Lisa Koodie

Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells

We have derived from normal human, mouse, and rat postnatal bone marrow primitive, multipotent adult progenitor cells (MAPCs) that can differentiate into most mesodermal cells and neuroectodermal cells in vitro and into all embryonic lineages in vivo. Here, we show that MAPCs can also differentiate into hepatocyte-like cells in vitro. Human, mouse, and rat MAPCs, cultured on Matrigel with FGF-4 and HGF, differentiated into epithelioid cells that expressed hepatocyte nuclear factor-3beta (HNF-3beta), GATA4, cytokeratin 19 (CK19), transthyretin, and alpha-fetoprotein by day 7, and expressed CK18, HNF-4, and HNF-1alpha on days 14-28. Virtually all human, as well as a majority of rodent cells stained positive for albumin and CK18 on day 21; 5% (rodent) to 25% (human) cells were binucleated by day 21. These cells also acquired functional characteristics of hepatocytes: they secreted urea and albumin, had phenobarbital-inducible cytochrome p450, could take up LDL, and stored glycogen. MAPCs, which can be expanded in vitro and maintained in an undifferentiated state for more than 100 population doublings, can thus differentiate into cells with morphological, phenotypic, and functional characteristics of hepatocytes. MAPCs may therefore be an ideal cell for in vivo therapies for liver disorders or for use in bioartificial liver devices.

Modulation of Immune Function by Morphine: Implications for Susceptibility to Infection

The outcome of microbial infection in an organism is a dynamic process that depends on factors derived from both the microorganism and the host. In chronic human infections, the kind of immune response made in response to pathogens may be of vital importance to host defense. An inappropriate immune response may not only result in lack of protection, but even contribute to disease severity. Chronic morphine use and abuse has been documented to result in severe immune consequence (Roy and Loh 1996; Dinda et al. 2005; Friedman and Eisenstein 2004) and thus may pose a significant risk factor to opportunistic infection. It is therefore not surprising that epidemiological studies show increased prevalence of opportunistic infections such as tuberculosis, HIV infection, and pneumonia in opioid abusers (Quaglio et al. 2002; Nath et al. 2002; Georges et al. 1999). Besides the sharing of unsterilized, contaminated needles, the occurrence of infections in these patients has been attributed to the immune modulatory effect of morphine. In animal studies where confounding variables such as nutritional status, environmental influences, history of drug use, and genetic variability can be controlled, morphine treatment resulted in significant immune deficits. Defense against microbes is mediated by the early reactions of innate immunity and the later response of adaptive immunity. Chronic morphine has been shown to affect both these arms of immune defense (Vallejo et al. 2004). This review on the immunological effects of morphine summarizes the effects of this drug on innate and adaptive immunity, identifies the role of the mu opioid receptor in these functions, and finally discusses how changes in these parameters increase the risk for opportunistic infection.

Opioid Drug Abuse and Modulation of Immune Function: Consequences in the Susceptibility to Opportunistic Infections

Infection rate among intravenous drug users (IDU) is higher than the general public, and is the major cause of morbidity and hospitalization in the IDU population. Epidemiologic studies provide data on increased prevalence of opportunistic bacterial infections such as TB and pneumonia, and viral infections such as HIV-1 and hepatitis in the IDU population. An important component in the intravenous drug abuse population and in patients receiving medically indicated chronic opioid treatment is opioid withdrawal. Data on bacterial virulence in the context of opioid withdrawal suggest that mice undergoing withdrawal had shortened survival and increased bacterial load in response to Salmonella infection. As the body of evidence in support of opioid dependency and its immunosuppressive effects is growing, it is imperative to understand the mechanisms by which opioids exert these effects and identify the populations at risk that would benefit the most from the interventions to counteract opioid immunosuppressive effects. Thus, it is important to refine the existing animal model to closely match human conditions and to cross-validate these findings through carefully controlled human studies. Better understanding of the mechanisms will facilitate the search for new therapeutic modalities to counteract adverse effects including increased infection rates. This review will summarize the effects of morphine on innate and adaptive immunity, identify the role of the mu opioid receptor in these functions and the signal transduction activated in the process. The role of opioid withdrawal in immunosuppression and the clinical relevance of these findings will also be discussed.

