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Development of a multidisciplinary platform for the screening of new preventive and therapeutic strategies against oral mucositis

Final Report Summary - MUCOSITIS PLATFORM (Development of a multidisciplinary platform for the screening of new preventive and therapeutic strategies against oral mucositis)

Oral mucositis is a serious and common side effect of anticancer therapies resulting in a decreased quality of life, hospitalization and in worst-case, interruption of the therapy. Oral mucositis is still largely considered as an inflammatory reaction of the mucosal tissues in the mouth, and the involvement of the oral microbiota in the pathobiology of the disease is largely underexplored. Therefore, the general aim of this fellowship was to develop an integrated mucositis platform to further explore the role of the microbiome in the context of oral chemo- and radiotherapy-induced mucositis, following the strategy depicted below (scheme).
First objective was the development of an in vitro oral mucosa model that allows the study of microbe-epithelial interactions in the context of chemotherapy-induced oral mucositis. The second objective was to validate the data generated by this model in vivo based of new and existing data of rat studies. The integration of in vitro and rat models of mucositis in the mucositis platform allowed in a further stage of the project the identification of new biomarkers, risk factors and mucositis modulating factors as well as the screening of candidate anti-mucositis molecules (third objective).
Results and impact can be summarised as follows:
- Using our newly developed in vitro oral mucosa model, we were able to show the importance of the host-microbe interactions in a wound healing. Wound recovery seems to be negatively impacted by the presence of microbiota and the extent of the effect depended largely on the microbial load and the type of species. Klebsiella oxytoca and Lactobacillus salivarius clearly had a negative effect on wound recovery while Streptococcus mitis and Streptococcus oralis simulated healing. We further showed evidence that microbial quorum sensing molecules might play an role in inhibiting wound recovery although we were unable to identify a particular quorum sensing molecule. Hence, new strategies to interfere with the quorum sensing molecules may lead to new therapies for mucositis.
- Via the platform, we next showed that a even single dose of radiotherapy affected the abundance of Rothia, Granulicatella and Gemella in reconstructed biofilms, biofilm formation and virulence of microbiota as was shown for Klebsiella oxytoca. These results highlight the effects of low dose irradiation on the composition and functional behaviour of resident microbiota, which might be linked to a general negative impact on epithelial wound healing in the context of radiation-induced mucositis especially when the microbial load on the wounds is high.
- Further, we observed shifts in the microbial community of the oral cavity of head and neck cancer patients during radiotherapy in a clinical pilot study. Shifts in the buccal and tongue mucosa occurred after the first 2-3 weeks of radiotherapy. Within the buccal microbiota, the relative abundance of buccal Bacteroidetes and Fusobacteria spp. were shown to increase due to radiotherapy. In contrast, the Actinobacteria tended to decrease at the end of the therapy. At the genus level, the number of Abiotrophia significantly decreased due to radiation, whereas the abundance of Lachnospiraceae_incertae_sedis and unclassified Prevotellaceae spp. increased towards the end of the therapy. The abundances of unclassified Streptococcaceae were shown to fluctuate during radiotherapy. Compared to the buccal microbiota, no clear shifts in abundances could be noticed in the tongue samples after radiotherapy. Importantly, a significant correlation between the shifts in the buccal microbial community and the patients’ normal oral functioning (pain, nutrition) was found.
- We were also able to identify microbial biomarkers of oral mucositis. For the buccal oral microbiota, the data from the cohort study showed that when Gemella spp. were present at an abundance of > 2.5 % and Atopobium spp. were absent, patients did not suffer from severe mucositis. It was further noticed that patients who suffered from severe mucositis showed a significantly higher abundance of unclassified Prevotellaceae and absence of unclassified Enterobacteriaceae in the follow-up period after radiotherapy. When we evaluated the link between the oral microbial composition during the therapy and the mucositis grade at those specific time points, a significant increase in 13 genera was found, of which Bifidobacterium, Fusobacterium, Peptostreptococcus, Porhyromonas and Prevotella spp. were of interest as they increased steadily with mucositis grade. For the tongue, patients with higher abundances of Leptotrichia, Atopobium and an unclassified group of Lachnospiraceae at the start of their treatment were more likely to develop severe mucositis during therapy. In the follow-up period, Streptococcus spp. and unclassified Pasteurellaceae were more abundant on the tongue surface of patients that suffered from severe mucositis during radiotherapy. During and at the end of the radiotherapy, 7 low abundant genera were significantly linked with the mucositis grade of which Parvimonas and Peptostreptococcus spp. were the most interesting as these genera became at least 10 times more abundant when patients suffered from mucositis grade 2 and 3. These results show that abundance of certain microbiota, such as Prevotella spp, increased steadily with the severity of oral mucositis and hence might have the potential to be used as a microbial biomarker for mucositis. Moreover, the abundance of specific microbiota, such as Atopobium spp., at start of the therapy was shown to be predictive for severe oral mucositis. Those genera should be investigated more in detail both in vitro and in patients, as they may provide potentially new therapeutic targets in the combat against mucositis. We are now exploring the radiosensitivity of different oral species, both as pure cultures and in complex biofilms, to identify potential pathogens present in the oral cavity that may be activated following radiotherapy.
- We also identified a cellular biomarker of oral mucositis. During the development of mucositis, the epithelial barrier is disrupted which allows microbiota to invade underlying tissues. The E-cadherin/catenin complex at the membrane of normal epithelial cells is instrumental in maintaining cell-cell adhesion as this complex is part of the adherent junctions. Therefore, we examined if soluble E-cadherin (sE-cadherin) could be used in the clinic as a biomarker of mucositis or as a therapeutic target. Our results from a cohort study showed that salivary sE-cadherin levels decreased during radiotherapy. We further found that sE-cadherin can be used as a predictive marker of severe mucositis in the oropharynxcarcinoma patients in the first week of treatment. Measuring salivary sE-cadherin levels could therefore be useful to identify patients that are prone to develop severe oral mucositis in order to provide preventive/curative measurements and/or more frequent follow-up.
- Using the platform, we screened a range of candidate anti-mucositis drugs. We showed that the plant-derived polyphenolic compound 8-PN delayed the onset of radiation-induced oral mucositis in an established mouse model for oral radiotherapy-induced mucositis. We hypothesize that 8-PN stabilizes the E-cadherin mediated cell–cell adhesion in the mucosal layers, thereby delaying further damage and protecting the mucosa from microbial translocation. We further showed that Vitamin D has the potential to increase the viability of epithelial cells that were exposed to the chemotoxic drug irinotecan and we are currently exploring this molecule further in our integrated platform.
To conclude, results demonstrate an association between microbiota composition and mucositis severity and suggest that, upon confirmation, these data can be used to guide stratified patient management strategies. Initial clinical impact will be via targeted patient education and early intervention. The identification of patients at risk of severe toxicity may permit selection for treatment aimed at reducing symptom severity, interventions to improve bacterial overgrowth or alternative cancer treatments otherwise applied for selected indications due to costs.
Contact: Barbara Vanhoecke, PhD