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We sought to identify tumor-secreted factors that altered the frequency of MDSCs and correlated with clinical outcomes in advanced melanoma patients. We focused our study on several of the many factors involved in the expansion and mobilization of MDSCs. These were identified by measuring circulating concentrations of 13 cytokines and growth factors in stage IV melanoma patients (n = 55) and healthy controls (n = 22). Based on these results, we hypothesized that IL-6 and IL-8 produced by melanoma tumor cells participate in the expansion and recruitment of MDSCs and together would be predictive of overall survival in melanoma patients. We then compared the expression of IL-6 and IL-8 in melanoma tumors to the corresponding plasma concentrations and the frequency of circulating MDSCs. These measures were correlated with clinical outcomes. Patients with high plasma concentrations of either IL-6 (40%) or IL-8 (63%), or both (35%) had worse median overall survival compared to patients with low concentrations. Patients with low peripheral concentrations and low tumoral expression of IL-6 and IL-8 showed decreased frequencies of circulating MDSCs, and patients with low frequencies of MDSCs had better overall survival. We have previously shown that IL-6 is capable of expanding MDSCs, and here we show that MDSCs are chemoattracted to IL-8. Multivariate analysis demonstrated an increased risk of death for subjects with both high IL-6 and IL-8 (HR 3.059) and high MDSCs (HR 4.265). Together these results indicate an important role for IL-6 and IL-8 in melanoma patients in which IL-6 potentially expands peripheral MDSCs and IL-8 recruits these highly immunosuppressive cells to the tumor microenvironment. This study provides further support for identifying potential therapeutics targeting IL-6, IL-8, and MDSCs to improve melanoma treatments.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid cells that are increased in the peripheral blood of cancer patients and limit productive immune responses against tumors. Immunosuppressive MDSCs are well characterized in murine splenic tissue and are found at higher frequencies in spleens of tumor-bearing mice. However, no studies have yet analyzed these cells in parallel human spleens. We hypothesized that MDSCs would be increased in the spleens of human cancer patients, similar to tumor-bearing mice. We compared the frequency and function of MDSC subsets in dissociated human spleen from 16 patients with benign pancreatic cysts and 26 patients with a variety of cancers. We found that total MDSCs (Linneg CD11bpos CD33pos HLA-DRneg), granulocytic MDSCs (additional markers CD14neg CD15pos), and monocytic MDSCs (CD14pos CD15neg) were identified in human spleen. The monocytic subset was the most prominent in both spleen and peripheral blood and the granulocytic subset was expanded in the spleen relative to matched peripheral blood samples. Importantly, the frequency of CD15pos MDSCs in the spleen was increased in patients with cancer compared to patients with benign pancreatic cysts and was associated with a significantly increased risk of death and decreased overall survival. Finally, MDSCs isolated from the spleen suppressed T cell responses, demonstrating for the first time the functional capacity of human splenic MDSCs. These data suggest that the human spleen is a potential source of large quantities of cells with immunosuppressive function for future characterization and in-depth studies of human MDSCs.

Solid allograft transplantation can be lifesaving at the point of organ failure. However, long-term allograft and patient survival depends on sustained drug-induced immunosuppression. Acute cellular rejection (ACR) occurs in around 25% of heart transplant recipients in the first-year posttransplant despite optimal levels of currently available immunosuppressive therapy, accounting for 10% of mortality in the

Cardiovascular diseases (CVD) are a leading cause of human death worldwide. Over the past two decades, the emerging field of cardioimmunology has demonstrated how cells of the immune system play vital roles in the pathogenesis of CVD. MicroRNAs (miRNAs) are critical regulators of cellular identity and function. Cell-intrinsic, as well as cell-extrinsic, roles of immune and inflammatory cell derived miRNAs have been, and continue to be, extensively studied. Several ‘’immuno-miRNAs’’ appear to be specifically expressed or demonstrate greatly enriched expression within leucocytes. Identification of miRNAs as critical regulators of immune system signalling pathways has posed the question of whether and how targeting these molecules therapeutically, may afford opportunities for disease treatment and/or management. As the field of cardioimmunology rapidly continues to advance, this review discusses findings from recent human and murine studies which contribute to our understanding of how leucocytes of innate and adaptive immunity are regulated—and may also regulate other cell types, via the actions of the microRNAs they express, in the context of CVD. Finally, we focus on available information regarding microRNA regulation of regulatory T cells (TREGS) and argue that targeted manipulation of microRNA regulated pathways in these cells may hold therapeutic promise for the treatment of CVD and associated risk factors.

