Effect of NR-V04 on immune cells in the TME. (A–D) Tumor-bearing mice were treated with 1.8 mg/kg NR-V04 or vehicle via i.p. injection when tumor size reached 1 cm in diameter, with two treatments on day 1 and day 4. Tumors were collected and single cells were isolated from tissues for flow cytometry analysis. (A and B) NR-V04 treatment increases B cell percentage in the TME, but not in spleen and blood in mice inoculated with (A) B16F10, P = 0.002 (tumor), seven tumors per group, one experiment, or (B) Yumm1.6 melanomas, P = 0.0134, seven tumors per group, one experiment. (C) NR-V04 treatment increased CD8 Tem cell percentage in B16F10 melanoma. P = 0.0926 (tumor) and P = 0.0169 (spleen), seven tumors per group, two experiments. (D) NR-V04 treatment decreased m-MDSC percentage in tumor and blood, but not in spleen in B16F10 melanoma. P = 0.05 (tumor) and P = 0.0006 (blood), seven tumors per group, two experiments. (E) NR4A1 depletion induces B cell proliferation. B cells isolated from spleen were labeled with Cell Trace Violet, untreated or treated with B16F10 lysis, following with the co-treatment of DMSO, 250, or 500 nM NR-V04 for 24 h. NR4A1 degradation (left) and B cell proliferation (right) were determined by flow cytometry. P < 0.0001 (left) and P = 0.0002 (right), three biological repeats, two experiments. Representative images shown in Fig. S4, E and F. (F) NR-V04 fails to inhibit B16F10 melanoma growth in B6.129S2-Ighmtm1Cgn/J mice deficient of mature B cells. Five tumors per group, one experiment. Two-way ANOVA was performed for (F) tumor growth curve with P value indicated. Others are shown as the mean ± SD. A two-sided unpaired t test was performed, with P values indicated. NS is non-significant.