Stat3 mediates myeloid cell-dependent tumor angiogenesis in mice

Abstract

The underlying molecular mechanisms that cause immune cells, mediators of our defense system, to promote tumor invasion and angiogenesis remain incompletely understood. Constitutively activated Stat3 in tumor cells has been shown to promote tumor invasion and angiogenesis. Therefore, we sought to determine whether Stat3 activation in tumor-associated inflammatory cells has a similar function. We found that Stat3 signaling mediates multidirectional crosstalk among tumor cells, myeloid cells in the tumor stroma, and ECs that contributes to tumor angiogenesis in mice. Myeloid-derived suppressor cells and macrophages isolated from mouse tumors displayed activated Stat3 and induced angiogenesis in an in vitro tube formation assay via Stat3 induction of angiogenic factors, including VEGF and bFGF. Stat3-regulated factors produced by both tumor cells and tumor-derived myeloid cells also induced constitutive activation of Stat3 in tumor endothelium, and inhibiting Stat3 in ECs substantially reduced in vitro tumor factor-induced endothelial migration and tube formation. In vivo assays demonstrated the requirement for Stat3 signaling in tumor-associated myeloid cells for tumor angiogenesis. Our results indicate that, by virtue of the ability of Stat3 in tumor cells and tumor-derived myeloid cells to upregulate expression of factors that activate Stat3 in ECs, Stat3 mediates multidirectional crosstalk among tumor cells, tumor-associated myeloid cells, and ECs that contributes to tumor angiogenesis.

Figures

Figure 1. Role of Stat3 in the contribution of tumor-associated MDSCs to tumor angiogenesis.

(A) Flow cytometric analysis of cells isolated from spleens and B16 tumors shows accumulation of Gr1+CD11b+ cells in the tumors and spleens of tumor-bearing mice. Cells were labeled with antibodies for CD11b, CD11c, and Gr1, as indicated. (B) Top: Western blot analysis of VEGF protein levels in Gr1+CD11b+ cells isolated from B16 melanoma. Gr1+CD11b+ cells were purified by flow cytometric cell sorting, using spleens and/or tumors from mice with Stat3+/+ and Stat3–/– hematopoietic systems. Bottom: Flow cytometric analysis of CD11b+ cells isolated from B16 tumors indicates increased Stat3 activation in Stat3+/+ but not in Stat3–/– cells. (C) ECs form tube structures when coincubated with Stat3+/+ but not Stat3–/– MDSCs (Gr1+CD11b+CD11c–) isolated directly from B16 melanoma (tumor-associated MDSCs; TM-MDSC). Tumor supernatant (spnt) was also added to the EC culture as a positive control. Graph shows mean ± SEM of 2 independent experiments (n = 3). *P < 0.05.

Figure 2. Role of Stat3 activity and different populations of myeloid cells in angiogenesis.

(A) In vitro collagen tube formation assays using different subsets of CD11b+ myeloid cells. Both Gr1+CD11b+CD11c– cells and Gr1–CD11b+CD11c– cells in the presence of 2.5% tumor-conditioned medium successfully induced ECs to form tube-like structures. Gr1–CD11b+CD11c+ DCs did not induce efficient EC tube formation. Graph shows mean ± SEM of 2 independent experiments. (B) VEGF and bFGF expression in CD11b+Gr1+ myeloid cells contribute to angiogenesis. Splenic CD11b+Gr1+ myeloid cells were exposed to tumor-conditioned medium. Left: Flow cytometric analysis of Stat3+/+ and Stat3–/– CD11b+Gr1+ myeloid cells isolated from spleens of tumor-bearing mice for phospho-Stat3 (p-Stat3) and VEGF levels. Levels of bFGF in the indicated cells were determined by the Luminex system (see Methods). Graph shows mean ± SEM of 2 independent experiments. Right: Neutralization of VEGF and VEGF together with bFGF partially but significantly reduced myeloid cell–mediated angiogenesis. Graph shows mean ± SEM of 2 independent experiments with triplicates. (C) VEGF and bFGF are important for Stat3-dependent, tumor-derived MDSC–mediated angiogenesis. MDSCs purified from B16 tumors were used for tube formation assays in the presence of the indicated antibodies. Data are mean ± SEM of 3 independent experiments, each involving 10–15 pooled mice per group done in triplicates. (D) Stat3 activity in tumor-associated myeloid cells contributes to elevated expression of multiple angiogenic factors. Stat3+/+ and Stat3–/– CD11b+ myeloid cells were directly isolated from B16 tumors, and real-time RT-PCR was performed to detect RNA levels of the indicated genes. RNA levels of each indicated gene relative to a housekeeping gene (either 18s or GAPDH) in Stat3+/+ myeloid cells were assigned as 1. Data are mean ± SEM (n = 3). *P < 0.05; **P < 0.01.

Figure 3. Stat3 is constitutively activated in ECs exposed to the tumor milieu in vivo and in vitro.

