Characterizing The Morphological And Immunohistochemical Features Of Cancer Stem Cells And Their Clinical Implications In Nasopharyngeal Carcinoma

Background and objectiveNasopharyngeal carcinoma (NPC) is a distinctive type of head and neck cancer with distinct ethnic and geographic distributions. It occurs with the highest incidence in Southern China, approximately20to50new cases per100,000populations each year. In contrast with other head and neck cancers, most cases of NPCs are undifferentiated squamous cell carcinomas with a high tendency to invade adjacent tissues and metastasize to regional lymph nodes at an early stage. Furthermore, the majority of NPC patients died from local recurrence and distant metastases. It is known that genetic susceptibility, Epstein-Barr virus (EBV) infection and dietary factors play important roles in NPC pathogenesis. However, to date, the molecular mechanisms mediating the progression and metastasis of NPC have not been fully clarified.Cancer stem cells (CSCs) are being the hot spots in recent cancer research. The CSC concept postulates that, similar to the growth of embryonic stem cells (ESCs), the growth of tumors is fueled by limited numbers of dedicated stem cells that are capable of self-renewal and mutli-lineage. It has been considered that CSCs might be responsible for cancers’relapse and metabasis as well as insensitivity to the chemotherapy and radiotherapy. More interestingly, these stem and tumorigenic characteristics have been proved to be driven by epithelial-mesenchymal transition (EMT) induction. Increasing evidence has shown that EMT is crucial for the metastatic dissemination of epithelial cancer cells, providing tumor cells with the capacity to escape from the primary site and invade the surrounding stroma. In NPC, we have often found that neoplastic spindle cells are particularly located at the invasive tumor front (ITF). or migrate into the surrounding stroma. Moreover, we have recently demonstrated for the first time that that ALDH1(aldehyde dehydrogenase1) could be a functional marker for isolation and identification of CSCs in NPC.Notely, tumor cells in the invasive front or infirlatring into the stromal generally displayed high expression of ALDH1, and often with spindle-shaped morphologies. In the early1960s, Lang PC et al. have pointed out that these spindle cells tend to escape from the primary site with highly migratory and metastatic potential. However, to date, molecular features and biological functions about the invasive spindle cells still remain poorly understood. Therefore, we performed the following experiments:①To test whether these CSCs-like neoplastic spindle cells are associated with EMT in NPC;②To investigate the potential association of these EMT and CSCs-related proteins with clinicopathological factors and patient survival period;③In addition, gene-expression profiles used by cDNA microarrays go further to screen EMT-related genes, and④detect the impacts of the candidate genes on the invasion and metastasis of NPC.MethodsPart Ⅰ Neoplastic spindle cells in nasopharyngeal carcinoma possess features with epithelial-mesenchymal transition and cance stem cells 1. Immunohistochemical method was utilized to analyze the expression of Pan-cytokeratin, E-cadherin, N-cadherin,(3-catenin, vimentin, fibronectin, Snail. Slug, aldehyde dehydrogenase1(ALDH1); SOX2; OCT4, and Nanog in115neoplastic spindle cell samples and47neoplastic squamous cell samples.2. Tumor budding was investigated in105patients with NPC by immunohistochemistry and immunofluorescence.Part Ⅱ Expression levels of epithelial-mesenchymal transition and cancer stem cell markers and their clinical implications in nasopharyngeal carcinoma1. Tissue microarray (TMA) was constructed by biopsy speciment from122NPC patients and29non-cancer patients..2. Expression of EMT-associated proteins E-cadherin, N-cadherin,β-catenin. vimentin, Snail, and CSCs-related proteins ALDH1, SOX2, OCT4, Nanog and Nestin their prognostic significance in122NPC patients by immunohistochemistry. Immunofluorescence was used to confirm the definite localization of proteins.3. Hierarchical clustering analysis of expression of EMT-associated proteins and CSCs-related proteins in122NPCs.Part Ⅲ Screening and identification of the EMT-related markers of human nasopharyngeal carcinoma1. Total RNA from25pathologically-confirmed cases of poorly-differentiated NPC and from8clonic nasopharyngitis (CNP) were brought to a human22K cDNA array platform.2. Statistical identification of candidate expression genes of NPC biomarkers was based on SAM analysis. Real-time RT-PCR was performed for the validation of our microarray data. On