Chronic Morphine Administration Delays Wound Healing by Inhibiting Immune Cell Recruitment to the Wound Site

Patients prescribed morphine for the management of chronic pain, and chronic heroin abusers, often present with complications such as increased susceptibility to opportunistic infections and inadequate healing of wounds. We investigated the effect of morphine on wound-healing events in the presence of an infection in an in vivo murine model that mimics the clinical manifestations seen in opioid user and abuser populations. We show for the first time that in the presence of an inflammatory inducer, lipopolysaccharide, chronic morphine treatment results in a marked decrease in wound closure, compromised wound integrity, and increased bacterial sepsis. Morphine treatment resulted in a significant delay and reduction in both neutrophil and macrophage recruitment to the wound site. The delay and reduction in neutrophil reduction was attributed to altered early expression of keratinocyte derived cytokine and was independent of macrophage inflammatory protein 2 expression, whereas suppression of macrophage infiltration was attributed to suppressed levels of the potent macrophage chemoattractant monocyte chemotactic protein-1. When the effects of chronic morphine on later wound healing events were investigated, a significant suppression in angiogenesis and myofibroblast recruitment were observed in animals that received chronic morphine administration. Taken together, our findings indicate that morphine treatment results in a delay in the recruitment of cellular events following wounding, resulting in a lack of bacterial clearance and delayed wound closure.

Morphine Suppresses Tumor Angiogenesis through a HIF-1α/p38MAPK Pathway

Morphine, a highly potent analgesic agent, is frequently prescribed for moderate to severe cancer pain. In this study, morphine was administered at a clinically relevant analgesic dose to assess tumor cell-induced angiogenesis and subcutaneous tumor growth in nude mice using mouse Lewis lung carcinoma cells (LLCs). Implantation of mice with a continuous slow-release morphine pellet achieved morphine plasma levels within 250-400 ng/ml (measured using a radioimmunoassay, Coat-A-Count Serum Morphine) and was sufficient to significantly reduce tumor cell-induced angiogenesis and tumor growth when compared with placebo treatment. Morphometric analysis for blood vessel formation further confirmed that morphine significantly reduced blood vessel density (P < 0.003), vessel branching (P < 0.05), and vessel length (P < 0.002) when compared with placebo treatment. Morphines effect was abolished in mice coadministered the classical opioid receptor antagonist, naltrexone, and in mu-opioid receptor knockout mice, supporting the involvement of the classical opioid receptors in vivo. Morphines inhibitory effect is mediated through the suppression of the hypoxia-induced mitochondrial p38 mitogen-activated protein kinase (MAPK) pathway. Our results suggest that in vitro morphine treatment of LLCs inhibits the hypoxia-induced nuclear translocation of hypoxia-inducible transcription factor 1alpha to reduce vascular endothelial growth factor transcription and secretion, in a manner similar to pharmacological blockade with the p38 MAPK-specific inhibitor, SB203585. These studies indicate that morphine, in addition to its analgesic function, may be exploited for its antiangiogenic potential.

Morphine Inhibits Migration of Tumor-Infiltrating Leukocytes and Suppresses Angiogenesis Associated with Tumor Growth in Mice

Tumor cells secrete factors that stimulate the migration of peripheral blood leukocytes and enhance tumor progression by affecting angiogenesis. In these studies, we investigated the effect of morphine, a known immunosuppressant, on leukocyte migration and recruitment to conditioned media derived from long-term cultures of mouse Lewis lung carcinoma cells. Our results indicate that morphine treatment reduced the migration and recruitment of tumor-infiltrating leukocytes into Matrigel plugs and polyvinyl alcohol sponges containing conditioned media derived from long-term cultures of mouse Lewis lung carcinoma cells when compared with placebo. A reciprocal increase in peripheral blood leukocytes was observed at the time of plug or sponge removal in morphine-treated mice. Decreased angiogenesis was observed in conditioned media derived from long-term cultures of mouse Lewis lung carcinoma cells Matrigel plugs taken from morphine-treated wild-type mice when compared with placebo but was abolished in morphine-treated μ-opioid receptor knockout mice. In addition, in vitro studies using trans-well and electric cell substrate impedance sensing system studies reveal for the first time morphines inhibitory effects on leukocyte migration and their ability to transmigrate across an activated endothelial monolayer. Taken together, these studies indicate that morphine treatment can potentially decrease leukocyte transendothelial migration and reduce angiogenesis associated with tumor growth. The use of morphine for cancer pain management may be beneficial through its effects on angiogenesis.

