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Macrophage gating strategy and chemoattractant expression levels

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.

Semaphorin-7a (SEMA7A), best known as a neuroimmune molecule, plays a diverse role in many cellular processes and pathologies. Here, we show that SEMA7A promotes anoikis resistance in cultured mammary epithelial cells through integrins and activation of pro-survival kinase AKT, which led us to investigate a role for SEMA7A during postpartum mammary gland involution—a normal developmental process where cells die by anoikis. Our results reveal that SEMA7A is expressed on live mammary epithelial cells during involution, that SEMA7A expression is primarily observed in α6-integrin expressing cells, and that luminal progenitor cells, specifically, are decreased in mammary glands of SEMA7A−/− mice during involution. We further identify a SEMA7A-α6/β1-integrin dependent mechanism of mammosphere formation and chemoresistance in mammary epithelial cells and suggest that this mechanism is relevant for recurrence in breast cancer patients.

Postpartum mammary gland involution is a mammalian tissue remodeling event that occurs after pregnancy and lactation to return the gland to the pre-pregnant state. This event is characterized by apoptosis and lysosomal-mediated cell death of the majority of the lactational mammary epithelium, followed by remodeling of the extracellular matrix, influx of immune cell populations (in particular, T helper cells, monocytes, and macrophages), and neo-lymphangiogenesis. This postpartum environment has been shown to be promotional for tumor growth and metastases and may partially account for why women diagnosed with breast cancer during the postpartum period or within 5 years of last childbirth have an increased risk of developing metastases when compared to their nulliparous counterparts. The lymphatics and macrophages present during mammary gland involution have been implicated in promoting the observed growth and metastasis. Of importance are the macrophages, which are of the “M2” phenotype and are known to create a pro-tumor microenvironment. In this report, we describe a subset of postpartum macrophages that express lymphatic proteins (PoEMs) and directly interact with lymphatic vessels to form chimeric vessels or “macphatics”. Additionally, these PoEMs are very similar to tumor-associated macrophages that also express lymphatic proteins and are present at the sites of lymphatic vessels where tumors escape the tissue and enter the lymphatic vasculature. Further characterizing these PoEMs may offer insight in preventing lymphatic metastasis of breast cancer, as well as provide information for how developmental programming of lymphatic endothelial cells and macrophages can contribute to different disease progression.

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.

Breast cancer (BC) is the 2nd leading cause of cancer-related death in women. Increased lymphatic vessel density (LVD), lymphovascular invasion (LVI), and lymph node positivity (LN+) are associated with increased metastasis in BC patients. LVD, LVI, and LN+ are promoted by angiogenesis and lymphangiogenesis, which can be induced by tumor-associated macrophages (TAMs). TAMs are implicated in creating a pro-tumor tumor microenvironment (TME) in various cancers. We have shown that semaphorin7A (SEMA7A)—a signaling molecule that activates integrin-mediated signaling cascades—is associated with decreased survival and increased metastasis in BC patients. In mouse models, SEMA7A expression is associated with increased LVD and TAM presence. SEMA7A can also induce expression of lymphatic endothelial cell (LEC)-associated proteins, such as podoplanin (PDPN), on macrophages to polarize them into “PoEMs” (PDPN-expressing macrophages). Furthermore, PoEMs intercalate into lymphatic vessels to form PoEM-LEC chimeric vessels; tumor cells are found within these vessels at the PoEM-LEC junctions. We have also shown that SEMA7A promotes programmed death ligand-1 (PD-L1) expression on TAMs, LECs, PoEMs, and BC cells to inhibit anti-tumor immune responses and promote a pro-tumor TME. In neuronal cells, SEMA7A expression is regulated by steroid hormones in a B1-integrin mediated manner. Given the link between SEMA7A and hormones, we hypothesized that SEMA7A expression in tumors could be induced by estrogen (E2) and that SEMA7A and E2 may cooperate to create a pro-tumor immune TME (iTME). To test this, we orthotopically implanted E0771-DDK (Ctrl) and E0771-DDK-SEMA7A (SEMA7A overexpressing [OE]) cells into female mice with or without E2 supplementation. E0771 cells have recently been characterized as the luminal B subtype—which is ER+ and highly proliferative—and E0771 cells are dependent upon E2 supplementation for growth in vivo. SEMA7A OE tumors without E2 overcame the need for E2 and grew as well as the Ctrl+E2 tumors, but SEMA7A+E2 significantly accelerated tumor growth. Tumors were harvested for flow cytometry (FC) to reveal that E2 increased SEMA7A expression in Ctrl tumors to similar levels observed in the SEMA7A OE and SEMA7A+E2 significantly increased expression of PD-L1 expression on tumor cells. Additionally, SEMA7A+E2 was the only condition to increase the presence of intratumoral LECs; however, E2, SEMA7A, and SEMA7A+E2 all induced PD-L1 expression on LECs. Additionally, SEMA7A and SEMA7A+E2 groups had more TAMs and PoEMs compared to controls, and significantly more of those TAMs/PoEMs were PD-L1+. Lastly, we treated Ctrl and SEMA7A OE tumors (in the presence of E2) with anti-PD1 or anti-PD-L1 therapies and only the SEMA7A OE tumors responded. These results indicate that E2 can induce SEMA7A expression in tumors and that SEMA7A can cooperate with E2 to inhibit anti-tumor immunity. Citation Format: Alan Elder, Lyndsey Crump, Alexander Stoller, Traci Lyons. Semaphorin7A and estrogen work in concert to promote mammary tumor growth and alter the immune tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2791.

