Fig. 6

Immunization against NY-BR-1 delays tumor growth and diminishes accumulation of TAMs with reduced immunosuppressive phenotype. HLA-DRB1*0401tg mice were immunized i.p. with 5 × 10⁸ pfu Ad.NY-BR-1 (n = 10) or with 5 × 10⁸ pfu Ad. Control (n = 10) and 2 × 10⁵ EONY#17 cells were injected s.c. into the right flank 14 days post immunization. Tumor growth was monitored for 30 days followed by excision of tumors and isolation of tumor-infiltrating leukocytes. a Schematic representation of the experimental procedure. b Tumor area was measured and statistical analysis was performed using a mixed linear model with random intercept for animal. Difference in treatment was highly significant (p < 0.0001). Analysis was performed using SAS Version 9.4 (SAS Institute Inc., Cary, NC, USA.). c Tumor weight was also measured. d-h CD45⁺CD11b⁺F4/80⁺ macrophages were isolated by FACS and RNA was extracted for gene expression analysis. d Frequency of CD11b⁺F4/80⁺ macrophages among CD45⁺ cells, e frequency of HLA-DR4⁺ cells among macrophages and f level of HLA-DR4 surface expression on TAMs. c-f Statistical analysis was performed using Student’s t test and p values are indicated (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). Expression of g M2- and h M1-associated genes in TAMs was quantified by qRT-PCR. Log fold expression change of a gene normalized to the house-keeping gene is shown. Error bars represent SEM and Bonferoni-Holm adjusted p values were calculated (* p < 0.05; ** p < 0.01; *** p < 0.001)