BackgroundCancer stem cells (CSCs) play an important role in tumor initiation, progression, and metastasis and are responsible for high therapeutic failure rates. Identification and characterization of CSC are crucial for facilitating the monitoring, therapy, or prevention of cancer. Great efforts have been paid to develop a more effective methodology. Nevertheless, the ideal model for CSC research is still evolving. In this study, we created a nonadhesive culture system to enrich CSCs from human oral squamous cell carcinoma cell lines with sphere formation and to characterize their CSC properties further.MethodsA nonadhesive culture system was designed to generate spheres from the SAS and OECM-1 cell lines. A subsequent investigation of their CSC properties, including stemness, self-renewal, and chemo- and radioresistance in vitro, as well as tumor initiation capacity in vivo, was also performed.ResultsSpheres were formed cost-effectively and time-efficiently within 5 to 7 days. Moreover, we proved that these spheres expressed putative stem cell markers and exhibited chemoradiotherapeutic resistance, in addition to tumor-initiating and self-renewal capabilities.ConclusionsUsing this nonadhesive culture system, we successfully established a rapid and cost-effective model that exhibits the characteristics of CSCs and can be used in cancer research.

Head and neck squamous cell carcinoma (HNSCC) is one of the leading causes of cancer-related death worldwide. The prognosis of HNSCC is usually poor because of its propensity for extensive invasion, local recurrence and frequent regional lymph node metastasis, even at initial diagnosis. Carcinoma-associated fibroblasts (CAFs), a major type of tumour-surrounding stromal cell, generate mediators through which they interact with tumours and contribute to cancer progression. The orchestration between CAFs and cancer cells is complex. Despite recent studies demonstrating the paracrine effect of stromal cells in the tumour microenvironment on initiation and progression of cancer cells, the major mediator related to CAFs and its underlying mechanism remain unknown. In the present study, we used organotypic culture to investigate CAFs that promote aggressive behaviour of HNSCC cells. Using microarray analysis, we detected abundant expression of interleukin-33 (IL-33) in CAFs and identified IL-33 as a critical mediator in CAF-induced invasiveness. Counteracting IL-33 activity diminished the aggressive phenotype of cancer cells induced by CAFs. Administration of IL-33 promoted cancer cell migration and invasion through induction of epithelial-to-mesenchymal transdifferentiation and increased IL-33 gene expression in cancer cells. In 40 patients with HNSCC, IL-33 expression in CAFs correlated with IL-33 expression in cancer cells. Most cases with a low invasion pattern grading score (IPGS) showed low or no expression of IL-33, whereas most HNSCC cases with high IPGS displayed over-expression of IL-33 in CAFs and cancer cells. High IL-33 expression associated with poor prognosis in terms of nodal metastasis-free survival. These results indicate that CAFs promote cancer invasiveness via paracrine and autocrine effects on microenvironmental IL-33 signalling, and suggest that IL-33 is a potential prognostic biomarker that could be considered in therapeutic strategies for the treatment of patients with HNSCC.

BACKGROUNDCranial nerve palsy is a rare complication after patients with nasopharyngeal carcinoma (NPC) receive radiotherapy using a technique that delivers 180–200 centigrays (cGy) per day. Cranial neuropathy is of particular clinical interest in terms of making a differential diagnosis, because it is also a common presenting manifestation in patients with NPC. Cranial neuropathy may lead to distressing signs and symptoms in these patients, and their treatment has not been addressed in previous reports. This article presents the authors' experience with radiotherapy‐related cranial nerve palsy in patients with NPC.METHODSNineteen patients were diagnosed with radiation‐related neuropathy. Patients with recurrent tumors or with a suspicion of persistent or recurrent tumors were excluded. Most patients were treated using 180 cGy or 200 cGy per fraction per day. The total dose was 7000–13,000 cGy to the nasopharynx and 5000–9000 cGy to the neck. Unilateral vocal cord paralysis alone and hearing loss were not included in the analysis.RESULTSThere were 15 male patients and 4 female patients. The latency before palsy occurred was 12–240 months. Single nerve palsy developed in four patients, including two patients with hypoglossal palsy and two patients with recurrent laryngeal palsy. Two patients had three nerve palsies each. The other 13 patients presented with 2 nerve palsies each. Vagus and hypoglossal palsy appeared to be a frequent combination and occurred in 11 patients. Overall, there were 17 patients with hypoglossal palsy (7 bilateral, 8 left‐sided, and 2 right‐sided), 11 patients with vagus palsy (2 bilateral, 7 left‐sided, and 2 right‐sided), 6 patients with recurrent laryngeal nerve palsy (5 bilateral), and 2 patients with accessory palsies (all bilateral). Marked neck fibrosis was present in 12 patients. Patients who had vocal cord paralysis suffered from easy choking and hoarseness. Severe respiratory difficulty occurred in two patients who had bilateral vocal cord palsy. Surgical procedures included laryngoplasty, tracheostomy, and gastrostomy. Quality of life improved considerably after patients underwent surgery.CONCLUSIONSRadiotherapy‐related cranial nerve palsy may occur in patients with NPC after they receive conventional radiotherapy. Hypoglossal nerve palsy was found the most frequently in this series, followed by vagus nerve palsy and recurrent laryngeal nerve palsy. Neck fibrosis and the course of the three nerves through the neck may be important risk factors for the development of palsy. The diagnosis must be made only after the possibilities of tumor‐induced palsy and idiopathic palsy are excluded. Surgery is helpful in improving the quality of life in many patients. Cancer 2002;95:404–9. © 2002 American Cancer Society.DOI 10.1002/cncr.10668

