1. Hematopoietic cells regulate the angiogenic switch during tumorigenesis
by Rika Okamoto, Masaya Ueno, Yoshihiro Yamada, Naoko Takahashi, Hideto Sano, Toshio Suda, and Nobuyuki Takakura
Blood
Volume 105(7):
April 1, 2005
©2005 by American Society of Hematology

2. Localization of HCs in colon26 tumor tissues.
Localization of HCs in colon26 tumor tissues. (A-D) Sections of colon26 tumor tissues from tumors with a volume of (A) 30 to 40 mm3, (B) 60 to 70 mm3, (C) 100 and 120 mm3, and (D) more than 500 mm3 were stained with anti–PECAM-1 mAb (dark blue) and anti-CD45 mAb (red). The dotted line (A-C) shows the border between the fibrous cap surrounding the tumor mass and the tumor mass. The area beneath the dotted line is the tumor mass. (E-H) Sections from colon26 tumors of 60 to 70 mm3 that were stained with anti-CD11b (Mac-1) (E), anti-B220 (F), a mixture of anti-CD4 and anti-CD8 (G), or anti–Ly-76 (Ter119) (H) mAb. Insets in panels A-D show high-power views of the area indicated by box. Bar indicates 50 μm.
Rika Okamoto et al. Blood 2005;105:
©2005 by American Society of Hematology

3. Suppression of hematopoiesis inhibits vascular network formation of ECs in P-Sp culture system.
Suppression of hematopoiesis inhibits vascular network formation of ECs in P-Sp culture system. (A-D) P-Sp explants were cultured on OP9 stromal cells in the presence of anti-B220 mAb (20 μg/mL) as a control (A-B) or anti–c-Kit neutralizing antibody, ACK2 (20 μg/mL) (C-D), and the development of HCs and ECs was observed. (A,C) Phase contrast view of P-Sp cultures. The dotted line shows the presumptive border between the vascular bed (vb) and the vascular network area (vn). (B,D) PECAM-1 staining of culture plates after 10 days of culture. Bar in panel A indicates 50 μm (A,C). Original magnification, ×4 (B,D). (E) Total number of CFU-c's observed in the P-Sp culture system in the presence of anti-B220 mAb (20 μg/mL) or ACK2 (5 or 20 μg/mL). Results are expressed as the mean number of CFU-c's in 3 experiments (± SEM). (F) Length of elongated ECs (sprouting) from the vb. Results are expressed as mean length at 10 random points. Standard deviations are indicated.
Rika Okamoto et al. Blood 2005;105:
©2005 by American Society of Hematology

4. Suppression of tumor angiogenesis in colon26 tumor mass by leukocytopenia.
Suppression of tumor angiogenesis in colon26 tumor mass by leukocytopenia. (A-F) Whole mount staining of colon26 tumor mass with anti–PECAM-1 mAb (dark blue) (A,B,D,E) or anti–PECAM-1 (dark blue) and anti-CD45 (red) mAbs (C,F). The tumor mass along with skin was dissected 3 days after inoculation of colon26 tumor cells from mice after treatment with the anti–c-Kit mAb, ACK2 (D-F), or anti-B220 mAb as a control (A-C). The dotted line shows the mass of inoculated tumor cells (A,D). (B,E) High-power view of the location indicated by the arrow in panels A,D, respectively. (G,H) Gross appearance of tumor mass. The photographs of the dissected tumors were taken 5 days after inoculation of colon26 tumor cells from ACK2-treated mice (H) or anti-B220–treated mice (G). (I) Number of white blood cells (WBCs) in the peripheral blood after treatment with ACK2 (blue line) or anti-B220 mAb (red line; control [CTL]) for 4 days from day –4 to day –1. Results are expressed as the mean number of WBCs in 3 experiments (± SEM). (J) Tumor volume in mice that had been treated with ACK2 (blue line) or anti-B220 mAb (red line; CTL). Day 0 in this graph corresponds to the day 0 indicated in panel I. The tumor volumes measured on day 5, day 7, and day 14 were as follows: ACK2 (c-Kit): 52 ± 13 mm3 at day 5, 65 ± 15 mm3 at day 7, and 1020 ± 358 mm3 at day 14; B220 (CTL): 86 ± 15 mm3 at day 5, 890 ± 211 mm3 at day 7, and 1856 ± 325 mm3 at day 14. Results are expressed as the mean volume of 6 experiments (± SEM).
Rika Okamoto et al. Blood 2005;105:
©2005 by American Society of Hematology

