Development and validation of a gene model predicting lymph node metastasis and prognosis of oral squamous cell carcinoma based on single-cell and bulk RNA-seq analysis.

BACKGROUND: Lymph node metastasis can independently predict oral squamous cell carcinoma patients' survival. This study would investigate the genetic and cellular differences between oral squamous cell carcinoma with positive and negative lymph node metastases. METHODS: We gathered single-cell RNA sequencing and bulk gene expression data from the Cancer Genome Atlas and Gene Expression Omnibus databases. Sixty lymph node-metastasis-related genes were discovered with refined single-cell RNA sequencing data analysis, and consensus clustering provided three molecular subtypes of oral squamous cell carcinoma. Least absolute shrinkage and selection operator analyses were then utilized to establish a five-gene risk model. CIBERSORT analysis revealed the immune infiltration profile of different risk subgroups. RESULTS: Oral squamous cell carcinoma patients were classified into three subtypes based on the 60 lymph node-metastasis-related key genes identified by single-cell RNA sequencing data. Patients in Subtype 3 showed a tendency for lymph node metastasis and poorer prognosis. Moreover, five biomarkers were selected from the 60 genes to construct a five-gene risk model evaluating the risk of lymph node metastasis. A lower probability of lymph node metastasis and a better prognosis was observed in the low-risk group. The immune infiltration of three different risk groups was explored with CIBERSORT. Besides, further analysis implied different sensitivities of anticancer drugs, including immunotherapy drugs and targeted compounds, in the three risk groups. CONCLUSION: In view of intratumoral heterogeneity, we found 60 genes associated with lymph node metastasis of oral squamous cell carcinoma. Subsequently, we constructed a five-gene signature that could improve the prediction of lymph node metastasis, clinical outcome, and promote individualized treatment strategies for oral squamous cell carcinoma.

[The prognosis of neck dissection with sternocleidomastoid muscle preservation and resection in advanced oral squamous cell carcinoma: a retrospective cohort analysis].

PURPOSE: To investigate the prognosis of advanced oral squamous cell carcinoma (AOSCC) patients undergoing neck dissection with sternocleidomastoid muscle (SCM) preservation and resection. METHODS: From January 2013 to June 2017, a total of 235 AOSCC patients(stage Ⅲ and stage Ⅳ) who were diagnosed and underwent neck dissection at the Department of Oral and Maxillofacial Surgery, College and Hospital of Stomatology, Guangxi Medical University, were collected and followed-up. The differences in overall survival（OS）, local recurrence-free survival (LRFS) and regional recurrence-free survival (RRFS) were compared between different surgical procedures. SPSS 25.0 software package was used for statistical analysis. RESULTS: Among 235 patients with postoperative follow-up, 101 patients retained the SCM during operation, and 134 patients had SCM removed. There was no significant difference in 5-year survival rate and 5-year regional recurrence rate between the SCM preservation group and the SCM resection group. Kaplan-Meier method of univariate analysis showed that SCM preservation or resection had no significant difference in OS, LRFS and RRFS. Cox multivariate regression analysis results showed that there was no significant difference between different surgical procedures in OS, LRFS and RRFS, while N stage and postoperative chemoradiotherapy were independent influencing factors for OS, LRFS and RRFS in AOSCC patients. CONCLUSIONS: Neck dissection with SCM preservation in AOSCC patients has no effect on survival and recurrence (including local recurrence and regional recurrence). It is feasible for AOSCC patients to undergo SCM-preserving neck dissection when metastatic cervical lymph nodes do not invade SCM. N stage and postoperative chemoradiotherapy affect the prognosis of AOSCC patients.

[Maintenance of the stemness in CD133+primary oral squamous cell carcinoma cells under different culture conditions].