(A) Immunohistochemistry staining of mouse B16 melanoma tissue sections with pY705-Stat3 antibody. Dashed lines indicate tumor blood vessels. (B) Immunofluorescent staining (pY705-Stat3 and goat anti-rabbit Alexa Fluor 488 antibodies) of ECs cultured alone or with mouse C4 melanoma cells at a 1:5 ratio. ECs were labeled with CellTracker Orange (red) prior to coculturing. Arrows indicate ECs. Below are examples of nuclear translocation of phospho-Stat3 in cells from the same experiment visualized with confocal microscopy. (C) Tumor-derived CD11b+ myeloid cells induce Stat3 activation in ECs. Stat3+/+ and Stat3–/– CD11b+ myeloid cells isolated from B16 tumors were added to the EC culture. ECs were labeled with CellTracker Orange (red), and pY705-Stat3 was detected using rabbit antibody and goat anti-rabbit Alexa Fluor 488 antibody (green). Arrows indicate ECs. (D) Stat3-positive myeloid cells interact with ECs in human breast cancer tissues. Human breast cancer tissue sections were first stained with rabbit pY705-Stat3 antibody and mouse anti-CD68 antibody to detect human myeloid cells, followed by staining with secondary antibodies goat anti-rabbit Alexa Fluor 488 (green) for Stat3 and goat anti-mouse Alexa Fluor 555 (red) for CD68. Insets show higher magnification of the boxed region, both nuclear counterstaining (Hoechst 33342, blue) and CD68 staining (red). Arrows indicate CD68+ cells; arrowhead indicates blood vessel. Original magnification, ×400 (A; B, top; C; and D); ×1,000 (B, bottom); ×1,250 (D, insets).

Figure 4. Stat3 activity in tumor cells affects Stat3 activity and tube formation potential in ECs.

(A) Tumor-conditioned media generated after blocking Stat3 activity in mouse C4 melanoma cells reduced Stat3 DNA binding in ECs, as shown by EMSA. C, DMSO control. (B) Inhibition of VEGF expression in mouse C4 melanoma cells by transfection with siRNA or Stat3 inhibitor CPA7, as detected by real-time PCR. (C) Collagen tube formation assay of ECs exposed to tumor-conditioned medium containing tumor supernatant collected from C4 melanoma cells exposed to vehicle or CPA7. The control cells were cultured in RPMI medium with 1% FBS. Shown are representative microphotographs of 3 independent experiments. Original magnification, ×100. (D) Quantification of tube-like structures per well of 48-well plates for EC lines derived from colon and prostate exposed to tumor-conditioned media. Data are mean ± SEM of 3 independent experiments. *P < 0.05.

Figure 5. Stat3 signaling in ECs is important for tumor factor–induced tube formation.

(A) Representative microphotographs of tube formation assay of ECs treated with vehicle or Stat3 inhibitor CPA7 for 16 h in the presence of control (RPMI medium with 1% FBS) or 10% tumor-conditioned media. Quantification of tube-like structures per well (48-well plate) exposed to control medium or 3 different concentrations of tumor-conditioned media with or without Stat3 inhibitor treatment is shown. Data are mean ± SEM of 3 independent experiments. EMSA results of Stat3 DNA-binding of nuclear extracts isolated from ECs are also shown. (B) Representative microphotographs of tube formation assay of ECs transfected with scrambled or Stat3 siRNA and exposed to control (RPMI medium with 1% FBS) or 10% tumor-conditioned media. Quantification of tube-like structures per well (48-well plate) exposed to control medium or 10% tumor-conditioned media is shown. Data are mean ± SEM of 3 independent experiments. Transfection efficiency, as shown by Western blot analysis of total protein lysates using indicated antibodies, is also shown. *P < 0.05. Original magnification, ×40 (A); ×100 (B).

Figure 6. Stat3 activity is required for tumor factor–induced migration of ECs.

(A) Representative microphotographs of wound healing assay of ECs transfected with scrambled or Stat3 siRNA and exposed to 10% tumor-conditioned media at the beginning and end of incubation (0 and 16 h, respectively). Quantification of cells that migrated into the wound is also shown. (B) Representative microphotographs of wound healing assay of ECs treated with vehicle or Stat3 inhibitor CPA7 and exposed to 10% tumor-conditioned media at 0 and 16 h of incubation. Quantification of cells that migrated into the wound is also shown. (C) Representative microphotographs of Transwell migration assay inserts of ECs transfected with scrambled or Stat3 siRNA after 6 h of migration toward control medium (RPMI medium; 1% FBS) or 10% tumor-conditioned media, stained with Harris hematoxylin solution. Quantification of cells that migrated toward control and tumor-conditioned media is also shown. Data are mean ± SEM of 3 independent experiments; for each, 4 images were analyzed. *P < 0.05. Original magnification, ×100.

Figure 7. Stat3 is critical for myeloid cell–induced tumor angiogenesis by in vivo.

(A) Matrigel plugs containing a mixture of B16 melanoma cells and CD11b+CD11c– myeloid cells isolated from spleens of tumor-bearing mice were implanted into mice with Stat3–/– hematopoietic system. Both Stat3+/+ and Stat3–/– myeloid cells were used for the Matrigel assays. Plugs were harvested for hemoglobin content measurement 6 d after implanting in vivo. Data are mean ± SEM of 3 independent experiments combined. *P < 0.05 (see Methods, Table 1, and Table 2 for detailed statistical analysis). (B) Representative microphotographs of Matrigel plug frozen sections stained with CD31 (red) and pY705-Stat3 (green) antibodies. Arrows indicate newly formed vessels. Original magnification, ×100. (C) Stat3 activity allows multidirectional crosstalk in the tumor stroma. Stat3 activity in tumor cells propagates, through Stat3-regulated factors (circles), to myeloid cells and ECs, and Stat3 activity in myeloid cells can further impact Stat3 activity in ECs, contributing to tumor angiogenesis.