Semaphorin 7A Promotes Macrophage-Mediated Lymphatic Remodeling during Postpartum Mammary Gland Involution and in Breast Cancer

Abstract

Postpartum mammary gland involution is a tissue remodeling event that occurs in all mammals in the absence of nursing or after weaning to return the gland to the pre-pregnant state. The tissue microenvironment created by involution has proven to be tumor promotional. Here we report that the GPI-linked protein semaphorin 7A (SEMA7A) is expressed on mammary epithelial cells during involution and use preclinical models to demonstrate that tumors induced during involution express high levels of SEMA7A. Overexpression of SEMA7A promoted the presence of myeloid-derived podoplanin (PDPN)-expressing cells in the tumor microenvironment and during involution. SEMA7A drove the expression of PDPN in macrophages, which led to integrin- and PDPN-dependent motility and adherence to lymphatic endothelial cells to promote lymphangiogenesis. In support of this mechanism, mammary tissue from SEMA7A-knockout mice exhibited decreased myeloid-derived PDPN-expressing cells, PDPN-expressing endothelial cells, and lymphatic vessel density. Furthermore, coexpression of SEMA7A, PDPN, and macrophage marker CD68 predicted for decreased distant metastasis-free survival in a cohort of over 600 cases of breast cancer as well as in ovarian, lung, and gastric cancers. Together, our results indicate that SEMA7A may orchestrate macrophage-mediated lymphatic vessel remodeling, which in turn drives metastasis in breast cancer.Signficance: SEMA7A, which is expressed on mammary cells during glandular involution, alters macrophage biology and lymphangiogenesis to drive breast cancer metastasis. Cancer Res; 78(22); 6473-85. ©2018 AACR.

Figure 1:. LYVE-1 expressing macrophages during involution.

A) Vessel-like structures that stain positive for LYVE-1 (green) and F4/80 (red) are abundant during involution. B) Merged image and 3D reconstruction of a LYVE-1+F4/80+ vessel at involution day 6. C) Quantification of LYVE-1+F4/80+ vessel density in mouse mammary glands from virgin mice and mice at various stages of involution. D) Macrophage (CD68-pink) and lymphatic (PDPN-green) interaction in breast tissue biopsied from a women less than one year after pregnancy. Unpaired t-test: **p<0.01, ***p<0.001, ****p<0.0001. Arrows point to vessels that were quatntitated. Scale bars=50μm.

Figure 2:. Podoplanin expression on macrophages drives lymphangiogenesis.

Flow cytometry analysis of cells isolated from nulliparous or involution day 6 mouse mammary tissues reveals A) increased lymphatic endothelial cells, B) PDPN+ leukocytes, C) PDPN+ myeloid cells, D-F) monocytes and macrophages, as well as G&H) PDPN-expressing F4/80+ macrophages. I&J) F4/80+ macrophages isolated from nulliparous and involution day 6 mouse mammary glands enhance in vitro lymphangiogenesis. Unpaired t-test: *p<0.05, **p<0.01, ***p<0.001.

Figure 3:. Semaphorin 7a promotes tumor-associated macrophages and lymphangiogenesis.

A) In an in vivo matrigel plug assay, 66cl4 tumor cells were mixed with macrophages isolated from nulliparous or recently weaned mice (at involution day 6). The resulting plugs were harvested and F4/80/LYVE-1 co-staining quantitated. B) Growth of 66cl4 cells engineered to overexpress SEMA7A compared to empty vector controls injected into nulliparous and involution day 1 mouse mammary tissues. C) SEMA7A expression by IHC in tumors from B. D) CD45+CD31-F4/80+PDPN+ cells in tumors from B. E) %F4/80+ cells and F) # LYVE-1+ vessel density in tumors from B. G) F4/80+PDPN+ cells in E0771 SEMA7A tumors in nulliparous mice. H) LYVE-1+ density in tumors from G with the addition of tumors from SEMA7A−/− hosts injected with E0771 cells. Unpaired t-test: *p<0.05, **p<0.01, ***p<0.001.

Figure 4:. Semaphorin 7a promotes macrophage-mediated lymphatic mimicry.

A) SEMA7A is expressed on EpCAM+ cells at involution day 6. In mammary glands from SEMA7A−/− mice we observe decreased B) LECs, C) PDPN+ immune cells, D) monocytes, and E&F) monocytes and macrophages expressing PDPN. G) LYVE-1 (green) and F4/80 (brown) IHC on involution day 6 mouse mammary glands from WT (top) and SEMA7A−/− (KO—bottom). Inset, a-c and e-g are mammary gland and d and h are lymph node. Unpaired t-test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bars=50μm.

Figure 5:. Semaphorin 7a drives PDPN expression, macrophage motility, and adherence to endothelial cell monolayers.

SEMA7A promotes macrophage A) expression of PDPN, B) migration and C) adherence to endothelial cell monolayers in 2D, and D&E) inclusion in vessel like structures in 3D. Induction of F) motility and G) adherence to LEC monolyaers in 2D is inhibited by blocking β−1 integrin (motility and adherence) and PDPN (adherence). H) Macrophage incorporation into 3D LEC vessel-like structures is inhibited by function blocking antibodies against β−1 integrin (9EG7) and PDPN (pMAB). Unpaired t-test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6:. Lymphatic trafficking of tumor cells is driven by involution and Semaphorin 7a.

A) LVD in mouse mammary tissues injected with tumor cells at involution day 1 compared to those injected into nulliparous hosts. B) Representative image of lymphatics at involution day 6. C) Clonogenic assay reveals presence of micrometastasis/axillary lymph node during active involution. D) Quantitative IHC for SEMA7A expression in normal mammary tissue from never-been-pregnant women or women within 5 years of most recent childbirth. E) CD68, PDPN, and SEMA7A in a patient diagnosed within 1 year postpartum, who was also posititve for LVI and LN involvement. Overall survival analysis using KmPlot for F) all (n=626), G) LN negative (n=122), and H) LN positive (n=177) patients. I) Distant-metastatsis-free survival for patients co-expressing SEMA7A, PDPN, and CD68. J) Progression-free survival in ovarian cancer, K&L) overall survival in lung and gastric cancer. Unpaired t-test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bars=50μm.