Search

Lysosomal acid lipase (LAL) is a key enzyme in the metabolic pathway of neutral lipids. In the blood of LAL-deficient (Lal-/-) mice, increased CD11c+ cells were accompanied by upregulated programmed cell death ligand 1 (PD-L1) expression. Single-cell RNA sequencing of Lal-/- CD11c+ cells identified 2 distinctive clusters with a major metabolic shift toward glucose utilization and reactive oxygen species overproduction. Pharmacologically blocking pyruvate dehydrogenase in glycolysis not only reduced CD11c+ cells and their PD-L1 expression but also reversed their capabilities of T cell suppression and tumor growth stimulation. Colony-stimulating factor 1 receptor (CSF1R) played an essential role in controlling Lal-/- CD11c+ cell homeostasis and function and PD-L1 expression. Pharmacological inhibition of LAL activity increased CD11c, PD-L1, and CSF1R levels in both normal murine myeloid cells and human blood cells. Tumor-bearing mice and human patients with non-small cell lung cancer also showed CD11c+ cell expansion with PD-L1 and CSF1R upregulation and immunosuppression. There were positive correlations among CD11c, PD-L1, and CSF1R expression and negative correlations with LAL expression in patients with lung cancer or melanoma using The Cancer Genome Atlas database and patient samples. Therefore, CD11c+ cells switched their functions to immune suppression and tumor growth stimulation through CSF1R/PD-L1 upregulation and metabolic reprogramming.

BackgroundMyeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells in tumor microenvironment, which suppress antitumor immunity. Expansion of various MDSC subpopulations is closely associated with poor clinical outcomes in cancer. Lysosomal acid lipase (LAL) is a key enzyme in the metabolic pathway of neutral lipids, whose deficiency (LAL-D) in mice induces the differentiation of myeloid lineage cells into MDSCs. TheseLal-/-MDSCs not only suppress immune surveillance but also stimulate cancer cell proliferation and invasion. Understanding and elucidating the underlying mechanisms of MDSCs biogenesis will help to facilitate diagnosis/prognosis of cancer occurrence and prevent cancer growth and spreading.MethodsSingle-cell RNA sequencing (scRNA-seq) was performed to distinguish intrinsic molecular and cellular differences between normal versusLal-/-bone marrow–derived Ly6G+myeloid populations in mice. In humans, LAL expression and metabolic pathways in various myeloid subsets of blood samples of patients with non-small cell lung cancer (NSCLC) were assessed by flow cytometry. The profiles of myeloid subsets were compared in patients with NSCLC before and after the treatment of programmed death-1 (PD-1) immunotherapy.ResultsscRNA-seq ofLal-/-CD11b+Ly6G+MDSCs identified two distinctive clusters with differential gene expression patterns and revealed a major metabolic shift towards glucose utilization and reactive oxygen species (ROS) overproduction. Blocking pyruvate dehydrogenase (PDH) in glycolysis reversedLal-/-MDSCs’ capabilities of immunosuppression and tumor growth stimulation and reduced ROS overproduction. In the blood samples of human patients with NSCLC, LAL expression was significantly decreased in CD13+/CD14+/CD15+/CD33+myeloid cell subsets. Further analysis in the blood of patients with NSCLC revealed an expansion of CD13+/CD14+/CD15+myeloid cell subsets, accompanied by upregulation of glucose-related and glutamine-related metabolic enzymes. Pharmacological inhibition of the LAL activity in the blood cells of healthy participants increased the numbers of CD13+and CD14+myeloid cell subsets. PD-1 checkpoint inhibitor treatment in patients with NSCLC reversed the increased number of CD13+and CD14+myeloid cell subsets and PDH levels in CD13+myeloid cells.ConclusionThese results demonstrate that LAL and the associated expansion of MDSCs could serve as targets and biomarkers for anticancer immunotherapy in humans.

