Exploring Spatial Heterogeneity of Immune Cells in Nasopharyngeal Cancer

Nasopharyngeal cancer (NPC) is a malignant tumor of the upper pharynx. In this study, we used multiplex tissue analysis combined with digital spatial profiling to map the immune heterogeneity of NPC. Forty-seven specified regions-of-interest (ROIs) with 49 target proteins were profiled across 30 cases of NPC for quantitative assessment of proteins related to CD45+ cells present in NPC tissue. Protein markers associated with B cells, NK cells, macrophages, and regulatory T cells were expressed at higher levels in 'immune-rich cancer cell islets' compared to the 'surrounding stromal leukocyte' regions. In contrast, biomarkers associated with suppressive populations of myeloid cells and exhausted T cells were overexpressed in 'surrounding stromal leukocytes' compared to 'immune-rich cancer cell islet' regions of the tumor. Moreover, findings regarding defined cancer phenotypes, such as 'inflamed', 'immune-excluded', and 'desert', were highlighted. In the 'inflamed' phenotype, compared with the other two phenotypes, markers associated with B cells, NK cells, macrophages, and myeloid cells were expressed at a higher level. On the other hand, in the 'immuneexcluded' phenotype, markers associated with suppressive populations of myeloid cells and T cells were expressed at a higher level compared with 'inflamed' and 'desert' groups, while the 'desert' profile had greater levels of granulocyte markers and immune-regulatory markers. The results shed light on the cellular composition of NPCs and may aid in stratifying patients to treatment based on their immune microenvironment. Nasopharyngeal cancer (NPC) is a malignant tumor. In a recent publication, we described the presence and distribution of CD8+ T cells in NPC and used the information to identify 'inflamed', 'immune-excluded', and 'desert' immune phenotypes, where 'inflamed' and 'immune-excluded' NPCs were correlated with CD8 T cell infiltration and survival. Arguably, more detailed and, in particular, spatially resolved data are required for patient stratification and for the identification of new treatment targets. In this study, we investigate the phenotype of CD45+ leukocytes in the previously analyzed NPC samples by applying multiplexed tissue analysis to assess the spatial distribution of cell types and to quantify selected biomarkers. A total of 47 specified regions-ofinterest (ROIs) were generated based on CD45, CD8, and PanCK morphological staining. Using the GeoMx (R) Digital Spatial Profiler (DSP), 49 target proteins were digitally quantified from the selected ROIs of a tissue microarray consisting of 30 unique NPC biopsies. Protein targets associated with B cells (CD20), NK cells (CD56), macrophages (CD68), and regulatory T cells (PD-1, FOXP3) were most differentially expressed in CD45+ segments within 'immune-rich cancer cell islet' regions of the tumor (cf. 'surrounding stromal leukocyte' regions). In contrast, markers associated with suppressive populations of myeloid cells (CD163, B7-H3, VISTA) and T cells (CD4, LAG3, Tim-3) were expressed at a higher level in CD45+ segments in the 'surrounding stromal leukocyte' regions (cf. 'immune-rich cancer cell islet' regions). When comparing the three phenotypes, the 'inflamed' profile (cf. 'immuneexcluded' and 'desert') exhibited higher expression of markers associated with B cells, NK cells, macrophages, and myeloid cells. Myeloid markers were highly expressed in the 'immune-excluded' phenotype. Granulocyte markers and immune-regulatory markers were higher in the 'desert' profile (cf. 'inflamed' and 'immune-excluded'). In conclusion, this study describes the spatial heterogeneityof the immune microenvironment in NPC and highlights immune-related biomarkers in immune phenotypes, which may aid in the stratification of patients for therapeutic purposes.