Differential effects of gram-positive and gram-negative bacterial products on morphine induced inhibition of phagocytosis

Opioid drug abusers have a greater susceptibility to gram positive (Gram (+)) bacterial infections. However, the mechanism underlying opioid modulation of Gram (+) versus Gram (−) bacterial clearance has not been investigated. In this study, we show that opioid treatment resulted in reduced phagocytosis of Gram (+), when compared to Gram (−) bacteria. We further established that LPS priming of chronic morphine treated macrophages leads to potentiated phagocytosis and killing of both Gram (+) and Gram (−) bacteria in a P-38 MAP kinase dependent signaling pathway. In contrast, LTA priming lead to inhibition of both phagocytosis and bacterial killing. This study demonstrates for the first time the differential effects of TLR4 and TLR2 agonists on morphine induced inhibition of phagocytosis. Our results suggest that the incidence and severity of secondary infections with Gram (+) bacteria would be higher in opioid abusers.

Blocking aggrecanase cleavage in the aggrecan interglobular domain abrogates cartilage erosion and promotes cartilage repair

This study demonstrates that a CD34(-), vascular endothelial cadherin(-) (VE-cadherin(-)), AC133(+), and fetal liver kinase(+) (Flk1(+)) multipotent adult progenitor cell (MAPC) that copurifies with mesenchymal stem cells from postnatal human bone marrow (BM) is a progenitor for angioblasts. In vitro, MAPCs cultured with VEGF differentiate into CD34(+), VE-cadherin(+), Flk1(+) cells - a phenotype that would be expected for angioblasts. They subsequently differentiate into cells that express endothelial markers, function in vitro as mature endothelial cells, and contribute to neoangiogenesis in vivo during tumor angiogenesis and wound healing. This in vitro model of preangioblast-to-endothelium differentiation should prove very useful in studying commitment to the angioblast and beyond. In vivo, MAPCs can differentiate in response to local cues into endothelial cells that contribute to neoangiogenesis in tumors. Because MAPCs can be expanded in culture without obvious senescence for more than 80 population doublings, they may be an important source of endothelial cells for cellular pro- or anti-angiogenic therapies.

413. Effect of Adenoviral Death Protein on NIS-Based Iodine Therapy and Imaging for Pancreatic Cancer

In this study, we assess our novel Oncolytic Adenovirus (OAd) expressing a dual function therapeutic and imaging transgene, the sodium-iodide symporter (NIS) as a promising alternative for pancreatic cancer treatment. We hypothesized that NIS expression in pancreatic cancer will induce uptake of radioiodine and allow noninvasive SPECT/CT imaging with 123I. We designed our OAd-NIS to overcome the low efficacy of previous vectors. These viruses have a modified Ad5/Ad3 fiber-knob shown to improve the poor transduction of pancreatic cancer cells. Viral replication is controlled under the Cox2 promoter allowing specific delivery of viral genes, and most of adenoviral E3 genes are deleted and replaced with NIS and Adenovirus Death Protein (ADP) genes. We have previously demonstrated significantly improved ability of ADP-overexpressing OAds to enhance viral release and cytolytic activity. Although improved oncolysis enhances viral release and improve the therapeutic effect in solid tumors, it can potentially reduce radioiodide tracer uptake. To test this possibility we designed and compared identical vectors with ADP (OAd-NIS-ADP) and without ADP (OAd-NIS-noADP) in vitro and in vivo. We first compared virus killing ability in pancreatic cancer cell lines. The ADP+ vector was significantly more cytolytic that no-ADP counterpart and showed improved viral replication and spread. Cox2-controlled OAd did not produce cell death in Cox2-negative control, confirming selectivity. We next assessed OAds ability to induce NIS protein expression and radioiodine uptake. NIS-OAds efficiently produce glycosylated NIS multimers as early as 2-days post infection. Infection with no-ADP vectors resulted in a significantly greater radioiodine uptake (125I) compared to ADP+ viruses. Importantly, ex vivo uptake test in human patient tissues confirmed the high level of NIS in pancreatic adenocarcinoma samples, and revealed no 125I uptake in normal pancreas. We further assessed the ability of our vectors to visualize human pancreatic cancer xenografts in a mouse model by monitoring 99mTcO4− accumulation with SPECT-CT. The OAd-NIS-noADP showed an earlier and more sustained radioisotope uptake when compared to ADP+ supporting its use as a more sensitive diagnostic tool for pancreatic cancer. Of contrary, the ADP+ vector significantly outperformed OAd-NIS-noADP in tumor shrinkage. These results suggest that while OAd-NIS-noADP produces a greater radiotracer uptake and can be used as a diagnostic tool, its ADP+ counterpart results in a better therapeutic effect when applied as a monotherapy. It is now essential to estimate the therapeutic ability of both vectors upon combination with I-131 (ongoing study). Further investigation will include the imaging and therapeutic studies in patient-derived xenografts. Ultimately, the goal of our research is to design a multimodal therapy with radiation and oncolytic virus for diagnosis and therapy of cancers.

