EMT and CSCs-associated markers are predominately expressed in the main subgroup of NPC.

<p>(A) Unsupervised clustering of 18 proteins in 122 NPC (non-keratinizing carcinoma) samples classified phenotypically distinct subgroups of tumors. Rows, experimental samples; columns, individual genes. High expression levels are in red and low expression levels in green, as indicated in the scale bar below. Tumors were divided into two major clusters (cluster A and B, segregated by a black line). Elevated expression of EMT and CSCs-related markers occurred more frequently in Cluster B than Cluster A in tumors. Of note, the majority of the tumors in cluster B displayed a spindle-shaped phenotype (≥20%). (B) Immunohistochemical staining of selected markers in tumors, especially in the spindle cells component.</p

Neoplastic spindle cells exhibit properties of stem cells.

<p>(A) Stem cells-like markers SOX2, OCT4, Nanog and ALDH1 were markedly expressed in neoplastic spindle cells compared with neoplastic non-spindle cells (DNKC) in NPC. (B) Different expression levels of SOX2, OCT4, Nanog and ALDH1 proteins in human NPC C666-1 (undifferentiated, spindle-like morphology) and CNE1 (well differentiated, epithelial-like morphology) cell lines detected by immunofluorescence analysis. </p

Molecular Characterization and Clinical Implications of Spindle Cells in Nasopharyngeal Carcinoma: A Novel Molecule-Morphology Model of Tumor Progression Proposed

<div><p>Up to now, the precise molecular and morphological changes underlying the invasive and metastatic properties of nasopharyngeal carcinoma (NPC) remain largely unresolved. We speculate that neoplastic spindle cells, which are prominently found in the invasive tumor front and the surrounding stroma, might be responsible for the aggressive patterns. Expression profiling of various biomarkers relevant to cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) was performed by tissue microarray-based immunohistochemistry in NPC samples. The expression of EBER and LMP1 was detected by in situ hybridization and immunohistochemistry, respectively. We found that overexpression of CSCs-related markers (ALDH1, Nanog and ABCG2) and up-regulation of EMT markers (Fibronectin, MMP-2, Periostin, SPARC, Snail and Slug), together with E- to N-cadherin switching, occurred preferentially in tumors containing a large proportion of spindle-shaped malignant cells. Furthermore, CSCs-like properties were highly present in spindle cells compared with non-spindle cells of tumors, and correlated strongly with EMT features. In addition, EBV-related factors EBER and LMP1 were highly expressed and correlated strongly with CSCs and EMT characteristics in neoplastic spindle cells. Importantly, high proportion of spindle cells (≥20%) correlated significantly with various aggressive aspects including lymph node metastasis (<i>P</i> = 0.031) and local recurrence (<i>P</i> = 0.014). Patients with high proportion of spindle cells had poor survival (<i>P</i> = 0.004), though it was not an independent value. In conclusion, we demonstrate that spindle cells could be valuable morphological indicators of tumor progression and unfavorable prognosis of NPC. An integrated molecule-morphology model of NPC firstly constructed may shed significant light on the metastatic cascade and clinical relevance of patients. </p> </div

Spindle cells contribute to tumor aggressiveness and poor survival in NPC.

<p>(A) Representative examples showed that neoplastic spindle cells were frequently located at the invasive tumor edge or the invading surroundings (Pan-cytokeratin staining). (B) Correlation between the clinicopathologic characteristics and spindle cells in NPC. Columns: number of cases with >20% spindle cells vs <20. (C) Cumulative overall survival curves of 122 NPC patients according to the degree of spindle cells (≥20% vs <20%). Patients with high percentage of spindle cells (≥20%) had significantly shorter overall survival. <i>P</i>-values were calculated by the log-rank test. </p

A molecule-morphology model of tumor progression and metastasis in NPC firstly put forward.

