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Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by bone marrow (BM) failure and associated with aging. We have previously shown that reversal of BM microenvironment (BMME) dysfunction in MDS mitigates MDS associated marrow failure and delays progression to acute leukemia. However, the exact mechanisms driving BMME dysfunction in MDS remain unknown. We recently reported that interleukin-1 (IL1) Receptor Type1 (IL1R1) signaling is a driver in myeloid bias via disruption of BMME in aging. In addition, we have found that IL1R1 signaling is involved in disease progression of AML. Therefore, to assess the role of IL1R1 signaling in MDS associated BMME dysfunction and marrow failure, we employed an age appropriate murine transplant model for MDS utilizing NUP98-HOXD13 (NHD13) transgenic mice. Methods: BM cells (NHD13 transgenic or wild type (WT), 7 weeks) and competitor cells were transplanted into irradiated aged recipients (WT or IL1R1 KO, 60 weeks), and subsequently monitored for development of marrow failure. When marrow failure developed, mice were euthanized and peripheral blood, BM, BM extracellular fluid (BMEF), and collagenase-1 digested bone associated cells were analyzed including flow cytometry, colony forming units (CFU) assay, and cytokine analyses. Next, BM from NHD13 (8-10 weeks) and competitor cells were transplanted into lethally irradiated aged recipients (WT, 50-60 weeks). At onset of marrow failure, mice were treated with inhibitors of IL1/Toll-like receptor signaling (IL1R antagonist, MCC950, or IL1R-associated kinase 4 protein (IRAK4) inhibitor) for fourteen days, and then euthanized and analyzed as above. Finally, we evaluated cytokine profile in the BM serum from the patients with MDS and normal donors. Results: Transplant of NHD13 BM cells into aged IL1R1 wt recipients (NHD13→IL1R1 wt) was not associated with a significant difference in survival rates or levels of NHD13 engraftment compared to NHD13 into IL1R1 ko recipients (NHD13→IL1R1 ko). IL1R1 wt developed macrocytic anemia compared to IL1R1 ko recipient (Hb 11.3±0.57 v.s 13.1±0.42 g/dL, n=12 and 9, p Conclusions: Collectively, our findings demonstrate that IL1R1 signaling alters the BMME and contributes to the disease phenotype of MDS and that the effects of targeting IL1R1 pharmacologically have differing effects based on the modality of inhibition as well as the cell population. IL1R1 signaling can be a promising target to alleviate the complexity of MDS via improving inflammatory status in BMME. Disclosures No relevant conflicts of interest to declare.

Targeted irradiation (TR) is widely used for tumor treatment in the clinic. TR benefits tumor therapy through direct effects as well as poorly understood systemic (abscopal) effects. Recent studies suggest that the systemic innate and acquired immune responses to TR contribute to elimination of tumor cells, but also cause systemic inflammation with prolonged tissue injury that may result in secondary malignancies. To elucidate and eventually target the mechanisms underlying these systemic effects of TR, we utilized a murine model using the small animal radiation research platform (SARRP). To define the dynamics of cytokine production and immune responses after TR, we administered local irradiation to a single tibia of 6-8 week old C57BL/6 male mice using a single dose of 15 Gy. We analyzed bone marrow (BM) and BM extracellular fluid (BMEF) from both the irradiated (TR) and non-irradiated, contralateral (CONT) tibiae at 2, 6, 48 hours, 1 and 3 weeks post-TR, performing phenotypic (flow cytometry) and cytokine analyses. As a tumor-bearing model, we utilized 3-4 weeks old C57BL/6 mice injected with Rhabdomyosarcoma (RMS) in one hind limb, and treated with (1) one dose i.p injection of 1mg/Kg Vincristine (Vin) as chemotherapy model, (2) 4.8GyX5times fractionated TR to the tumor area and (3) combination (TR+Vin) therapy. Analysis of peripheral blood (PB), BM, BMEF was performed 3 weeks after the final TR dose (n = 5-13 mice/time point). We found that multiple inflammatory cytokines and chemokines, such as IL-1b, IL-18, CCL2, CCL3, CXCL2, CXCL9, CXCL10 were upregulated from very early phase (2hrs) up to 48hrs in BMEF of the radiated tibiae. Consistent with the dynamics of these cytokines, we observed influx of myeloid cells in both TR and CONT side and expansion of T cells peaking at 6hrs in BM. At the same time of these immune responses, Norepinephrine (NE) was elevated in BMEF even in CONT side. In the tumor-bearing model of RMS, fractionated TR eliminated the tumor while systemically expanding CD8+ cytotoxic T cells and reducing neutrophils. Vin alone did not eliminate the tumor and was associated with systemic decrease of lymphoid cells and expansion of neutrophils. In Vin+TR, tumor control and CD8+ cell expansion were restored, with normalization of neutrophils. These data suggest that TR in the setting of tumor differentially activates lymphoid and myeloid cells. Since recent studies showed catecholamine production from myeloid cells may augment cytokine production in the setting of infection, we hypothesized that BM myeloid cells respond to radiation-induced cell damage by producing catecholamines that trigger a systemic inflammatory response after TR. To test this hypothesis, we utilized standard long-term bone marrow cultures (LT-BM) that reproduce three-dimensional BM structures with myeloid-skewing in vitro, and irradiated them to look at inflammatory changes induced by radiation at 2, 6 and 24hrs. In this experimental model, 5Gy of radiation led to the elevation of NE along with the production of chemokines CCL2, CCL3, CXCL2, CXCL9 mostly peaking at 6hrs in the cell culture supernatants. In contrast, these responses could not be reproduced in spleen cultures, which also had a much lower baseline NE production compared to LT-BMs. These data indicate that radiation induced-chemokine elevations might come from myeloid cells stimulated by NE, independent of systemic innervation. To define the contribution of catecholamines to cytokine production in LT-BM, we directly stimulated culture-LT-BM with NE and Isoproterenol, a pan beta stimulant. While both agents showed similar effect and increased CXCL2, CXCL9, CCL2 and CCL3 at 6hrs, they decreased CXCL10 level, suggesting that catecholamine mostly stimulate myeloid cells but rather inhibit lymphoid activation through chemokine production. Together, these data show that local irradiation initiates global immune responses, and identify local BM production of NE as its potential trigger. Blocking local catecholamine production in the bone marrow could therefore be a positive adjuvant to TR in tumor treatment by inhibiting unfavorable effects of radiation, such as chronic inflammation with systemic increases of neutrophils, while facilitating expansion and recruitment of the cytotoxic T cells which play an essential beneficial role in tumor immunity. Disclosures No relevant conflicts of interest to declare.

