Inhibition of neovascularization to simultaneously ameliorate graft-vs-host disease and decrease tumor growth

Abstract

BACKGROUND Blood vessels are formed either by sprouting of resident tissue endothelial cells (angiogenesis) or by recruitment of bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs, vasculogenesis). Neovascularization has been implicated in tumor growth and inflammation, but its roles in graft-vs-host disease (GVHD) and in tumors after allogeneic BM transplantation (allo-BMT) were not known. METHODS We analyzed neovascularization, the contribution of endothelial cells and EPCs, and the ability of anti-vascular endothelial-cadherin antibody, E4G10, to inhibit neovascularization in mice with GVHD after allo-BMT using immunofluorescence microscopy and flow cytometry. We examined survival and clinical and histopathologic GVHD in mice (n = 10-25 per group) in which GVHD was treated with the E4G10 antibody using immunohistochemistry, flow cytometry, and cytokine immunoassay. We also assessed survival, the contribution of green fluorescent protein-marked EPCs to the tumor vasculature, and the ability of E4G10 to inhibit tumor growth in tumor-bearing mice (n = 20-33 per group) after allo-BMT using histopathology and bioluminescence imaging. All statistical tests were two-sided. RESULTS We found increased neovascularization mediated by vasculogenesis, as opposed to angiogenesis, in GVHD target tissues, such as liver and intestines. Administration of E4G10 inhibited neovascularization by donor BM-derived cells without affecting host vascularization, inhibited both GVHD and tumor growth, and increased survival (at 60 days post-BMT and tumor challenge with A20 lymphoma, the probability of survival was 0.29 for control antibody-treated allo-BMT recipients vs 0.7 for E4G10-treated allo-BMT recipients, 95% confidence interval = 0.180 to 0.640, P < .001). CONCLUSIONS Therapeutic targeting of neovascularization in allo-BMT recipients is a novel strategy to simultaneously ameliorate GVHD and inhibit posttransplant tumor growth, providing a new approach to improve the overall outcome of allogeneic hematopoietic stem cell transplantation.

Figures

Figure 1

Neovascularization by donor bone marrow (BM)–derived endothelial cells (ECs) during graft-vs-host disease (GVHD). Lethally irradiated BALB/c mouse recipients were transplanted with 5 × 106 T cell–depleted BM (TCD-BM) cells, and in some mice, but not others, 1 × 106 T cells from B6 donors were simultaneously injected to induce GVDH. A) Representative image of increased neovascularization in inflamed intestine during GVHD (right) compared with noninflamed intestine 7 days after BM transplantation (BMT) (left). Sections of the liver, ileum, and colon of both groups of mice were stained with a fluorescent antibody to CD34 (green) to detect blood vessels and counterstained with a nuclear stain, 4′,6-diamidino-2-phenylindole (DAPI) (blue). B) Quantification of the neovascularization (vascular area/total area) in GVHD target organs by microscopy (as in A) at 7 and 14 days after allogeneic BMT, n = 10 per group, for statistical comparisons two-sided t tests were used. Means and 95% confidence intervals are shown. C) Quantification of host resident tissue ECs and donor BM-derived ECs by flow cytometric analysis. Donor H-2kb (B6) TCD-BM was transplanted into host H-2kd (BALB/c) mice with or without addition of donor T cells as above (n = 5 per group). Livers were excised from recipient mice on days 7 and 14, disaggregated, and (VEGFR2+/CD45−/CD11b−/TER119−) ECs were sorted and quantified according to H2k type. A stratified t test (stratified by day posttransplant) was used to for statistical comparisons.

