Primary tumor–derived systemic nANGPTL4 inhibits metastasis

Figures

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  Figure 1.. Tracing the distribution of ANGPTL4 and its fragments in local tumor tissues and serum samples from melanoma patients. (A) Graph depicts the significance (log-rank [Mantel–Cox] test) of Kaplan–Meier plots comparing high (>75 percentile) and low (<25 percentile) gene expression of 38 angiogenic molecules with overall survival of patients from 32 cancer entities. HR, hazard ratio. (B) Representative immunofluorescent (IF) images of heathy skin, AK, primary cutaneous SCCs (cSCC), nevus, and melanoma tissue sections stained with ANGPTL4 antibody. (C) Quantitation of no, low, moderate, and high ANGPTL4 protein expression in healthy skin (n = 7), AK (n = 107), cSCC (n = 130), nevus (n = 7), and melanoma (n = 160) tissue sections. (D–G) Top: Western blotting with a cANGPTL-4–specific antibody (D and F) and a nANGPTL-4–specific antibody (E and G) in indexed melanoma tumors (D and E) and serum samples (F and G). Bottom: Graphs showing the densitometry quantitation of protein levels of the corresponding Western blots. Protein levels were normalized to actin in melanoma tumors and to transferrin in patients’ sera (n = 6; mean ± SD). *, P < 0.05; **, P < 0.01, Mann–Whitney U test. Source…

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  Figure S1.. Overall survival analysis of key angiogenic molecules in 32 tumor entities. (A–F) Kaplan–Meier plots depicting the overall survival of molecules belonging to (A) angiopoetin-like signaling pathway, (B) VEGF-A signaling pathway, (C) Delta-Notch signaling pathway, (D) PDGF signaling pathway, (E) angiopoetin/Tie signaling pathway, and (F) FGF signaling pathway.

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  Figure S2.. Tracing and characterizing the functional role of ANGPTL4 in clinical and preclinical settings. (A) Representative IF images of healthy colon, adenoma, and different grades of colon adenocarcinoma tissue sections (G1, G2, and G3) stained with ANGPTL4 antibody. (B) Quantification of no, low, moderate, and high ANGPTL4 protein expression in healthy colon (n = 20), adenoma (n = 25), and different grades of colon carcinoma (n = 69) tissue sections. (C) Representative stitched tile scanned IHC images of lung adenocarcinoma, breast cancer, cSCC, and corresponding healthy tissue sections stained with ANGPTL4 antibody. (D) Schematic illustration of the amino acid sequence of ANGPTL4. The protein sequence was visualized with the Protter software. (E) Graphs showing the densitometry quantitation of the Western blots from Fig. 1, E and G. Protein levels were normalized to actin in melanoma tumors and to transferrin in patients’ sera (n = 6; mean ± SD). **, P < 0.01, Mann–Whitney U test. (F and G) Western blotting with a cANGPTL4-specific antibody and an nANGPTL4-specific antibody in indexed lung cancer tumors and serum samples (n = 3). (H and I) Western blotting of melanoma patients’ sera…

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  Figure 2.. nANGPTL4 levels decrease in melanoma patients in the transition from stable to progressive disease. (A) Schematic representation of patient status at time point A (stable disease) and B (progressive disease). The primary melanoma is colored in black. Micrometastases are multicolored, and macrometastases are colored in black. The figure was generated with BioRender.com. (B) Representative MRI images of melanoma patients with either stable or progressive disease status. Arrows are pointing toward metastatic areas which are progressing from stable disease (SD) to progressive disease (PD). (C) Western blotting with an nANGPTL4-specific antibody in serum samples of melanoma patients with either stable or progressive disease status. Densitometry quantitation of protein levels on Western blot was performed with Fiji. (D and E) The graphs show the level of ANGPTL4 (D) and NT1/ANGPTL4 ratio (E) normalized to transferrin in patients’ sera with either stable disease or progressive disease diagnosis (n = 10). *, P < 0.05, two-tailed paired Student’s t test. (F) Schematic representation of patient status during longitudinal serum collection. The primary melanoma and macrometastases are…