Utilization of proper preclinical models accelerates development of immunotherapeutics and the study of the interplay between human malignant cells and immune cells. Lysosomal acid lipase (LAL) is a critical lipid hydrolase that generates free fatty acids and cholesterol. Ablation of LAL suppresses immune rejection and allows growth of human lung cancer cells in lal(−/−) mice. In the lal(−/−) lymph nodes, the percentages of both T- and B-regulatory cells (Tregs and Bregs, respectively) are increased, with elevated expression of programmed death-ligand 1 and IL-10, and decreased expression of interferon-γ. Levels of enzymes in the glucose and glutamine metabolic pathways are elevated in Tregs and Bregs of the lal(−/−) lymph nodes. Pharmacologic inhibitor of pyruvate dehydrogenase, which controls the transition from glycolysis to the citric acid cycle, effectively reduces Treg and Breg elevation in the lal(−/−) lymph nodes. Blocking the mammalian target of rapamycin or reactivating peroxisome proliferator-activated receptor γ, an LAL downstream effector, reduces lal(−/−) Treg and Breg elevation and PD-L1 expression in lal(−/−) Tregs and Bregs, and improves human cancer cell rejection. Treatment with PD-L1 antibody also reduces Treg and Breg elevation in the lal(−/−) lymph nodes and improves human cancer cell rejection. These observations conclude that LAL-regulated lipid metabolism is essential to maintain antitumor immunity.

In this study, a novel asymmetric amine-based strongly basic anion exchange resin SE-1 was synthesized successfully via the reaction of chloromethylated styrene–divinylbenzene copolymer with N, N-dimethyloctylamine. The sorption performance of SE-1 for selective removal of nitrate in aqueous solution was compared to a commercially available nitrate specialty resin, namely Purolite A 520E (A 520E). It was found that the kinetic data could be described better by the pseudo-second-order model, and SE-1 indicated a faster sorption kinetics than A 520E resin. The Langmiur model was more appropriate for explicating the sorption isotherm. Importantly, SE-1 exhibited a greater sorption capacity for nitrate regardless of the absence or presence of competing anions in solutions. The result of column tests reinforced the feasibility of SE-1 for practical application in groundwater treatment.

Summary Objective To investigate the role of Ca2+/calmodulin-dependent protein kinase 2 (CaMKK2) in post-traumatic osteoarthritis (PTOA). Methods Destabilization of the medial meniscus (DMM) or sham surgeries were performed on 10-week-old male wild-type (WT) and Camkk2−/− mice. Half of the DMM-WT mice and all other cohorts (n = 6/group) received tri-weekly intraperitoneal (i.p.) injections of saline whereas the remaining DMM-WT mice (n = 6/group) received i.p. injections of the CaMKK2 inhibitor STO-609 (0.033 mg/kg body weight) thrice a week. Study was terminated at 8- or 12-weeks post-surgery, and knee joints processed for microcomputed tomography imaging followed by histology and immunohistochemistry. Primary articular chondrocytes were isolated from knee joints of 4–6-day-old WT and Camkk2−/− mice, and treated with 10 ng/ml interleukin-1β (IL)-1β for 24 or 48 h to investigate gene and protein expression. Results CaMKK2 levels and activity became elevated in articular chondrocytes following IL-1β treatment or DMM surgery. Inhibition or absence of CaMKK2 protected against DMM-associated destruction of the cartilage, subchondral bone alterations and synovial inflammation. When challenged with IL-1β, chondrocytes lacking CaMKK2 displayed attenuated inflammation, cartilage catabolism, and resistance to suppression of matrix synthesis. IL-1β-treated CaMKK2-null chondrocytes displayed decreased IL-6 production, activation of signal transducer and activator of transcription 3 (Stat3) and matrix metalloproteinase 13 (MMP13), indicating a potential mechanism for the regulation of inflammatory responses in chondrocytes by CaMKK2. Conclusions Our findings reveal a novel function for CaMKK2 in chondrocytes and highlight the potential for its inhibition as an innovative therapeutic strategy in the prevention of PTOA.

