Nasopharyngeal Tumor Cells

Nestled in the upper region of the pharynx, the nasopharynx is a vital part of the human anatomy that plays a crucial role in respiratory and auditory functions. Nasopharyngeal carcinoma (NPC) is a rare and aggressive form of head and neck cancer with a distinct geographical distribution. Nasopharyngeal tumor cells originate from the epithelial lining of the nasopharynx, a region located at the back of the nasal cavity.

Cellular Characteristics of Nasopharyngeal Tumor Cells

• Histological classification. Nasopharyngeal tumor cells can be broadly classified into two main histological subtypes: undifferentiated and differentiated. The undifferentiated subtype is characterized by a complete lack of cellular differentiation, resulting in a highly aggressive and rapidly proliferating phenotype. These tumor cells exhibit a pleomorphic appearance, with large, irregular nuclei and a high nuclear-to-cytoplasmic ratio, often appearing as a homogeneous sheet of cells under the microscope. In contrast, the differentiated subtype displays a more organized cellular structure, with varying degrees of squamous or glandular differentiation. These tumor cells tend to exhibit a lower proliferative rate and a more favorable prognosis compared to their undifferentiated counterparts.
• Immunohistochemical profiling. The overexpression of cytokeratins, such as CK5/6 and CK19, is a hallmark of nasopharyngeal tumor cells, reflecting their epithelial origin. Additionally, the presence of Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) and Epstein-Barr nuclear antigen 1 (EBNA1) in the majority of these tumor cells underscores the critical role of the EBV in their pathogenesis.
• Molecular characteristics. Through cutting-edge genomic analyses, some research has identified a complex landscape of genetic aberrations, including recurrent mutations in genes such as TP53, KRAS, and PIK3CA, as well as chromosomal abnormalities like 3q and 12p amplifications. Importantly, the deregulation of key signaling pathways, such as the PI3K/AKT/mTOR, MAPK, and Wnt/β-catenin cascades, has been widely documented in nasopharyngeal tumor cells. These molecular alterations not only contribute to the aggressive nature of these tumors but also present potential targets for the development of personalized therapeutic strategies.

Development of Targeted Therapies

Nasopharyngeal tumor cells are used to identify novel therapeutic targets and develop targeted therapies. For instance, mutations in the PIK3CA gene, which encodes a protein involved in cell growth and survival, have been identified in nasopharyngeal tumor cells. Inhibitors targeting the PI3K/AKT signaling pathway are currently under investigation as potential treatments for NPC.

Biomarker Discovery

The analysis of nasopharyngeal tumor cells has led to the identification of biomarkers that can be used for diagnosis, prognosis, and treatment stratification. EBV-encoded small RNA (EBER) and viral capsid antigen (VCA) are examples of EBV-related biomarkers that are being evaluated for their utility in NPC detection and monitoring.

Tumor Microenvironment Research

Understanding the interactions between nasopharyngeal tumor cells and their microenvironment is crucial for developing new treatments. Several studies investigate the role of stromal cells, immune cells, and extracellular matrix components in tumor growth and progression.

Drug Resistance and Sensitivity Testing

Nasopharyngeal tumor cells are used to study drug resistance mechanisms and identify novel combination therapies. High-throughput screening assays are conducted to help in the identification of drugs that can overcome resistance and improve patient outcomes.

Single-Cell Transcriptomic Profiling of the Multicellular Ecosystem of NPC

To systematically survey the cellular diversity of NPC, full-length scRNA-seq was performed on 1476 cells from 3 EBV⁺ NPC samples, including matched primary tumors and lymph node metastases. 3' droplet-based scRNA-seq (10× Genomics) was conducted on 102,525 cells from 17 EBV⁺ NPC and 7 non-cancerous nasopharyngeal samples (Fig. 1a) . All NPC biopsies were histologically examined by H&E and EBERs staining. NPC36 was studied using both scRNA-seq approaches to validate reproducibility. Quality checks of detected unique molecular identifier (UMI)/gene numbers and batch effects were performed.