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  Figure 3.. ANGPTL4 enhances angiogenesis via its C-site. (A) Growth kinetics of sh control (non-targeting) and ANGPTL4-silenced (sh-1 and sh-2) A375 tumors (n = 5–6; mean ± SEM). **, P < 0.01; ****, P < 0.0001, two-way ANOVA with Bonferroni’s multiple comparison test. (B) Representative IF images of control and ANGPTL4-silenced A375 primary tumor sections stained with CD31 (in green) and DAPI (in blue). Scale bar: 100 µm. (C) Quantitation of vascularized area in sh control (non-targeting) and ANGPTL4-silenced (sh-1 and sh-2) A375 primary tumors (n = 5; mean ± SD). **, P < 0.01, Mann–Whitney U test. (D) Growth kinetics of control, nANGPTL4-, and cANGPTL4-expressing LLC tumors (n = 7–12; mean ± SEM). ****, P < 0.0001, two-way ANOVA with Bonferroni’s multiple comparison test. (E) Representative IF images of tumor sections from LLC co-stained with CD31 (green), Desmin (red), and DAPI (in blue). Scale bar: 100 µm. (F) Quantitation of vascularized area in control, nANGPTL4-, and cANGPTL4-expressing LLC primary tumors (n = 7–9; mean ± SD). ***, P < 0.001, Mann–Whitney U test.

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  Figure S3.. Differential functions of ANGPTL4 and its cleavage fragments. (A) Representative images of the spheroid sprouting assay with either VEGF-A or ANGPTL4 mono-stimulation. Scale bar: 200 µm. (B) Quantitation of the cumulative sprout length (n = 3–5; mean ± SD). *, P < 0.05; **, P < 0.01, two-tailed paired Student’s t test. (C) Representative pictures of the spheroid sprouting assay with 400 ng/ml ANGPTL4 in combination with a subcritical dose of VEGF-A. Scale bar: 200 µm. (D) The graph shows the quantitation of the cumulative sprout length (n = 3; mean ± SD). **, P < 0.01, two-tailed paired Student’s t test. (E) Representative IF images of the aortic ring assay stained with FITC-lectin. Scale bar: 100 µm. (F) The graph depicts the quantitation of the number of sprouts per aortic ring (n = 11–17; mean ± SD). *, P < 0.05, two-tailed unpaired Student’s t test. (G) Angptl4 expression in control, nANGPTL4-, and cANGPTL4-overexpressing LLC tumor cells quantified by RT-qPCR (n = 3; mean ± SEM). (H) Angptl4 expression in control, nANGPTL4-, and cANGPTL4-overexpressing B16F10 tumor cells quantified by RT-qPCR (n = 2; mean ± SEM). (I) Quantitation of cellular proliferation of control,…

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  Figure 4.. nANGPTL4 and cANGPTL4 have independent and opposing functions on sprouting angiogenesis. (A) Quantitation of the cumulative sprout length of HUVEC) spheroids stimulated with the indicated cytokines (n = 5; mean ± SD). *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-tailed paired Student’s t test. (B) Representative stitched tile scanned IF images of the corneas isolated from C57BL6/J mice stimulated with either PBS or the indicated cytokines. Corneas were stained for the EC marker CD31 (green). Scale bar: 500 µm. (C) Quantitation of vascularized corneal area (n = 4–9; mean ± SD). **, P < 0.01; ***, P < 0.001, unpaired Student’s t test. (D) Representative images of IgG or integrin-blocking antibody-treated EC spheroids stimulated with the indicated cytokines. Scale bar: 200 µm. (E) Quantitation of the cumulative sprout length of IgG or integrin-blocking antibody treated EC spheroids stimulated with the indicated cytokines (n = 4; mean ± SD). *, P < 0.05, paired Student’s t test. (F) Representative images of sh control (non-targeting) and SDC4-silenced EC spheroids stimulated with the indicated cytokines. Scale bar: 200 µm. (G) Quantitation of the cumulative sprout length of EC…

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  Figure 5.. nANGPTL4 inhibits metastases formation in preclinical mouse tumor models. (A) Schematic illustration of the experimental design to investigate the effect of systemically released ANGPTL4 and nANGPTL4 on lung metastasis. (B) Representative images of lung metastatic foci imaged under a stereomicroscope. Scale bar: 2 mm. (C) Quantitation of B16F10 lung metastatic foci (n = 10–17; mean ± SEM). **, P < 0.01, Mann–Whitney U test. (D) Pearson correlation of nANGPTL4/ANGPTL4 ratio identified by targeted mass spectrometry in sera of control (black dots) and ANGPTL4 tumor–bearing mice (blue dots) and numbers of macroscopic metastases. The black dotted lines depict the 95% confidence interval of the regression line (n = 19, r = −0.4725). *, P < 0.05, Pearson correlation. (E) Schematic representation of the LLC postsurgical metastasis model. (F) Representative PET–CT images, 2 wk after tumor resection in mice implanted with control or nANGPTL4-expressing LLC. (G) Kaplan–Meier plot showing the percentage survival of mice implanted with control or nANGPTL4-expressing LLC tumor cells following primary tumor resection (n = 10). ***, P < 0.001, log-rank (Mantel–Cox) test.

