Dual functions of angiopoietin-like protein 2 signaling in tumor progression and anti-tumor immunity

Figure 1.. Tumor stroma-derived ANGPTL2 suppresses tumor progression. (A) Gene expression profiling of HK-2/PRCC-TFE3, HK-2/NonO-TFE3, and HK-2/PSF-TFE3 cells treated with (+) or without (−) doxycycline (Dox) based on microarray analysis. Venn diagram shows number of genes up-regulated approximately twofold in doxycycline-treated versus doxycycline-untreated cells. (B) Comparison of ANGPTL2 mRNA levels between doxycycline-treated and doxycycline-untreated cells based on microarray analysis. Levels in doxycycline-untreated cells were set to 1. (C) Representative immunoblotting of ANGPTL2 and TFE3 chimeric proteins (HA-tag) in HK-2/PRCC-TFE3, HK-2/NonO-TFE3, and HK-2/PSF-TFE3 cells treated with (+) or without (−) Dox. GAPDH served as a loading control. (D) Schematic representation of the mouse model used. In kidney tissues of Chd16-Cre/RosaLSL-PRCC-TFE3/+ mice, Angptl2 is expressed in both renal tubular epithelial cells and stromal cells. However, in Chd16-Cre/RosaLSL-PRCC-TFE3/+/Angptl2F/F mice, Angptl2 is specifically deleted in renal tubular epithelial cells. In addition, Chd16-Cre/RosaLSL-PRCC-TFE3/+/Angptl2−/− mice lack Angptl2 in both renal tubular epithelial cells and stromal cells.…

Figure 2.. Host ANGPTL2 deficiency enhances tumor growth. (A) Comparison of tumor volumes in female WT and Angptl2−/− mice bearing B16-OVA cells. Data are means ± SEM; n = 8 tumors per group. (*) P < 0.05, two-way ANOVA test. (B,C) Gross appearance (B) of tumors extirpated from WT and Angptl2−/− mice 14 d after implantation and tumor weight (C). Data are means ± SD; n = 6 tumors per group. (***) P < 0.001, unpaired t-test. Scale bar, 10 mm. (D) Kaplan–Meier tumor-free survival curves of WT and Angptl2−/− mice bearing B16-OVA tumors (n = 10 per WT group, n = 17 mice per Angptl2−/− group). Results are pooled from two independent experiments. (**) P < 0.01, log-rank test.

Figure 3.. PDGFRα+ fibroblast-derived ANGPTL2 inhibits tumor growth. (A,B) Immunofluorescent staining for ANGPTL2 (red) and indicated marker proteins (green) in tumor tissues of WT mice bearing B16-OVA tumors, 14 d after implantation. Nuclei are counterstained with DAPI (blue). Scale bar, 100 µm. (C) Schematic illustrating the experimental design of coinjection of B16-OVA cells and PDGFRα+ cells. PDGFRα+ cells were isolated from tumor tissues of WT mice (PDGFRα+; Angptl2WT) or Angptl2−/− mice (PDGFRα+; Angptl2−/−) bearing B16-OVA tumors 21 d after implantation. Female Angptl2−/− mice were coinjected with B16-OVA cells plus PDGFRα+; Angptl2WT or PDGFRα+; Angptl2−/− cells. (D) Comparison of tumor volumes in Angptl2−/− mice coinjected with B16-OVA cells plus PDGFRα+; Angptl2WT or PDGFRα+; Angptl2−/− cells. Data are means ± SEM; n = 6 tumors per group. (**) P < 0.01, two-way ANOVA test. (E) Kaplan–Meier tumor-free survival curves of Angplt2−/− mice coinjected with B16-OVA cells plus PDGFRα+; Angptl2WT or PDGFRα+; Angptl2−/− cells (n = 10 mice for PDGFRα+; Angptl2WT group, n = 8 mice for PDGFRα+; Angptl2−/− group). Results are pooled from two independent experiments. (**) P < 0.01, log-rank test.

