Fig 2: Interferon-stimulated gene 15 (ISG15)-induced CCL18 secretion by macrophages was dependent on the ISG15 receptor, leukocyte function-associated antigen-1 (LFA-1), and SRC family kinase (SFK) signaling. (A) Immunofluorescence demonstrated that the ISG15 receptor CD11a/CD18, also called LFA-1, was expressed on the membranes of human macrophages. Macrophages were treated with 295nM rISG15 for 24 h. Scale bars, 10 μm. (B) ELISA results showed small molecular inhibitors of LFA-1, A286982, inhibited CCL18 secretion by macrophages. (C) Small molecular inhibitors of LFA-1 and SRC, A286982 and PP2, hindered the ISG15-induced activation of SFK signaling. (D) PP2 reduced the CCL18 secretion by ISG15-treated macrophages in a dose-dependent manner, as determined by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Fig 3: Clinical implication of tumor-associated macrophages (TAMs) and ISG15-expressing TAMs in NPC. (A) Intracellular free and conjugated ISG15, and ISGytion E1 enzyme (UBE1L) and E2 enzymes (UBE2E2) were assessed by Western blotting on total cell lysates of rISG15 treated macrophages. (B) Kaplan-Meier survival curve of overall survival (OS) and disease-free survival (DFS) in the TMA contained 90 NPC biopsy specimens. (C) Representative immunohistochemistry data with double staining of CD163 (in wine red) and ISG15 (in brown) in early (T1N0M0) and advanced (T3N2M1) Tumor Node Metastasis (TNM) stage in NPC samples. Scale bars, 50 μm. (D) Kaplan-Meier survival curve of OS and DFS in NPC patients with distinct ISG15+ CD163+ TAMs count.

Fig 4: Interferon-stimulated gene 15 (ISG15) induced the M2-like phenotype and promoted NPC cell migration and tumorigenicity. (A, B) Human peripheral blood mononuclear cell (PBMC)-derived macrophages treated with 59 nM and 295 nM rISG15 for 24 h, and then were detached for fluorescence activated cell sorting (FACS) analysis, or were extracted mRNA for qPCR analysis. (A) Representative data of the switched surface markers profile of M2 (CD163)/M1 (HLA-DR) in ISG15-treated macrophages measured by FACS. (B) rISG15 induced macrophage mRNA expression of M2 cytokines, such as IL-10 and TGF-β, but not of M1 cytokines, such as iNOS, by qPCR analysis. IFN-γ/LPS and IL-4 served as positive control for M1 and M2 polarization. Heat-inactivated recombinant ISG15 (HI ISG15) protein was boiled within 100°C for 15 min. (C) Cytokine panel indicated the increased secretion of M2-polarizing cytokine IL-4 in culture supernatants of ISG15-treated macrophages, but not of M1-polarizing cytokine IL-6 and TNF-α. The culture supernatants were harvested from the macrophages upon rISG15 treatment at indicated dose for 24 h and replaced with serum-free culture for 24 h. (D) Representative photography of transwell filters in HK1 NPC cells cultured with the culture medium (CM) of macrophages. Quantification analysis revealed the increased number of transwell cell in the presence of ISG15 treatment. Data are expressed as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p< 0.0001. (E) Photographic illustration of the grow tumors established by a tumor model within BALB-c mice through subcutaneous inoculation with a mixture of human HK1 NPC cells and PBMC-derived macrophages (at ratio of 3:1) with or without rISG15 pre-treatment. All (10 in 10) mice with ISG15 treatment had tumor grow, but only 8 of 10 mice in the control group had tumor grow. The growth curve showed a significant larger tumor volume in the ISG15 treatment group compared with that of the control. ns, not significant.

Fig 5: Interferon-stimulated gene 15 (ISG15) is released from nasopharyngeal carcinoma (NPC) cells and functions as a cytokine for macrophages in NPC. (A) Upregulated interferon (IFN)-α and β mRNA were detected in NPC biopsy tissues. (B) Increased ISG15 protein was determined in HK1 and C666-1 NPC cells treated with IFN-β. (C) SDS-PAGE analysis of immunoprecipitated free ISG15 protein from conditional media (CM) of NPC cells. Control as RPMI-1640 CM, and HK1-IFN-β as HK1 cells treated with 50 unit/ml IFN-β for 16 h, and C666-1 as EBV-positive NPC cells. (D) Representative confocal images showing double staining of ISG15 and CD163+ in NPC and non-cancerous nasopharyngeal epithelium (NPE) sample (left panel). Scale bars, 20 μm in 40X or 10 μm in 100X. The quantification of the number of ISG15+ CD163+ cells in NPC (n=6) and in NPE (n=3) (right panel). Data are expressed as mean ± SD. Statistical significance was calculated using two-tailed Student’s t test and is indicated by *p < 0.05, **p < 0.01, and ***p < 0.001.