Introduction: Cell therapy using unselected mononuclear cell populations has had modest benefits in patients with critical limb ischemia (CLI). We hypothesized that tissue-remodeling monocytes, identified by their expression of CD16 (CD16+ Mo), may be a novel cellular therapy for CLI. Methods and Results: Flow cytometry showed that the proportion of circulating CD16+ Mo was greater in CLI patients (n=25) compared with matched controls (n=15, P<0.0001). Removal of ischemia following revascularization or amputation resulted in a fall in CD16+ Mo to control levels (P<0.05). CLI CD16+ Mo expressed higher levels of the adhesive proteins VLA, ICAM-1 and CD11c compared with controls (P<0.05). Conditioned media from these cells contained higher levels of HB-EGF, PlGF, endoglin, VEGF-C and VEGF-D (P<0.05) and induced greater endothelial cell tubule formation (P<0.05) compared with CD16- Mo from the same patients (n=9). CD16+ Mo preferentially migrated towards ischemic muscle supernatants isolated from CLI patients (n=7, P<0.02). CD16+ and CD16- Mo were isolated from 12 CLI patients and 1x10 6 cells injected into the adductor muscles of nude athymic mice following femoral artery excision. More ischemic hindlimbs were salvaged when treated with CD16+ compared with CD16- Mo (83% [10/12] vs 17% [2/12] limbs, P<0.05) and this was associated with enhanced arteriogenesis (αSMA-stained vessels, P<0.05). Conclusion: Circulating CD16+ Mo from CLI patients have increased expression of adhesion markers, are preferentially retained within ischemic muscle and promote robust arteriogenesis and limb salvage in experimental HLI. This monocyte subset may be an effective cellular therapy for CLI.

Women diagnosed with breast cancer within five years postpartum have worse prognosis than age-matched nulliparous women, independent of tumor stage1,2. While lymph node involvement, lymphatic vessel presence at the tumor margin, and invasion of tumor cells into peritumor lymphatics are all poor prognostic factors for breast cancer patients1-3; it is unknown if lymphatic vessel invasion plays a role in the poor prognosis of postpartum breast cancer. To address this question, we utilized a cohort of young women's breast cancer cases balanced for patient age, tumor stage, and tumor biologic subtype. We observe increased lymph vessel density (LVD) and lymph vessel specific invasion (LSI) in the peritumor region of tumors from postpartum cases compared to nulliparous cases, consistent with a role for lymphatic vessel invasion in postpartum breast cancer metastasis. In addition, we observe increased LVD in normal mammary tissue from postpartum women suggesting that lymphangiogenesis is activated in the postpartum gland. We hypothesize that expansion of the lymphatic vasculature occurs during normal postpartum mammary gland regression, known as postpartum involution, and that tumor cells exposed to the involution microenvironment similarly upregulate lymphangiogenesis, providing increased opportunity for metastasis. To test this hypothesis, we measured LVD in normal rodent mammary tissues and observed a significant increase in LVD during postpartum mammary gland involution. We then utilized our mouse xenograft model of postpartum breast cancer to test whether postpartum tumors have increased LVD and LSI. In this model, postpartum tumors invade the surrounding mammary stroma and peripheral blood, and infiltrate the lung at increased frequency compared to nulliparous tumors4. Here, we also observe increased LVD and LSI in the peritumor region in the postpartum group. Furthermore, we utilized COX-2 inhibitors to show that COX-2 activity during involution is required for the increased LVD, LSI, and lung infiltration associated with postpartum tumors. COX-2 inhibitors also significantly reduced LVD in the normal gland during involution without interfering with morphological gland regression. Finally, tumor cells exposed to postpartum involution appear to be stably altered, as transplantation of postpartum tumor cells into nulliparous mice show elevated levels of tumor associated lymphatics. We conclude that tumor cells exploit the normal lymphangiogenesis programs of postpartum involution to persistently upregulate lymphangiogenesis, which may explain the increased metastasis observed in postpartum tumors. This work was funded by DOD, ACS, Komen, and Cancer League of Colorado. References: 1. Schedin P. Pregnancy-associated breast cancer and metastasis. Nature reviews 2006;6:281-91. 2. Lyons TR, Schedin PJ, Borges VF. Pregnancy and breast cancer: when they collide. Journal of mammary gland biology and neoplasia 2009;14:87-98. 3. Jain RK, Padera TP. Prevention and treatment of lymphatic metastasis by antilymphangiogenic therapy. Journal of the National Cancer Institute 2002;94:785-7. 4. Lyons TR, O'Brien J, Borges VF, et al. Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2. Nat Med 2011;17:1109-15. Citation Format: Traci R. Lyons, Courtney Betts, Sonali Jindal, Puja Kapoor, Virginia Borges, Pepper Schedin. Postpartum mammary gland involution drives COX-2-dependent lymphangiogenesis and lymphatic vessel invasion in a model of postpartum breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B79.

