Vascular endothelial growth factor secreted by activated stroma enhances angiogenesis and hormone-independent growth of estrogen receptor-positive breast cancer

Abstract

"Reactive" or activated stroma characterizes many malignancies including breast cancers. Recently, we isolated a reactive mouse mammary gland stromal cell line called BJ3Z. These cells express alpha-smooth muscle actin and stromal cell-derived factor 1 (SDF-1) and are tumorigenic when injected into mice. Here we show that, in vivo, BJ3Z cells influence the angiogenesis and proliferation of xenografted estrogen receptor (ER)-positive MCF-7 human breast cancer cell-derived solid tumors. The growth-promoting effects of BJ3Z cells are equivalent to those of estradiol (E(2)). BJ3Z cells also increase the proliferation of normal mouse mammary luminal cells adjacent to tumors. In vitro, BJ3Z cells reorganize and increase the proliferation of cocultured malignant MCF-7 and normal human breast MCF10A cells grown as organoids in three-dimensional culture. The effects of BJ3Z cells on MCF-7 cells are equivalent to those of E(2). In contrast, BJ3Z cells do not alter the growth of highly aggressive ER-negative MDA-MB-231 human breast cancer cells. We show that BJ3Z cells secrete vascular endothelial growth factor (VEGF). The growth of MCF-7 organoids induced by BJ3Z can be inhibited by antagonists of VEGF and SDF-1. Conversely, recombinant VEGF stimulates the proliferation of MCF-7, but not MDA-MB-231, organoids. We conclude that, in addition to angiogenesis, VEGF released by activated stroma increases the growth of ER-positive malignant epithelial cells and of adjacent normal epithelium. Because activated stroma can substitute for E(2) and fosters hormone-independent growth of ER-positive tumors, we suggest that breast cancers exhibiting intrinsic hormone resistance may respond to antiangiogenic therapies.

Figure 1. BJ3Z cells are tumorigenic, stimulate angiogenesis and promote estrogen-independent tumor growth

A. mammary glands of ovx nude mice were injected with RedTomato tagged BJ3Z cells (top panels), ZsGreen tagged MCF-7 cells (central panels) or co-injected with both BJ3Z and MCF-7 cells (bottom panels). Mice were implanted with cellulose control (C; left) or 17β-Estradiol (E₂) releasing silastic pellets. Tumors were photographed and representative pictures are shown. B. Tumor growth was monitored for 10 weeks for C (n=5) and E₂ (n=10) for each tumor type. Statistically significant differences (* p<0.05) were calculated by two-way ANOVA. C. Neovascularization of tumors was quantified by IHC of fluorescent CD34 positive cells in tumors, in the absence or presence of E₂; * p<0.05.

Figure 2. BJ3Z cells enhance proliferation of MCF-7 cells and of adjacent normal mammary tissue in the absence of E₂

A. Ovx nude mice were implanted in mammary glands with MCF-7 cells alone, or MCF-7/BJ3Z cell mixtures. Mice were supplemented with cellulose (C) or E₂-releasing pellets. Tumors were grown for 10 weeks. Slides were stained by IHC against CK18 (red) and BrdU (green). Magnification bar is 50μm. B. BrdU incorporation was quantified; * and # p<0.05 tested by ANOVA, followed by Tukey's post-test. C. Tumors were grown as above in mice receiving BJ3Z cells only, MCF-7 cells only or BJ3Z/MCF-7 cell mixtures; all in the absence of E₂. Slides from tumors were stained with H&E (left). Serial sections were stained by IHC for normal E-cadherin positive cells (red) and BrdU (green nuclei). D. Quantification of BrdU incorporation; * p<0.05.

Figure 3. BJ3Z cells enhance proliferation of MCF-7 and MCF10A but not MDA-MB-231 in 3D organoids

Eight-well slides were coated with Growth Factor Reduced Matrigel™. MCF-7, MCF10A, MDA-MB-231 and BJ3Z cells were plated on Matrigel alone or in the combinations shown and grown for 7 days. Organoids were paraffin embedded, and sections were cut for IHC. A. Left panels, light microscopy images of organoids at 7 days. Magnification bar is 100μm. Middle panels show dual IHC against CK18 (red) and BrdU (green) for MCF-7 cells; B. CK14 (red) and BrdU (green) for MCF10A cells; and C. CD44 (red) and BrdU (green) for MDA-MB-231 cells. Magnification bar is 50μm. Right panels show quantification of BrdU incorporation; * p<0.05.

Figure 4. BJ3Z cells grown in tumors and 3D organoids show similar expression profiles for stromal makers and secrete VEGF and TIMP-1

A. BJ3Z cells were grown in vivo as solid tumors in the mammary glands of ovx nude mice (tumor), or as 3D organoids in vitro. Sections of tumors and organoids were stained by IHC for Fibroblast Activation Protein (FAP); alpha-smooth muscle actin (α-SMA); stromal derived factor 1 (SDF-1); vimentin and with an anti-mouse epithelial panCK. Magnification bar is 50μm. B. BJ3Z cells were cultured in 3D as described for 2 days, then switched to serum-free MEM for 48 hrs. Controls consisted of a Matrigel-coated chamber without cells. Supernatants from control or BJ3Z cells were collected and hybridized to a TranSignal™ mouse angiogenesis antibody array. Lower panel lists the arrayed factors.

Figure 5. BJ3Z cells enhance proliferation of MCF-7 cells through VEGF and SDF-1; VEGF activates p-ERK

MCF-7 or MDA-MB-231 cells were grown alone or together with BJ3Z cells as 3D organoids. A. MCF-7/BJ3Z co-cultures were incubated with a blocking anti-mouse VEGF monoclonal antibody (VEGF MAb) or Avastin (Bevacizumab) or with the CXCR-4 blocker AMD 3100 for 7 days. On day 7, cells were processed as described and the proliferation rate was calculated using the CK18/BrdU proliferation assay; * p<0.05. B. BrdU incorporation in MCF-7 and MDA-MB-231 monocultures treated with rVEGF for 7 days; * p<0.05. C. Seven day-old MCF-7 or MDA-MB-231 organoids were treated 10 min with vehicle (control) or rVEGF then processed and stained by IHC against p-ERK; magnification bar is 50μm.