Fig. 1.

NI-1701 modulates the tumor microenvironment by promoting accumulation of immune cells associated with anti-tumorigenic functions and triggers enhanced engulfment of tumor cells by TAMs and monocytes. Immunodeficient NOD SCID mice were implanted with Raji GFP^hi lymphoma tumor cells and treatment was initiated when tumor volume reached 100 mm³. Tumors were excised 14 and 25 days after treatment initiation to evaluate impact of NI-1701 on the tumor microenvironment. a Design of the experiment. b Mean tumor volume at days 14 and 25 after treatment with human IgG1 isotype control Ab or NI-1701. Data are presented as mean ± SD, with n = 7–9 mice per group. c Analysis by flow cytometry of tumor-infiltrating total mouse leukocytes, myeloid cell subsets (TAMs, monocytes, G-MDSCs, dendritic cells) and NK cells. Gating strategy to identify subpopulations in the TME is displayed in Additional file 1: Fig. S1. Data are presented as mean ± SD, with n = 4–9 mice per group. d Analysis by flow cytometry of MHCII⁺ TAMs. e Representative flow cytometry plots of phagocytic TAMs or monocytes, determined as F4/80⁺GFP⁺ or Ly6C⁺GFP⁺ respectively, from Raji GFP^hi tumors dissected 14 days after treatment initiation. The threshold of GFP positivity and GFP internalization in TAMs and monocytes were determined based on methods and analysis depicted in supplementary experimental procedures. f The percentage of phagocytosis of GFP^hi Raji tumor cells by TAMs and monocytes, assessed at day 14 and day 25 post-treatment initiation, is shown for each individual mouse. Data are presented as mean ± SD, with n = 4–9 mice per group. Significance was determined by unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001