the basis of this, we wanted to screen the genes related to EMT in NPC tissues by MILANO analysis and PubGene, and further to identify their functions by GO analysis and signaling pathways by KEGG analysis.ResultsPart I Neoplastic spindle cells in nasopharyngeal carcinoma possess features with epithelial-mesenchymal transition and cance stem cells1. Compared to malignant squamous cells, there was no significant difference in the expression of Pan-cytokeratin observed in neoplastic spindle cells (P=0.644), whereas E-cadherin expression was repressed, and the expression of β-catenin, N-cadherin, vimentin. fibronectin, Snail, Slug, ALDH1, SOX2, OCT4and Nanog was upregulated in the spindle cells (all, P<0.001). Furthermore, E-cadherin expression was negatively associated with β-catenin (P<0.001), vimentin (P<0.001), fibronectin (P<0.001), Slug (P<0.001), ALDH1(P<0.001), OCT4(P <0.001) and Nanog (P<0.001) in neoplastic spindle cells, but did not correlate with the expression of N-cadherin (P=0.086)、Snail (P=0.093) and SOX2(P=0.0875). More interestingly, the percentages of neoplastic spindle cells were further found to be associated significantly with T classification (P=0.036), N classification (P=0.031), lymphatic invasion (P=0.025), clinical stage (P0.037) and poor survival ((P=0.004).2. Of the105patients, low grade tumor budding was detected in53(50.5%) samples and high grade in52(49.5%) samples. The intensity of budding correlated strongly with T classification (P=0.008), lymphatic invasion (P<0.001), vascular invasion (P=0.029), lymph node metastasis (P<0.001) and clinical stage (P=0.010). Univariate analysis revealed that patients with high budding grade had poorer survival than those with low grade (P=0.002). Multivariate analysis showed that tumor budding was an independent predictor (P=0.001). Furthermore, budding cells highly expressed cancer stem cell marker aldehyde dehydrogenase1(ALDH1). Budding with high ALDH1expression contributed to several aggressive behaviors including T classification (P=0.034), lymphatic invasion (P=0.002), vascular invasion (P=0.017), lymph node metastasis (P=0.035) and clinical stage (P<0.001) and poor survival (P<0.001).Part Ⅱ Expression levels of epithelial-mesenchymal transition and cance stem cells markers and their clinical implications in nasopharyngeal carcinoma1. Our results showed that both membranous and cytoplasmic localization of E-cadherin staining were associated with lymph node metastasis (P<0.001and0.005, respectively) and clinical stage (P<0.001and0.007, respectively). High cytoplasmic β-catenin correlated significantly with larger tumor size (P=0.020), lymph node metastasis (P<0.001) and advanced clinical stage (P=0.036). However, no significant difference was observed between membranous β-catenin and clinicopathologic features (P>0.05). High nuclear vimentin expression correlated significantly with positive lymph node metastasis (P<0.001) and advanced clinical stage (P<0.001). Multivariate analysis showed that nuclear vimentin and cytoplasmic E-cadherin were independent prognostic factors (P0.016and0.001, respectively), as well as M classification(P=0.001). More importantly, patients with high coexpression of nuclear vimentin and cytoplasmic E-cadherin had shorter survival time (P<0.001). Furthermore, high coexpression of these two proteins was closely associated with lymph node metastasis (P<0.001) and advanced clinical stage (P<0.001).2. Cytoplasmic N-cadherin was observed in42.6%and nuclear N-cadherin was observed in45.1%of NPC tissues. High expression of cytoplasmic and nuclear N-cadherin was associated with a majority of the clinicopathologic variables, including lymph node metastasis, distant metastasis and clinical stage. Cytoplasmic N-cadherin was positively associated with nuclear N-cadherin expression (P<0.001). However, neither expression of cytoplasmic N-cadherin nor nuclear N-cadherin correlated with E-cadherin expression (P>0.05). In univariate analysis, cytoplasmic N-cadherin showed no significant impact on patient prognosis. On the contrary, the overall survival was significantly shorter in patients with high nuclear N-cadherin than low staining (P=0.002). High expression nuclear N-cadherin could predict worse survival in patients with late stage(P=0.033), but not those with early tumor stage. In addition, multivariate analysis showed that nuclear N-cadherin was an independent prognostic marker for NPC patients (P=0.024).3. Cytoplasmic Snail was detected in37.7%and nuclear staining was detected in49.2%of primary tumors, respectively. No significant associations were found between cytoplasmic Snail and the clinicopathologic variables except lymph node