<p>(A) The diagram depicts that spindle cells with CSCs and EMT characteristics are crucial for NPC aggressive progression. (B) One representative case (NO.52277) with NPC provides the histological evidence for cancer pathogenesis and metastasis (Pan-cytokeratin staining). Towards central areas of primary tumors, cancer cells exhibited epithelial-like phenotypes and polarized (1). In contrast, cancer cells acquired mesenchymal-like phenotypes and lost cell polarity during tumor progression, including budding cells moving from the invasive front (2), invading the adjacent stroma (3) and the microvasculature (4). <b>Spindle cells might be valuable morphological predictors of “mobile/migratory CSCs” and “invasive/metastatic NPC”.</b></p

Stem-like spindle cells are strongly associated with EMT -like phenotypic changes and EBV infection.

<p>(A) Serial sections from the same patient showed aberrant expression of EMT indicators E-cahderin, N-cadherin and Vimentin in stem-like spindle cells. (B) Spindle-like C666-1 cells had strong fluorescence staining of N-cadherin and Vimentin, and weak staining of E-cadherin compared with epithelial-like CNE-1 cells. (C) Representative images show that high expression (dark brown) of EBV-encoded small RNA (EBER) was observed in neoplastic spindle cells in comparison with non-spindle cells in the same tumor. (D) The significant relationship was found between EBER, EBV-encoded protein LMP1 and CSCs/EMT-related markers. Differences statistically significant at <i>P</i> values <0.05.</p

Associations between the clinicopathologic factors and SOX2, OCT4 and Nanog at the tumor invasive front in 122 NPCs.

<p>Associations between the clinicopathologic factors and SOX2, OCT4 and Nanog at the tumor invasive front in 122 NPCs.</p

Immunohistochemical staining of embryonic stem cells (ESCs) proteins SOX2, OCT4, Nanog and Nestin in non-cancerous nasopharyngeal tissues and nasopharyngeal carcinoma (NPC).

<p>Nuclear SOX2 expression was low in non-tumoral epithelium (A), whereas it was highly expressed in NPC tissues (B). Nuclear staining of OCT4 was limited to basal cells of non-tumoral epithelium (D; arrows indicated) and markedly expressed in tumor cells (E). Low cytoplasmic expression of Nanog was observed in non-tumoral epithelium (G) and strongly displayed in tumor tissues (H). Nestin expression was completely absent in non-cancerous epithelium (J) and tumor cells (K), whereas it was strongly stained in the cytoplasm of endothelial cells in cancer tissues (K; arrows indicated). Immunofluorescent labeling of both SOX2 (C) and OCT4 (C) showed nuclear staining (red), Nanog (I) and Nestin (L) showed cytoplasmic localization (red) of tumor cells and endothelial cells, respectively, DAPI (blue).All images, ×400.</p

Influence of SOX2, OCT4 and Nanog expression on overall survival of NPC patients.

<p>There was no significant difference in the overall survival between low and high nuclear SOX2 expression(A). Patients showed worse overall survival with high nuclear OCT4 (B), cytoplasmic Nanog (C) and coexpression of OCT4 and Nanog (D) in tumors. Patients with high expression of nuclear SOX2 (E), nuclear OCT4 (F), cytoplasmic Nanog (G) and coexpression of OCT4 and Nanog (H) in the invasive front of tumors showed worse overall survival. <i>P</i>-values were calculated by log-rank test.</p

Immunohistochemical expression levels of SOX2, OCT4, Nanog and Nestin in the invasive front of NPC (arrows indicated).

<p>Strong staining of nuclear SOX2 (A, B) was mostly found at the tumor invasive front. High staining of nuclear OCT4 (C, D) was observed in the invasive front of tumors. Cytoplasmic Nanog expression (E, F) was particularly evident at the invasive edge of tumors. <b>Of note</b>, these cells often exhibited a fibroblast-like, spindle-shaped phenotype. On the other hand, Nestin expression in blood vessels (G, H) were distributed predominantly at the invasive front of tumors. (A, C, E, G×100; B, D, F, H×400, respectively).</p