Ammonia is widely distributed in sulfate-reducing bioreactor dealing with sulfate wastewater, which shows potential effect on the metabolic pathway of sulfate and ammonia. This study investigates the sulfate-reducing efficiency and microbial community composition in the sulfate-reducing EGSB reactor with the increasing ammonia loading. Results indicated that, compared with low ammonia loading (166–666 mg/L), the sulfate and organic matter removal efficiencies were improved gradually with the appropriate ammonia loading (1000–2000 mg/L), which increased from 63.58 ± 3.81 to 71.08 ± 1.36% and from 66.24 ± 1.32 to 81.88 ± 1.83%, respectively. Meanwhile, with the appropriate ratio of ammonia and sulfate (1.5–3.0) and hydraulic retention time (21 h), the sulfate-reducing anaerobic ammonia oxidation (SRAO) process was occurred efficiently, inducing the accumulation of S0 (270 mg/L) and the simultaneous ammonia removal (70.83%) in EGSB reactor. Moreover, the key sulfate-reducing bacteria (SRB) (Desulfovibrio) and denitrification bacteria (Pseudomonas and Alcaligenes) were responsible for the sulfate and nitrogen removal in these phases, which accounted for 3.66–5.54 and 3.85–9.13%, respectively. However, as the ammonia loading higher than 3000 mg/L (phases 9 and 10), the sulfate-reducing efficiency was decreased to only 28.3 ± 1.26% with the ammonia removal rate of 18.4 ± 3.37% in the EGSB reactor. Meanwhile, the predominant SRB in phases 9 and 10 were Desulfomicrobium (1.22–1.99%) and Desulfocurvus (4.0–5.46%), and the denitrification bacteria accounted for only 0.88% (phase 10), indicating the low nitrogen removal rate.

For a full understanding of microbial function in simultaneous nitrification and denitrification (SND), comprehensive genetic information in activated sludge from two parallel oxidation ditches (Ditches A and B) was investigated by high-throughput sequencing and quantitative real time PCR (q-PCR). Results showed that over 90% of the ammonia was removed by both oxidation ditches, and Ditch A had a higher average value of SND efficiency. Both ditches shared a similar bacterial composition, and Proteobacteria and Bacteroidetes were the dominant phyla. However ditch A had a microbial community with higher Shannon and Chao1 indices than Ditch B. At genus level, Nitrosomonas, Thauera, Arcobacter and Zoogloea were mainly involved in the SND of oxidation ditches. And more types and higher abundances of (de)nitrifiers were detected in Ditch A. The results were verified by q-PCR analysis. According to functional analysis, the metabolism of carbohydrates and proteins, clustering-based subsystems, as well as amino acids and derivatives were the major functions of activated sludge in oxidation ditches. SND was accomplished in oxidation ditches by means of conversion and incorporation of nitrogenous compounds. Furthermore, more types of Level 3 subsystems and higher efficiency of ammonia assimilation were found in the nitrogen metabolism of Ditch A. These findings provide an insight into genetic pattern of SND in the municipal sewage treatment plants.

Lysosomal acid lipase (LAL), a key enzyme in the metabolic pathway of neutral lipids, has a close connection with inflammation and tumor progression. One major manifestation in LAL-deficient ( Lipa −/− ) mice is an increase of tumor growth and metastasis associated with expansion of myeloid-derived suppressor cells. In the lung, LAL is highly expressed in alveolar type II epithelial cells. To assess how LAL in lung epithelial cells plays a role in this inflammation-related pathogenic process, lung alveolar type II epithelial cell–specific expression of human LAL (hLAL) in Lipa −/− mice was established by crossbreeding of CCSP-driven rtTA transgene and (TetO) 7 -CMV-hLAL transgene into Lipa −/− mice (CCSP-Tg/KO). hLAL expression in lung epithelial cells not only reduced tumor-promoting myeloid-derived suppressor cells in the lung, but also down-regulated the synthesis and secretion of tumor-promoting cytokines and chemokines into the bronchoalveolar lavage fluid of Lipa −/− mice. hLAL expression reduced the immunosuppressive functions of bronchoalveolar lavage fluid cells, inhibited bone marrow cell transendothelial migration, and inhibited endothelial cell proliferation and migration in Lipa −/− mice. As a result, hLAL expression in CCSP-Tg/KO mice corrected pulmonary damage, and inhibited tumor cell proliferation and migration in vitro , and tumor metastasis to the lung in vivo . These results support a concept that LAL is a critical metabolic enzyme in lung epithelial cells that regulates lung homeostasis, immune response, and tumor metastasis.

