Anti-VEGF-A affects the angiogenic properties of tumor-derived microparticles.

Tumor derived microparticles (TMPs) have recently been shown to contribute to tumor re-growth partially by inducing the mobilization and tumor homing of specific bone marrow derived pro-angiogenic cells (BMDCs). Since antiangiogenic drugs block proangiogenic BMDC mobilization and tumor homing, we asked whether TMPs from cells exposed to an antiangiogenic drug may affect BMDC activity and trafficking. Here we show that the level of VEGF-A is reduced in TMPs from EMT/6 breast carcinoma cells exposed to the anti-VEGF-A antibody, B20. Consequently, these TMPs exhibit reduced angiogenic potential as evaluated by a Matrigel plug and Boyden chamber assays. Consistently, BMDC mobilization, tumor angiogenesis, microvessel density and BMDC-colonization in growing tumors are reduced in mice inoculated with TMPs from B20-exposed cells as compared to mice inoculated with control TMPs. Collectively, our results suggest that the neutralization of VEGF-A in cultured tumor cells can block TMP-induced BMDC mobilization and colonization of tumors and hence provide another mechanism of action by which antiangiogenic drugs act to inhibit tumor growth and angiogenesis

TMPs from cells exposed anti-VEGF-A antibody do not promote angiogenesis in tumors.

<p>(A) Matrigel plugs containing an equal number of TMPs (0.5×10⁶) from untreated or B20-exposed EMT/6 cells were implanted into the flanks of 8–10 week old BALB/c mice. Matrigel plugs containing PBS were used as a negative control. Ten days later, plugs were removed and prepared as single cell suspensions. The extracted cells were immunostained for endothelial cells, hemangiocytes and MDSCs and analyzed by flow cytometry. Results are presented as the number of cells per 1 mg Matrigel. (B–E) Eight to ten week old BALB/c mice (n = 4 mice/group) were implanted with 0.5×10⁶ EMT/6 cells into the flanks. When tumors reached a size of approximately 50 mm³, injections with 0.5×10⁶ TMPs from untreated or B20-exposed EMT/6 cells were performed twice weekly. Control mice were injected with PBS. (B) Tumor growth was assessed by a Vernier caliper using the formula, width²×length×0.5. Tumors were removed at endpoint, and subsequently were either (C) stained for CD31 (in red), CD45 (in green), and Hoechst (in blue) for the evaluation of (D) microvessel density and perfusion (scale bar = 100 µm), or (E) prepared as single cell suspensions for the evaluation of MDSCs and hemangiocytes colonization of tumors using flow cytometry. **, 0.01>p>0.001; ***, p<0.001.</p

Exposing tumor cells to anti-VEGF-A antibodies reduces the level of VEGF-A in TMPs without affecting the number of TMPs.

<p>(A) A representative flow cytometry dotplot for TMP quantification. TMPs are approximately 1 µm, and counting beads are 7.35 µm. The number of TMPs per sample was calculated as the ratio between the number of events collected in the counting beads gate and the number of events collected in the TMPs gate over the total number of counting beads loaded in the sample. (B) EMT/6, 4T1 and MDA-MB231 breast carcinoma cells were either left untreated or exposed to 2 µg/ml B20 antibody for 48 h. TMPs were purified from conditioned medium and quantified by flow cytometry. Shown are the means ± S.D. of triplicates. (C) An equal number of TMPs (100,000) from untreated or B-20-exposed EMT/6, 4T1 and MDA-MB231 breast carcinoma cells were used to quantify the level of VEGF-A by ELISA. In some experiments control for B20 antibodies was used in a form of IgG in culture. Shown are the means ± S.D. of triplicates. **, 0.01>p>0.001.</p

TMPs from cells exposed to anti-VEGF-A antibody exhibit reduced ability to promote endothelial cell activity.

<p>Matrigel plugs containing an equal number of TMPs (0.5×10⁶) from untreated or B20-exposed EMT/6 cells were implanted into the flanks of 8–10 week old BALB/c mice. Matrigel plugs containing PBS were used as a negative control. Ten days later, plugs were removed and then sectioned. (A) Slides were stained with H&E or immunostained with the endothelial cell marker CD31 (designated in red) (scale bar = 100 µm). (B) Microvessel density in the plugs was calculated by counting vessel structures. (C–D) An equal number of TMPs (5×10⁶) from untreated or B20-exposed EMT/6 cells were tested for HUVEC migration (C) and invasion (D) using the modified Boyden chamber assay. PBS was used as a negative control. Cells invading the membrane of the Boyden chamber were stained with Crystal Violet and images were captured using a Leica CTR 6000 microscope. The number of cells invading the membrane were counted and plotted (n>8/group). (E) Aortic rings from BALB/c mice (n = 4/group) were cultured in medium containing 0.1×106 TMPs from untreated or B20-exposed EMT/6 cells. Endothelial cell medium (ECGS) was used as a positive control. Images were captured using an inverted light microscope system (Leica CTR 6000 system) (Scale bar = 500 µm). *, 0.05p>0.001; ***, p<0.001.</p

TMPs from cells exposed to anti-VEGF-A antibody do not induce viable CEP and hemangiocyte mobilization.

<p>(A) An equal number of TMPs (0.5×10⁶) from untreated (CONT) or B20-exposed EMT/6 cells was injected into the tail vein of 8–10 week old non-tumor bearing BALB/c mice (n = 4 mice/group). Control mice were injected with PBS (PBS). One hour later, blood was drawn from the retro-orbital sinus for the evaluation of viable CEPs (CD45−/VEGFR2+/CD117+/7AAD−), MDSCs (Gr1+/CD11b+), and hemangiocytes (CD11b+/CXCR4+/VEGFR1+) using flow cytometry. (B) Half a million TMPs from untreated (CONT) or B20-exposed cells were tagged with PKH26, and subsequently injected into the tail vein of BALB/c mice (n = 4 mice/group). Control mice were injected with PBS. One hour later, blood was drawn by cardiac puncture and total BMDCs (CD45+), viable CEPs, hemagiocytes, and MDSCs were analyzed by flow cytometry. The percentage of the different cell types positive for tagged TMPs was plotted. **, 0.01>p>0.001; ***, p<0.001.</p

Representative flow cytometry plots of viable CEPs, hemangiocytes, and myeloid derived suppressor cells.

<p>An example of the analysis of flow cytometry data obtained from peripheral blood of BALB/c mice is presented. Viable CEPs are determined as (a) positive for VEGFR2 and negative for CD45 as well as (b) positive for CD117 and negative for 7-AAD. Hemangiocytes are determined as (c) positive for CD45 and CXCR4 as well as (d) positive for VEGFR1. Myeloid derived suppressor cells (MDSCs) are determined as positive for (e) both Gr-1 and CD11b.</p