Background Tumor associated macrophages (TAMs) promote an immunosuppressive and protumor immune microenvironment reducing the efficacies of cancer immunotherapies. Altering TAM phenotypes from immunosuppressive to proinflammatory should stimulate TAMs directly and lymphocytes indirectly to attack cancer cells and potentially increase the efficacies of check point inhibitor therapies, like anti-CTLA4. Mannosylated Amine Dextrans (MADs) are synthetic ligands for CD206 that can carry small molecule payloads on degradable linkers to CD206 expressing cells, i.e. TAMs. Three MAD constructs were made carrying payloads of paclitaxel or one of two novel bisphosphonates and were evaluated in human GM-CSF macrophages for their abilities to durably alter macrophage phenotypes towards pro-inflammatory. The paclitaxel MAD was tested in the CT26 tumor model with or without anti-CTLA4. Methods >Human monocytes from 3 donors were differentiated to CD206+ macrophages in GM-CSF supplemented RPMI1640 with 10% FBS (fresh medium) for 5 days. >Medium was replaced with fresh media with varying concentrations of MAD + drug constructs or molar equivalents of free drugs or zoledronate and incubated for 24 hours. >Saline or MAD without payload were controls. >Drug containing media were replaced with fresh medium and incubated for 3 days (for durable responses) >Cells were assessed by flow cytometry (MFI) for viability, CD206, CD163, CD80, CD86, MHC1, MHC2, SIRPalpha, and PD1. >CT26 tumors implanted in Balb/c mice, treatment began when tumors were 100mm2. >Mice treated with MAN-Paclitaxel (127 micrograms/dose), free paclitaxel, saline, or MAD no payload twice/week for 5 doses. Tumor volumes measured with calipers were the output. Results Cell/FlowAssays >None of the constructs reduced macrophage viability significantly. >All three MAD-Drug constructs reduced expression of CD206 and CD163 (immunosuppression markers) and increased expression of CD80 and CD86 (pro-inflammatory markers). >PD1 expression increased. >MHC1 and MHC2 expression was not significantly changed. >Both MAD-bisphosphonates – but not free drugs or MAD-paclitaxel – significantly reduced expression of SIRPalpha (p<.0001). CT26 Studies >MAD-Paclitaxel and free paclitaxel were equally effective at tumor growth reduction. >MAD-Paclitaxel plus anti-CTLA4 reduced tumor growth by 76%. Anti-CTLA4 alone by 52%. Conclusions > All 3 MAD-drug constructs shifted macrophage phenotypes towards pro-inflammatory and more effectively the free drugs. > Both MAD-Bisphosphonated constructs significantly reduced expression of SIRPalpha (checkpoint) >MAD-paclitaxel and free paclitaxel increased the efficacy of anti-CTLA4, but >CD206 targeted delivery can avoid off target toxicities of paclitaxel. Shifting TAM phenotypes towards a pro-inflammatory state in a targeted fashion has the potential to increase the efficacies of other immunotherapies to reduce tumor burdens. Acknowledgements Macrophage cell culture assays were performed at Discovery Life Sciences (Huntsville, AL). CT26/Balb/c studies were performed by Charles River Laboratories (North Carolina). Ethics Approval All studies performed at Charles River Laboratories were reviewed and approved by an institutional IACUC committee.