Figure 2

Contribution of bone marrow–derived endothelial progenitor cells (EPCs) to neovasculature in graft-vs-host disease (GVHD) target tissues. A) Identification of EPCs by flow cytometric analysis. CD11b−/TER119− cells in peripheral blood and in bone marrow of B6 mice are shown. The c-Kit+/VEGFR+/CD11b−/TER119− cell population (red square) contains EPCs. B) Incorporation of GFP+ EPC-derived endothelial cells (ECs) surrounding the luminal space in neovasculature of the colon during inflammation. We transferred 20 000 GFP+ EPCs (from C57BL/6 GFP+ [B6 GFP+] [H-2b] mice) along with GFP− bone marrow and GFP− T cells (from C57BL/6 GFP− [B6 GFP−] [H-2b] mice) into lethally irradiated H-2kd (BALB/c) allo-BMT recipients on the day of bone marrow transplant. Colon sections from recipient mice were stained with a fluorescent antibody to the endothelial marker CD34 (red) and with the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI) (blue). The location of GFP+ (green) cells was evaluated by high-resolution confocal microscopy. Scale bar: 50 μm. C) Overlay of GFP+ ECs with CD34 staining, in experiments described in (B). Scale: 1 U = 2.5 μm. D) Overlay of GFP+ ECs (indicated by white arrows in the left lower panel) in colon sections from similar mice stained with an alternative endothelial marker, Meca-32 (red) and DAPI (blue). Scale bar: 100 μm.

Figure 3

Effect of the anti–VE-cadherin antibody E4G10 on endothelial progentior cells (EPCs) during graft-vs-host disease (GVHD). A) Flow cytometric analysis showing specific binding of E4G10 to EPCs. This experiment was done by flow cytometric analysis of bone marrow cells of C57BL/6 mice. Cells were stained with conjugated monoclonal rat anti-mouse antibodies against CD45, CD11b, TER119, c-Kit, and VEGFR2 as well as with conjugated E4G10 antibody (or rat IgG2a isotype control). The upper right panel shows that E4G10 binds to H5V endothelioma cells, which was used as a positive control (H5V is known to express VE-cadherin). The upper left panel shows TER119−/CD11b− cells from the bone marrow (BM) of B6 mice. The lower panels show that E4G10 does not bind to c-Kit+/VEGFR2−/CD11b−/TER119− BM cells, containing hematopoietic stem cells (arrow leading to lower left panel). In contrast, E4G10 binds to c-Kit+/VEGFR2+/CD11b−/TER119− BM cells, containing EPCs (arrows leading to lower right panel). The red line shows E4G10 binding, and the blue shaded area indicates the isotype control. B) Quantification of EPCs during GVHD. Lethally irradiated BALB/c recipients were transplanted with 5 × 106 TCD-BM cells + 1 × 106 T cells from B6 donors (n = 10 per group). Recipients were treated i.p. on days 0, 2, 4, 6, 8, and 10 after allo-BMT with 1 mg of either E4G10 or rat IgG, a control antibody. Donor BM-derived EPCs in peripheral blood and BM during GVHD were quantified by staining with monoclonal rat anti-mouse antibodies against CD45, CD11b, TER119, c-Kit, and VEGFR2 followed by flow cytometry. Combined data from two experiments are shown, n = 5 in each experiment, n = 10 total per group; for statistical comparisons, two-sided t tests were used. Means and 95% confidence intervals are shown.

Figure 4

Effect of E4G10 administration on vasculogenesis during graft-vs-host disease (GVHD). Lethally irradiated BALB/c (H-2kd) recipients were transplanted with 5 × 106 T cell–depleted bone marrow (TCD-BM) cells and 1 × 106 T cells from B6 (H-2kb) donors. Recipients were treated intraperitoneally with 1 mg E4G10 or rat IgG control antibody on days 0, 2, 4, 6, 8, and 10 after allogeneic BM transplantation (allo-BMT). A) Neovascularization in GVHD target organs as measured by microscopy at 7 and 14 days after allo-BMT. In these experiments, livers and intestines were collected on day 7 or 14 after BMT (n = 5 for each group, each of these days), and neovascularization was quantified by sectioning each organ and staining 10 representative sections with anti-CD34 antibodies (endothelial marker), followed by measurement of the CD34+ area (vascular area) vs total area; for statistical comparisons, two-sided t tests were used. B) Quantification of host resident tissue endothelial cells (ECs) vs donor BM-derived ECs by flow cytometric analysis during hepatic GVHD. In these experiments, livers were collected on day 7 (n = 5 per group), cells were disaggregated and stained with anti-H-2 antibodies as well as antibodies to endothelial markers before quantification of (VEGFR2+/CD45−/CD11b−/TER119− ECs by flow cytometry. A stratified t test (stratified by day posttransplant) was used to for statistical comparisons. Means and 95% confidence intervals are shown.