Preclinical models of breast cancer have established mechanistic links between psychological stress and cancer progression. However, epidemiological evidence linking stress and cancer is equivocal. We tested the impact of stress exposure in female mice expressing the mouse mammary tumor virus polyoma middle-T antigen (MMTV-PyMT), a spontaneous model of mammary adenocarcinoma that mimics metastatic hormone receptor–positive human breast cancer development. MMTV-PyMT mice were socially isolated at 6 to 7 weeks of age during premalignant hyperplasia. To increase the potency of the stressor, singly housed mice were exposed to acute restraint stress (2 hours per day for 3 consecutive days) at 8 to 9 weeks of age during early carcinoma. Exposure to this dual stressor activated both major stress pathways, the sympathetic nervous system and hypothalamic-pituitary-adrenal axis throughout malignant transformation. Stressor exposure reduced mammary tumor burden in association with increased tumor cleaved caspase-3 expression, indicative of increased cell apoptosis. Stress exposure transiently increased tumor vascular endothelial growth factor and reduced tumor interleukin-6, but no other significant alterations in immune/inflammation-associated chemokines and cytokines or changes in myeloid cell populations were detected in tumors. No stress-induced change in second-harmonic generation-emitting collagen, indicative of a switch to a metastasis-promoting tumor extracellular matrix, was detected. Systemic indicators of slowed tumor progression included reduced myeloid-derived suppressor cell (MDSC) frequency in lung and spleen, and decreased transforming growth factor β (TGF-β) content in circulating exosomes, nanometer-sized particles associated with tumor progression. Chronic β-adrenergic receptor (β-AR) blockade with nadolol abrogated stress-induced alterations in tumor burden and cleaved caspase-3 expression, lung MDSC frequency, and exosomal TGF-β content. Despite the evidence for reduced tumor growth, metastatic lesions in the lung were not altered by stress exposure. Unexpectedly, β-blockade in nonstressed mice increased lung metastatic lesions and splenic MDSC frequency, suggesting that in MMTV-PyMT mice, β-AR activation also inhibits tumor progression in the absence of stress exposure. Together, these results suggest stress exposure can act through β-AR signaling to slow primary tumor growth in MMTV-PyMT mice.

The non-invasive, in vivo measurement of microvascular blood flow has the potential to enhance breast cancer therapy monitoring. Here, longitudinal blood flow of 4T1 murine breast cancer (N=125) under chemotherapy was quantified with diffuse correlation spectroscopy based on layer models. Six different treatment regimens involving doxorubicin, cyclophosphamide, and paclitaxel at clinically relevant doses were investigated. Treatments with cyclophosphamide increased blood flow as early as 3 days after administration, whereas paclitaxel induced a transient blood flow decrease at 1 day after administration. Early blood flow changes correlated strongly with the treatment outcome and distinguished treated from untreated mice individually for effective treatments.

A significant difference between a treated and a control group of mice with breast cancer were observed in hemodynamic parameters quantified with diffuse optics at day 3 after cyclophosphamide treatment.