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  Figure S4.. Proteolytically cleaved fragments of ANGPTL4 differentially regulate metastasis. (A) Schematic representation of experimental design to study the effect of systemic cANGPTL4 on lung metastasis. (B) The graph represents the quantitation of metastatic lung foci (n = 9; mean ± SEM). *, P < 0.05, Mann–Whitney U test. (C) Tumor volume at the time of tumor resection of control and nANGPTL4-overexpressing LLC primary tumors (n = 10; mean ± SEM). (D) Schematic representation of the LLC postsurgical metastasis model. (E) Tumor volume at the time of tumor resection of control and cANGPTL4-overexpressing LLC primary tumors (n = 10–18; mean ± SD). (F) Kaplan–Meier plot showing percent survival of mice implanted with either control or cANGPTL4-overexpressing LLC tumor cells after primary tumor resection (n = 10–18). *, P < 0.05, log-rank (Mantel–Cox) test. (G and H) Vector map of empty backbone plasmid (pAceBac-EGT; G) and (H) plasmid construct used for generating recombinant mouse nANGPTL4. (I) SDS-PAGE analysis of nANGPTL4-expressing insect cells lysate, cell culture medium, flow through (FT) from StrepTactinXT column, wash solution used in StrepTactinXT column and successive eluates…

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  Figure 6.. Therapeutic administration of nANGPTL4 inhibits metastasis formation. (A) Schematic representation of the experimental metastasis model to evaluate the therapeutic efficacy of recombinant nANGPTL4. Mice were injected with either recombinant nANGPTL4 (7 µg/shot) or PBS as depicted in the scheme. (B) Representative images of metastatic lung foci from mice treated with PBS or nANGPTL4. Scale bar: 2 mm. (C) Graph representing the quantitation of metastatic foci (n = 9–10; mean ± SEM). ****, P < 0.0001, Mann–Whitney U test. (D) Schematic representation of the experimental metastasis model to evaluate the therapeutic efficacy of nANGPTL4 AAV. Mice were injected with 1011 nANGPTL4 or control AAV. (E) Representative images of metastatic lung foci from mice treated with control or nANGPTL4 AAV. Scale bar: 2 mm. (F) Graph representing the quantitation of metastatic foci (n = 9–11; mean ± SD). *, P < 0.05, Mann–Whitney U test. (G) Schematic representation of the LLC postsurgical metastasis model. (H) Kaplan–Meier plot showing the percentage survival of control or nANGPTL4 AAV-injected mice implanted with LLC tumor cells following primary tumor resection (n = 8–11). Log-rank (Mantel–Cox)…

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  Figure S5.. nANGPTL4 perturbs angiogenesis and WNT signaling at the metastatic site. (A) Schematic representation of the RNAseq experiment with AAV mediated upregulation of nANGPTL4 or control. (B) GSEA of pathways downregulated after nANGPTL4 treatment. NES, normalized enrichment score. (C) GSEA plots showing regulated hallmark pathways in nANGPTL4-treated lung EC. (D) Schematic representation of the B16F10 primary tumor experiment with AAV mediated upregulation of nANGPTL4 or control. (E) Representative images of lung sections co-stained with CD31 (green) and active β-catenin (red). Scale bar: 100 µm. (F and G) Quantitation of active β-catenin positive ECs (F) and CD31 (G) in lungs of mice with systemic overexpression of control or nANGPTL4 AAV (n = 6; mean ± SD). *, P < 0.05, Mann–Whitney U test. (H) Representative images of B16F10 tumors sections stained co-stained with CD31 (green) and active β-catenin (red). Scale bar: 100 µm. (I and J) Immunofluorescence based quantitation of active β-catenin positive ECs and CD31 (J) in primary tumors of mice with systemic overexpression of control or nANGPTL4 AAV (n = 6; mean ± SD).