Figure 5

Treatment of graft-vs-host disease (GVHD) with E4G10 antibody. In the first two panels (A and B), lethally irradiated BALB/c or B6D2F1 mice were transplanted with 5 × 106 T cell–depleted bone marrow (TCD-BM) cells plus variable doses of T cells (as indicated in the figure) from B6 mice. Recipients were treated intraperitoneally with 1 mg E4G10 or rat IgG control antibody on days 0, 2, 4, 6, 8, and 10 after allogeneic BM transplantation (allo-BMT). A) Survival of E4G10- vs control-treated allo-BMT recipients in different GVHD models with variable T-cell doses, as shown by Kaplan–Meier plots. Combined data from five experiments (A1) or two experiments (A2, A3, and A4) are shown. Sample sizes: A1 group 1+2 n = 25, A1 group 3+4 n = 60; A2 group 1+2 n = 10, A2 group 3+4 n = 15; A3 group 1+2 n = 10, A3 group 3+4 n = 20; A4 group 1+2 n = 10, A4 group 3+4 n = 15. Survival data were analyzed with the log-rank test. The numbers of mice at risk on days 0, 20, 40, 60, and 80 post-BMT were as follows: for A1 control, 60, 40, 20, 9, 4; for A1 E4G10, 60, 48, 38, 16, 12; for A2 control, 15, 15, 13, 7, 3; for A2 E4G10, 15, 13, 13, 10, 9; for A3 control, 12, 12, 4, 1, 0; for A3 E4G10, 12, 12, 11, 4, 3; for A4 control, 15, 13, 0, 0, 0; for A4 E4G10, 15, 14, 6, 3, 1. B) Clinical GVHD scores of E4G10- vs control-treated allo-BMT recipients at 21 days posttransplantation in the experiments shown in (A). Group sizes: B1 n = 60; B2 n = 15; B3 n = 20; B4 n = 15. The Wilcoxon rank sum test was used to assess the differences in clinical GVHD scores between treatment groups. In the second two panels (C and D), lethally irradiated BALB/c recipients were transplanted with 5 × 106 TCD-BM cells and 1 × 106 T cells from B6 donors. Organs were harvested at day +21 after allo-BMT, fixed in formalin, embedded in paraffin, stained with hematoxylin and eosin, and investigated microscopically to assess organ damage due to GVHD. C) Histopathologic GVHD scores showing inflammation in multiple organs after E4G10 vs control treatment (liver and intestine: n = 10 per group; skin: n = 5 per group); for statistical comparisons, two-sided t tests were used. Means and 95% confidence intervals are shown. D) T-cell infiltration of liver and intestine during GVHD after E4G10 vs control treatment, as quantified by immunohistochemistry with anti-CD3 (n = 5 per group); for statistical comparisons, two-sided t tests were used.