Post-partum breast cancer patients, or breast cancer patients diagnosed within 10 years of last childbirth, are ~3–5 times more likely to develop metastasis in comparison to non-post-partum, or nulliparous, patients. Additionally, post-partum patients have increased tumor-associated lymphatic vessels and LN involvement, including when controlled for size of the primary tumor. In pre-clinical, immune-competent, mouse mammary tumor models of post-partum breast cancer (PPBC), tumor growth and lymphogenous tumor cell spread occur more rapidly in post-partum hosts. Here we report on PD-L1 expression by lymphatic endothelial cells and CD11b+ cells in the microenvironment of post-partum tumors, which is accompanied by an increase in PD-1 expression by T cells. Additionally, we observed increases in PD-L1 and PD-1 in whole mammary tissues during post-partum mammary gland involution; a known driver of post-partum tumor growth, invasion, and metastasis in pre-clinical models. Importantly, implantation of murine mammary tumor cells during post-partum mammary gland involution elicits a CD8+ T cell population that expresses both the co-inhibitory receptors PD-1 and Lag-3. However, upon anti-PD-1 treatment, during post-partum mammary gland involution, the involution-initiated promotional effects on tumor growth are reversed and the PD-1, Lag-3 double positive population disappears. Consequently, we observed an expansion of poly-functional CD8+ T cells that produced both IFNγ and TNFα. Finally, lymphatic vessel frequency decreased significantly following anti-PD-1 suggesting that anti-PD-1/PD-L1 targeted therapies may have efficacy in reducing tumor growth and dissemination in post-partum breast cancer patients.

Postpartum breast cancers are a highly metastatic subset of young women’s breast cancers defined as breast cancers diagnosed in the postpartum period or within 5 years of last child birth. Women diagnosed with postpartum breast cancer are nearly twice as likely to develop metastasis and to die from breast cancer when compared with nulliparous women. Additionally, epidemiological studies utilizing multiple cohorts also suggest that nearly half of all breast cancers in women aged

