Inhibition of vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after irradiation in mice (original) (raw)
Irradiation promotes recruitment of BMDCs into GBM. To investigate our hypothesis, we established an orthotopic brain tumor in a mouse model using human U251 GBM cells that were retrovirally transduced with the firefly luciferase gene in order to monitor tumor growth in real time. As shown in Figure 1, A and B, intracranially (i.c.) implanted U251 tumors grew progressively, and 5 daily doses of 2 or 4 Gy delivered locally to the tumors by irradiating the whole brain caused the tumors to regress and then regrow in a dose-dependent manner. We confirmed the tumor growth, infiltration into the surrounding normal brain, and regrowth after irradiation by histology and by MRI (Supplemental Figure 1, A and B; supplemental material available online with this article; doi:10.1172/JCI40283DS1).
Irradiation promotes homing of BM-derived CD11b+ myeloid monocytic cells into GBM. (A) Representative bioluminescent images of mouse heads with or without fractionated irradiation. Firefly luciferase–transduced U251 (U251/pFB-Luc) tumors were implanted i.c. and were irradiated from day 11 to day 15. 5 × 2 Gy, 2 Gy irradiation 5 times daily. (B) Tumor radioresponse was measured by BLI (n = 5 per group). Error bars indicate SD. (C) Radiation induced the influx of BMDCs into the U251 i.c. tumor. GFP-BM–transplanted nude mice received whole brain irradiation at 0, 8, or 15 Gy on day 22. Scale bar: 50 μm. (D) Quantification of GFP-BM influx into the tumor. Error bars indicate SEM. ***P < 0.001 versus control. (E) Representative images of IHC for GFP-BM cells and CD11b+ myeloid monocytic cells in U251 i.c. tumor before and after irradiation. Arrowheads indicate CD11b+ GFP-BM cells. Scale bar: 50 μm. (F) Quantification of CD11b and GFP-BM influx into the tumor. Error bars indicate SEM. ***P < 0.001. (G) Characterization of CD11b+ infiltrating cells into tumor after irradiation. Representative images of IHC staining for DAPI (blue), CD11b (red), and other markers (VEGFR1, VEGFR2, Tie-2, or Gr-1; green) in i.c. tumors after irradiation. Scale bar: 50 μm. IR, irradiation.
We next examined whether local irradiation induced the recruitment of BMDCs into the tumors. Tumor-bearing nude mice that had been transplanted earlier with GFP-expressing BM (34) were sacrificed when the tumors grew back to their preirradiation size after irradiation at either 8 or 15 Gy. We found by both immunohistochemistry (IHC) and quantitative real-time PCR (qPCR) that irradiation induced BMDC influx in a dose-dependent manner (Figure 1, C and D, and Supplemental Figure 2A). If the cells that colonized the irradiated tumor were circulating EPCs derived from the BM, we would expect that the proportion of endothelial cells arising from the BM would be increased in tumors regrowing after radiation. However, we found that although the GFP BMDCs in the tumors were often associated with CD31-staining endothelial cells, they were not colocalized (<1%, Supplemental Figure 2B). This is in agreement with our previous study (22) that showed no increase in EPCs in tumors growing in preirradiated sites and is also consistent with several recent reports (16, 35–37). Significantly, we found that most of the induced influx of BMDCs into the tumors were CD11b+ myelomonocytes (Figure 1, E and F). However, non-GFP CD11b+ cells were modestly increased after irradiation (from 3 to 10 GFP–CD11b+ cells compared with 6 to 72 GFP+CD11b+ cells per high-power field [HPF]). CD11b+ cells levels in normal brain were very low compared with those in the irradiated tumor (Supplemental Figure 2C). Further characterization of the CD11b+ cells in the irradiated tumors revealed that most of them did not express VEGFR1 and VEGFR2. Approximately 20% of the CD11b+ cells expressed Gr-1. Interestingly, approximately 50% of the CD11b+ cells were Tie2 positive, although they were not found in nonirradiated tumors (Figure 1G and Supplemental Figure 2D).