Although tumor cell dissemination via the lymphatic vasculature is thought to be a common pathway of metastasis for solid human cancers, the mechanisms of lymphatic mediated metastasis remain poorly understood1. For breast cancer patients, lymph node involvement remains a very important indicator of patient prognosis and is utilized clinically for therapeutic choices2,3. A recent study has shown that the incidence of being diagnosed with stage IV breast cancer, i.e. breast cancer that has spread outside the breast, is increasing in young women4. Furthermore, young women's breast cancers diagnosed within 5 years postpartum are more likely to be metastatic5-7. Given that 35-45% of young women's breast cancers are likely to be diagnosed within 5 years of giving birth, we predict that postpartum cases may be driving the observed increase in breast cancers with distant involvement in young women. Here, we hypothesized that the postpartum period promotes lymphangiogenesis, which leads to breast tumor metastasis. To test this hypothesis we have generated xenograft and isograft mouse models of postpartum breast cancer that show increased metastasis in postpartum animals8,9. We utilized these rodent models and a cohort of young women's breast tissues to investigate lymphatic mediated metastasis in the postpartum period. In our rodent models, we show that 1) lymphangiogenesis is enhanced during normal postpartum involution, 2) tumor cells can utilize the lymphatic vasculature to escape the mammary gland during postpartum involution, 3) postpartum tumors display increased lymphatic vessel density and lymphatic vessel invasion in the tumor periphery, and 4) that postpartum tumor cells promote lymphangiogenesis and express high levels of pro-lymphangiogenic molecules VEGF-C and Sem7a ex vivo. Importantly, we show that both normal and tumor associated lymphangiogenesis in vivo are dependent upon COX-2 and that COX-2 inhibitors can block metastasis of postpartum tumors. In our young women's breast cancer cohort we also report increased mammary lymphangiogenesis tissues from women within one year of childbirth, increased lymphatic vessel density at the tumor periphery of postpartum breast cancers, as well as a positive correlation between lymphatic vessel density and lymphatic vessel invasion. Thus, we suggest that lymphatic mediated metastasis in young women may be decreased by COX-2 inhibitor or NSAID use. 1. Stacker, S.A., Baldwin, M.E. & Achen, M.G. The role of tumor lymphangiogenesis in metastatic spread. Faseb J 16, 922-934 (2002). 2. Alitalo, K. & Carmeliet, P. Molecular mechanisms of lymphangiogenesis in health and disease. Cancer cell 1, 219-227 (2002). 3. Pepper, M.S., Tille, J.C., Nisato, R. & Skobe, M. Lymphangiogenesis and tumor metastasis. Cell Tissue Res 314, 167-177 (2003). 4. Johnson, R.H., Chien, F.L. & Bleyer, A. Incidence of breast cancer with distant involvement among women in the United States, 1976 to 2009. Jama 309, 800-805 (2013). 5. Callihan, E.B., et al. Postpartum diagnosis demonstrates a high risk for metastasis and merits an expanded definition of pregnancy-associated breast cancer. Breast Cancer Res Treat (2013). 6. Johansson, A.L., Andersson, T.M., Hsieh, C.C., Cnattingius, S. & Lambe, M. Increased Mortality in Women with Breast Cancer Detected during Pregnancy and Different Periods Postpartum. Cancer Epidemiol Biomarkers Prev 20, 1865-1872 (2011). 7. Stensheim, H., Moller, B., van Dijk, T. & Fossa, S.D. Cause-specific survival for women diagnosed with cancer during pregnancy or lactation: a registry-based cohort study. J Clin Oncol 27, 45-51 (2009). 8. McDaniel, S.M., et al. Remodeling of the mammary microenvironment after lactation promotes breast tumor cell metastasis. The American journal of pathology 168, 608-620 (2006). 9. Lyons, T.R., et al. Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2. Nat Med 17, 1109-1115 (2011). Citation Format: Traci R. Lyons, Virginia F. Borges, Courtney B. Betts, Puja Kapoor, Holly A. Martinson, Sonali Jindal, Pepper Schedin. Postpartum mammary gland involution promotes COX-2 dependent tumor cell invasion of lymphatics. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B099.