Figure 3.
Increased circulating and tissue-bound EGFL7 in the murine Vk*MYC model of MM. (A-F) MACS-isolated CD138+ Vk*MYC BM and spleen cells were injected into C57BL/6J mice via the tail vein. C57BL/6J mice transplanted with Vk*MYC tumor cells (Vk*MYC mice) and control mice (WT) that did not receive tumor cells were analyzed 25 days after cell inoculation. (A) Representative macroscopic images of whole spleens (left panel) and total spleen weight (right panel; n = 6/group) of WT and Vk*MYC mice. (B) Spleen and BM cells from transplanted Vk*MYC recipient or WT mice were stained for the plasma cell marker CD138 and the B-cell marker B220. Percentage of CD138+ cells per total BM or spleen cells and percentage of B220+ cells per total BM cells are given in the left panels. Representative FACS blots of CD138+ cells in BM and spleen are shown in the right panels. (C) Immunohistochemistry analysis of EGFL7 in BM and spleen sections of WT and VK*MYC mice (scale bar of the insert, 10 µm). Hematoxylin and eosin–stained sections (H&E; scale bar of the insert, 20 µm) show massive infiltration of MM cells in the tissue section of Vk*MYC mice. (D) Representative western blot for EGFL7 and b-ACTIN in BM and spleen cell lysates of WT and Vk*MYC mice. Two independent experiments were performed. (E) Fold change of EGFL7 expression in total BMNCs, MACS-isolated CD31+ BM cells, and splenocytes of Vk*MYC mice when compared with the EGFL7 expression in spleen and BM of nontumor-bearing mice (n = 2/group). Transcripts normalized to β-ACTIN. Graphs represent averages from 3 independently prepared templates per condition. Experiments were repeated twice, with similar results. (F) Plasma mouse EGFL7 as determined by enzyme-linked immunosorbent assay (n = 6/group). (G-J) Vk*MYC BM and spleen cells of donor mice with a tumor load of 30% in the BM were injected into C57BL/6J mice via the tail vein. VK*MYC transplanted mice were left untreated for 21 days to ensure sufficient tumor load. Then, 3 mice were treated with neutralizing Abs against EGFL7 (anti-EGFL7) or with isotype (nonbinding) control Ab at 1.5 mg/kg IV. On day 42, mice were euthanized. (G) Spleen weight was determined. (H) Frequency of B220−CD138+ MM cells in splenocytes as determined by FACS. (J) Hematoxylin and eosin–stained sections (scale bar of the insert, 50 µm) showing mild infiltration of MM cells in the BM. (I) Frequency of B220−CD138+ MM cells in BM of Vk*MYC mice treated with anti-EGFL7 Ab or isotype controls (n = 3/group). The data represent the data of 1 experiment. A similar experiment was performed independently, showing similar results. Graphs show mean ± SEM. Significance was calculated by Student t test, * P ≤ .05; ** P ≤ .01.

Increased circulating and tissue-bound EGFL7 in the murine Vk*MYC model of MM. (A-F) MACS-isolated CD138+ Vk*MYC BM and spleen cells were injected into C57BL/6J mice via the tail vein. C57BL/6J mice transplanted with Vk*MYC tumor cells (Vk*MYC mice) and control mice (WT) that did not receive tumor cells were analyzed 25 days after cell inoculation. (A) Representative macroscopic images of whole spleens (left panel) and total spleen weight (right panel; n = 6/group) of WT and Vk*MYC mice. (B) Spleen and BM cells from transplanted Vk*MYC recipient or WT mice were stained for the plasma cell marker CD138 and the B-cell marker B220. Percentage of CD138+ cells per total BM or spleen cells and percentage of B220+ cells per total BM cells are given in the left panels. Representative FACS blots of CD138+ cells in BM and spleen are shown in the right panels. (C) Immunohistochemistry analysis of EGFL7 in BM and spleen sections of WT and VK*MYC mice (scale bar of the insert, 10 µm). Hematoxylin and eosin–stained sections (H&E; scale bar of the insert, 20 µm) show massive infiltration of MM cells in the tissue section of Vk*MYC mice. (D) Representative western blot for EGFL7 and b-ACTIN in BM and spleen cell lysates of WT and Vk*MYC mice. Two independent experiments were performed. (E) Fold change of EGFL7 expression in total BMNCs, MACS-isolated CD31+ BM cells, and splenocytes of Vk*MYC mice when compared with the EGFL7 expression in spleen and BM of nontumor-bearing mice (n = 2/group). Transcripts normalized to β-ACTIN. Graphs represent averages from 3 independently prepared templates per condition. Experiments were repeated twice, with similar results. (F) Plasma mouse EGFL7 as determined by enzyme-linked immunosorbent assay (n = 6/group). (G-J) Vk*MYC BM and spleen cells of donor mice with a tumor load of 30% in the BM were injected into C57BL/6J mice via the tail vein. VK*MYC transplanted mice were left untreated for 21 days to ensure sufficient tumor load. Then, 3 mice were treated with neutralizing Abs against EGFL7 (anti-EGFL7) or with isotype (nonbinding) control Ab at 1.5 mg/kg IV. On day 42, mice were euthanized. (G) Spleen weight was determined. (H) Frequency of B220−CD138+ MM cells in splenocytes as determined by FACS. (J) Hematoxylin and eosin–stained sections (scale bar of the insert, 50 µm) showing mild infiltration of MM cells in the BM. (I) Frequency of B220−CD138+ MM cells in BM of Vk*MYC mice treated with anti-EGFL7 Ab or isotype controls (n = 3/group). The data represent the data of 1 experiment. A similar experiment was performed independently, showing similar results. Graphs show mean ± SEM. Significance was calculated by Student t test, * P ≤ .05; ** P ≤ .01.

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