Metabolomics, Transcriptome and Single-Cell RNA Sequencing Analysis of the Metabolic Heterogeneity between Oral Cancer Stem Cells and Differentiated Cancer Cells.

Understanding the distinct metabolic characteristics of cancer stem cells (CSC) may allow us to better cope with the clinical challenges associated with them. In this study, OSCC cell lines (CAL27 and HSC3) and multicellular tumor spheroid (MCTS) models were used to generate CSC-like cells. Quasi-targeted metabolomics and RNA sequencing were used to explore altered metabolites and metabolism-related genes. Pathview was used to display the metabolites and transcriptome data in a KEGG pathway. The single-cell RNA sequencing data of six patients with oral cancer were analyzed to characterize in vivo CSC metabolism. The results showed that 19 metabolites (phosphoethanolamine, carbamoylphosphate, etc.) were upregulated and 109 metabolites (2-aminooctanoic acid, 7-ketocholesterol, etc.) were downregulated in both MCTS cells. Integration pathway analysis revealed altered activity in energy production (glycolysis, citric cycle, fatty acid oxidation), macromolecular synthesis (purine/pyrimidine metabolism, glycerophospholipids metabolism) and redox control (glutathione metabolism). Single-cell RNA sequencing analysis confirmed altered glycolysis, glutathione and glycerophospholipid metabolism in in vivo CSC. We concluded that CSCs are metabolically inactive compared with differentiated cancer cells. Thus, oral CSCs may resist current metabolic-related drugs. Our result may be helpful in developing better therapeutic strategies against CSC.

KLF7 is associated with poor prognosis and regulates migration and adhesion in tongue cancer.

OBJECTIVE: The present study was performed to determine the clinical relevance of KLF7 in tongue squamous cell carcinoma (TSCC) and to characterize its potential function and mechanism of action. MATERIALS AND METHODS: KLF7 expression was measured by RT-qPCR in 21 tongue cancer samples. The clinical relevance of KLF7 was analyzed in another cohort of 127 TSCC samples from a public database. Then, we performed RNA sequencing analysis in KLF7-overexpressing TSCC (SCC9 and CAL27) cells to define significantly altered pathways. The possible changes in migration and adhesion were then analyzed in KLF7-overexpressing and knockdown TSCC cells. RESULTS: Our results showed that KLF7 mRNA expression was upregulated in TSCC and was significantly associated with the T and N stages. Patients with high-KLF7 expression had worse overall survival. RNA sequencing and KEGG enriched pathway analysis showed that altered genes were enriched in extracellular matrix-receptor interactions and focal adhesions in both cell lines. KLF7-overexpressing TSCC cell lines showed enhanced migration capacity and cell adhesion ability, and knockdown of KLF7 expression decreased TSCC migration and adhesion ability. CONCLUSIONS: We concluded that KLF7 was overexpressed in TSCC and has prognostic value. KLF7 promoted TSCC migration and increased cell adhesion.

CDK4 and TERT amplification in head and neck mucosal melanoma.

BACKGROUND: Recent high-throughput sequencing studies have revealed frequent CDK4 and TERT amplification in mucosal melanoma, suggesting that they are potential therapeutic targets. In this study, we investigated the statuses of CDK4 and TERT in head and neck mucosal melanoma (HNMM) with the aim of providing preclinical data to support future clinical trials. METHODS: In total, 29 HNMM samples were collected, including 16 oral mucosal melanoma (OMM) samples and 13 nasal cavity/sinuses melanoma (SNMM) samples. Fluorescence in situ hybridization was used to analyze CDK4 and TERT amplification, and immunohistochemistry was used to analyze CDK4 and TERT protein expression patterns. CDK4 expression was knocked down in the ME cells (an OMM cell line), and changes in cell cycle were analyzed. Cell viability assays were performed to determine the sensitivity of ME to abemaciclib (a CDK4 inhibitor) combined with dacarbazine (an anti-melanoma chemotherapy drug). RESULTS: We detected five samples exhibited CDK4 amplifications and nine samples exhibited TERT amplifications in our HNMM series, and found that CDK4 amplification tended to occur in combination with TERT amplification. Amplifications of CDK4 and TERT were more common in OMM than in SNMM. Amplifications of CDK4 and TERT were associated with greater CDK4 and TERT protein expression levels. CDK4 knockdown led to delayed G1/S phase transition in ME cells. Furthermore, ME cells were sensitive to abemaciclib (IC50  = 5.23 nM). Abemaciclib and dacarbazine synergistically inhibited ME cells' viability. CONCLUSION: We confirmed high frequencies of CDK4 and TERT amplification in OMM. Combined therapy with a CDK4/6 inhibitor and anti-melanoma chemotherapeutic agents will be a reasonable strategy for future clinical trials concerning unresectable or metastatic OMM.