Irradiation causes vascular damage and increased tumor hypoxia. We postulated that the CD11b+ BMDC recruitment is a result of the fact that local tumor irradiation sterilizes sufficient numbers of endothelial cells in and adjacent to the tumor to abrogate the sprouting and proliferation of endothelial cells from adjacent normal tissues and in tumor vessels, thereby forcing the tumor to rely on the vasculogenesis pathway stimulated by increased tumor hypoxia (7, 22, 24). To test this, we determined the level of tumor endothelial cells, blood flow, and hypoxia by measuring CD31 expression, lectin perfusion, and pimonidazole staining after local tumor irradiation. We found that CD31-stained cells were reduced to some 25% of control levels by 2 weeks after 15 Gy of irradiation (Supplemental Figure 2E) and that this was accompanied by a large decrease in tumor perfusion and a large increase in tumor hypoxia (Figure 2, A and B). These data suggest that the increased tumor hypoxia caused by the severe vascular damage and decreased vessel perfusion after tumor irradiation could be the stimulus leading to the recruitment of BMDCs.
HIF-1 activity is increased in irradiated tumors and is necessary for influx of BMDCs and tumor recurrence after irradiation. (A) Representative images of IHC staining for hypoxia (pimonidazole [pimo]) and functional vessels (lectin perfusion [perf]) in i.c. tumors after irradiation. Scale bar: 50 μm. (B) Quantification of hypoxic areas and vessels in the tumors of A. Error bars indicate SEM. *P < 0.05, **P < 0.01 versus control. Note the log scale on the ordinate. (C) BLI of 5HRE-Luc or pFB-Luc U251 i.c. tumors after a single dose (arrowhead) of irradiation on day 32. The divergence of the 2 curves starting at about 2 weeks after irradiation indicates increased HIF-1 activity. Error bars indicate SD. (D) Effect of NSC-134754 on inhibition of HIF-1 activity in vivo. 5HRE-Luc or pFB-Luc U251 tumor–bearing mice were treated with 15 Gy of irradiation, followed by daily injection of NSC-134754 for 3 weeks. Images were taken on the day of irradiation and 3 weeks later. (E) IHC staining for leukocyte (CD45) and monocyte (CD11b, top row) or macrophage (F4/80, bottom row) infiltration into i.c. tumors. Tumors were harvested on the day of irradiation for controls and 17 days after irradiation in treatment groups. Scale bar: 50 μm. (F) Quantification of CD11b+ and F4/80+ cell influx in tumors of E. Error bars indicate SEM. **P < 0.01, ***P < 0.001 versus control. (G) Growth curves of i.c. tumors treated with irradiation, NSC-134754, irradiation+NSC-134754, and control (n = 5 per group). Error bars indicate SD. *P < 0.05. (H) Growth curves of orthotopic U251/pSR (vector) and U251 HIF1KD1+4 tumors using BLI (n = 7). A whole brain irradiation was given on day 22. Error bars indicate SD. *P < 0.05.
HIF-1 is a key factor responsible for BMDC influx by radiation and tumor recurrence. Consistent with our results (Figure 2, A and B), other investigators have shown that tumors recurring following irradiation or growing in an irradiated normal tissue are more hypoxic (25, 26, 38) and would therefore be expected to have increased HIF-1 levels. To determine HIF-1 activity in real time in our brain-implanted U251 GBM, we stably expressed the HIF-1 reporter construct 5HRE-Luc (39) in U251 cells and monitored luciferase activity in control and irradiated tumors. By comparing the bioluminescence from the tumors expressing the constitutive pFB-Luc (proportional to total cells) with that of those expressing 5HRE-Luc (proportional to total HIF-1 activity), we could monitor tumor HIF-1 activity as a function of tumor size. HIF-1 levels were relatively high immediately after inoculation and then decreased, most likely as tumor vessels formed (data not shown). After that, HIF-1 levels increased as the tumor grew. As shown in Figure 2C, HIF-1 activity paralleled tumor growth in nonirradiated tumors but increased more rapidly than tumor growth starting at about 2 weeks after 15 Gy irradiation, indicating increased HIF-1 levels coincident with the increase in tumor hypoxia at this time.