The mechanisms by which hypoxic tumors evade immunological pressure and anti-tumor immunity remain elusive. Here, we report that two hypoxia-responsive microRNAs, miR25 and miR93, are important for establishing an immunosuppressive tumor microenvironment by down-regulating expression of the DNA-sensor cGAS. Mechanistically, miR25/93 targets NCOA3, an epigenetic factor that maintains basal levels of cGAS expression, leading to repression of cGAS upon hypoxia. This allows hypoxic tumor cells to escape immunological responses induced by damage-associated molecular pattern molecules (DAMPs), specifically the release of mtDNA. Moreover, restoring cGAS expression results in an anti-tumor immune response. Clinically, decreased levels of cGAS are associated with poor prognosis for patients with breast cancer harboring high levels of miR25/93. Together, these data suggest that inactivation of the cGAS pathway plays a critical role in tumor progression, and reveals a direct link between hypoxia-responsive miRNAs and adaptive immune responses to the hypoxic tumor microenvironment, thus unveiling potential new therapeutic strategies.

Hypoxia is a hallmark of solid tumors that drives malignant progression by altering epigenetic controls. In breast tumors, aberrant DNA methylation is a prevalent epigenetic feature associated with increased risk of metastasis and poor prognosis. However, the mechanism by which hypoxia alters DNA methylation or other epigenetic controls that promote breast malignancy remains poorly understood. We discovered that hypoxia deregulates TET1 and TET3, the enzymes that catalyze conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), thereby leading to breast tumor-initiating cell (BTIC) properties. TET1/3 and 5hmC levels were closely associated with tumor hypoxia, tumor malignancy, and poor prognosis in breast cancer patients. Mechanistic investigations showed that hypoxia leads to genome-wide changes in DNA hydroxymethylation associated with upregulation of TNFa expression and activation of its downstream p38-MAPK effector pathway. Coordinate functions of TET1 and TET3 were also required to activate TNFa-p38-MAPK signaling as a response to hypoxia. Our results reveal how signal transduction through the TET-TNFa-p38-MAPK signaling axis is required for the acquisition of BTIC characteristics and tumorigenicity in vitro and in vivo, with potential implications for how to eradicate BTIC as a therapeutic strategy. Cancer Res; 75(18); 3912-24. Ó2015 AACR.

The purpose of this report is to describe a novel technique for endoscopic vidian neurectomy (EVN) based on preoperative computed tomography (CT) classification of the vidian canal (VC), and to present anatomical and surgical findings from an initial series. Retrospective study, consisting of medical chart review and patient interviews, of all preoperative CT-guided EVN procedures was performed from 2006 to 2010 at a tertiary-care medical center. A total of 89 patients with intractable rhinorrhea (77 males and 12 females, mean age 29 years, age range 16-57 years) underwent bilateral EVN. Configuration of the VC was classified into three types based on preoperative CT findings. The technique for surgical access of each of these configurations is presented. The most common configuration of the VC was type 2 (47%). A wide, direct, and safe exposure of the vidian nerve was achieved in all cases. 84 of 89 patients completed the questionnaires regarding the postoperative improvement in quality of life. Follow-up ranged from 2 to 42 months, with an average of 19.6 months. 77 of 84 (91.7%) patients were satisfied with their surgical result. Two patients underwent revision ETSVN due to relapsed symptoms. With the help of a preoperative CT scan of the paranasal sinuses, the vidian nerve can be identified precisely via an endoscopic intrasphenoidal or transsphenoidal approach, which provides an easy and reliable way to perform vidian neurectomy.

