Table 1

Studies investigating the molecular and/or immunological mechanisms during mammary gland involution in PPBC in rodent models

Research subject and methods Findings Ref
I. Studies investigating ECM remodeling and tumor
  •  ECM from mammary glands of np and involuting rats.

  •  Invasive potential in metastatic MDA-MB-435 cells.

  •  Increased motility and invasion of ECM in MDA-MB-435 cells.

  •  Increased fibronectin and MMP activity in in vitro cultured cells of the involuting mammary ECM.

  •  ECM from mammary glands of np, pregnant, lactating, and involuting rats.

  •  Mouse mammary epithelial tumor cell line TM-6 with hormone withdrawal-induced death.

  •  Increased fibronectin and fibronectin fragment levels in involuting ECM associated with apoptosis in TM-6.

  •  Fibronectin fragments induce ECM proteases and contribute to epithelial cells loss and dissolution of mammary alveoli in involuting ECM.

  •  Mammary glands from np, pregnant, lactating. and involuting mice.

  •  Affymetrix microarrays.

  •  Increased expression of components of apoptosis, inflammatory cytokines, and acute-phase response genes during involution.

  •  Isolated ECM from mammary glands of np, pregnant, lactating, and involuting rats.

  •  Histological, IHC, and western blotting analyzes.

  •  Increased epithelial cell proliferation; differentiation, death, and reorganization in ECM isolated from involuting rats.

  •  Changed ECM function and fibronectin, laminin, clusterin, and MMP composition in parous mammary gland.

  •  ECM from mammary glands of np and involuting rats.

  •  In vivo models of MDA-MB-231 cells injected into mammary fat pad.

  •  Deceased ductal organization and increased invasiveness in tumor MDA-MB-231 cells of involuting ECM.

  •  Increased metastasis to the lung, liver, and kidney in the involution group.

  •  Normal mammary glands, mammary tumors, and explants in 3D culture of involuting mice.

  •  Epithelial-stromal interactions via histology, electron microscopy, and optical imaging.

  •  Increased orientation along certain aligned collagen fibers in involuting mice, facilitating tumor invasion.

  •  Primary tumor explants realigned collagen fibers to migrate.

  •  In vivo rodent model of PPBC: injecting 4T1 and D2A1cells into the portal vein of involuting mice/rats.

  •  Flow cytometry and MS of mouse/rat livers.

  •  Induced maternal liver involution characterized by hepatocyte cell death and stromal remodeling in post-weaning mice.

  •  Increased liver metastasis in post-weaning mice.

II. Studies investigating lymphangiogenesis
  •  In vivo rodent model of PPBC: injecting DCIS-GFP, D2A1, or 66cl4-LUC cells into mammary fat pad of SCID or BALC/c mice on INV1.

  •  Lymphatic vessel density by LYVE1 +vessel count and mRNA gene expression levels.

  •  COX-2 knock down cells injected into postpartum host.

  •  Increased PPBC tumor sizes, LN positivity, lung micro-metastases, and LYVE1 +mammary lymphatic vessel density 3–4 days after weaning.

  •  Increased gene expression of Lyve1, vegfrd, vegfr2, and vegfr3 during involution.

  •  Decreased peritumor lymphatic vessel density (60%) and decreased invasion of lymphatics (85%) in shCOX-2 PPBC.

  •  In vivo rodent model of PPBC: injecting 66cl4-DDK, E0771-DDK, 66cl4-SEMA7A, or E0771-SEMA7A-overexpressing-cells in Balb/c mice on INV1.

  •  IHC, flow cytometry, lymphangiogenesis and macrophage migration, endothelial cell adhesion, and expression of PDPN assays.

  •  Increased expression of SEMA7A and PDPN-expressing cells in involuting tumors.

  •  Increased expression of PDPN in macrophages during involution.

  •  Increased motility and adherence of PDPN-expressing macrophages to lymphatic endothelial cells that promoted lymphangiogenesis.

III. Studies investigating the immune
  •  Normal mammary glands from involuting Balb/c mice that were weaned after 7 days.

  •  Affymetrix microarrays for transcript analysis of involution.

  •  Protein extracts, western blotting, and IHC.

  •  Upregulated expression of 145 genes during first 4 days of involution, including:

    •  49 immunoglobulin genes

    •  12 acute-phase response genes (STAT3, CD14, LBP)

  •  Increased infiltration of neutrophils, plasma cells, macrophages, and eosinophils in involuting mammary tissue.

  •  In vivo rodent model of involution: weaned rats and C57BL/6 mice at day 10 (rats) or 14 (mice) of lactation.

  •  IHC and quantification of CD68, CSF-1R, and F4/80.

  •  ECM isolation, chemoattractant & zymogen assay, collagen detection and quantification, and collagen western blot.