PURPOSE: CD133+/-cells were isolated and purified from primary oral squamous cell carcinoma(OSCC) to explore the effects of different culture conditions on the maintenance and biological characteristics of CD133+ primary OSCC. METHODS: CCK-8 was used to detect the ability of proliferation and cisplatin resistance between CD133+/-cell subsets. Transwell assay was used to compare the invasive ability of two cell subsets under the action of cisplatin. Flow cytometry was used to detect the proportion of CD133+ cells cultured by serum free medium(SFM) (with or without leukemia inhibitory factor, LIF) or serum supplied medium (SSM). Subcutaneous tumor model in nude mice was used to verify the difference in tumorigenicity of CD133+/- cell subsets. The transplanted tumor was removed for H-E staining and immunohistochemistry (IHC). SPSS 25.0 software package was used for statistical analysis. RESULTS: Compared with CD133- cell subsets, CD133+ cell subsets had stronger ability of proliferation in vitro(P<0.05) and cisplatin tolerance(P<0.001). Cisplatin had a stronger effect on the invasive ability of CD133- cell subsets than CD133+ cell subsets (P<0.01). No significant difference in the proportion of CD133+ cell between LIF-SFM and no-LIF-SFM was found (P>0.05); but compared with SSM culture method, SFM culture method could maintain the proportion of CD133+ cell better(P<0.05). CD133+ cell subsets showed stronger tumorigenic ability with fewer cells than CD133- cell subsets in nude mice(P<0.05). CONCLUSIONS: Serum free culture method can better maintain the characteristics of primary OSCC stem cells, but the addition of LIF has no significant effect on the maintenance of stemness of primary OSCC cells.

Expression of beta adrenergic receptor in oral squamous cell carcinoma and its significance to the prognosis.

The aim of this study was to detect the expression of ß-AR (Beta Adregenic Receptor) in Oral squamous cell carcinoma (OSCC), para-cancerous and normal oral mucosa and to investigate the relationship between the expression intensity and the characteristics and prognosis of oral cancer. 100 cases of OSCC were collected; 20 cases of paraneoplastic tissues and 10 cases of normal oral mucosa were taken as control. The expression of ß-AR was detected by immunohistochemical method and the average optical density determination using Image J software. Finally, the difference of ß-AR expression and the correlation with the clinicopathological factors were analyzed statistically. The expression of ß-AR in OSCC was higher than that in paracarcinoma and normal mucosa (P<0.01). The expression intensity of ß1, ß2-AR in preoperative lymph node metastasis group was higher than that in patients without lymph node metastasis (P<0.01). The expression intensity of ß3-AR was not related to pathological grade and tumor size (P>0.05). ß1 and ß2-AR in early stage of OSCC were higher than those in early stage (P<0.05). Lymph node metastasis, recurrence, TNM clinical stage, and the expression intensity of ß1-AR all had an effect on the cumulative survival rate. All the ß1, 2, 3-AR were expressed in OSCC. ß1 and ß2-AR were involved in lymphatic metastasis and had influence on clinical staging. Metastasis, recurrence, TNM stage and expression of ß1-AR had an effect on the cumulative survival rate of tumor. The expression of ß3-AR in OSCC was not associated with the pathological grades and tumor growth.

Propranolol inhibits angiogenesis via down-regulating the expression of vascular endothelial growth factor in hemangioma derived stem cell.

BACKGROUND: Oral propranolol (PRN) has recently been shown to be highly effective for infantile hemangiomas (IHs), and is currently recommended as the first-line treatment of complicated IHs. However, the therapeutic mechanism(s) still remain unclear. METHODS: In this study, we tested hemangioma-derived stem cells for expression of vascular endothelial growth factor (VEGF) in vitro and studied the inhibition of VEGF expression. We used PCR, Elisa, Western blotting and immunohistochemistry in vivo and in vitro trial. RESULTS: The study demonstrated that application of PRN at a "normal" concentration equivalent to plasma concentration did not inhibit proliferation or promote apoptosis of hemangioma derived stem cells (HemSCs) isolated from IH patients. PRN suppressed expression of vascular endothelial growth factor (VEGF) and basic Fibroblast Growth Factor (bFGF) in HemSCs in vitro. Morphological, histological and immunohistological improvement were observed in vivo using murine IH model in which HemSCs pre-treated with PRN were implanted into BALB/c-nu mice. In the pre-treated HemSC grafts, mean micro-vessel density (MVD) significantly decreased and protein levels of VEGF markedly decreased, while bFGF was still detectable. CONCLUSIONS: The results suggested PRN inhibited angiogenesis via down-regulating the expression of vascular endothelial growth factor in hemangioma derived stem cell. These findings provide critical insight into the potential mechanisms of PRN action on IH.

A practical guide to treatment of infantile hemangiomas of the head and neck.

Infantile hemangiomas are the most common benign vascular tumors in infancy and childhood. As hemangioma could regress spontaneously, it generally does not require treatment unless proliferation interferes with normal function or gives rise to risk of serious disfigurement and complications unlikely to resolve without treatment. Various methods for treating infant hemangiomas have been documented, including wait and see policy, laser therapy, drug therapy, sclerotherapy, radiotherapy, surgery and so on, but none of these therapies can be used for all hemangiomas. To obtain the best treatment outcomes, the treatment protocol should be individualized and comprehensive as well as sequential. Based on published literature and clinical experiences, we established a treatment guideline in order to provide criteria for the management of head and neck hemangiomas. This protocol will be renewed and updated to include and reflect any cutting-edge medical knowledge, and provide the newest treatment modalities which will benefit our patients.