A comparative study of the surgical outcomes between video-assisted and open lateral neck dissection for papillary thyroid carcinoma with lateral neck lymph node metastases.

PURPOSE: Video-assisted lateral neck dissection (VALND) for papillary thyroid carcinoma (PTC) with lateral neck lymph node metastases (LNM) has been described previously, however, the advantages and drawbacks of VALND have not been demonstrated in previous studies. The aim of this study was to compare the surgical outcomes of video-assisted and open lateral neck dissection for PTC with lateral neck LNM. MATERIALS AND METHODS: Between May 2013 and November 2014, 92 consecutive patients with PTC and lateral neck lymph node metastases underwent total thyroidectomy with central compartment neck dissection and unilateral lateral neck dissection. These included 54 individuals who underwent video-assisted surgery, and 38 in whom an open approach was used. The two groups were retrospectively compared with respect to their clinicopathological characteristics, surgical outcomes and oncological completeness. RESULTS: The mean follow-up period was 18.6months. The mean tumor size, tumor stage, mean numbers of retrieved lymph nodes, mean postoperative serum thyroglobulin levels, complication rates, and mean postoperative hospital stay were similar between the two groups. The mean operation time was longer (p=0.0001) and mean age was lower (p=0.0354) in the video-assisted group. The cosmetic results, evaluated by numerical scale and verbal response scale, were in favor of the video-assisted group (p=0.0003 and p<0.0001, respectively). CONCLUSIONS: The safety and oncological completeness of VALND was similar to that of open procedures, but the VALND resulted in improved cosmetic results. VALND is an effective treatment for the selected cases of PTC with lateral neck LNM.

Predictive value of pAKT/PTEN expression in oral squamous cell carcinoma treated with cetuximab-based chemotherapy.

OBJECTIVE: Molecular alterations in downstream effectors of epidermal growth factor receptor may confer resistance to epidermal growth factor receptor inhibitors. Our aim is to investigate whether PTEN/pAKT expression predicts response to cetuximab-based chemotherapy in oral squamous cell carcinoma. STUDY DESIGN: We analyzed a cohort of 50 patients with oral squamous cell carcinoma treated with cetuximab-based induction chemotherapy. PTEN expression and pAKT expression were assessed by immunohistochemistry and their correlation with treatment outcome was analyzed. RESULTS: Of the study patients, 18.4% had low PTEN expression, and 38.8% had high pAKT expression. Lower pAKT expression were associated with pathologic remission (P = .034) and better disease-free survival (P = .031). CONCLUSION: Our study demonstrates that pAKT expression is a predictive biomarker of cetuximab-based induction chemotherapy in OSCC.

Risk factors for posterior to right recurrent laryngeal nerve lymph node metastasis in papillary thyroid carcinoma.

OBJECTIVES: To identify the risk factors for posterior right recurrent laryngeal nerve lymph node metastasis (PRRLN-LNM) in papillary thyroid carcinoma (PTC). METHODS: We conducted a retrospective study of 389 patients with primary PTC who underwent right lobectomy or total thyroidectomy, and comprehensive right or bilateral central compartment dissection (CCD) with or without lateral neck dissection (LND) between January 2010 and May 2013 at the Department of Head and Neck Surgery, Institute of Micro-Invasive Surgery of Zhejiang University, Zhejiang, China. The clinicopathological findings were investigated, and relative risk factors for PRRLN-LNM were analyzed. RESULTS: Central compartment LNM were present in 50.9% (198/389), and PRRLN-LNM were present in 12.6% (49/389) of patients, wherein 3.1% (12/389) had PRRLN-LNM only. A multivariate analysis revealed that younger age (≤ 35 years), extrathyroidal extension (ETE), lateral compartment LNM, prelaryngeal LNM, pretracheal, and right paratracheal LNM were independent predictors of PRRLN-LNM. CONCLUSION: This study revealed that younger age (≤ 35 years), ETE, prelaryngeal LNM, lateral compartment LNM, and pretracheal and right paratracheal lymph nodes (anterior to the right recurrent laryngeal nerve [level VIa]), LNM were independent factors of PRRLN-LN (level VIb). Therefore, comprehensive right CCD should be routinely performed for such patients.

