Nasopharyngeal carcinoma (NPC) is characterized by significant intratumoral heterogeneity and a propensity for recurrence driven by therapy-resistant Cancer Stem Cells (CSCs). While the genomic landscape of adult NPC is increasingly understood, the cellular architecture of paediatric NPC and its distinct stem cell hierarchies remain largely unexplored. To overcome the limitations of short-read sequencing in resolving complex transcriptomes, we performed high-resolution, long-read single-cell RNA sequencing (scRNA-seq) using Oxford Nanopore Technologies on fresh biopsy specimens from a paediatric and an adult NPC patient. Transcriptional profiling and intercellular communication networks were reconstructed to dissect the tumour microenvironment (TME). Our analysis revealed fundamental differences in stemness programs dependent on patient age. Adult NPC stem cells exhibited a classic basal-like phenotype (KRT15hi/TP63+/KRT5+), relying on basement membrane interactions. In contrast, paediatric CSCs formed a distinct quiescent reservoir (SOX2+/ALDH1A1+/MKI67neg) that was transcriptionally separated from a transient amplifying progenitor population. Despite these structural differences, both stem cell subtypes orchestrated a conserved, multi-layered immune evasion strategy. Network analysis identified NPC stem cells as the dominant hubs for (1) CD99 signalling, which facilitates structural cohesion and suppresses innate immunity via the PILRα inhibitory receptor; (2) MIF secretion, promoting tolerogenic myeloid polarization; and (3) the NECTIN1-CD96 checkpoint axis, which drives T-cell exhaustion despite the presence of intact MHC Class I and II antigen presentation machinery. Disruption of the CD96, CD99, and MIF signalling axes represents a promising universal therapeutic approach to restore anti-tumor immunity in high-risk NPC.
