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.

 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.

 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.

 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.