5. Localization of HCs in PC3 tumor tissues.
Localization of HCs in PC3 tumor tissues. (A-E) Sections of PC3 tumor tissues from PC3 tumors with a volume of (A) more than 500 mm3, (B-C) 60 to 70 mm3, and (D-E) 100 to 120 mm3 were double stained with anti–PECAM-1 mAb (dark blue) and anti-CD45 mAb (red) (A,B,D) or with anti–PECAM-1 mAb (dark blue) and anti–c-Kit mAb (red) (C,E). Arrows in panel C indicate a small capillary at the tumor edge. Panels C,E are serial sections of panels B,D, respectively. (F) Colon26 tumor tissue section from a tumor with a volume of 60 to 70 mm3 was stained with anti–PECAM-1 mAb (dark blue) and anti–c-Kit mAb (red). Arrows indicate cells that are positive for c-Kit. The dotted line (B-F) shows the border between the fibrous cap surrounding the tumor mass and the tumor mass. Insets (D-F) show high-power views of the area indicated by box. The bar in panel A indicates 50 μm (A-C). The bar in panel D indicates 100 μm (D-F).
Rika Okamoto et al. Blood 2005;105:
©2005 by American Society of Hematology

6. Phenotype of c-Kit+ HCs located in the tumor.
Phenotype of c-Kit+ HCs located in the tumor. (A) Fluorescence-activated cell sorter (FACS) analysis of cells in PC3 tumor tissue. The numbers in each quadrant represent the percentage of gated Lin– cells. (B) Phenotype of c-Kit+ HCs sorted from PC3 tumor tissue by FACS. Cells fixed on glass were analyzed by May-Grünwald-Giemsa staining. M indicates mast cell. Bar indicates 10 μm. (C) FACS analysis of migrated BM cells by tumor lysates. The number in the upper right quadrant indicates the total percentage of migrated c-Kit+ cells. (D) Chemotaxis of c-Kit+ HCs in the presence of the lysates of various tumor cells. Colon26 indicates cell lysate from in vitro culture of colon26 cells; colon26-t, lysate of cells from colon26 tumor; PC3, cell lysate from in vitro culture of PC3 cells; PC3-t, lysate of cells from PC3 tumor. Results are expressed as the mean number in 3 experiments (± SEM).
Rika Okamoto et al. Blood 2005;105:
©2005 by American Society of Hematology

7. Suppression of tumor angiogenesis in PC3 tumor mass by anti–c-Kit mAb.
Suppression of tumor angiogenesis in PC3 tumor mass by anti–c-Kit mAb. The anti–c-Kit mAb, ACK2, was injected for 4 days (1 mg/d per mouse) into nude mice as described in the legend to Figure 3. (A) Photographs of mice taken 14 days after inoculation with PC3 cells. Arrows indicate the tumor mass in mice that had been treated with ACK2 (anti–c-Kit) or anti-B220 mAb (control). (B) Tumor volume on day 14 in mice that had been treated with ACK2 (anti–c-Kit) or anti-B220 mAb (control). The tumor volumes measured on day 14 were 486 ± 53 mm3 (ACK2: n = 8), and 1927 ± 258 mm3 (B220: n = 8). Results are expressed as the mean volume of 8 experiments (± SEM). (C-D) Tissue sections of the tumors from the mice observed in panel A, stained with anti–PECAM-1 mAb and counterstained with fast red. Tissue sections of the tumors from mice that had been injected with anti-B220 mAb (C) or ACK2 (D) are shown. The dotted line shows the border between the fibrous cap surrounding the tumor mass and the tumor mass. The area above the dotted line is the tumor mass. Arrow indicates a capillary that migrated into the tumor. The arrowheads show a vascular bed observed in the fibrous cap surrounding the tumor mass. Bar indicates 150 μm in panels C,D.
Rika Okamoto et al. Blood 2005;105:
©2005 by American Society of Hematology

8. Analysis of the survival and migration of ECs into tumor tissues after injection of anti–c-Kit mAb in tumor-bearing mice.
Analysis of the survival and migration of ECs into tumor tissues after injection of anti–c-Kit mAb in tumor-bearing mice. Colon26 tumor cells (A-C) or PC3 tumor cells (D-F) were subcutaneously inoculated into mice. Then, anti-B220 control mAb (A,D), anti–c-Kit mAb (B,E), or CPM (C,F) was injected into the mice. Anti-B220 mAb or anti–c-Kit mAb (1 mg/d per mouse) was injected daily from day 2 to day 5 after inoculation of tumor cells, and CPM (170 mg/kg per single injection) was injected on day 0 and day 6. Tumors were dissected on day 7. Sections were stained with anti–PECAM-1 mAb (red) and were subsequently analyzed for the presence of apoptotic cells by the TUNEL assay (dark blue). Arrowheads in panels B,E indicate round, apoptotic cells, and arrows in panels C,F indicate ECs. The inset in each panel shows a high-power view of the area indicated by the box. Bar in panel A indicates 40 μm in panels A-C and 60 μm in panels D-F.
Rika Okamoto et al. Blood 2005;105:
©2005 by American Society of Hematology