  •  Increased macrophage influx (8-fold) during involution that exhibited an M2-phenotype with expression of IL-4 and IL-13.

  •  Increased chemotactic capacity for macrophages from involuting ECM.

  •  Increased fibrillar collagen levels and proteolysis during involution.

  •  Increased chemoattractant capacity for denatured collagen I during involution.

  •  In vivo rodent model of PPBC: injecting D2A1 cells into mammary fat pad of mice on INV1.

  •  In vivo treatment model of PPBC: intraperitoneal injection of anti-IL-10 or rat IgG on L10, INV2, INV4, INV6, and INV8.

  •  Flow cytometry, IHC, and western blot.

  •  Increased tumor sizes (6-fold), decreased CD4 +, and CD8+T cell infiltrates and increased number of macrophages in involuting mice.

  •  Reduced tumor growth in IL-10 targeted mice.

  •  In vivo rodent model of involution: weaned Balb/c mice at day 9 to 13 of lactation.

  •  In vivo rodent model of PPBC: injecting FACS sorted fibroblasts and D2A1 cells in postweaning Balb/c mice.

  •  RT-qPCR, RNA sequencing, IF, and IHC analyzes.

  •  Increased fibroblast activation during involution.

  •  Increased growth and decreased CD8 +T cell infiltration and tumor cell death in mammary tumors in the involuting-fibroblast group.

  •  Suppressed involution-fibroblast activation and tumor promotional capacity by ibuprofen treatment.

  •  In vivo rodent model of involution: weaned Balb/c-C57Bl/6 mice at day 9 to 14 of lactation.

  •  IHC, flow cytometry, Ag assays, and adoptive T-cell transfer.

  •  Elevated mucosal CD4 +T cells within lactating and involuting glands.

  •  Increased accumulation of Th17-Treg CD4 +T cells and elevated levels of Gata3+, FoxP3+, and PD-1 +CD4+T cells in the involuting mammary gland.

IV. Studies investigating treatment options
  •  In vivo rodent model of PPBC: injecting human breast tumor MCF10DCIS cells into the mammary fat pads on INV1.

  •  Treatment with celecoxib and ibuprofen.

  •  Increased characterization of fibrillar collagen, high cyclooxygenase-2 (COX-2) expression, and invasive phenotype in PPBC tumors.

  •  Reduced promotional effects of celecoxib and ibuprofen.

  •  In vivo rodent model of involution: weaned rats at day 10 of lactation and treated with NSAIDs on INV4, INV5, and INV6.

  •  In vivo xenograft model: injecting D2.OR cells mixed with ECM in fat pad of np mice.

  •  RT-PCR, western blot, ELISA, IHC, and imaging.

  •  Reduced tumor growth of cells mixed with NSAID-involution ECM in PPBC mice compared with control-involution ECM in np mice.

  •  Identified tenascin-C as potential mediator of tumor progression during involution that is decreased by NSAID treatment.

  •  In vivo rodent model of PPBC: injecting D2A1 cells into fat pad of Balb/c-C57Bl/6 mice on INV1.

  •  In vivo multiparity mouse model of involution: weaned Balb/c-C57Bl/6 mice at day 10 of lactation (ibuprofen treated) – repeated 2 x.

  •  Flow cytometry, mass spec, immunoblot, bone marrow assays, RT-PCR, RNA seq, multiplex IHC, and TCGA.

  •  Increased tumor growth in PPBC mice with a distinct immune milieu compared with tumor of np mice.

  •  Increased monocytes and reduced number of T-cells in PPBC mice, which is reversed on ibuprofen treatment.

  •  Enhanced Th1-associated cytokines and T-cell accumulation by ibuprofen treatment.

  •  Ibuprofen does not impede normal involution.

  •  In vivo rodent model of involution: weaned Balb/c-C57Bl/6 mice at day 10 to 14 of lactation.

  •  In vivo rodent model of PPBC: injecting 66cl4 or E0771 carcinoma cells in Balb/c or C57Bl/6 mice on INV1.

  •  Lymphatic vessel density, multiplex staining, flow cytometry, and cytokine staining

  •  Increased PD-1, PD-L1 expression, and PD-L1 T-cells in mouse mammary tissues during normal postpartum involution.

  •  Increased expression of CD8 +T cells expressing co-inhibitory receptors PD-1 and Lag-3 in PPBC mice models.

  •  Reduced tumor growth in postpartum mice using PD-1 targeted therapies.

  •  Reduced lymphatic vessel frequency using PD-1 therapies.

  • ECM, extracellular matrix; IF, immunofluorescence; IHC, immunohistochemistry; INV1, involution day 1; LN, lymph node; MMP, matrix-metalloproteinase; MS, mass spectrometry; np, nulliparous; PDPN, podoplanin; PPBC, postpartum breast cancer.