Preliminary study on plasma RPN concentration of patients with infantile hemangioma treated with propranolol.

Propranolol (PRN) has recently been recommended as the first-line medicine for complicated infantile hemangiomas (IHs), because of the significant effect. However, no pharmacokinetic parameters have ever been reported for infants who receive PRN treatment for IH. In this study, we show that plasma PRN concentration is affected by the frequency of administration of PRN. A single daily administration of PRN (1 mg/kg/d) resulted in an early elevation of plasma PRN compared to a twice a day administration of the same dose. In contrast, the twice a day application resulted in a more prolonged expression at a later time-point. Our findings provide pharmacokinetic parameters of PRN action in IH for clinic.

Guidelines for the treatment of head and neck venous malformations.

Venous malformation is one of the most common benign vascular lesions, with approximately 40% of cases appearing in the head and neck. They can affect a patient's appearance and functionality and even cause life-threatening bleeding or respiratory tract obstruction. The current methods of treatment include surgery, laser therapy, sclerotherapy, or a combined. The treatment of small and superficial venous malformations is relatively simple and effective; however, the treatment of deep and extensive lesions involving multiple anatomical sites remains a challenge for the physicians. For complex cases, the outcomes achieved with one single treatment approach are poor; therefore, individualized treatment modalities must be formulated based on the patient's condition and the techniques available. Comprehensive multidisciplinary treatments have been adapted to achieve the most effective results. In this paper, based on the national and international literature, we formulated the treatment guidelines for head and neck venous malformations to standardize clinical practice. The guideline will be renewed and updated in a timely manner to reflect cutting-edge knowledge and to provide the best treatment modalities for patients.

CD133 selected stem cells from proliferating infantile hemangioma and establishment of an in vivo mice model of hemangioma.

BACKGROUND: Infantile hemangioma (IH) is the most common benign tumor in children with prevalence in the face and neck. Various treatment options including oral propranolol have been described for IH, but the mechanism of drugs remains enigmatic. The aim of this study was to investigate the pathogenesis and establish a reliable in vivo model of IH which can provide platform for drug exploration. METHODS: Stem cells from the proliferating hemangiomas (HemSCs) were isolated by CD133-tagged immunomagnetic beads. Their phenotype and angiogenic property were investigated by flow cytometry, culturing on Matrigel, real-time polymerase chain reaction (PCR), immunofluorescent staining and injection into BALB/c-nu mice. RESULTS: HemSCs had robust ability of proliferating and cloning. The time of cells doubling in proliferative phase was 16 hours. Flow cytometry showed that HemSCs expressed mesenchymal markers CD29, CD44, but not endothelial/hematopoietic marker of CD34 and hematopoietic marker CD45. The expression of CD105 was much lower than that of the reported hemangioma derived or normal mesenchymal stem cell (MSC). Real-time PCR showed that the mRNA levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and matrix metalloproteinase-1 (MMP-1) of HemSCs were higher than that of neonatal human dermal fibroblasts (NHDFs) and human umbilical vein endothelial cells (HUVECs). After HemSCs were cultured on Matrigel in vitro, they formed tube-like structure in a short time (16 hours) and differentiated into endothelial cells in 7 days. After 1 - 2 weeks of implantation into immunodeficient mice, HemSCs generated glucose transporter 1 positive blood vessels. When co-injected with HUVECs, the vascularization of HemSCs was greatly enhanced. However, the single implantation of HUVECs hardly formed blood vessels in BALB/c-nu mice (P < 0.05). CONCLUSIONS: HemSCs may be some kinds of primitive mesoderm derived stem cells with powerful angiogenic ability, which can recapitulate human hemangioma by co-injecting into immunodeficient mice with HUVECs.

Efficacy of pingyangmycin injection for the treatment of cervical epidermoid cysts.

Epidermoid cysts are benign lesions commonly seen in the head and neck region. Here, we describe the case of an epidermoid cyst in a 20-year-old man with a history of trauma presenting with a complaint of mass in the right cervical region. Upon closer examination, a 3cm long scar was noticed within the swollen region of the right submandibular area. MRI revealed a soft tissue mass involving the right neck and fine-needle aspiration biopsy showed that the content of the cyst was homogenous, confirming the diagnosis of epidermoid cyst. We used a pingyangmycin injection to manage the cyst with excellent short-term results, but the lesion reappeared after 3 months. The patient ultimately underwent surgical enucleation of the mass under general anesthesia.