BackgroundTreatment failure in oral squamous cell carcinoma (OSCC) leading to local recurrence(s) and metastases is mainly due to drug resistance. Cancer stem cells (CSCs) are thought be responsible for the development of drug resistance. However, the correlations between CSCs, drug resistance, and new strategy against drug resistance in OSCC remain elusive.MethodsA drug-resistant sphere (DRSP) model was generated by using a nonadhesive culture system to induce drug-resistant cells from SCC25 oral cancer cells. A comparative analysis was performed between the parent control cells and DRSPs with a related treatment strategy focusing on the expression of epithelial–mesenchymal transition (EMT)-associated markers, drug-resistance-related genes, and CSC properties in vitro, as well as tumorigenicity and the regimen for tumor regression in vivo.ResultsOur data show the presence of a phenomenon of EMT with gradual cellular transition from an epithelioid to mesenchymal-like spheroid morphology during induction of drug resistance. The characterization of DRSPs revealed the upregulation of the drug-resistance-related genes ABCG2 and MDR-1 and of CSC-representative markers, suggesting that DRSPs have greater resistance to cisplatin (Cis) and stronger CSC properties compared with the control. Moreover, overexpression of phosphorylated heat-shock protein 27 (p-Hsp27) via the activation of p38 MAPK signaling was observed in DRSPs. Knockdown of Hsp27 decreased Cis resistance and induced apoptosis in DRSPs. Furthermore, an inhibitor of Hsp27, quercetin (Qu), suppressed p-Hsp27 expression, with alterations of the EMT signature, leading to the promotion of apoptosis in DRSPs. A xenographic study also confirmed the increase of tumorigenicity in DRSPs. The combination of Qu and Cis can reduce tumor growth and decrease drug resistance in OSCC.ConclusionsThe p38 MAPK–Hsp27 axis plays an important role in CSCs-mediated drug resistance in OSCC. Targeting this axis using Qu combined with Cis may be a treatment strategy to improve prognosis in patients with OSCC.

BackgroundMetastasis occurs in a series of discrete steps involving invasion, angiogenesis, lymphovascular space permeation, and establishment of secondary tumors. Malignant pleural effusion (MPE), a type of tumor metastasis, is usually a poor prognostic sign for patients with pulmonary adenocarcinoma, although its underlying mechanism has received less attention than other types of metastases have. The objective of the current study was to confirm whether cancer stem cells (CSCs) in MPE contribute to the “metastatic cascade” through the epithelial – mesenchymal transition (EMT), anoikis, and adaptation in the microenvironment.MethodsPulmonary tissue and corresponding cell blocks of MPE samples from 20 patients with primary adenocarcinoma were analyzed by immunohistochemical staining with CSC-representative markers (CD133, Nanog, and OCT-4) and EMT-associated markers (E-cadherin and vimentin). Correlations between these variables and clinico-pathological parameters were analyzed. Primary cultures from eight cases of MPE were investigated to characterize the CSC properties, including marker expression, sphere formation, and differentiation.ResultsExpressions of CSC-representative markers for 20 cases of MPE cell blocks were quite diverse and variable ranging from 15% to 90%. Stronger expression of CSC-representative markers and alteration of EMT-associated markers were found at the invasive fronts and in MPEs compared with the expression in primary pulmonary tumor tissues. The expression of OCT-4 in MPEs significantly related to distant metastasis and stage, as well as inversely correlated with patient survival. Primary cultures confirmed the CSC properties in MPE. Five of eight cases of MPE yielded adequate cell clusters, which also showed variable expressions of CSC markers in addition to sphere formation and the ability for differentiation and metastasis.ConclusionThis pilot study offers a better understanding of the metastatic cascade. Establishing a model of MPE will provide further insight into the role of CSCs in metastasis and may explain the high therapeutic failure rates for patients with MPE.