and have been used as a marker of keratinocytes (Bucchieri et al, 2012; Thomsen et al, 2022). Their expression at the basal stratum, along with the absence of other cytokeratins that are associated to keratinocyte differentiation, confirms the stemness character of cells allocated at this stratum, and suggests that no differences between POM and OOM may exist at this level.…”

Section: Discussionmentioning

confidence: 99%

Histological characterization of the human masticatory oral mucosa. A histochemical and immunohistochemical study

Ibáñez‐Cortés,

Martín‐Piedra,

Blanco‐Elices

et al. 2023

Microscopy Res & Technique

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BackgroundHistology of human oral mucosa is closely related with its function and anatomical location, and a proper characterization of the human masticatory oral mucosa could be very useful in periodontal pathology.ObjectiveIn the present work, we have carried out a comprehensive study in order to determine the main histological features of parakeratinized (POM) and orthokeratinized (OOM) masticatory human oral mucosa using light and electron microscopy.MethodsTo perform this, we have used several histological, histochemical and immunohistochemical methods to detect key markets at the epithelial, basement membrane and connective tissue levels.ResultsOur results demonstrated that POM and OOM share many histological similarities, as expected. However, important differences were observed at the epithelial layer of POM, that was significantly thicker than the epithelial layer found in OOM, especially due to a higher number of cells at the stratum spinosum. The expression pattern of CK10 and filaggrin revealed intense signal expression in OOM as compared to POM. Collagen and proteoglycans were more abundant in OOM stroma than in POM. No differences were found for blood vessels and basement membrane.ConclusionThese results may contribute to a better understanding of the pathological conditions affecting the human masticatory oral mucosa. In addition, these findings could be useful for the generation of different types of oral mucosa by tissue engineering techniques.Research highlights Microscopical features of parakeratinized and orthokeratinized masticatory human oral mucosa showed important differences at both, epithelial and stromal levels. Parakeratinized masticatory human oral mucosa exert thicker epithelial layer, especially, at the stratum spinosum in comparison to orthokeratinized human oral mucosa. Cytokeratin 10 and filaggrin human epithelial markers were intensively expressed in orthokeratinized masticatory human oral mucosa in comparison to parakeratinized masticatory human oral mucosa. At the stromal level, orthokeratinized masticatory human oral mucosa exhibit higher levels of collagen and proteoglycans than parakeratinized masticatory oral mucosa. The deep knowledge of histological features of masticatory oral mucosa could lead to a better understanding of oral mucosa pathology and advanced treatments.

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Section: Discussionmentioning

confidence: 99%

Histological characterization of the human masticatory oral mucosa. A histochemical and immunohistochemical study

Ibáñez‐Cortés,

Martín‐Piedra,

Blanco‐Elices

et al. 2023

Microscopy Res & Technique

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“…To generate primary GBM cell lines, the patient-derived tumor tissue was cut into small pieces, transferred into a 6-well plate, and covered with medium-filled agarose cups (provided by Dr. Andreas Thomsen, University of Freiburg [ 28 ]). After 1–2 weeks of incubation, the cells were transferred to culture flasks and cultivated in RPMI 1640 medium supplemented with 10% FCS and 1% penicillin/streptomycin at 37 °C, 5% CO 2 , and 95% humidity.…”

Section: Methodsmentioning

confidence: 99%

“…In order to establish a reliable 3D model of patient-derived GBM cells for analysis of long-term tumor growth and radiation response, we used a conical agarose 3D microwell system [ 28 , 29 ]. The microwells are made of agarose, which allows the diffusion of molecules, and the non-adhesive characteristic prevents cells from attaching to the surface.…”

Section: Introductionmentioning

confidence: 99%

Establishment of a 3D Model to Characterize the Radioresponse of Patient-Derived Glioblastoma Cells