To test our hypothesis that the increased HIF-1 levels were responsible for the increased influx of CD11b+ cells into the tumors, we used the HIF-1 inhibitor NSC-134754 (40). This compound prevented the increased HIF-1 activity produced by hypoxia in vitro (data not shown) and by tumor irradiation in vivo (Figure 2D). When this inhibitor was given daily for 2 weeks, starting immediately after irradiation, the increased tumor levels of CD11b+ monocytes (and also the more mature macrophages, staining for F4/80) observed at 17 days after 15 Gy were abrogated (Figure 2, E and F). As shown in Supplemental Figure 3, A and B, approximately 75% of infiltrating CD11b+ cells also expressed F4/80. These data indicate that HIF-1 plays a major role in the recruitment of BMDCs into tumors after radiotherapy.
To test our hypothesis that the recruitment into the tumor of CD11b+ and other proangiogenic cells promoted tumor growth after irradiation, we treated the mice with NSC-134754 in combination with local tumor irradiation. We started the drug delivery after irradiation to avoid any drug-induced changes in oxygen levels that would affect tumor radiosensitivity. Although the growth of the unirradiated tumor was not affected by NSC-134754, the response of the irradiated tumor was profoundly changed, with constant shrinkage and no regrowth over the time of the experiment (Figure 2G), even after the end of the treatment with NSC-134754.
We also determined the effect of HIF-1 on the radiosensitivity of GBM by genetic means, using both brain-implanted HIF-1α–knockdown (KD) U251 cells and _Hif1a_-KO mouse GBM cells. Increased tumor radiosensitivity was found in the HIF-1–KD tumors that depended on the KD levels of HIF-1 in i.c. U251 (Figure 2H and Supplemental Figure 4, A and B). Hif-1–null murine GBM tumors were also more sensitive to irradiation (Supplemental Figure 4, C and D), resulting in longer survival of the Hif-1–KO tumor-bearing mice compared with the control tumor-bearing mice (Supplemental Figure 4E). This increase in tumor radiosensitivity of the HIF-1α–KD tumors was not the result of any change in radiosensitivity or growth of the cells in vitro (data not shown). These data suggest that blocking the HIF-1 signal caused by local irradiation attenuates recruitment of CD11b+ BMDCs, thereby preventing tumor vasculogenesis and tumor regrowth.
HIF-1–induced SDF-1 activates its receptor and sustains tumor blood flow. Several investigators have shown that BMDCs are recruited to and retained in hypoxic normal tissues and in tumors by the hypoxia-dependent secretion of SDF-1, which binds to its receptor, CXCR4, on BMDCs (7, 29–32). Indeed, we found that SDF-1 secretion was upregulated under hypoxic conditions in vitro, and this upregulation was abolished by NSC-134754 (data not shown). In our brain-implanted GBM, SDF-1 levels in the tumors rose to a maximum level by 2 weeks after irradiation and then returned to control levels at 4 weeks (Figure 3, A and B). In addition, we found that local irradiation increased phosphorylation of CXCR4 in the tumor, indicating interaction of SDF-1 with CXCR4 (Figure 3, C and D). Approximately 20% of the BMDCs recruited into the tumor after irradiation exhibited phospho-CXCR4, whereas very low levels of phosphorylation of BMDCs were found prior to irradiation (Supplemental Figure 5A).
