Fig 2: Serum SFRP2 levels are elevated in mice with metastatic osteosarcoma compared to non-tumor-bearing mice and responds to therapy. (A) Serum levels of SFRP2 were compared between C57BL/6 mice with metastatic RF420 OS treated with IgG1 control and non-tumor-bearing C57BL/6 mice with ELISA. There were significantly higher levels of SFRP2 in the serum of mice with metastatic RF420 OS versus mice without tumors (n = 3, * p < 0.01). (B) ELISA was used to compare the serum levels of SFRP2 in all treatment groups of the C57BL/6 mice with metastatic RF577 OS and C57BL/6 mice without tumors. The serum level of SFRP2 was significantly higher in the group of tumor-bearing mice treated with IgG1 compared to non-tumor-bearing mice (* p < 0.01) (n = 9 and n = 8, respectively). The serum level of SFRP2 was significantly decreased in the mPD-1 mAb (n = 8), hSFRP2 mAb (n = 12) and Combo (n = 12) treatment groups compared to IgG1-treated mice (+ p < 0.01).

Fig 3: Humanized SFRP2 mAb inhibits TCR/TGFß-induced elevation of CD38 and PD-1 in T-cells. (A) Western blot showing the levels of SFRP2 protein in T-cells treated either with IL-2 (6000 U/mL) alone, IL-2 + TCR, IL-2 + TCR + TGFß (5 ng/mL) or IL-2 + TCR + TGFß + hSFRP2 mAb (30 nM). Loading control: GAPDH. Int. ratio: intensity ratios listed below SFRP2 Western blot were calculated after normalization to both GAPDH and the reference sample (indicated by 1.00). T-cell activation by TCR/TGFß increases the levels of SFRP2 protein, which are decreased by hSFRP2 mAb treatment. (B) FACS analysis showing the levels of CD38 in CD8+/CD4+ T-cells after incubation with IL-2 alone, IL-2 + TCR/TGFß or IL-2 + TCR/TGFß + hSFRP2 mAb. CD38 protein levels increase upon activation by TCR/TGß, compared to control (n = 3, * p < 0.001), and this effect is abrogated by treatment with hSFRP2 mAb (n = 3, * p < 0.001). (C) NAD+ concentration was measured using an NAD+/NADH cell-based assay kit on T-cells exposed to IL-2 alone, IL-2 + TCR + TGFß or IL-2 + TCR + TGFß + hSFRP2 mAb. T-cell activation by TCR/TGß led to a decrease in NAD+ concentration compared to IL-2, which was restored by hSFRP2 mAb treatment (n = 3, ** p = 0.02). PD-1 levels were measured by FACS in (D) CD4+ and (E) CD8+ T-cells exposed to IL-2 alone, IL-2 + TCR + TGFß or IL-2 + TCR + TGFß + hSFRP2 mAb. In both cell types, T-cell activation by TCR/TGFß induced an increase in PD-1 protein levels (n = 3, * p < 0.001), which was reduced by hSFRP2 mAb treatment (n = 3, + p <0.05) in CD4+ cells (D) and n = 3, * p < 0.001 in CD8+ cells (E). (F) Results obtained by FACS analysis on T-cells exposed to IL-2 alone, IL-2 + TCR + TGFß or IL-2 + TCR + TGFß + hSFRP2 mAb were confirmed by Western blot for the indicated markers. TCR/TGFß activation led to an increase in SFRP2, CD38 and PD-1 protein levels, which were reduced by hSFRP2 mAb treatment. Loading control: actin. Int. ratio: intensity ratios listed below SFRP2, CD38 and PD-1 Western blots were calculated after normalization to both actin and the reference sample (indicated by 1.00).

Fig 4: SFRP2 increases CD38 and PD-1 mRNA and protein levels in T-cells. (A) Splenic T-cells cultured in IL2-containing medium were treated with or without SFRP2 (30 nM) for 1 h, and the mRNA levels for CD38 and PD-1 were measured by qRT-PCR (n = 8). GAPDH was used as an internal control. (B–F) All T-cells were treated with IL-2. Samples were probed using Western blot with antibodies to the indicated protein markers. Actin was the loading control for cytoplasmic fraction; histone H3 and TATA were loading controls for nuclear fractions. (B,C,E,F) Int. ratio: intensity ratios listed below Western blots for (B–F) were calculated after normalization to both loading control and reference samples (indicated by 1.00). (B,C) Cytoplasmic fractions of wild-type splenic T-cells were isolated and protein levels of CD38 (B) and PD-1 (C) were increased in SFRP2-treated T-cells, compared to untreated cells. (D) Western blot probing for FZD5 protein in mouse splenic T-cells. % actin: total band intensity was normalized to actin. (E) Splenic T-cells were untreated or treated with recombinant SFRP2 protein for 1 h. The nuclear fraction was isolated from the T-cells, and nuclear NFATc3 and ß-catenin protein levels were measured. Nuclear fractions demonstrated increased NFATc3 protein levels but no change in ß-catenin levels with SFRP2 treatment. (F) Splenic T-cells were activated with antigen gp100 (0.87 µM) with or without 10 µM of hSFRP2 mAb treatment. Nuclear fractions demonstrated decreased NFATc3 protein levels in hSFRP2 mAb-treated cells activated by antigen.