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Another important macrophage regulator during postpartum involution and breast cancer is COX-2.
Previous studies in breast cancer models have shown that COX-2 expression increases with cancer stage,
and its expression levels can indicate breast cancer progression, recurrence, and metastasis [137] . Recently,
COX-2 expressing tumor associated macrophages (TAMs) have been shown to promote the metastatic
potential of breast cancer cells via secretion of IL-6 and subsequent activation of AKT signaling in cancer
cells [138] . Further, expression of COX-2 in stromal TAMs results in upregulation of COX-2 in breast cancer
cells, thereby shifting polarization of local macrophages toward the M2 phenotype. In addition to its
association with tumor promotional CD163+ TAMs, COX-2 expression in the stroma is further associated
with increased collagen alignment in invasive breast cancer [139] . TAMs are known to associate with dense
regions of collagen in breast cancer in the same way M2 macrophages associate with fibrillar collagen during
involution. In the MMTV-PyMT model, macrophages associated with fibrillar collagen have been shown by
intravital imaging to migrate across collagen fibers, suggesting that one mechanism by which macrophages
promote metastasis is by supporting the migration of tumor cells across collagen networks [21,140] . SEMA7A,
COX-2, and collagen all represent important effectors of macrophage-mediated tumor cell growth, survival,
and metastasis. As macrophages are considered essential for successful metastasis, targeting the molecules
responsible for alternative macrophage activation, survival, and chemotaxis may be critical for the successful
treatment of metastatic disease.

ENDOTHELIAL VESSEL FORMATION
Blood and lymphatic vessels form two similar, yet distinct, organ systems that assemble into extensive
networks throughout the body to support development and survival. Blood vessels provide tissues with
oxygen and nutrients, while the primary functions of lymphatic vessels are immune cell trafficking and
removal of excess interstitial fluid from tissues. Blood and lymphatic vessels are lined with blood endothelial
cells and lymphatic endothelial cells, respectively, and are both surrounded by a thin layer of smooth muscle.
Some lymphatic vessels have unique “button-like” junctions that differ from the more continuous “zipper-
like” junctions of the blood vasculature and established lymphatic vessels. These specialized junctions are
covered with a flap that opens and closes to allow fluid and cells to pass without affecting vascular integrity.
Vascular networks are highly dynamic, expanding and retracting as tissues change in response to normal
developmental processes or pathologies. Indeed, a widely accepted hallmark of cancer is the ability of tumors
[59]
to induce angiogenesis, or the development of new blood vessels from existing vasculature . Tumors must
acquire pro-angiogenic abilities in order to grow beyond 1-2 mm 3[141] ; otherwise, the tumor will die by
necrosis or apoptosis [142,143] . In rat mammary tissues, we observe an overall net increase in lymphatic vessel
[16]
density (LVD) during involution when compared to a lactation timepoint . In contrast, blood vessel density
(BVD) drops dramatically after lactation, suggesting an initial period of regression before increasing in a
manner similar to LVD [144] . During involution, the highest vasculature densities peak at day 10, followed
by a slight decrease in the fully regressed gland [Figure 1]. This is consistent with published studies from
our group and others describing increased pro-angiogenic and pro-lymphatic signaling during postpartum
involution, and in postpartum tumors compared to non-postpartum controls [14,16,22,145] . Interestingly, fibrillar
collagen, COX-2, and SEMA7A all have established roles in endothelial vessel formation. Studies using
artificial collagen matrices have shown that collagen increases angiogenic responses from endothelial cells
by providing the support needed for sustained endothelial cell growth and the formation of endothelial
networks [146] . Angiogenesis can also be regulated by mechanical stiffness within the small microvessel
environment [147] , and breast tumors are often stiffer than neighboring normal tissues by up to 6-fold [84,148] . For
[149]
a more comprehensive review on collagen and angiogenesis, see Fang et al. . These studies demonstrate
how the ECM in the TME can modulate vessel formation and alter the tumor’s blood supply.

If a tumor outgrows its blood supply and loses its access to oxygen, it can become hypoxic. Under normal
oxygen content conditions, termed “normoxia,” the transcriptional regulator hypoxia-inducible factor-1