Overall, the transcriptomes of 9 major cell types were captured according to the expression of canonical gene markers (Fig. 1b) in the 10× Genomics dataset. These cells included T cells, B cells, myeloid cells, mast cells, fibroblasts, endothelial cells (ECs), plasmacytoid dendritic cells (pDCs) and so on, which was consistent with the multiplex immunohistochemistry (multi-IHC) results (Fig. 1c). The cell clustering derived from tumors and nonmalignant tissues exhibited obvious differences, as exemplified by T cells, myeloid cells and fibroblasts (Fig. 1d). Using graph-based clustering, all cells were categorized into 19 clusters. Each of the 19 clusters harbored differentially expressed genes (DEGs) representing distinct cell types or subtypes. To distinguish malignant from nonmalignant cells, large-scale copy number variations (CNVs) were inferred based on scRNA-seq data. Putative malignant cells were grouped with extensive CNVs across the whole genome (Fig. 1e). These inferred CNVs were highly consistent with those generated from paired bulk whole-genome sequencing, such as chromosome deletions (3p, 9p, 14q, and 16q) and copy number gains (11q and 12p) (Fig. 1e), which is consistent with previous reports.

Among the 3 cases with full-length scRNA-seq data, EBV-derived sequencing reads were detected primarily located in the 0-6 kb and 150-170 kb regions of the EBV genome, covering virus latent genes such as LMP1, LMP2A/2B, and BARTs (BARF0, A73, and RPMS1), as well as lytic genes, including LF1, LF2, and BALF3 (Fig. 1f). Interestingly, typical latent and lytic genes of EBV were co-expressed within single host cells. Furthermore, potential host-virus cross-talk in malignant cells at single-cell resolution. The expression of EBV genes BNLF2a/2b was highly correlated with host genes associated with the immune response, including C3 and CFH in NPC16, OASL and ISG20 in NPC17, and IFTM1 and SERPING1 in NPC36.

Fig. 1 Expression profiling of ~104,000 single cells from 26 samples. (Jin S, et al, 2020)

Radiation-Resistant NPC Cells Show a Metabolic Signature of Active FAO

A metabolomics approach was used to profile the global metabolic changes of radiation-responsive (CNE2) and radiation-resistant (CNE2-IR) NPC cells. The contents of 260 biochemical compounds were quantified (Fig. 2A), and obvious differences between the metabolic profiles of CNE2 and CNE2-IR cells. Relative to CNE2 cells, 130 biochemical factors were altered in CNE2-IR, and the general changes are shown as a pie chart (Fig. 2B). Among these changes, 54 metabolic factors belong to lipid metabolism (Fig. 2C). The phosphoglycerides degradation products, glycerol 3-phosphate, glycerol phosphorylcholine and multiple lysolipids, as well as the membrane sphingolipid metabolites, palmitoyl sphingomyelin and stearoyl sphingomyelin, were elevated in CNE2-IR cells. These observations may reflect an increased lipid turnover or active lipid breakdown in CNE2-IR cells.

More importantly, several acylcarnitine levels are substantially higher in CNE2-IR cells (Fig. 2D). During the oxidation of fatty acids, carnitine acts as an acyl-CoA carrier facilitating the movement of fatty acids into the matrices of the mitochondria. To further explore the utilization of fatty acids through the carnitine shuttle system, a ¹³C isotopomer tracing experiment was undertaken. Cells were incubated with uniformly labeled ¹³C₁₆-palmitate, and the abundance of ¹³C-labeled palmitoyl-carnitine (C16), myristoyl-carnitine (C14), and lauroyl-carnitine (C12) was measured by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS; Fig. 2E). ¹³C enrichment in acylcarnitines was higher in CNE2-IR and HK1-IR cells in response to a dose of 4 Gy radiation, indicating that radiation-resistant cells utilize palmitate and may have active fatty acid oxidation (FAO). To determine the activity of FAO, the palmitate (PA)-based oxygen consumption rate (OCR) was examined. Following radiation treatment, an obvious increase in OCR was observed in radiation-resistant cells after PA stimulation, which was then inhibited by ethyl 2-[6-(4-chlorophenoxy) hexyl] oxirane-2-carboxylate (Etomoxir, ETO), a specific FAO inhibitor (Fig. 2F). In contrast, neither PA nor ETO treatment affected the OCR of radiation-responsive cells. These results in combination with the metabolomics analysis indicate an enhanced lipid turnover and FAO in radiation-resistant NPC cells, which suggests that lipid reprogramming might play a crucial role in mediating radiation resistance.

Fig. 2 Lipid turnover and fatty acid oxidation are enhanced in radiation-resistant NPC cells. (Tan Z, et al., 2016)