Despite new targeted therapies and improved screening, breast cancer is the second leading cause of cancer-related death of women in the United States. This emphasizes the need to better understand the molecular pathways contributing to breast cancer progression and metastasis and the need for identification of novel therapeutic targets. This is especially true for triple-negative breast cancers (TNBCs), which in comparison to hormone receptor-positive (ER+/PR+) and HER2+ subtypes lack a targeted therapy. Without targeted therapy TNBC patients have limited options for treatment, resulting in decreased overall survival. Our lab identified semaphorin 7a (SEMA7A) as a novel protein that promotes TNBC progression. SEMA7A is a member of the semaphorin family, a group of proteins originally described for their developmental roles in axon guidance. SEMA7A is a unique member of the semaphorin family as it is the only GPI-linked semaphorin and can be shed into the extracellular space. Since breast cancer patients mainly die from metastatic disease, we aim to uncover the role of SEMA7A in TNBC cell invasion and metastasis. To study the role SEMA7A may have in promoting cellular invasion, we utilized an shRNA-mediated knockdown of SEMA7A in human TNBC cell lines. Our results show that SEMA7A plays an important role in many steps of the metastatic cascade, with shSEMA7A cells showing increased cell death in both attached and force suspension conditions (anoikis), reduced mesenchymal phenotypes, and less invasion in 3D matrices. We also observed decreased growth and invasion of shSEMA7A tumors in vivo and decreased synthesis of fibronectin and matrix-metalloproteinase 2 ex vivo from shSEMA7A tumors. Taken together, these results show that SEMA7A is a critical molecule for cellular invasion by modulating not only intrinsic cellular pathways, but also the tumor microenvironment. Citation Format: Sarah Elizabeth Tarullo, Alan Elder, Ryan Hill, Kirk Hansen, Traci Lyons. Investigating the role of semaphorin 7a in triple-negative breast cancer cell invasion [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B55.

Infectious prions traverse epithelial barriers to gain access to the circulatory system, yet the temporal parameters of transepithelial transport and persistence in the blood over time remain unknown. We used whole-blood real-time quaking-induced conversion (wbRT-QuIC) to analyze whole blood collected from transmissible spongiform encephalopathy (TSE)-inoculated deer and hamsters throughout the incubation period for the presence of common prion protein-conversion competent amyloid (PrP C -CCA). We observed PrP C -CCA in the blood of TSE-inoculated hosts throughout the disease course from minutes postexposure to terminal disease.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, affecting human and animal species can be transmitted from TSE-infected individuals to naive susceptible hosts during the long asymptomatic (years to decades) and symptomatic disease stages. The presence of infectious hematogenous prions in asymptomatic TSE-infected hosts demonstrates the highly infectious nature of blood-borne prions in hosts lacking overt clinical symptoms. It is currently unknown when and how infectious prions first enter the blood following initial exposure. We have previously shown that the whole-blood real-time quaking-induced conversion assay (wbRT-QuIC) possesses 100% specificity and >92% sensitivity, making it an ideal tool to address this question. Here, we use wbRT-QuIC to analyze whole blood collected from oral, extranasal or aerosol TSE-exposed hosts for blood-borne prions. Our results demonstrate that conversion competent prions in the inoculum are capable of crossing mucosal surfaces and entering the circulatory system within 30 min—no matter the route of exposure. Detection of the inoculum minutes post exposure is followed by a steady decline in the detection of blood-borne prions up to 3 days which is followed by a progressive increase in the detection of nascent conversion competent prions between 1 and 17 months post exposure. These data provide the first evidence for the facile transport of mucosally acquired prions into the circulatory system, providing evidence for multiple routes of inter- and intra- host prion trafficking and shedding.

Blood-borne transmission of infectious prions during the symptomatic and asymptomatic stages of disease occurs for both human and animal transmissible spongiform encephalopathies (TSEs). The geographical distribution of the cervid TSE, chronic wasting disease (CWD), continues to spread across North America and the prospective number of individuals harboring an asymptomatic infection of human variant Creutzfeldt-Jakob Disease (vCJD) in the United Kingdom has been projected to be ~1 in 3000 residents. Thus, it is important to monitor cervid and human blood products to ensure herd health and human safety. Current methods for detecting blood-associated prions rely primarily upon bioassay in laboratory animals. While bioassay provides high sensitivity and specificity, it requires many months, animals, and it is costly. Here we report modification of the real time quaking-induced conversion (RT-QuIC) assay to detect blood-borne prions in whole blood from prion-infected preclinical white-tailed deer, muntjac deer, and Syrian hamsters, attaining sensitivity of >90% while maintaining 100% specificity. Our results indicate that RT-QuIC methodology as modified can provide consistent and reliable detection of blood-borne prions in preclinical and symptomatic stages of two animal TSEs, offering promise for prionemia detection in other species, including humans.