Abstract 1861: Estrogen modulation of fibroblast growth factor signaling in non-small cell lung cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The estrogen signaling pathway induces proliferation in non-small cell lung cancer (NSCLC) and represents a novel therapeutic target for lung cancer. Estrogen receptor β (ERβ) is the predominant ER isoform in lung cancer and we have previously shown that high cytoplasmic ERβ combined with low progesterone receptor (PR) expression is a poor prognostic marker for NSCLC patients. To further elucidate the biological differences between ERβ high/PR low versus ERβ low/PR high expressing lung tumors, a mRNA microarray analysis using the Illumina HT-12 V4 Bead Chip platform was performed using RNA extracted from 64 cases. 165 genes were differentially expressed between these two groups at P<001. Fibroblast growth factor receptor 1 (FGFR1) was a highly over-expressed gene associated with ERβ high tumors (2.1-fold, P<0.004), while the decoy receptor for FGFR ligands, fibroblast growth factor receptor like-1 (FGFR5), was down-regulated (0.54-fold, P<0.0001). Ingenuity Pathway analysis revealed a network among the 165 differentially expressed genes that linked FGFR1 and FGFR5 to the ER. To analyze cross-talk between the FGF and E2 pathways, we determined the FGF/FGFR profile in a panel of three FGFR1 amplified and five non-amplified NSCLC cell lines. FGF2, 3, 10, and 19 were the most highly secreted FGFs in the cell line panel. FGFR1 amplified cells secreted statistically more FGF3, 6 and 10 compared to non-amplified cells. β-estradiol (E2) induced secretion of FGF2 in a cell line without FGFR1 amplification. FGFR3 and 5 were ubiquitously expressed across all cell lines while FGFR1, 2 and 4 expression was higher in amplified cell lines. There was no relationship between ERβ protein expression and FGFR1 amplification status or FGFR1-5 protein expression. Because FGF signaling has been shown to increase stem cell-like phenotypes in breast cancer, we determined basal and E2-stimulated expression of the stem cell markers, SOX2 and OCT4. Basal SOX2 and OCT4 were more highly expressed in FGFR1 amplified cells compared to non-amplified cells. SOX2 and OCT4 protein expression were increased by E2 treatment in FGFR1 non-amplified cells, with lesser effects in FGFR1 amplified H520 cells. SOX2 expression was also down-regulated in lung sections from mice treated with a combination of the anti-estrogen fulvestrant and the aromatase inhibitor anastrozole. Finally, we have shown that in FGFR1 non-amplified cells, the anti-proliferative effect of a pan-FGFR inhibitor, AZD4547, can be enhanced in the presence of the anti-estrogen fulvestrant. These results suggest that there is an interaction between the estrogen and FGF signaling pathways in lung cancer and provides a rationale for combination treatment of NSCLC tumors. Further characterization of the molecular types of NSCLC that will benefit from joint therapy is warranted. This work was supported by P50 CA090440. Citation Format: Laura P. Stabile, Natalie J. Rothenberger, Marjorie Romkes, Lisa Koodie, Mariya Farooqui, Sanja Dacic, Jill M. Siegfried. Estrogen modulation of fibroblast growth factor signaling in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1861. doi:10.1158/1538-7445.AM2015-1861