[Guided bone regeneration at a dehiscence-type defect using chitosan/collagen membranes in dogs].

OBJECTIVE: To compare a developed absorbable chitosan/collagen membrane (CCM) with a standard biodegradable collagen membrane for the treatment of implant dehiscence-type defect in dog model. METHODS: The right four mandibular premolars and the first molar were extracted in each of 10 beagle dogs.Four months later, acute buccal dehiscence-type defects were surgically created following implant site preparation in each dog. Using self-control, defects were randomly assigned to four different groups: CCM-1 (with the ratio of chitosan and collagen of 40: 1), CCM-2(with the ratio of chitosan and collagen of 20: 1), Bio-Gide collagen membrane (BG collagen), control. The animals were sacrificed after 4 (3 animals), 8 (3 animals) and 12 (4 animals) weeks of healing interval for histological observation and histomorphometrical analysis including defect length (DL), new bone height (NBH), bone-to-implant contact (BIC) and area of new bone fill (BA). RESULTS: Newly formed bone was observed in all the groups and became mature with time. At 8 weeks, increased mean NBH and BIC values were obtained for all the groups, the mean NBH values of the CCM-1, CCM-2 and BG groups [( 1.1 0 ± 0.11)∼(1.48 ± 0.07) mm]were significantly higher than that of the control [(0.74 ± 0.12) mm] (P < 0.05). At 12 weeks, the membranes treated groups obtained more mean NBH,BIC and BA values compared with the control. The CCM-1 groups showed the highest mean NBH value [(1.91 ± 0.25) mm], which was significantly higher than the control [(1.20 ± 0.34) mm](P < 0.05).However, no statistically significant differences in BIC and BA were found between membrane groups and control and among the membranes treated groups. CONCLUSIONS: The results of this study demonstrated that the developed CCM can enhance bone regeneration and obtaine similar amounts of newly formed bone compared with defects regenerated with a standard collagen membrane.

Guided bone regeneration using chitosan-collagen membranes in dog dehiscence-type defect model.

PURPOSE: The purpose of the present study was to compare a newly developed chitosan-collagen membrane (CCM) with a standard collagen membrane (SCM) regarding their effects on guided bone regeneration. MATERIALS AND METHODS: The right mandibular premolars and first molar were extracted from 12 beagle dogs. Four months later, acute buccal dehiscence-type defects (4 × 3 mm in height and width) were surgically created after implant site preparation. The defects were randomly assigned to 4 different groups: CCM-1 (weight ratio of chitosan to collagen of 40:1), CCM-2 (weight ratio of chitosan to collagen of 20:1), SCM, and vehicle control. The dogs were sacrificed after 4, 8, and 12 weeks of healing for radiographic examination, histologic observation, and histometric analysis. RESULTS: The membrane-treated sites showed more bone formation than the control sites, although no statistically significant differences were found between the membrane-treated sites and the control sites for new bone-to-implant contact and new bone-filled area at any point. At 8 weeks, the new bone height for the membrane-treated sites was significantly greater statistically than that of the untreated group (P < .05). At 12 weeks, the CCM-1 group showed significantly greater new bone height (1.91 ± 0.25 mm) than the untreated group (1.20 ± 0.34 mm; P < .05). However, the CCMs did not show any statistically significant differences compared with the SCMs for any assessed parameter. CONCLUSIONS: The results of the present study have shown that the developed CCMs can enhance bone regeneration and could be a candidate for use in guided bone regeneration.