CXCR4 or CXCR7 antagonists treat endometriosis by reducing bone marrow cell trafficking

Abstract

Adult stem cells have a major role in endometrial physiology, including remodelling and repair. However, they also have a critical role in the development and progression of endometriosis. Bone marrow-derived stem cells engraft eutopic endometrium and endometriotic lesions, differentiating to both stromal and epithelial cell fates. Using a mouse bone marrow transplantation model, we show that bone marrow-derived cells engrafting endometriosis express CXCR4 and CXCR7. Targeting either receptor by the administration of small molecule receptor antagonists AMD3100 or CCX771, respectively, reduced BM-derived stem cell recruitment into endometriosis implants. Endometriosis lesion size was decreased compared to vehicle controls after treatment with each antagonist in both an early growth and established lesion treatment model. Endometriosis lesion size was not effected when the local effects of CXCL12 were abrogated using uterine-specific CXCL12 null mice, suggesting an effect primarily on bone marrow cell migration rather than a direct endometrial effect. Antagonist treatment also decreased hallmarks of endometriosis physiopathology such as pro-inflammatory cytokine production and vascularization. CXCR4 and CXCR7 antagonists are potential novel, non-hormonal therapies for endometriosis.

Keywords: AMD3100; BMDSC; CCX771; CXCR4; CXCR7; bone marrow-derived stem cells; endometriosis.

Figure 1

A model of 5‐FU submyeloablation to characterize the bone marrow cell trafficking into endometriotic lesions in mice. Schematic model: Treatment for bone marrow conditioning (BMC) started on day −36 before uterine fragment grafting and 6 days before bone marrow transplantation (BMT) (A). Ectopic lesions were harvested 15 and 30 days (each group n = 6) after grafting uterine fragments into the peritoneal. Representative images of flow cytometry analysis of ectopic lesion cells on day 15 and 30 after uterine fragment grafting demonstrating the percentage of GFP‐positive cells in endometriosis. B, Control group (n = 6 mice), represented by GFP‐negative endometriosis lesions, was used to set a gate. Fluorescence confocal microscopy analysis of mouse endometriosis sections (C, D and E). Representative images of IF and quantitative analyses of cells expressing CXCR4, CXCR7 or CXCL12 and GFP, as a per cent of total endometriosis cells. Murine lesions were stained with anti‐GFP antibody (green) and co‐stained with CXCR4 antibody (red) (C), with CXCR7 antibody (red) (D) and with CXCL12 antibody (red) (E). Nuclei were stained by DAPI and are shown in blue. BMC, Bone morrow conditioning; BMT, Bone morrow transplantation; PLC, percentage of labelled cells. Results shown as mean ± SE. *P < .05 vs vehicle. Scale bar: 20 μm

Figure 2

Immunostaining of CXCR4, CXCR7 and CXCL12 and BMDCs. A, Representative immunofluorescence images of CXCR4, CXCR7 and CXCL12 staining in the epithelial cells of murine endometriosis. In our model, no GFP (bone marrow‐derived cells) was identified in the epithelium. Scale bar: 10 μm. B, Engrafted BMDCs express CD45 and F4/80. BMDCs: Engrafted bone marrow‐derived immune cells (BMDCs) were stained with DAPI or immunofluorescence for CD45 and F4/80. Engrafted BMDCs shows the presence of non‐immune (CD45 negative) and immune cells (CD45 positive), some of which co‐express F4/80, a marker of macrophages. Included is a cell that is a bone marrow‐derived (GFP+), CD45+, F4/80+ macrophage (arrow) Scale bar: 10 μm

Figure 3

Targeting CXCR4 or CXCR7 reduced BMDCs engraftment of endometriosis. Schematic model: treatment for bone marrow conditioning (BMC) started on day −36 before grafting uterine fragments and 6 days before bone marrow transplantation (BMT) (A). Treatment with AMD3100, CCX771 or vehicle (three groups, each group n = 6 mice) started on day 1 after grafting uterine fragments into the peritoneal cavity (day 0). Ectopic lesions were harvested after 15 days of treatment. B, Representative images of flow cytometry analysis of ectopic lesion cells after treatment showing the percentage of GFP‐positive cells in endometriotic lesions. Control, represented by GFP‐negative endometriosis lesion stained with CD45 isotype, was used to set gates (B). Fluorescence confocal microscopy analysis of mouse endometriosis sections. Representative images of IF and quantitative analyses of cells expressing CD45 (C) or CD31 (D) and GFP, as a per cent of total cells, following treatment. Nuclei were stained by DAPI and are shown in blue. PLC, percentage of labelled cells. BMC, Bone morrow conditioning. BMT: Bone morrow transplantation. Results shown as mean ± SE. *P < .05 vs vehicle. Scale bar: 20 μm

Figure 4

Targeting CXCR4 or CXCR7 reduced endometriosis development. Early growth model: AMD3100 or CCX771 or vehicle (three groups, each group n = 6 mice) injection started on day 1 after uterine fragments into the peritoneal cavity (day 0) of wild‐type animals (A). Ectopic lesions were harvested after 15 days of treatment. Representative pictures of ectopic lesions isolated from wild‐type mice with endometriosis subcutaneously treated with either vehicle or receptor antagonists (B). Lesion volume measurement after 15 days of treatment. IHC and quantitative analyses of the expression patterns of Ki‐67 in epithelial or stromal cells of ectopic lesions of wild‐type mice after treatment (C). Scale bar: 50 μm. IHC, and quantification of microvessel density of ectopic lesions of wild‐type mice treated with ligands or vehicle using CD31 staining (D). Scale bar: 100 μm. Expression of IL‐6, TNF‐α, IL‐1β, TGF‐β, COX‐2, VEGEF‐A and MMP2, MMP9 in ectopic lesions was analysed by quantitative reverse transcription PCR (qRT‐PCR) after 15 days of treatment. mRNA levels are expressed relative to transcript level in ectopic lesions of mice treated with vehicle (E), set at 1.0. Results showed as mean ± SE. *P < .05 vs vehicle

Figure 5

Targeting CXCR4 or CXCR7 induced the regression of endometriosis. Treatment model: AMD3100 or CCX771 or vehicle was subcutaneously injected into three groups of mice separately (each group n = 6 mice) of mice starting on day 15 after grafting uterine fragment into the peritoneal cavity (day 0) of wild‐type animals (A). Ectopic lesions were harvested after 15 days of treatment (day 30). Representative pictures of ectopic lesions isolated from wild‐type mice with endometriosis subcutaneously treated with either vehicle or receptor antagonists (B). Lesion volume measurement after 15 days of treatment. IHC, and quantification of microvessel density of ectopic lesions of wild‐type mice treated with ligands or vehicle using CD31 staining (C). Scale bar: 100 μm. IHC and quantitative analyses of the expression patterns of Ki‐67 in epithelial or stromal cells of ectopic lesions of wild‐type mice after treatment (D). Scale bar: 50 μm. Representative pictures and quantification of deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end labelling (TUNEL) assays in epithelial and stroma cells (E) (the number of TUNEL‐positive stained cells are shown in the bar graphs as percentage to the total cells). Scale bar: 50 μm. Expression of IL‐1β, IL‐6, TNF‐α, COX‐2 and MMP‐2 and MMP‐9 in ectopic lesions was analysed by quantitative reverse transcription PCR (RT‐PCR) after 15 days of treatment (F). mRNA levels are expressed relative to transcript level in ectopic lesions of mice treated with vehicle, set at 1.0. Results show mean ± SE. *P < .05 vs vehicle