Figure 1. Curcumin reduces tumor burden and proportion of MDSCs with upregulation of T cell immunity in vivo.

(A) BALB/c mice (n = 5/group) bearing 7-day subcutaneous 4T1 mammary carcinoma, 4–6 mm in diameter, were injected intraperitoneally with 25 or 50 mg/kg of curcumin (CUR), DMSO control or PBS three times weekly for three weeks and tumor size was evaluated with vernier calipers every 2–3 days. Experiments were performed at least three times. Data shown are the mean tumor volumes (mm³) ± standard error (SE). (B) Spleen cells or tumor-infiltrating mononuclear cells pooled from each group of mice at day 28 were analyzed by flow cytometry. The middle panel and bottom panel show the percentage of Gr1⁺CD11b⁺ (MDSCs) cells and the top panel shows the percentage of monocytic (M)-MDSCs (Ly6C^hiLy6G^low) and granulocytic (G)-MDSCs (Ly6C^loLy6G^hi) cells among the Gr1⁺ cells. (C) Pooled splenocytes from each group at day 28 were stained with Gr1-PE, CD11b-APC, CCR7-FITC or stained with Gr1-PE, CD11b-APC, Dectin-1-FITC to assess for CCR7 and Dectin-1 expression of MDSCs by flow cytometry. Data represents the mean percentages ± SE of CCR7⁺Gr1⁺CD11b⁺ MDSCs and Dectin-1⁺Gr1⁺CD11b⁺ MDSCs from three independent experiments (*P < 0.05). To assess ROS production, another set of splenocytes were cultured with 3 μM dichlorodihydrofluorescein diacetate (DCFDA) followed by staining with antibodies to CD11b and Gr1, and analyzed by flow cytometry. Data shown are gated on CD11b⁺Gr1⁺ cells. (D) Splenocytes from each group at day 28 were stained with antibodies to CD8 or CD4 and IFN-γ. Data shown are the mean cell number ± SE of IFN-γ-positive CD8⁺ or CD4⁺ cells (*P < 0.05). (E) Splenocytes from each group at day 28 were plated at varying ratios with ⁵¹Cr-labeled 4T1 tumor targets. After 18 h at 37 °C, supernatants were harvested and the percentage of specific ⁵¹Cr release was determined by the following equation: (experimental cpm − spontaneous cpm)/(total cpm incorporated − spontaneous cpm) × 100. All determinations were done in triplicate, and the SE of was calculated and was typically 5% of the mean or less. All in vitro data are from a representative experiment of 3 experiments performed.