Figure 6

Effects of E4G10 administration on tumor growth and graft-vs-host disease (GVHD). A) Contribution of endothelial progenitor cells (EPCs) to neovasculature during GVHD. We transferred 2 × 104 sorted B6 GFP+ endothelial progenitor cells (EPCs) 5 × 106 GFP− bone marrow (BM) cells, and 1 × 105 mouse renal carcinoma (RENCA) cells by intravenous injection to BALB/c recipients on the day of BM transplantation (BMT). RENCA lung metastases were excised on day 14 after allo-BMT. Sections of lung metastases were stained using anti-CD34 antibodies as an endothelial marker (red) or 4′,6-diamidino-2-phenylindole (DAPI) as a pan-nuclear marker (blue). Confocal images (scale bar: 50 μm) showed incorporation of GFP+ EPC-derived endothelial cells surrounding the luminal space (L) in neovasculature of the RENCA lung metastases (TC = tumor cell). The portions of the microscopic images boxed by dashes are further magnified in the bottom panels (scale: 1 U = 2.5 μm). In later experiments (B and C), lethally irradiated recipients were transplanted with 5 × 106 donor BM cells; challenged intravenously with RENCA cells, C1498 mouse acute myeloid leukemia (AML) cells, or A20 mouse B-lymphoma cells at day 0; and treated intraperitoneally with 1 mg E4G10 or rat IgG control antibody on days 0, 2, 4, 6, 8, and 10 after allo-BMT. Combined data from two to four experiments are shown. B) Effect of E4G10 administration on three kinds of tumor growth in mice given T cell–depleted BMT. Lethally irradiated BALB/c allo-BMT recipients were challenged intravenously with 1 × 105 RENCA (renal carcinoma) or with 2.5 × 105 A20 (lymphoma) TCs. Both cell lines were previously transfected to express luciferase, to enable weekly bioluminescence imaging of tumor growth. In an alternative model, lethally irradiated C57BL/6 allo-BMT recipients were challenged intravenously with 1 × 105 C1498 (AML). The left panels show survival curves after tumor challenge with RENCA (n = 30 per group), C1498 (n = 20 per group), and A20 (n = 13 per group). The survival of allo-BMT recipients treated with the E4G10 antibody is shown as blue triangles; the survival of control rat IgG-treated allo-BMT recipients is shown in red diamonds. Survival data were analyzed with the log-rank test. The numbers of mice at risk on days 0, 20, 40, 60, and 80 post-BMT were as follows: for RENCA control, 30, 26, 1, 0, 0; for RENCA E4G10, 30, 29, 11, 1, 0; for A20 control, 13, 11, 8, 0, 0; for A20 E4G10, 13,13, 7, 2, 1; for C1498 control, 17, 17, 0, 0, 0; for C1498 E4G10, 20, 19, 1, 1, 1. The picture shows bioluminescent imaging, at different time points, of allo-BMT recipients challenged with RENCA tumors (n = 15, shown is one out of two experiments with n = 15 each). The bar graphs below the picture shows the quantification of the fluorescence intensity assessed by bioluminescent imaging of the experiments using the luciferase-transfected cell lines RENCA (n = 15) and A20 (n = 13); for statistical comparisons, two-sided t tests were used. Means and 95% confidence intervals are shown. C) Effect of E4G10 administration on survival of tumor-bearing allo-BMT recipients with GVHD in different models. Lethally irradiated BALB/c allo-BMT recipients were intravenously injected with 5 × 106 B6 T cell–depleted BM cells, 1 × 105 B6 T cells and 2 × 105 RENCA (renal carcinoma) cells or with 5 × 105 A20 (lymphoma) TCs. In an alternative model, lethally irradiated C57BL/6 allo-BMT recipients were challenged intravenously with 5 × 106 B10BR T cell–depleted BM cells, 1 × 105 B10BR T cells as well as 2 × 105 C1498 (AML) cells. The left panels show survival curves after tumor challenge with RENCA (C1; n = 20 per group), C1498 (C2; n = 20 per group), and A20 (C3; n = 33 per group). The survival of allo-BMT recipients with GVHD treated with the E4G10 antibody is shown as blue triangles, the survival of control rat IgG treated allo-BMT recipients with GVHD is shown in red diamonds, and the survival of allo-BMT recipients without GVHD (no transfer of T cells, thereby eliminating the anti-tumor effect of T cells) is shown in black squares. Survival data were analyzed with the log-rank test. The numbers of mice at risk on days 0, 20, 40, 60, and 80 post-BMT were as follows: for C1 control, 20, 16, 11, 1, 0; for C1 E4G10, 20, 17, 17, 7, 6; for C2 control, 28, 22, 18, 8, 6; for C2 E4G10, 33, 32, 31, 23, 20; for C3 control, 19, 15, 4, 0, 0; for C3 E4G10, 20, 18, 7, 6, 5. The tables to the right show the reason of deaths in the survival experiments. All allo-BMT recipients without GVHD that were challenged with tumors (the black squares in the survival curves) died of tumors. Survival data were analyzed with the log-rank test.