Sox17 promotes tumor angiogenesis and destabilizes tumor vessels in mice

Abstract

Little is known about the transcriptional regulation of tumor angiogenesis, and tumor ECs (tECs) remain poorly characterized. Here, we studied the expression pattern of the transcription factor Sox17 in the vasculature of murine and human tumors and investigated the function of Sox17 during tumor angiogenesis using Sox17 genetic mouse models. Sox17 was specifically expressed in tECs in a heterogeneous pattern; in particular, strong Sox17 expression distinguished tECs with high VEGFR2 expression. Whereas overexpression of Sox17 in tECs promoted tumor angiogenesis and vascular abnormalities, Sox17 deletion in tECs reduced tumor angiogenesis and normalized tumor vessels, inhibiting tumor growth. Tumor vessel normalization by Sox17 deletion was long lasting, improved anticancer drug delivery into tumors, and inhibited tumor metastasis. Sox17 promoted endothelial sprouting behavior and upregulated VEGFR2 expression in a cell-intrinsic manner. Moreover, Sox17 increased the percentage of tumor-associated CD11b+Gr-1+ myeloid cells within tumors. The vascular effects of Sox17 persisted throughout tumor growth. Interestingly, Sox17 expression specific to tECs was also observed in highly vascularized human glioblastoma samples. Our findings establish Sox17 as a key regulator of tumor angiogenesis and tumor progression.

Figure 1. Sox17 expression specific to tECs is coincident with increased angiogenesis.

LLC and B16F10 tumors grown in Sox17^GFP/+ mice (A–C and F) or wild-type mice (D, E, and G) were analyzed. (A–C) Images showing Sox17 expression (GFP) in tumor vessels (PECAM), but not in tumor-associated macrophages (CD11b⁺) or pericytes (α-SMA⁺), 2 weeks after implantation. (D) Sox17 transcripts specific to tECs purified from tumors 2 weeks after implantation. FACS plot shows cell populations purified from tumors. tHCs, tumor hematopoietic cells; OCs, other cells. (E) Sox17 expression in tECs at 1, 2, and 3 weeks after implantation. (F) Sox17 expression in individual tECs, as determined by FACS. Percentages of Sox17^high tECs are shown. Cells from Sox17^+/+ mice were used to establish background expression. (G) Images of tumor vessels showing different patterns of angiogenesis in LLC and B16F10 tumors at 1, 2, and 3 weeks after implantation. (A–C and G) Scale bars: 100 μm. (D–F) n = 3 (D and E) or 3–5 (F) per group. *P < 0.05, ^†P < 0.01 versus LLC tECs; ^#P < 0.001 versus tECs.

Figure 2. Sox17 deletion in tECs inhibits tumor progression, necrosis, and hypoxia.

LLC tumors grown in control and Sox17^iΔEC mice were analyzed. (A) Tumor growth (volume and mass) was inhibited in Sox17^iΔEC mice. Red arrows denote tamoxifen administration to elicit Sox17 deletion. (B) Tumor growth rates showed increased tumor volume relative to that 2 days earlier. (C) H&E staining of tumor sections showed hemorrhage (arrows) and necrosis (arrowheads) in controls. Boxed regions are shown at higher magnification. HIF1-α staining and cleaved caspase-3 staining of tumor sections are also shown. (D) H&E staining of lung sections showed clustered metastatic LLC tumors (arrows). Boxed regions are shown at higher magnification. (E) Quantitation of metastatic nodules >200 μm in diameter in mouse lungs. n.d., not detectable. (A, B, and E) n = 5–6 (A and B) or 3 (E) per group. *P < 0.01 versus control. (C and D) Scale bars: 2 mm (C, top); 400 μm (C, all others); 4 mm (D, top); 200 μm (D, bottom).

Figure 3. Sox17 deletion in tECs reduces tumor angiogenesis and vascular abnormalities.

Tumor vessels were analyzed in LLC tumors grown in control and Sox17^iΔEC mice. (A) Images of tumor vessels. (B) Filopodial protrusions, indicated by yellow arrows on the outside surfaces of tumor vessels. (C) Dextran release from the tumor vessels. (D) VE-cadherin staining of tumor vessels. (E) Col4 staining showing the basement membrane in tumor vessels. (F) α-SMA staining showing pericytes in tumor vessels. Higher-magnification images are shown below to better visualize the association of pericytes and endothelium. (G and H) Quantitation of blood vessel (BV) density and branches in tumors. (I) Quantitation of filopodial extensions on tumor vessels. (J) Quantitation of extravasated Evans blue in tumors. (K and L) Quantitation of tumor vessels surrounded by Col4 matrix and α-SMA⁺ pericytes. (A–F) Scale bars: 100 μm. (G–L) n = 3 per group. *P < 0.01 versus control.

Figure 4. Gain of Sox17 in tECs promotes tumor progression, tumor angiogenesis, and vascular destabilization.

B16F10 tumors grown in control and Sox17^iGOF mice were examined. (A) Tumor growth (volume and mass). Tetracycline was withdrawn (red arrow) to allow for Sox17 overexpression. (B) Quantitation of metastatic nodules >200 μm in diameter in mouse lungs. (C) Images of tumor vessels showing increased tumor angiogenesis in Sox17^iGOF mice. (D) Filopodial extensions (yellow arrows) on the outside surfaces of tumor vessels. (E and F) Col4 and α-SMA staining of tumor vessels. (G and H) Quantitation of tumor vessel density and branches. (I and J) Quantitation of tumor vessels surrounded by Col4 matrix and α-SMA⁺ pericytes. (A, B, and G–J) n = 3 (A, B, G, and H) or 3–4 (I and J) per group. ^#P < 0.05, *P < 0.01 versus control. (C–F) Scale bars: 100 μm.