89 Objectives: Activated macrophages are a critical component of the inflammatory etiology of RA due to prolonged RA joint inflammation and destruction through the release of pro-inflammatory cytokines and chemokines. At present, there are no reliable means to non-invasively monitor disease activity in these patients. TCT is a radiopharmaceutical imaging agent that binds to the macrophage mannose receptor CD206 that reside on macrophages with high affinity. Previous clinical studies demonstrated the safety and tolerability of TCT in the subcutaneous (SC) route of administration. This Phase I/Phase II study of IV administered TCT was completed in RA subjects and contrasting healthy controls (HC) presenting initial safety and tolerability findings, as well as a powered determination of an optimal clinical dose and timepoint for imaging. Methods: A total of 39 subjects (33 active RA/6 HC) divided into 11 groups received various combinations of IV Tc 99m at 1, 5 and 10 mCi with 50, 200, or 400 µg of tilmanocept (see Figure 1) and had standard gamma camera whole- body planar imaging as well as AP spot view of the hands and wrists at 1 and 3 hours post-injection. Twelve of the 39 subjects (6 RA/6 HC) underwent whole-body planar scans for radiodosimetry assessment and pharmacokinetic analysis. An independent panel of 3 blinded readers performed a visual assessment of joint-space uptake for determination of inter-reader agreement. Quantitative assessment of count activity in individual joint spaces of the hands and wrists was extracted from each subject by placing separate ROI on 1 and 3-hour planar anterior images and used to model the optimal timepoint and dose for detecting TCT uptake in involved RA joints. Results: No adverse drug reactions (ADRs) or serious adverse events (SAEs) were observed in any dose group. Planar imaging revealed that TCT localizes specifically to inflamed joints in RA subjects when compared to HC subjects (see Figure 2). Qualitative and quantitative data analysis and modeling revealed an optimal mass dose of 134 µg tilmanocept/10 mCi of Tc 99m stable over an imaging time frame of 1-3 hours post-injection. Conclusions: IV administration of TCT was well-tolerated and demonstrated joint-specific localization in RA subjects, revealing potentially significant immunodiagnostic information about CD206-expressing synovial macrophage involvement in RA patients. Additionally, increased TCT activity via IV administration may enhance localization and anatomic delineation in tilmanocept-positive joints of RA patients compared to SC administration studied in a prior Phase I trial. Study data analysis revealed an optimal mass dose of 134 µg of tilmanocept radiolabeled with 10 mCi Tc 99m with an imaging time frame of 1-3 hours post injection. For the purposes of accuracy and streamlining practice, a mass dose of 150 µg and imaging time frame 1-3 hours post-injection is recommended for future use. These results provide the foundation for a non-invasive method to monitor disease activity in macrophage driven inflamed joints in patients with RA. Research Support: Supported by a grant from the National Inst Arthritis & Musculoskeletal & Skin Diseases/NIH, Grant R44AR067583 ClinicalTrials.gov Identifier: NCT02683421.

The paucity of cell culture models for childhood brain tumors prompted us to establish pediatric cell lines for use in biological experiments and preclinical developmental therapeutic studies. Three cell lines were established, CHLA-200 (GBM), CHLA-259 (anaplastic medulloblastoma) and CHLA-266 (atypical teratoid rhabdoid tumor, AT/RT). Consistent with an AT/RT origin, CHLA-266 lacked INI1 expression and had monosomy 22. All lines had unique DNA short tandem repeat "fingerprints" matching that of the patient's tumor tissue and were adherent on tissue culture plastic, but differed in morphology and doubling times. CHLA-200 had a silent mutation in TP53. CHLA-259 and CHLA-266 had wild-type TP53. All three lines were relatively resistant to multiple drugs when compared to the DAOY medulloblastoma cell line, using the DIMSCAN fluorescence digital image microscopy cytotoxicity assay. RNA expression of MYC and MYCN were quantified using RT-PCR (Taqman). CHLA-200 expressed MYC, DAOY and CHLA-259 expressed MYCN, and CHLA-266 expressed both MYCN and MYC. CHLA-200 was only tumorigenic subcutaneously, but CHLA-259 and CHLA-266 were tumorigenic both subcutaneously and in brains of NOD/SCID mice. Immunohistochemistry of the xenografts revealed GFAP staining in CHLA-200 and PGP 9.5 staining in CHLA-259 and CHLA-266 tumors. As expected, INI1 expression was lacking in CHLA-266 (AT/RT). These three new cell lines will provide useful models for research of pediatric brain tumors.

Background: Suppression of physiologic myocardial sequestration of glucose, and hence the 2-deoxy-18 fluorodeoxyglucose (FDG) is of critical importance to effectively evaluate intrinsic cardiac pathology and better delineate extra- cardiac FDG activity on Positron Emission Tomography (PET) imaging. In a rodent model, we studied the effect of duration of fasting with or without high fat diet (HFD) consumption on myocardial FDG uptake. Methods: 9 Sprague- Dawley rats underwent four different preparation protocols before obtaining micro PET imaging: Non-fasting (NF), 18-hrs/Prolonged fasting (PF), 12-hrs/Short fasting followed by High Fat Diet (SF-HFD) and 18-hrs/Prolonged fasting followed by High Fat Diet (PF-HFD). Region of interest were drawn on the myocardium (heart) and ascending aorta (blood pool) to generate maximum standard uptake values (SUVm) for the heart (H-SUVm) and blood pool (BP- SUVm). Results: PF-HFD and SF-HFD preparation protocols resulted in significantly lower H-SUVm as compared to PF and NF protocols with H-SUVm of 1.49, 1.56, 4.38 and 10.19 respectively. Conclusion: PF-HFD and SF-HFD preparation protocols provide superior suppression of myocardial FDG uptake in comparison to PF and NF protocols. These findings offer an approach to study intrinsic cardiac disorders (vascular, infiltrative etc) and also provide better visualization of extra-cardiac pathologic disorders.