Adsorption using anion exchange resins is an efficient method for the removal of aromatic sulfonic acids (ASAs) from industrial wastewater. In this study, a series of weak-base anion exchangers (SD1&ndash, SD5) were synthesized to investigate the effect of functional group density of resins on the adsorption of ASAs from wastewater containing competitive inorganic anions. p-Toluene sulfonic acid (PTSA) was selected as a target pollutant, and Na2SO4 was chosen as the competitive inorganic salt because of its widespread existence in industrial wastewater. Adsorption performances of these resins were evaluated and compared in terms of selectivity, kinetics, isotherms, regeneration, and dynamic adsorption behavior. Importantly, the PTSA uptake increased with the raising content of functional groups on resins in the absence of Na2SO4, however, in the presence of a high level of Na2SO4 (for example, &ge, 1%), a decrease in the functional group density could improve the adsorption capacity of resins for PTSA. Moreover, desorption and fixed bed column experiments were conducted in all resins, thereby confirming the effect of functional group density of resins on the PTSA adsorption in actual application. In brief, this research will provide a better understanding for the design and preparation of anion exchangers for the effective removal of ASA from wastewater.

Early detection of lung cancer offers an important opportunity to decrease mortality while it is still treatable and curable. Thirteen secretory proteins that are Stat3 downstream gene products were identified as a panel of biomarkers for lung cancer detection in human sera. This panel of biomarkers potentially differentiates different types of lung cancer for classification. Among them, the transthyretin (TTR) concentration was highly increased in human serum of lung cancer patients. TTR concentration was also induced in the serum, bronchoalveolar lavage fluid, alveolar type II epithelial cells, and alveolar myeloid cells of the CCSP-rtTA/(tetO)7-Stat3C lung tumor mouse model. Recombinant TTR stimulated lung tumor cell proliferation and growth, which were mediated by activation of mitogenic and oncogenic molecules. TTR possesses cytokine functions to stimulate myeloid cell differentiation, which are known to play roles in tumor environment. Further analyses showed that TTR treatment enhanced the reactive oxygen species production in myeloid cells and enabled them to become functional myeloid-derived suppressive cells. TTR demonstrated a great influence on a wide spectrum of endothelial cell functions to control tumor and immune cell migration and infiltration. TTR-treated endothelial cells suppressed T cell proliferation. Taken together, these 13 Stat3 downstream inducible secretory protein biomarkers potentially can be used for lung cancer diagnosis, classification, and as clinical targets for lung cancer personalized treatment if their expression levels are increased in a given lung cancer patient in the blood.

Lung cancer is a very aggressive malignant form of cancer, and is one of the biggest public health challenges facing the United States and many other countries. Although incidence rates have been stabilized, an estimated 154,050 Americans are expected to die from lung & bronchus cancer in 2018, accounting for approximately 25.3 percent of all cancer deaths. Lung cancer is a difficult disease to detect in its early stages, with greater than 50% of patients diagnosed with lung cancer presenting with metastatic disease. Early detection of lung cancer is an important opportunity for decreasing mortality while it is still treatable and curable. The overall 5-year survival rate is ~15 percent. Thus, it is essential to better understand the mechanisms that initiate lung carcinogenesis and find easy-use biomarkers for more accurate lung cancer detection. Due to heterogeneity of lung cancers, a panel of biomarkers should be used for more accurate lung cancer detection and classification. Signal transducer and activator of transcription 3 (Stat3) is well known for its lung cancer-promoting activity. To assess the consequences of STAT3 persistent activation in the lung, a doxycycline-controlled CCSP-rtTA/(tetO)7-Stat3C bitransgenic mouse model was generated that over-expresses STAT3C (a constitutively active form of STAT3) in alveolar type II (AT II) epithelial cells. In sequential steps, Stat3C over-expression up-regulated pro-inflammatory molecules, increased inflammatory cell infiltration and caused adenocarcinomas in the lung. Using this lung tumor model, thirteen secretory proteins that are Stat3 downstream gene products were identified as a panel of biomarkers for lung cancer detection in human sera. This panel of biomarkers potentially differentiates different types of lung cancer for classification. Among them, the transthyretin (TTR) concentration was highly increased in human serum of lung cancer patients. TTR concentration was also induced in the serum, bronchoalveolar lavage fluid, alveolar type II epithelial cells and alveolar myeloid cells of the CCSP-rtTA/(tetO)7-Stat3C lung tumor mouse model. Recombinant TTR stimulated lung tumor cell proliferation and growth, which were mediated by activation of mitogenic and oncogenic molecules. TTR possesses cytokine functions to stimulate myeloid cell differentiation, which are known to play roles in tumor environment. TTR demonstrated a great influence on a wide spectrum of endothelial cell functions to control tumor and immune cell migration and infiltration. TTR-treated endothelial cells suppressed T cell proliferation. Taken together, Stat3 downstream inducible secretory protein biomarkers potentially can be used as clinical targets for lung cancer personalized treatment if their expression levels are increased in a given lung cancer patient in the blood. Citation Format: Xinchun Ding. Stat3 downstream transthyretin stimulates tumor growth through regulation of tumor, immune and endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2048.

