Tumor growth and angiogenesis are dependent on the presence of immature dendritic cells

Abstract

Dendritic cells (DCs)--immunomodulatory cells that initiate adaptive immune responses--have recently been shown to exert proangiogenic effects when infiltrating the tumor microenvironment. As tumors that escape immune surveillance inhibit DC maturation, we explored whether maturation status determines their ability to promote angiogenesis and whether angiogenesis depends on the presence of DCs. Using mouse xenograft models of human tumors, we show that fast-growing "angiogenic" tumors are infiltrated by a more immature DC population than respective dormant avascular tumors. Accordingly, supplementation of immature DCs, but not mature DCs, enhanced tumor growth. When DCs were mixed with Matrigel and injected subcutaneously into mice, only immature DCs promoted the ingrowth of patent blood vessels. Notably, depletion of DCs in a transgenic mouse model that allows for their conditional ablation completely abrogated basic fibroblast growth factor-induced angiogenesis in Matrigel plugs, and significantly inhibited tumor growth in these mice. Because immature DCs actively promote angiogenesis and tumor growth, whereas DC maturation or ablation suppresses this response, we conclude that angiogenesis is dependent on the presence of immature DCs. Thus, cancer immunotherapies that promote DC maturation may act by both augmenting the host immune response to the tumor and by suppressing tumor angiogenesis.

Figure 1.

Immature DCs are enriched in fast-growing angiogenic tumors. A) Light micrographs showing explants of a fast-growing angiogenic human MDA-MB-436 breast cancer and an avascular dormant tumor after growth subcutaneously in mice for 28 d. Scale bars = 1 cm. B) Scatterplot of data obtained from flow cytometric analysis of cells after they were enzymatically digested from the spleen and from a vascularized breast cancer specimen, and stained with anti-CD11c and anti-IA/IE (MHCII) antibodies. Note the CD11c⁺MHCII⁺ DCs in both spleen and tumor. C) Histograms of MHCII expression on tumor infiltrating DCs (box and asterisk indicate DCs that were gated as CD11c⁺FSC^high cells) isolated from angiogenic cancers compared to avascular tumors; note that avascular tumors exhibit less immature DCs that express lower levels of MHCII. Data are representative of 4 separate experiments.

Figure 2.

Immature DCs enhance tumor growth when injected in vivo. A) Design of an experiment in which SCID mice (n=4/group) were inoculated first with 1 × 10⁶ intraperitoneal human ovarian carcinoma OVCAR5 cells, and then DCs that were generated by growing bone marrow cells in the presence of GM-CSF for 9 or 12 d were injected into the same animals, with or without overnight exposure to LPS (to stimulate maturation or not, respectively), 3 and 6 d after tumor inoculation. B) At d 10, mice were sacrificed, and the intra-abdominal tumors were removed and weighed; results are presented as mean ± sd weight (mg). Note that injection of immature DCs produced a significant increase in tumor mass compared to control tumors, or tumors injected with the same number of mature DCs. Two-tailed Student’s t test was applied to test for statistical significance.

Figure 3.

Immature DCs promote endothelial cell migration in vivo. Matrigel was mixed with 1 × 10⁶ DCs that were cultured in the presence or absence of LPS before being implanted subcutaneously into mice. After 14 d, Matrigel plugs were removed and subjected to enzymatic degradation. Single-cell suspensions were stained with anti-CD31 and anti-CD45 antibodies and analyzed by flow cytometry; endothelial cells are gated as CD45⁻CD31⁺ and hematopoietic cells as CD45⁺CD31⁻. Note that even though the amount of hematopoietic cells was lower in Matrigel plugs injected with immature DCs, these DCs led to a 10-fold increase in endothelial cell ingrowth as compared to mature DCs. Plot represents pooled single-cell suspensions from 2 Matrigel plugs/mouse, 3 mice/group (n=6/group). *P = 0.007; Fisher’s exact test.

Figure 4.

DC depletion abrogates angiogenesis in vivo. A) Flow cytometric analysis of single-cell suspensions derived from enzymatically digested spleens stained with anti-CD11c antibodies, which confirm that endogenous DCs (outlined region containing CD11c+GFP+ cells) were depleted within 1 d after of injection of CD11c⁺DTR-Tg mice with a single dose of DT (3 ng/g). B) Photographs of Matrigel plugs containing bFGF (100 ng/ml) that were implanted subcutaneously for 1 wk in wild-type and CD11c⁺DTR-Tg mice treated with DT (on d 1; n=3/group). Note the lack of blood, indicating decreased vascularity within the implants removed from animals in which DCs were depleted. C) Flow cytometric analysis of cells removed from Matrigel plugs after staining with anti-CD31 and anti-CD45 antibodies. D) Similar percentages of CD31⁺CD45⁻ endothelial cells (gated as in C) are recruited to Matrigel plugs regardless of the presence (wild type) or absence (CD11c⁺DTR-Tg) of DCs caused by DT treatment.

Figure 5.

DC depletion disrupts formation of a vascular network. Differential interference contrast microscopic images of capillary cells grown within whole mounts of Matrigel plugs removed from wild-type animals or CD11c⁺DTR-Tg mice treated with DT as described in Fig. 4. Cell nuclei are stained with DAPI (blue), and a z stack of images was acquired over a thickness of 200 μm. Scale bars = 150 μm. Note that well-developed microvascular networks can be seen throughout the Matrigel plug from the wild-type mouse, but only single rounded cells appear in the Matrigel plugs from DC-depleted mice (CD11c⁺DTR-Tg).