Figure 5. Sox17 promotes angiogenic behaviors of ECs.

Aortic rings and lung ECs were obtained from genetic mouse models. (A–D) The effect of Sox17 overexpression was examined by removing doxycycline in cultures of cells from control and Sox17^iGOF mice. (E–H) The effect of Sox17 deletion was studied by adding tamoxifen to cultures of cells from control and Sox17^iΔEC mice. (I–L) Control and Sox17-knockdown (KD) HUVECs were used. (A and E) Sprout density in the aortic ring assay. (B and F) Number of migrating cells in the wound scratch assay. (C and G) EC numbers increased during culture. (D and H) Percentage of BrdU-incorporated cells. (I) Trajectories of individual EC locomotion. The migration of individual ECs was captured using time-lapse microscopy and converted to a 2-dimensional graph. (J) Slow EC migration was observed upon Sox17 knockdown. (K) Whereas control ECs showed a spindle shape, Sox17-knockdown ECs exhibited a spreading shape (yellow arrows). (L) Sox17 knockdown increased the area of EC surface adherent on the matrix. (A–H, J, and L) n = 3 (A–H), 20–23 (J), or 32–34 (L) per group. ^#P < 0.05, *P < 0.01 versus control. (K) Scale bars: 100 μm.

Figure 6. Sox17 increases VEGFR2 expression in tECs.

(A and B) Transcript level of VEGFRs in (A) Sox17-knockdown HUVECs and (B) tECs from LLC tumors in Sox17^iΔEC mice and respective controls. Levels were normalized based on GAPDH mRNA levels and are shown as fold difference compared with control. (C) VEGFR2 staining in LLC tumor vessels in control and Sox17^iΔEC mice. (D) FACS plot showing 2 distinct subpopulations of tECs from LLC tumors grown in Sox17^GFP/+ mice. Sox17^high (red) and Sox17^low (blue) tECs were fractionated for subsequent analyses. (E) DNA content measured in individual tECs to determine cell cycle status. The percentage of cells in the S/G₂/M phases of the cell cycle is indicated. (F) FACS plot showing VEGFR2 expression on the surfaces of tECs, indicative of VEGFR2 upregulation on Sox17^high tECs. Data from 2 histograms from Sox17^high and Sox17^low tECs were merged. (G) Coincident expression of Sox17 (GFP) and VEGFR2 in LLC tumors grown in Sox17^GFP/+ mice. Arrows denote areas of tumor vessels with high expression of both Sox17 and VEGFR2. Arrowheads indicate areas with weak expression of both Sox17 and VEGFR2. The histogram shows the intensities of GFP, VEGFR2, and PECAM expression at each pixel along the dotted line shown in the merged image. (A, B, and E) n = 3 per group. *P < 0.05 versus control; ^#P < 0.01 versus Sox17^low. (C and G) Scale bars: 100 μm.

Figure 7. Sox17 deletion in tECs persistently inhibits tumor angiogenesis and vascular abnormalities.

Tumors were grown in control and Sox17^iΔEC mice, and tamoxifen was administered (red arrows) for early (A–F) or late (G–L) Sox17 deletion. (A and G) Tumor growth (volume and mass). Blue arrows denote the time point of tumor analysis. (B and H) Tumor angiogenesis was reduced in Sox17^iΔEC tumors. (C and I) VE-cadherin staining in tumor vessels. (D and J) Extravasated dextran, indicative of vascular leakage. (E and K) Col4 staining of the basement membrane in tumor vessels. (F and L) α-SMA staining of the pericytes in tumor vessels. (A and G) n = 4–5 per group. *P < 0.01 versus control. (B–F and H–L) Scale bars: 100 μm. For quantitative analysis of B, E, F, H, K, and L, see Supplemental Figure 10.

Figure 8. Sox17 promotes recruitment of inflammatory cells into tumors.

Tumors were grown in control and Sox17^iΔEC mice, and tamoxifen was administered for early or late Sox17 deletion. (A) FACS plots showing CD11b⁺Gr-1⁺ cells in peripheral blood, bone marrow, and tumors. (B–D) Percent CD11b⁺Gr-1⁺ cells in peripheral blood (PB; B), bone marrow (C), and tumor tissues (D). Peripheral blood cells were gated by CD45⁺. n = 3–5 per group.

Figure 9. Late Sox17 deletion inhibits tumor growth by improving the efficacy of chemotherapy.

Tumors were grown in control and Sox17^iΔEC mice. (A) Tumor growth (volume and mass). Red arrows indicate tamoxifen administration for delayed Sox17 deletion. Cisplatin (or PBS as vehicle) was administered 2 and 3 weeks after implantation (blue arrows). (B) H&E staining, showing tumor necrotic regions (dashed yellow outlines), and necrosis quantitation. (C) Effect of Sox17 deletion on tumor vessel morphogenesis. Tumor vessels (left) are characterized by excessive angiogenesis and poor integrity. Sox17 deletion in tECs (right) inhibits tumor angiogenesis and induces tumor vessel normalization. ECs are shown in red; pericytes are shown in green; basement membrane (BM) is shown in yellow. (A and B) n = 5 per group. *P < 0.05 versus all other groups; ^†P < 0.05; ^#P < 0.01. Scale bars: 4 mm.

Figure 10. Sox17 is highly expressed in tumor vessels in human glioblastoma.

Sox17 expression was analyzed in tissues from nontumor control human brain, low-grade human glioma, and high-grade human glioblastoma. H&E staining shows cancer grade. Sox17 immunostaining shows nuclear Sox17 in tECs of high-grade glioblastoma (arrows and arrowheads denote strong and weak Sox17 expression, respectively). n = 3 (control and low-grade human glioma) or 5 (high-grade human glioblastoma). Scale bars: 100 μm.