Abstract Abstract 293 The serine/threonine Pim kinases are known to play an important role in several signal transduction pathways, including those regulated by c-Myc, N-Myc, FLT3-ITD, BCR-ABL, HOXA9, and EWS fusions. Pim kinases are up regulated in some hematologic malignancies such as Acute Myeloid Leukemia (AML) and Chronic Lymphocytic Leukemia. Pim kinases were first identified as a proviral integration site in the mice that overexpress c-Myc and enhance lymphomagenesis. We have previously shown that SMI-4a, a novel benzylidene-thiazolidine-2,4-dione small molecule inhibitor of the Pim kinases inhibited the growth of precursor T-cell lymphoblastic leukemia in the mouse (Lin, YW. et al. Blood, 2010). We have also reported that SMI-4a in combination with a few MEK inhibitors or standard chemotherapeutic agents including daunorubicin, Ara-C, and 6-thioguanine synergistically killed myeloid and lymphoblastic leukemic cell lines as well as primary patient leukemic blasts (Lin, YW. et al. Blood, 2010, AACR 2010 abstract). In the current study, we found that the combination of Rapamycin and Pim kinase inhibitors including SMI-4a, SMI-20a, SMI-24a, and K00135 synergistically killed MV4-11, a myeloid leukemia cell line harboring FLT3-ITD. We also show that a combination of SMI-4a and Rapamycin significantly inhibited subcutaneous tumor growth of MV4-11 expressing firefly-luciferase in NOD/SCID mice, which was determined using conventional caliper measurement and bioluminescent analysis in the region of interest. Although the combination of those drugs caused a loss of appetite in NOD/SCID mice in the first week of the treatment, the mice became tolerable and did not lose weight from the second week. In addition, none of those drugs alone or in combination caused any adverse affects in wild type FVB mice. A combination of SMI-4a and Rapamycin also synergistically kills primary AML blasts including patients with or without expression of the FLT3-ITD. The treatment with SMI-4a or Rapamycin as a single agent down regulates phosphorylation of two substrates of the mTORC1 pathway, 4E-BP1 and S6K, while the combination significantly decreased their phosphorylation compared with single agent treatments, which is consistent with the synergistic effect. We found that SMI-4a increased phosphorylation of AMPK and decreased phosphorylation of mTOR at serine 2448 leading to down regulation of the mTORC1 pathway. In contrast, Rapamycin induced down regulation of 4E-BP1 and S6K without affecting phosphorylation of AMPK and mTOR. SMI-4a induced apoptotic cell death that was characterized by down regulation of MCL1, cleavage of Caspase 3, and nuclear condensation, whereas Rapamycin did not induce these changes. Together those two inhibitors modify different signaling pathways to synergistically kill AML blasts in vitro and in vivo. Disclosures: Tholanikunnel: Vortex Biotechnology: Employment. Kraft:Vortex Biotechnology: Consultancy.

Attempts to reduce the toxicity of hematopoietic stem cell transplantation have led to the use of various immunosuppressive, yet nonmyeloablative preparative regimens that often include low-dose irradiation. To determine the effects of low-dose irradiation on the dynamics of donor cell engraftment after bone marrow transplantation (BMT), we coupled standard endpoint flow cytometric analysis with in vivo longitudinal bioluminescence imaging performed throughout the early (<10 days) and late (days 10-90) post-BMT periods. To exclude the contribution of irradiation on reducing immunologic rejection, severely immune-deficient mice were chosen as recipients of allogeneic bone marrow. Flow cytometric analysis showed that sublethal doses of total body irradiation (TBI) significantly increased long-term (14 weeks) donor chimerism in the bone marrow compared with nonirradiated recipients (P < .05). Bioluminescence imaging demonstrated that the effect of TBI (P < .001) on chimerism occurred only after the first 7 days post-BMT. Flow cytometric analysis on day 3 showed no increase in the number of donor cells in irradiated bone marrow, confirming that sublethal irradiation does not enhance marrow chimerism early after transplantation. Local irradiation also significantly increased late (but not early) donor chimerism in the irradiated limb. Intrafemoral injection of donor cells provided efficient early chimerism in the injected limb, but long-term systemic donor chimerism was highest with i.v. administration (P < .05). Overall, the combination of TBI and i.v. administration of donor cells provided the highest levels of long-term donor chimerism in the marrow space. These findings suggest that the major effect of sublethal irradiation is to enhance long-term donor chimerism by inducing proliferative signals after the initial phase of homing.

