Physiologically activated mammary fibroblasts promote postpartum mammary cancer
Qiuchen Guo, … , Paul Spellman, Pepper Schedin
Qiuchen Guo, … , Paul Spellman, Pepper Schedin
Published March 23, 2017
Citation Information: JCI Insight. 2017;2(6):e89206. https://doi.org/10.1172/jci.insight.89206.
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Categories: Research Article Oncology

Physiologically activated mammary fibroblasts promote postpartum mammary cancer

Abstract

Women diagnosed with breast cancer within 5 years of childbirth have poorer prognosis than nulliparous or pregnant women. Weaning-induced breast involution is implicated, as the collagen-rich, immunosuppressive microenvironment of the involuting mammary gland is tumor promotional in mice. To investigate the role of mammary fibroblasts, isolated mammary PDGFRα+ cells from nulliparous and postweaning mice were assessed for activation phenotype and protumorigenic function. Fibroblast activation during involution was evident by increased expression of fibrillar collagens, lysyl oxidase, Tgfb1, and Cxcl12 genes. The ability of mammary tumors to grow in an isogenic, orthotopic transplant model was increased when tumor cells were coinjected with involution-derived compared with nulliparous-derived mammary fibroblasts. Mammary tumors in the involution-fibroblast group had increased Ly6C+ monocytes at the tumor border, and decreased CD8+ T cell infiltration and tumor cell death. Ibuprofen treatment suppressed involution-fibroblast activation and tumor promotional capacity, concurrent with decreases in tumor Ly6C+ monocytes, and increases in intratumoral CD8+ T cell infiltration, granzyme levels, and tumor cell death. In total, our data identify a COX/prostaglandin E2 (PGE2)–dependent activated mammary fibroblast within the involuting mammary gland that displays protumorigenic, immunosuppressive activity, identifying fibroblasts as potential targets for the prevention and treatment of postpartum breast cancer.

Authors

Qiuchen Guo, Jessica Minnier, Julja Burchard, Kami Chiotti, Paul Spellman, Pepper Schedin

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Figure 1

Mammary fibroblasts are activated during weaning-induced gland involution.

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Mammary fibroblasts are activated during weaning-induced gland involutio...
(A) Mammary gland thin sections from the Col1a1-GFP reporter mouse immunofluorescence stained for GFP+ collagen 1–expressing cells (red), fibroblast PDGFRα (green), and myoepithelial cell calponin (purple), show colocalization of GFP and PDGFRα (yellow), but not calponin. Nuclei stained with DAPI (blue). White scale bar: 50 μm. Yellow scale bar: 10 μm. Arrows show PDGFRα+GFP+calponin– cells. (B) Flow gate for isolating murine mammary PDGFRα+ cells. (C) RT-qPCR on FACS-isolated PDGFRα+ cells for fibroblast markers (Col1a1, Col3a1, Col5a1, and PDGFRα) and markers of potential contaminating cell populations: Cd45 (lymphocyte common antigen), F4/80 (mature macrophage), Cd31 (endothelial cell), Adipoq (adipocyte), and Ecad (epithelial cell). Sorted PDGFRα-negative cells were used as positive controls for Cd45, F4/80, Cd31, Adipoq, and mammary epithelial EpH4 cells were used as the Ecad-positive control. n = 3–4 per group. (D) RNA-Seq heatmap illustrates 870 significantly differentially expressed genes (>1.3-fold changes) between fibroblasts isolated from nulliparous and involution day 6 (InvD6) mammary glands. Red: relative high expression. Blue: relative low expression. (E) Pathway analysis of RNA-Seq data set showing extracellular matrix (ECM) regulation–related pathways that are upregulated in InvD6-fibroblasts. (F) PDGFRα+ fibroblasts reported as number per gram of mammary tissue observed in nulliparous and 4-, 6-, and 8-day postweaning mice; n = 3–10 per group. (G) Fibrillar collagens and Lox gene expression analysis by RT-qPCR in sorted PDGFRα+ mammary fibroblasts from nulliparous and 4-, 6-, and 8-day postweaning mice; n = 3–10 per group. (H) Mmp2, Mmp3, and Tgfb1 gene expression analysis by RT-qPCR in nulliparous and InvD6 fibroblasts, n = 6–9 per group. (I) Immunofluorescence staining shows non-colocalization of α-smooth muscle actin (red) and PDGFRα (green) in mammary glands of nulliparous and InvD6 mice. Boxes show images at high magnification. Arrow: PDGFRα+ cells. Arrowhead: αSMA+ cells. White scale bar: 50 μm. Yellow scale bar: 10 μm. (J) Pathway analysis of RNA-Seq data described in D showing immune regulation–related pathways that are upregulated in InvD6 fibroblasts. (K) RNA-Seq data show chemokine expression pattern of fibroblasts isolated from nulliparous mammary glands. (L) RT-qPCR gene expression for Cxcl12 in sorted PDGFRα+ fibroblasts from nulliparous and InvD6 mammary glands, n = 3–5 per group. Gene expression data are normalized to Actb and are from 2 to 6 independent studies. RNA-Seq data are archived at the NCBI Gene Expression Omnibus (GEO GSE94761). *P < 0.5, **P < 0.01, ***P < 0.001, #P < 0.0001 by unpaired 2-tailed t test. Data represent mean ± SEM.