The interaction of SDF-1 and CXCR4 promotes tumor influx of BMDCs and restoration of tumor vasculature. (A) Irradiation promotes the expression of SDF-1 in U251 i.c. tumor. Representative image of IHC staining for SDF-1. Scale bar: 50 μm. (B) Quantification of SDF-1 in the irradiated tumors. Error bars indicate SEM. ***P < 0.001 versus control. (C) Phosphorylation of CXCR4 on BMDCs in tumors was induced after irradiation. IHC staining for GFP-BM and pCXCR4 in U251 i.c. tumors 3 weeks after 15 Gy whole brain irradiation. Arrowheads indicate phospho-CXCR4 GFP-BM cells. Scale bar: 50 μm. (D) Quantification of pCXCR4 GFP-BM cells in U251 i.c. tumor after irradiation. Error bars indicate SEM. ***P < 0.001. (E) AMD3100 prevents the restoration of tumor blood flow (green) after irradiation. Representative ultrasound images from U251 s.c. tumors treated with 15 Gy irradiation, AMD3100, irradiation+AMD3100, and control. Scale bar: 1 mm. (F) Quantification of blood flow in tumor of E. Blood flow was reduced by irradiation but recovered by 3 weeks. AMD3100 plus IR prevents the recovery of tumor blood flow. Error bars indicate SD. *P < 0.05.
To determine the effect of inhibiting the interaction of SDF-1 with CXCR4 on tumor blood flow in the irradiated tumors, we used the clinically approved drug AMD3100, an inhibitor of the interaction of SDF-1 with CXCR4, utilizing the technique of ultrasound analysis with microbubbles (41) in the U251 s.c. tumor. As shown (Figure 3, E and F, and Supplemental Videos 1 and 2), AMD3100 had no effect on blood flow compared with control tumor but prevented the return of blood flow in the irradiated tumors. These data suggest that HIF-1–upregulated SDF-1 is an important factor for retention of BMDCs in the irradiated tumor and can promote the recovery of the tumor blood supply after its reduction by irradiation.
Blocking SDF-1 prevents tumor recurrence following irradiation and is significantly more effective than VEGF blockage. We next investigated the therapeutic effect of AMD3100 in blocking the interaction of SDF-1/CXCR4 stimulated by irradiation. Tumor-bearing mice were infused with AMD3100 starting immediately after irradiation. This had no significant effect on the growth of unirradiated tumors in the brain but completely inhibited the recurrence of the irradiated tumors after either a single dose of 15 Gy (Figure 4C) or the more clinically relevant scheme of 5 daily doses of 2 Gy (Figure 4, A and B). Of note is the fact that the tumors did not regrow after the end of the infusion period, and tumor recurrence was not observed at the end of the experimental period (more than 100 days; Supplemental Figure 5, C and D). We also confirmed the eradication of U251 i.c. tumor by histological and MRI analysis (Supplemental Figure 1C). Importantly, AMD3100 had no effect on the growth of the U251 cells treated in vitro with daily doses of 2 Gy (data not shown). As a check of the specificity of the effect of AMD3100 in blocking the SDF-1/CXCR4 interaction, we also performed the therapy study with 15 Gy using a neutralizing antibody against CXCR4 instead of the AMD3100 infusion. We found a similar abrogation of the recurrence of the irradiated tumors (Figure 4D).
Therapeutic effect of blocking the interaction of SDF-1 with CXCR4 after whole brain irradiation. (A) Growth curves of i.c. U251 early tumor model after fractionated irradiation (5 daily doses of 2 Gy starting on day 11 after transplantation). *P < 0.05. (B) BLI images after fractionated irradiation treated with or without AMD3100. (C and D) Growth curves of i.c. U251 advanced tumor model after a single dose of irradiation (15 Gy on day 22 after transplantation), treated with AMD3100 (21 day infusion; C, *P < 0.05) or with neutralizing anti-CXCR4 Ab (D, *P < 0.05), starting immediately after irradiation. (E) Growth curves of U251 i.c. tumor after 15 Gy irradiation, treated with DC101. Arrowheads indicate the treatment of DC101 (started immediately after irradiation and maintained for 21 days). (F) AMD3100 is more effective than DC101 in reducing tumor blood flow. Quantification of endothelial cells and functional vessels in U251 s.c. tumors after 15 Gy irradiation and combined with AMD3100 or DC101. Samples were taken 17 days after irradiation. Error bars indicate SEM. **P < 0.01, ***P < 0.001 versus IR.