Treatment guidelines of lymphatic malformations of the head and neck.

Lymphatic malformations, traditionally called lymphangiomas, are diseases caused by development errors of the lymphatic system. About 90% of the cases occur within 2years of age, except a few cases which occur in adulthood, and approximately 75% of the lesions are located in the head and neck region. The lesions can grow rapidly with infection, trauma or bleeding, resulting in disfigurement as well as severe impairment of respiration, swallow and speech. Although lymphatic malformations are benign lesions, they rarely resolve spontaneously, their infiltrating nature coupled with the difficulty in distinguishing involved vital structures of head and neck from adjacent normal tissues makes complete surgical resection even more difficult. The likelihood of postsurgical recurrence and complications is thus higher than other vascular lesions. Surgical resection, sclerotherapy and laser therapy are currently the main treatment modes of lymphatic malformations. Various treatment options have their advantages and disadvantages, the selection of treatment modalities should depend on the patient's individual status and available technology and expertise. The treatment protocol should be individualized, comprehensive as well as sequential in order to obtain the best treatment outcome. Based on published literatures and clinical experiences, we devised the treatment guideline for management of head and neck lymphatic malformations. This protocol will be reviewed and updated periodically to include cutting edge knowledge to provide the best treatment options to benefit our patients.

The potential role of progesterone during pregnancy as an induction of infantile hemangioma.

Infantile hemangioma(IH) is one of the most common benign tumors in infants characterized by occurrence within a few weeks after birth, rapid growth during the first year and spontaneous involution over a period of several years.Despite the high incidence rate of 5%-10% in infants of mixed European descent, detailed pathogenesis of IH remains elusive. Recent studies have indicated multipotential stem cells derived from hemangioma tissue(HemSCs) could recapitulate human infantile hemangioma in immunodeficient mice. Considering the effect of progesterone on regulation of cytokines and growth factors in endometrium as well as the inhibition of immune response, using progesterone during pregnancy might help the HemSCs escape from the immune response and reside in the tissue of embryo by the aid of increased MMPs and decreased TIMPs,then proliferation was stimulated by increased growth factors like VEGF and bFGF.Thus,IH is potentially produced.

[Cloning of human bone morphogenetic protein-2 gene and the construction of its eukaryotic expression vector].

OBJECTIVE: To clone human bone morphogenetic protein-2 (hBMP2) gene and construct its eukaryotic expression vector pcDNA3.1 -hBMP2. METHODS: Human BMP2 gene was amplified by RT-PCR method from human osteosarcoma cells and constructed into eukaryotic expression vector pcDNA3.1-hBMP2. The gene in the vector pcDNA3.1-hBMP2 was identified by PCR amplification, enzyme digestion and DNA sequencing. RESULTS: The cloned DNA was confirmed to be hBMP-2 gene. CONCLUSION: In this study, hBMP2 gene is successfully cloned and its eukaryotic expression vector pcDNA3.1-hBMP2 is constructed, which provides the foundation of using BMP2 gene therapy to accelerate new bone formation in distraction osteogenesis.

[The effect of dog bilateral distraction osteogensis in temporomandibular joint].

OBJECTIVE: To investigate the effect of bilateral mandibular distraction osteogenesis in the condyles. METHODS: 16 adult hybrid dogs were randomly divided into normal control group and experiment group. Experimental dogs underwent bilateral mandibular osteodistraction at a rate of 1 min/day. 4 dogs were killed respectively in distraction period, 2 and 8 weeks after completion of 10 days distraction. The bilateral condyles specimens were harvested and examined with histological and immunohistochemical methods. RESULTS: Compared with normal control group, various degrees of irregularities and erosion were found in fibrocartilage of condyle in experiment group, including damage in fibrous layer, hyperplasia layer and proliferative layer and osteogenic activity in cartilage layer. A significant increase of TGF-beta1 expression was also found in experiment groups. TGF-beta1 positive staining was noted in hypertrophic cell, matrix and chondroblast, osteoblast and matrix in osteogenic activity areas. These changes were the most obvious in 2 weeks after completion of distraction. CONCLUSION: Gradual bilateral mandibular distraction at a rate of 1 mm/day brought degenerative changes of condyle, but the changes are reversible.