Strand,

Schrickel,

Dobiasch

et al. 2023

Cancers

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Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. Despite modern, multimodal therapeutic options of surgery, chemotherapy, tumor-treating fields (TTF), and radiotherapy, the 5-year survival is below 10%. In order to develop new therapies, better preclinical models are needed that mimic the complexity of a tumor. In this work, we established a novel three-dimensional (3D) model for patient-derived GBM cell lines. To analyze the volume and growth pattern of primary GBM cells in 3D culture, a CoSeedisTM culture system was used, and radiation sensitivity in comparison to conventional 2D colony formation assay (CFA) was analyzed. Both culture systems revealed a dose-dependent reduction in survival, but the high variance in colony size and shape prevented reliable evaluation of the 2D cultures. In contrast, the size of 3D spheroids could be measured accurately. Immunostaining of spheroids grown in the 3D culture system showed an increase in the DNA double-strand-break marker γH2AX one hour after irradiation. After 24 h, a decrease in DNA damage was observed, indicating active repair mechanisms. In summary, this new translational 3D model may better reflect the tumor complexity and be useful for analyzing the growth, radiosensitivity, and DNA repair of patient-derived GBM cells.

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“…OM not only has a negative impact on quality of life (for example extreme pain and difficulty in swallowing) but is also a detrimental dose-limiting factor for RT and a reason to interrupt or even terminate radiation therapy ( 11 ). Several intrinsic and extrinsic factors, including individual radiosensitivity, determine the risk for the development of oral mucositis ( 12 , 13 ).…”

Section: Introductionmentioning

confidence: 99%

“…Besides cell death, γ-IR (0.1 Gy) was also shown to induce differentiation in Ca 2+ -activated keratinocytes, validated by increased mRNA and protein levels of involucrin, a marker for keratinocyte differentiation ( 25 ). Biopsy-derived oral keratinocytes were recently proposed as a model to investigate individual radiosensitivity, where irradiated keratinocytes showed a dose-dependent (2 Gy-8 Gy) decrease in their spread as well as their colony forming efficiency ( 13 ).…”

Section: Introductionmentioning

confidence: 99%

Towards unravelling biological mechanisms behind radiation-induced oral mucositis via mass spectrometry-based proteomics

et al. 2023

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ObjectiveHead and neck cancer (HNC) accounts for almost 890,000 new cases per year. Radiotherapy (RT) is used to treat the majority of these patients. A common side-effect of RT is the onset of oral mucositis, which decreases the quality of life and represents the major dose-limiting factor in RT. To understand the origin of oral mucositis, the biological mechanisms post-ionizing radiation (IR) need to be clarified. Such knowledge is valuable to develop new treatment targets for oral mucositis and markers for the early identification of “at-risk” patients.MethodsPrimary keratinocytes from healthy volunteers were biopsied, irradiated in vitro (0 and 6 Gy), and subjected to mass spectrometry-based analyses 96 h after irradiation. Web-based tools were used to predict triggered biological pathways. The results were validated in the OKF6 cell culture model. Immunoblotting and mRNA validation was performed and cytokines present in cell culture media post-IR were quantified.ResultsMass spectrometry-based proteomics identified 5879 proteins in primary keratinocytes and 4597 proteins in OKF6 cells. Amongst them, 212 proteins in primary keratinocytes and 169 proteins in OKF6 cells were differentially abundant 96 h after 6 Gy irradiation compared to sham-irradiated controls. In silico pathway enrichment analysis predicted interferon (IFN) response and DNA strand elongation pathways as mostly affected pathways in both cell systems. Immunoblot validations showed a decrease in minichromosome maintenance (MCM) complex proteins 2-7 and an increase in IFN-associated proteins STAT1 and ISG15. In line with affected IFN signalling, mRNA levels of IFNβ and interleukin 6 (IL-6) increased significantly following irradiation and also levels of secreted IL-1β, IL-6, IP-10, and ISG15 were elevated.ConclusionThis study has investigated biological mechanisms in keratinocytes post-in vitro ionizing radiation. A common radiation signature in keratinocytes was identified. The role of IFN response in keratinocytes along with increased levels of pro-inflammatory cytokines and proteins could hint towards a possible mechanism for oral mucositis.

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Biopsy-derived oral keratinocytes – A model to potentially test for oral mucosa radiation sensitivity

Cited by 3 publications

References 33 publications

Histological characterization of the human masticatory oral mucosa. A histochemical and immunohistochemical study

Histological characterization of the human masticatory oral mucosa. A histochemical and immunohistochemical study

Establishment of a 3D Model to Characterize the Radioresponse of Patient-Derived Glioblastoma Cells

Towards unravelling biological mechanisms behind radiation-induced oral mucositis via mass spectrometry-based proteomics

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