To test this strategy with another tumor, we used the U87 GBM cell line, which we retrovirally transduced with luciferase. Again, AMD3100 enhanced the radiosensitivity of the tumor, as demonstrated by tumor growth and by mouse survival time (Supplemental Figure 6, A and B).
It has been reported that tumor-produced VEGF not only promotes local angiogenesis but also mobilizes endothelial and hematopoietic progenitors from the BM (42, 43). As VEGF is also an HIF-1 target gene, we tested the inhibition of VEGF using the mouse VEGFR2-neutralizing monoclonal antibody DC101. DC101 treatment alone slightly reduced the tumor growth rate (Supplemental Figure 5B). As shown in Figure 4E, this treatment given for 3 weeks after irradiation delayed the regrowth of the tumors but was not as effective as AMD3100 (or the HIF-1 inhibitor NSC-134754) in preventing recurrence of the irradiated tumors. We also compared the effect of AMD3100 and DC101 on tumor vascularity and blood perfusion after irradiation. Whereas both agents decreased the number of endothelial cells in the irradiated tumors, only AMD3100 produced an essentially total blockage of tumor perfusion when combined with irradiation (Figure 4F).
We further tested whether AMD3100 prevented the vasculogenesis pathway by using the model system of transplanting tumors into an irradiated normal tissue to eliminate local angiogenesis. We found that HIF-1 KD or treatment of tumor-bearing mice with AMD3100 totally abrogated tumor growth in the preirradiated normal tissue (Supplemental Figure 6, A and B). These data show that tumor regrowth is absolutely dependent on the vasculogenesis pathway when angiogenesis is impaired by local irradiation and that blocking the HIF-1 or SDF-1/CXCR4 signal prevents tumor recurrence.
Myelomonocytic cells are critical for tumor regrowth after irradiation. We next tested the importance of myelomonocytic cells for tumor regrowth following irradiation. To do this, we depleted the monocyte/macrophage lineage using carrageenan (44). This treatment reduced the number of CD11b+ BMDCs in the U251 i.c. tumor (Figure 5B). We also found that this prevented tumor recurrence after irradiation, although depletion of myeloid cells did not reduce the growth rate of unirradiated i.c. tumor (Figure 5A and Supplemental Figure 8A). These data are consistent with previous reports suggesting involvement of myeloid lineage cells in tumor progression and vasculogenesis (20, 22, 35).
CD11b+ cells are associated with GBM tumor recurrences in U251 i.c. tumors in mice and in patients. (A) Growth of U251 i.c. tumor assessed by BLI. Tumor-bearing mice were treated with 15 Gy whole brain irradiation, carrageenan (CAR), or the combination of irradiation and CAR. The arrow indicates irradiation, and the bar indicates the period of CAR treatment. Error bars indicate SD. *P < 0.05. (B) IHC of U251 i.c. tumor of A for CD11b. The average of CD11b cells per HPF was 6.3 (control), 72.6 (IR), 2.6 (CAR), and 2.0 (CAR+IR). (C) IHC of GBM clinical samples staining for CD11b. (D) Significantly increased levels of CD11b+ cells in the recurrent human GBMs compared with the untreated tumors. Quantification of CD11b based on IHC with CD11b staining. Eight of twelve samples showed significant increases in CD11b+ cells in recurrent GBMs (P < 0.05). Error bars indicate SEM. Scale bars: 50 μm.
CD11b+ myelomonocytic cells are increased in recurrences of human GBM. Finally, we determined the level of CD11b+ cells in human GBM tumors both prior to therapy and when the tumors had recurred in the same patients. We observed higher levels of CD11b+ cells in the recurrent tumors compared with those in the untreated tumors in 10 of the 12 paired samples, which was statistically significant (P < 0.05) in 8 of the 12 pairs (Figure 5, C and D). CD11b+ cells were not found or were at undetectable levels in normal brain tissues, indicating that the presence of large number of infiltrating CD11b+ cells in recurrent tumors is stimulated by tumor-derived cytokines.