Stromal Cell-Derived Factor-1/CXCL12 Contributes to MMTV-Wnt1 Tumor Growth Involving Gr1+CD11b+ Cells

Abstract

BackgroundHistological examinations of MMTV-Wnt1 tumors reveal drastic differences in the tumor vasculature when compared to MMTV-Her2 tumors. However, these differences have not been formally described, nor have any angiogenic factors been implicated to be involved in the Wnt1 tumors.Methodology/Principal FindingsHere, we show that MMTV-Wnt1 tumors were more vascularized than MMTV-Her2 tumors, and this correlated with significantly higher expression of a CXC chemokine, stromal cell-derived factor-1 (SDF1/CXCL12) but not with VEGFA. Isolation of various cell types from Wnt1 tumors revealed that SDF1 was produced by both tumor myoepithelial cells and stromal cells, whereas Her2 tumors lacked myoepithelial cells and contained significantly less stroma. The growth of Wnt1 tumors, but not Her2 tumors, was inhibited by a neutralizing antibody to SDF1, but not by neutralization of VEGFA. Anti-SDF1 treatment decreased the proportion of infiltrating Gr1+ myeloid cells in the Wnt1 tumors, which correlated with a decrease in the percentage of endothelial cells. The involvement of Gr1+ cells was evident from the retardation of Wnt1 tumor growth following in vivo depletion of these cells with an anti-Gr1-specific antibody. This degree of inhibition on Wnt1 tumor growth was comparable, but not additive, to the effect observed with anti-SDF1, indicative of overlapping mechanisms of inhibition. In contrast, Her2 tumors were not affected by the depletion of Gr1+ cells.Conclusions/SignificanceWe demonstrated that SDF1 is important for Wnt1, but not for HER2, in inducing murine mammary tumor and the role of SDF1 in tumorigenesis involves Gr1+ myeloid cells to facilitate growth and/or angiogenesis.

Mammary Tumor Regression Elicited by Wnt Signaling Inhibitor Requires IGFBP5

“…Mainly, we isolated CD45 þ hematopoietic and CD45 À stroma cells and 2 distinct tumor epithelial cells referred to as basal/myoepithelial and luminal cells. Sorted epithelial cells have been characterized by the expression of cytokeratin isotypes (CK5 for basal/myoepithelial cells and CK18 for luminal epithelial cells) and basal/myoepithelial cell marker, P-cadherin and smooth muscle actin (2). By examining the transcripts from these isolated cell populations, we found that IGF-I was predominantly expressed by the tumor hematopoietic cells (CD45 þ ), and IGF-II was expressed by the CD45 À fibroblasts enriched stromal cells (Fig.…”

“…Immunofluorescence staining was conducted on formalinfixed paraffin-embedded tumor sections (2). For image analysis, SlideBook software was used to measure pixel intensities and calculated as previously described (16).…”

“…Real-time PCR was carried out by TaqMan reaction as previously described (2). All the primers used in this study were listed in the Supplementary Methods.…”

“…Mammalian Wnt ligand was originally identified as a protooncogene in viral induced mouse mammary gland tumors; overexpression of Wnt1 ligand under the control of the mouse mammary tumor virus (MMTV) promoter leads to hyperplasia and, ultimately, adenocarcinoma in the mammary gland (1). MMTV-Wnt1 tumors have characteristic histologic features and are composed of a variety of cell types, including distinct tumor luminal epithelial and basal/myoepithelial cells and host-derived stroma cells (2). Importantly, the existence of basal/myoepithelial cells in Wnt tumors is reminiscent of human basal-like breast cancers (3), which are often associated with a poor prognosis and the lack of effective treatment.…”

Development and Preclinical Characterization of a Humanized Antibody Targeting CXCL12

“…Whole tumors were minced into small pieces, followed by sequential digestion with Media I for 60 minutes and Media II for 15 minutes, as previously described (22). Single cells were first incubated at room temperature for 10 minutes with rat anti-mouse CD16/CD32 mAb (Fc block; BD Biosciences), followed by staining with the following fluorophore-conjugated antibodies: anti-CD11b (integrin aM chain, Mac-1, a-chain, M1/70; BD Pharmingen), anti-Ly6G (Gr1, RB6-8C5; eBioscience), anti-CD31 (PECAM-1; eBioscience), anti-human/mouse CXCR7 (8F11-M16; BioLegend), anti-F4/80 (BM8; eBioscience), and anti-CXCR4 (CD184; BioLegend).…”