Fig 2: 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.

Fig 3: hSFRP2 mAb reduces T-cell CD38 and PD-1 protein in vivo. Splenic CD4+ and CD8+ T-cells (top) and tumor infiltrating lymphocytes (TILs; bottom) isolated from RF420 tumor-bearing mice treated with IgG1 control (red) or hSFRP2 mAb (blue) for 21 days were stained for the following immune markers labeled with fluorochromes: CD38, PD-1, TNF-a, CD103 and CD5. Dot plot graphs show mean fluorescent intensity (MFI) analyzed by FACS for each of the immune markers of interest within the CD4+/CD8+ and TIL populations. For IgG1 and hSFRP2 mAb treatments, the number of independent samples (n) was 4. CD38 was significantly decreased with hSFRP2 mAb in both splenocytes (+ p < 0.05) and TILs (+ p < 0.05). PD-1 was significantly decreased in CD8+ TILs treated with hSFRP2 mAb (+ p < 0.05). Bottom tables: median MFIs (interquartile range) for each immune marker.

Fig 4: Schematic summary of OXT-PKC-CD38 signalling and Ca2+ related proteins dynamic in (a) hypothalamus of female and male (b) hippocampus of female and male (c) prefrontal cortex of female and male degus. Control (CTL), Partial Isolation (PI), Chronic Isolation (CI), and Re-socialization (CI-R) treatment groups.

Fig 5: hSFRP2 mAb reduces metastatic osteosarcoma growth in vivo. (A) Characterization of two osteosarcoma cell lines: RF420 (left) and RF577 (right) by Western blot analysis probing for SFRP2, FZD5, CD38 and PD-L1 levels. Loading control: actin and GAPDH. Western blot shows SFRP2, FZD5, PD-L1 and CD38 proteins detected in both cell lines. PD-L1 and CD38 proteins were detected in RF420 with lower levels than in RF577. Int. ratio: intensity ratios listed below SFRP2, FZD5, PD-L1, and CD38 Western blots were calculated in RF577 cells after normalization to both the loading control (actin or GAPDH) and reference sample in RF420 cells (indicated by 1.00). (B) In vivo monotherapy with hSFRP2 mAb in RF420 OS lung metastasis model. Osteosarcoma RF420 cells were injected iv via the tail vein in C57BL/6 mice. Treatments with IgG1 control (4 mg/kg weekly) or hSFRP2 mAb (4 mg/kg every 3 days) started 8 days after tumor cell injection. After 21 days of treatment, animals were euthanized, lungs were resected and surface nodules were quantified. Left: quantification of lung metastases in IgG1-treated control mice (n = 19) versus hSFRP2 mAb treated mice (n = 22) (* p < 0.001). Middle: representative lungs from a mouse treated with IgG1 control with multiple OS tumor metastases (arrows). Right: representative lungs from a mouse treated with hSFRP2 mAb with minimal OS metastases (C) Angiogenesis of metastases analyzed on paraffin-embedded lungs that underwent immunohistochemistry with antibody to CD31. Left: quantification of microvessel density measured on microphotographs of metastases from CD31-stained tissues, showing a reduction in hSFRP2 mAb-treated tumors (n = 10, * p = 0.001). Right: representative images of CD31-stained sections of lung tumors with negative control, IgG1 control-treated, and hSFRP2 mAb-treated. HPF: High Power Field. Scale bar: 100 µm.