Objective. To test the hypothesis that hematopoietic stem cells (HSCs) generate bone cells using bone marrow (BM) cell transplantation in a mouse model of osteogenesis imperfecta (OI). OI is a genetic disorder resulting from abnormal amount and/or structure of type I collagen and is characterized by osteopenia, fragile bones, and skeletal deformities. Homozygous OI murine mice (oim; B6C3Fe a/a-Col1a2(oim)/J) offer excellent recipients for transplantation of normal HSCs, because fast turnover of osteoprogenitors has been shown.Materials and Methods. We transplanted BM mononuclear cells or 50 BM cells highly enriched for HSCs from transgenic enhanced green fluorescent protein mice into irradiated oim mice and analyzed changes in bone parameters using longitudinal microcomputed tomography.Results. Dramatic improvements were observed in three-dimensional microcomputed tomography images of these bones 3 to 6 months post-transplantation when the mice showed high levels of hematopoietic engraftment. Histomorphometric assessment of the bone parameters, such as trabecular structure and cortical width, supported observations from three-dimensional images. There was an increase in bone volume, trabecular number, and trabecular thickness with a concomitant decrease in trabecular spacing. Analysis of a nonengrafted mouse or a mouse that was transplanted with BM cells from oim mice showed continued deterioration in the bone parameters. The engrafted mice gained weight and became less prone to spontaneous fractures while the control mice worsened clinically and eventually developed kyphosis.Conclusions. These findings strongly support the concept that HSCs generate bone cells. Furthermore, they are consistent with observations from clinical transplantation studies and suggest therapeutic potentials of HSCs in OI. Published by Elsevier Inc. on behalf of the ISEH - Society for Hematology and Stem Cells.

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The aim of this study was to develop and use in vivo bioluminescent imaging to track luciferase-labeled acute myelogenous leukemia (AML) stem cells in immunedeficient mice using AML blood samples collected from patients and subsequently frozen. The ability to utilize frozen pools as viable reservoirs for AML stem cells will allow the freezing back of samples from the clinic for later use as well as the use of previously frozen samples for engraftment and therapeutic studies. Using thawed, isolated cells, we assessed AML stem cell engraftment and quantitatively compared it to fresh AML stem cell engraftment in a xenogeneic transplant model using in vivo bioluminescent imaging. Frozen AML stem cell engraftment was also compared to both fresh and frozen normal human hematopoietic stem cell (HSC) engraftment. Human hematopoietic stem cells (CD34+CD38-) were isolated from both normal frozen cord blood and from peripheral blood of AML patients using modified Miltenyi Biotec (Auburn, CA) magnetic bead isolation kits. Isolated cells were transduced with a lentiviral vector (HIV-luc) pseudotyped with the VSV envelope able to express the firefly luciferase (fluc) gene in human HSC. Using a Caliper Life Sciences/Xenogen IVIS 200 optical imaging system, cells were assayed for fluc expression 48 hours after transduction. Transduced HSC and AML cells (6 × 104 each) were transplanted intravenously into sublethally irradiated adult NOD/SCID/IL2Rγnull mice and mice were imaged serially from day 1 up to day 160 post transplantation. Observed bioluminescent signal and patterns from thawed AML stem cell engraftment are similar to our results obtained using fresh AML stem cells in mice. Luminescent signal from peripheral blood and spleen engraftment of frozen AML stem cells was detectable within one day post-transplantation. Both fresh and frozen AML stem cell populations exhibited diffuse whole body signal, indicating luciferase-labeled stem cells in the peripheral blood, similar to a hallmark of the disease in humans. The presence of blast cells consistent with AML disease was observed by blood smears taken from mice engrafted with frozen AML stem cells. To our knowledge, we have demonstrated for the first time that frozen AML stem cells can be efficiently labeled with a luciferase enzyme for dynamic tracking in vivo. This work has resulted in a useful animal model that will enable us to elucidate the engraftment patterns of AML cells in living animals, learn the dynamics of the interplay of different stem cell populations, and provide information on the efficacy of new treatments in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4334.