metastasis (P=0.042). However, nuclear Snail was significantly associated with tumor stage (P=0.003), T classification (P=0.045), lymph node metastasis (P=0.019), distant metastasis (P=0.003) and reduced E-cadherin expression (P=0.021). Patients with high nuclear Snail expression, but not cytoplasmic staining, had a significantly shorter survival than those with low expression (P<0.001). Significantly, nuclear Snail was an independent prognostic predictor for NPC (P<0.001). Furthermore, the prognostic impact was largely limited to Ⅲ-Ⅳ patients.4. Our results found that SOX2, OCT4and Nanog were frequently upregulated in NPC, particularly in the tumor invasive front, and correlated significantly with epithelial-mesenchymal transition (EMT) biomarkers (E-cadherin/N-cadherin and Snail). In the invasive front, patients with high expression of SOX2, OCT4and Nanog had significantly reduced survival than others with low group (P=0.006,0.001and0.000; respectively). Multivariate analysis showed that OCT4and Nanog expression were independent prognostic factors (P=0.006and0.001; respectively). In the invasive front, patients with high coexpression of OCT4and Nanog had significantly shorter survival than other remaining samples(P=0.005). More importantly, at the invasive front, these molecules correlated strongly with cancer stem cells (CSCs) marker ALDH1expression, and Nestin expression in endothelial cells (P<0.001, all).5. High expression of ALDH1in the invasive front was associated significantly with aggressive behaviors of NPC including lymphatic invasion (P<0.001), T classification (P=0.001), M classification (P<0.001) and clinical stage (P<0.001). Moreover, in the invasive front of tumors, the expression levels of ALDH1correlated strongly with epithelial-mesenchymal transition (EMT) biomarkers E-cadherin (P=0.001), vimentin (P<0.001) and Snail (P<0.001). Univariate analysis showed that patients with high ALDH1expression in the invasive front had a significant worse survival compared with others with low ALDHl expression, though it was not independently predictive of prognosis on multivariate analysis. Furthermore, the prognostic impact was largely limited to patients with early stage (T1-T2or N0-N1)(P<0.001, P=0.002, respectively).6. Samples were clustered into two major groups (Group A and Group B) by EMT and CSCs-related proteins described above. Compared to Group A, expression of membranous E-cadherin was greatly inhibited, whereas expression of cytoplasmic E-cadherin, vimentin, Snail, OCT4and Nanog was increasedly upregulated in Group B. Furthermore, Group B correlated strongly with T classification, N classification, M classification, clinical stage and the percentage of spindle cells. More importantly, patients overall survival in Group B was significantly lower than those in Group A.Part Ⅲ Screening and identification of the EMT-related markers of human NPC 1. Stringent statistical filtering parameters identified306genes to be up-regulated and203genes to be down-regulated in NPC compared to CNP. These differentially expressed genes were involved in signal transduction, cell adhesion, proteolysis, cell cycle, cell proliferation and apoptosis.2. Combined with MILANO analysis and PubGene,50potential candidate genes were identified related to EMT, which were associated with regulation of transcription, cell adhesion, signal transduction, proteolysis, chemotaxis. organ morphogenesis. KEGG pathways showed that there were several pathways including Wnt signaling pathway, p53signaling pathway and MAPK signaling pathway. Moreover, clustering analysis showed that these genes correlated strongly with T stage, N stage, M stage and tumor stage.Conclusions1. Our findings demonstrate first that neoplastic spindle cells in NPC might have features of EMT and CSCs, and these spindle cells correlate strongly with the invasion and metastasis of NPC. In addition, we describe the presence of tumor budding possessing the features of CSCs in NPC. We propose that neoplastic spindle cells should be "metastasic NPC" morphologically and budding cells should represent the initial step of tumour invasion of NPC.2. The proteins related to EMT including vimentin, N-cadherin, and Snail, and related to CSCs including Nanog and OCT4were aberrantly expressed and these proteins might be predictors of the risks of metastasis and poor outcomes of NPC,3. Comparison of gene expressionn profiling of NPC and CNP identified50genes related to EMT, and this set of genes might be involved in Wnt, p53and MAPK signaling pathway. Furthermore, clustering analysis shows that these EMT-related genes could be valuable predictors of the invasion and metastasis of NPC.