Figure 3

NSD1 inactivation is associated with immune cell exclusion from the tumor microenvironment in HNSC: (a) Compared with other HNSC subtypes, the NSD1 subtype (red box) displayed significantly lower mean signature levels of overall tumor associated leukocytes (TALs), and specific TAL types including M1 tumor associated macrophages (TAMs), CD8+ cytotoxic T cells, and CD4+ memory T cells (All inferred using CIBERSORT³⁶). The NSD1 subtype had the low mean RNA expression of immunotherapy-relevant genes, including CD274 (PD-L1), PDCD1 (PD-1) and PDCD1LG2 (PD-L2), and a lower mean level of T cell signature based on expression of 13 T cell transcripts. (b) Control and NSD1 shRNA knockdown HNSC cells (1 × 10⁶) were injected into the subcutaneous compartments of the flanks of NOD-scid IL2Rgamma^null (NSG) mice. In each mouse, one flank was injected with control cells (black) and the other with NSD1 knockdown cells (red). After tumors were established (5 mm diameter), 100 × 10⁶ Ficoll-purified human PBMCs per mouse were injected via tail vein. After 10 days, tumors were dissociated, and tumor-infiltrating T cells (CD45⁺CD3⁺) were quantified by FACS. Cohorts were n = 5 per set of control and knock-down cell line, as indicated. *P < 0.05; **P < 0.005 (paired two-tailed Students t-test, error bars represent S.D.). (c) NSD1 knockdown in HNSC results in the decreased expression of multiple chemokine genes. Control and NSD1 shRNA knockdown HNSC cells were assessed for the expression of chemokine and chemokine-related genes using a qRT-PCR array. Log2 fold expression of of 35 chemokine-related genes upon NSD1 knockdown (Relative expression NSD1-shRNA/Control) in three established HNSC cell lines (PCI13, FADU, SCC6). Log2 fold expression is indicated by a color gradient, with NA values indicated in grey. Asterisks indicate genes that were upregulated* or downregulated** in the NSD1 subtype (relative to other subtypes) in the TCGA study.