Acute Tubular Necrosis (ATN) causes severe damage to the kidney epithelial tubular cells and is often associated with severe renal dysfunction. Stem-cell based therapies may provide alternative approaches to treating of ATN. We have previously shown that clonal c-kit(pos) stem cells, derived from human amniotic fluid (hAFSC) can be induced to a renal fate in an ex-vivo system. Herein, we show for the first time the successful therapeutic application of hAFSC in a mouse model with glycerol-induced rhabdomyolysis and ATN. When injected into the damaged kidney, luciferase-labeled hAFSC can be tracked using bioluminescence. Moreover, we show that hAFSC provide a protective effect, ameliorating ATN in the acute injury phase as reflected by decreased creatinine and BUN blood levels and by a decrease in the number of damaged tubules and apoptosis therein, as well as by promoting proliferation of tubular epithelial cells. We show significant immunomodulatory effects of hAFSC, over the course of ATN. We therefore speculate that AFSC could represent a novel source of stem cells that may function to modulate the kidney immune milieu in renal failure caused by ATN.

We used magnetic resonance spectroscopy to determine whether orthotopic mouse brain tumors grown as xenografts in immunocompromised mice either from human brain tumor cells implanted immediately after surgery or from cultured human tumor lines show metabolic profiles comparable to those of the original tumors. Using a 7 T scanner, spectra were acquired from mice with a human atypical teratoid/rhabdoid tumor (AT/RT) either implanted directly from the surgical specimen or first grown in culture, directly implanted choroid plexus carcinoma (CPC), and two medulloblastoma cell lines. The results were compared with spectra from these same tumors or tumor types in patients and with controls. Metabolic variability of tumors from a single cell line was also evaluated using the medulloblastoma lines. The main metabolic features of human tumors were qualitatively replicated in xenografts. AT/RTs in mice exhibited choline, creatine, and myo-inositol levels comparable to those observed in the patient. As in patients, choline was prominent in experimental CPC. Tumors from a single cell line were comparable. Significant correlations were found with key metabolites in humans and mice; however, differences including lower lipids in the implanted AT/RTs than in patient spectra and taurine observed in all animal spectra were also noted. The causes of these dissimilarities warrant further investigation.

Self-renewal capacity is rapidly lost during differentiation of hematopoietic stem cells to lineage-committed progenitors. We demonstrate here that regulated intracellular signaling through the cytokine receptor Mpl induces profound expansion of not only multipotent (ie, lymphomyeloid) but also lymphoid-committed human hematopoietic progenitors. A fusion protein containing the intracellular signaling domain of Mpl and a dimerization domain was constitutively expressed in populations enriched in human lymphomyeloid progenitor/stem cells (CD34(+)CD38(-)Lin(-)CD7(-)) and multilymphoid progenitors (CD34(+)CD38(-)Lin(-)CD7(+)). Intracellular dimerization of Mpl in target cells was induced by in vitro or in vivo administration of a diffusible synthetic ligand. In vitro, Mpl dimerization produced divisions of clonogenic, multilineage CD34(+) cells able to engraft immunodeficient mice. When dimerization was induced in vivo after transplantation of either lymphomyeloid or multilymphoid progenitors, donor-derived hematopoiesis was sustained for at least 12 weeks and primitive CD34(+)Lin(-) progenitors were expanded more than 1000-fold. Lineage potential of progenitors was not altered and differentiation was not prevented by synthetically induced Mpl signaling. These data demonstrate that dimerization of a single cytokine receptor can deliver a profound expansion signal in both uncommitted and lymphoid-committed human hematopoietic progenitors.

BACKGROUNDStudies in the elderly suggest a reciprocal relation between increased marrow adiposity and bone loss, supporting basic research data indicating that osteoblasts and adipocytes share a common progenitor cell. However, whether this relation represents a preferential differentiation of stromal cells from osteoblasts to adipocytes or whether a passive accumulation of fat as bone is lost and marrow space increases with aging is unknown. To address this question and avoid the confounding effect of bone loss, we examined teenagers and young adults.METHODSUsing computed tomography, we obtained measurements of bone density and cross-sectional area of the lumbar vertebral bodies and cortical bone area, cross-sectional area, marrow canal area, and fat density in the marrow of the femurs in 255 sexually mature subjects (126 females, 129 males; 15-24.9 yr of age). Additionally, values for total body fat were obtained with dual-energy x-ray absorptiometry.RESULTSRegardless of gender, reciprocal relations were found between fat density and measures of vertebral bone density and femoral cortical bone area (r = 0.19-0.39; all P values < or = .03). In contrast, there was no relation between marrow canal area and cortical bone area in the femurs, neither between fat density and the cross-sectional dimensions of the bones. We also found no relation between anthropometric or dual-energy x-ray absorptiometry fat values and measures for marrow fat density.CONCLUSIONSOur results indicate an inverse relation between bone marrow adiposity and the amount of bone in the axial and appendicular skeleton and support the notion of a common progenitor cell capable of mutually exclusive differentiation into the cell lineages responsible for bone and fat formation.