Figure 4.. Host ANGPTL2 deficiency decreases anti-tumor immune responses. (A) Representative H&E-stained images of tumor tissues from WT or Angptl2−/− mice bearing B16-OVA tumors, 14 d after implantation. Scale bar, 50 µm. (B) Immunofluorescent staining for CD45 (red) in tumor tissues from WT or Angptl2−/− mice bearing B16-OVA tumors, 14 d after implantation. Nuclei are counterstained with DAPI (blue). Scale bar, 50 µm. (C,D) The absolute number of CD45+ cells (C) and CD8+ T cells (D) infiltrating into the tumor tissues of WT and Angptl2−/− mice bearing B16-OVA tumors at 14 d after implantation, assessed by FACS analysis. Data are means ± SD, pooled from three independent experiments; n = 9 tumors for each group. (*) P < 0.05, Mann–Whitney test. (E,F) Frequency of MHC class I OVA tetramer+ CD8+ T cells (E) and CD8+ T cells expressing Granzyme B (GzmB) (F) in inguinal lymph nodes of WT or Angptl2−/− mice bearing B16-OVA tumors, 14 d after implantation, as assessed by FACS analysis. Frequency of both is shown as a percentage of total CD8+ T cells. Data are means ± SD, pooled from three independent experiments; n = 9 mice per group. (**) P < 0.01, (*) P < 0.05, unpaired t-test.

Figure 5.. ANGPTL2 stimulates dendritic cell-mediated immune responses. (A) In vitro cross-presentation of OVA proteins by LPS-stimulated or LPS-unstimulated bone-marrow-derived dendritic cells (BMDCs) derived from WT or Angptl2−/− mice using the CD8+ T-cell hybridoma B3Z. β-galactosidase activity in B3Z cells incubated with unstimulated BMDCs untreated with OVA proteins was set at 1. Data are means ± SD; n = 4 per group. (ns) Not significant (P > 0.05), one-way ANOVA test followed by Tukey's multiple comparison test. (B) Comparison of levels of Il12p40 transcripts in LPS-stimulated or LPS-unstimulated BMDCs derived from WT or Angptl2−/− mice. Levels in unstimulated WT BMDCs were set to 1. Data are means ± SD; n = 3 per group. (ns) Not significant (P > 0.05), one-way ANOVA test followed by Tukey's multiple comparison test. (C) In vitro cross-presentation of OVA proteins by WT BMDCs stimulated with or without recombinant ANGPTL2 (rANGPTL2) protein using B3Z cells. β-galactosidase activity in B3Z cells incubated with unstimulated BMDCs untreated with OVA proteins was set to 1. Data are means ± SD; n = 4 per group. (ns) Not significant (P > 0.05); (***) P < 0.001, one-way ANOVA test followed…

Figure 6.. ANGPTL2 activates dendritic cells via PIR-B-NOTCH signaling. (A) Comparison of Il12p40 transcript levels in ANGPTL2-stimulated WT BMDCs pretreated with control Ig (cIG) or anti-integrin antibodies. Levels in ANGPTL2-untreated WT BMDCs were set to 1. Data are means ± SD; n = 3 per group. (ns) Not significant (P > 0.05), one-way ANOVA test followed by Tukey's multiple comparison test. (B) Comparison of Il12p40 transcript levels in ANGPTL2-treated or ANGPTL2-untreated BMDCs derived from WT or Pirb−/− mice. Levels in ANGPTL2-untreated WT BMDCs were set at 1. Data are means ± SD; n = 3 per group. (***) P < 0.001, one-way ANOVA test followed by Tukey's multiple comparison test. (C,D) Frequency of CD11c+MHC IIhi cells (C) and relative expression levels (MFI) of costimulatory molecules (D) in ANGPTL2-treated or -untreated BMDCs derived from WT or Pirb−/− mice, as assessed by FACS analysis. Frequency is reported as a percentage of total CD11c+ cells. Expression levels of costimulatory molecules in ANGPTL2-untreated WT BMDCs were set at 1. Data are means ± SD; n = 3 per group. (***) P < 0.001, one-way ANOVA test followed by Tukey's multiple comparison test. (E) Representative…

Figure 7.. Model proposing regulation of anti-tumor immunity by host ANGPTL2 signaling. In the tumor microenvironment, PDGFRα+ fibroblasts abundantly express ANGPTL2. PDGFRα+ fibroblast-derived ANGPTL2 activates dendritic cells via PIR-B-NOTCH signaling, enhancing CD8+ T cell anti-tumor immune responses, contributing to tumor suppression.