Comparison of different DNA preservation solutions for oral cytological samples.

OBJECTIVE: The objective of this study was to compare the DNA preservation capacity of buccal mucosa exfoliated cells when stored in different solutions under varying time and temperature conditions. DESIGN: DNA preservation solutions, including Dimethyl sulphoxide disodium-EDTA-saturated NaCl (DESS), Tris-EDTA-NaCl-Tween20 buffer (TENT), Nucleic Acid Preservation Buffer (NAP), and phosphate-buffered saline (PBS), were prepared. Buccal mucosa cells from a single patient were collected, dispensed into these solutions, and stored at room temperature (RT) and 4 °C for 24 h, 72 h, 30 days, 90 days, and 180 days. DNA was extracted using the salting-out method and the QIAamp DNA Mini Kit. DNA concentration and purity were determined using the QuBit device and NanoDrop, while DNA integrity was assessed using the Agilent 4200 TapeStation system. The ability to amplify the IFNA primer was also evaluated by PCR. RESULTS: The salting-out method yielded better concentration and purity results, with PBS, TENT, and DESS buffers demonstrating superior concentration values when stored at 4 °C, resulting in mean values exceeding 10 ng/µL for up to 30 days. DESS consistently exhibited the best integrity values over time for both temperature conditions. Amplification capacity was enhanced when samples were stored at 4 °C. When stored at RT, PBS achieved 100% amplification within 24 h. NAP yielded the poorest results. CONCLUSION: In the context of long-term preservation, the DESS buffer emerges as the most effective solution, maintaining requisite DNA quality and quantity standards for up to 30 days at RT and up to 3 months at 4 °C.

The role of tumor acidification in aggressiveness, cell dissemination and treatment resistance of oral squamous cell carcinoma.

AIMS: To investigate the role of tumor acidification in cell behavior, migration, and treatment resistance of oral squamous cell carcinoma (OSCC). MAIN METHODS: The SCC4 and SCC25 cell lines were exposed to acidified (pH 6.8) cell culture medium for 7 days. Alternatively, a long-term acidosis was induced for 21 days. In addition, to mimic dynamic pH fluctuation of the tumor microenvironment, cells were reconditioned to neutral pH after experimental acidosis. This study assessed cell proliferation and viability by sulforhodamine B and flow cytometry. Individual and collective cell migration was analyzed by wound healing, time lapse, and transwell assays. Modifications of cell phenotype, EMT induction and stemness potential were investigated by qRT-PCR, western blot, and immunofluorescence. Finally, resistance to chemo- and radiotherapy of OSCC when exposed to acidified environmental conditions (pH 6.8) was determined. KEY FINDINGS: The exposure to an acidic microenvironment caused an initial reduction of OSCC cells viability, followed by an adaptation process. Acidic adapted cells acquired a mesenchymal-like phenotype along with increased migration and motility indexes. Moreover, tumoral extracellular acidity was capable to induce cellular stemness and to increase chemo- and radioresistance of oral cancer cells. SIGNIFICANCE: In summary, the results showed that the acidic microenvironment leads to a more aggressive and treatment resistant OSCC cell population.

Somatic mutations and promotor methylation of the ryanodine receptor 2 is a common event in the pathogenesis of head and neck cancer.

Genomic sequencing projects unraveled the mutational landscape of head and neck squamous cell carcinoma (HNSCC) and provided a comprehensive catalog of somatic mutations. However, the limited number of significant cancer-related genes obtained so far only partially explains the biological complexity of HNSCC and hampers the development of novel diagnostic biomarkers and therapeutic targets. We pursued a multiscale omics approach based on whole-exome sequencing, global DNA methylation and gene expression profiling data derived from tumor samples of the HIPO-HNC cohort (n = 87), and confirmed new findings with datasets from The Cancer Genome Atlas (TCGA). Promoter methylation was confirmed by MassARRAY analysis and protein expression was assessed by immunohistochemistry and immunofluorescence staining. We discovered a set of cancer-related genes with frequent somatic mutations and high frequency of promoter methylation. This included the ryanodine receptor 2 (RYR2), which showed variable promoter methylation and expression in both tumor samples and cell lines. Immunohistochemical staining of tissue sections unraveled a gradual loss of RYR2 expression from normal mucosa via dysplastic lesion to invasive cancer and indicated that reduced RYR2 expression in adjacent tissue and precancerous lesions might serve as risk factor for unfavorable prognosis and upcoming malignant conversion. In summary, our data indicate that impaired RYR2 function by either somatic mutation or epigenetic silencing is a common event in HNSCC pathogenesis. Detection of RYR2 expression and/or promoter methylation might enable risk assessment for malignant conversion of dysplastic lesions.

Effects of extracellular acidity on resistance to chemotherapy treatment: a systematic review.

Metabolic alterations in the tumor microenvironment have a complex effect on cancer progression. Extracellular acidity is a consequence of metabolic switch in cancer and results in cell phenotypes with higher resistance to chemotherapeutics. However, mechanisms underlying the relationship between the extracellular acidity and chemoresistance are not clearly understood. This systematic review was carried out by searching the databases PubMed and EMBASE using the keywords "cancer" and "acidosis" or "acidic" and "chemoresistance" or "drug resistance." In vitro and in vivo studies that evaluated the effects of acidification of the tumor microenvironment on chemotherapeutic treatments were included. Literature reviews, letters to the editor, and articles that were not published in English were excluded. The search resulted in a total of 352 articles. After discarding 75 duplicate references, 277 articles were analyzed by sequentially reading through their titles, abstracts, and finally full-text. A total of 14 articles was selected. Acidification of the tumor microenvironment can trigger resistance through different mechanisms, such as increase in drug efflux transporters, inhibition of proton pumps, induction of the unfolded protein response (UPR), and cellular autophagy.