// Xinchun Ding 1 , Wenjing Zhang 1 , Ting Zhao 1 , Cong Yan 1, 2 , Hong Du 1, 2 1 Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA 2 IU Simon Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA Correspondence to: Hong Du, email: hongdu@iupui.edu Cong Yan, email: coyan@iupui.edu Keywords: Rab7 GTPase, myeloid-derived suppressor cells, lipid metabolism, tumor growth Received: November 18, 2016 Accepted: March 09, 2017 Published: March 16, 2017 ABSTRACT Lysosomal acid lipase (LAL) is a critical neutral lipid metabolic enzyme that regulates metabolic reprogramming in myeloid-derived suppressor cells (MDSCs) through over-activation of mammalian target of rapamycin (mTOR). Affymetrix GeneChip microarray analysis of MDSCs from LAL deficient mouse ( lal –/– ) revealed upregulation of Rab7 GTPase protein, which belongs to a superfamily of small-molecular-weight GTPase known to regulate intracellular membrane trafficking from early to late endosomes and lysosomes. Here, the physical protein-protein interaction between Rab7 GTPase and mTOR has been detected by co-immunoprecipitation in the cell extract of wild type HD1A and lal –/– MDSC-like HD1B myeloid cell lines. The GST pull down assay using the recombinant GST-Rab7 GTPase fusion protein showed that Rab7 GTPase interacts with the mTOR N-terminal heat repeat domain. Rab7 GTPase siRNA knocking down reversed the altered lysosome/mTOR distribution and expression levels in HD1B cells. Rab7 GTPase siRNA knocking down in isolated bone marrow lal –/– MDSCs or HD1B cells not only reduced over-activation of mTOR and its downstream effector S6, but also decreased glucose consumption, decreased ROS over-production, and increased healthy mitochondria by membrane potential measurement. Inhibition of Rab7 GTPase led to reduced lal –/– MDSCs differentiation from bone marrow Lin – progenitor cells, reduced lal –/– MDSCs trans-endothelial migration, and reversed lal –/– MDSCs suppression of T cell proliferation. Furthermore, inhibition of Rab7 GTPase reduced lal –/– MDSCs ability to stimulate tumor cell proliferation in vitro , tumor growth in vivo , and tumor invasion. Together, these results showed that Rab7 GTPase is critically involved in MDSCs homeostasis and pathogenic functions.

Background Cigarette smoking has been verified as the risk factor of esophageal squamous cell carcinoma (ESCC). Overexpression of cyclooxygenase 2 (COX-2) is shown in ESCC. The objective of this study was to investigate the effects of cigarette smoking ethanol extract (EE) on the proliferation of the human ESCC cell lines, and to explore the correlation between the proliferation rate of human ESCC cell lines and the expression pattern of COX-2. Whether aspirin can inhibit the proliferation of the ESCC cell lines pretreated with EE, and regulate the mRNA expression levels of COX-2 are also examined. Methods Two human ESCC cell lines were selected. EC109 was poorly differentiated and EC9706 was highly differentiated. EC109 and EC9706 were treated with EE and aspirin for different time course. The cell growth of ESCC was measured by MTT reduction assay and the expression of COX-2 was measured by RT-PCR and Western blot analysis. Results EE promoted the proliferation of EC109 and EC9706 in dose- and time-dependent manners. In the concentration range (10−100 µg/ml for EE) and in the time range (24−72 hours) after addition of EE, the cell proliferation was prominent in an up-scaled manner respectively. Aspirin could inhibit the proliferation of cell lines EC109 and EC9706, pretreated with EE for 5 hours, in a dose-dependent manner. In the concentration range (0.5−8.0 mmol/L for aspirin), the cell growth inhibition was prominent in an up-scaled manner accordingly (P

Inflammation critically contributes to cancer metastasis, in which myeloid-derived suppressor cells (MDSCs) are an important participant. Although MDSCs are known to suppress immune surveillance, their roles in directly stimulating cancer cell proliferation and metastasis currently remain unclear. Lysosomal acid lipase (LAL) deficiency causes systemic expansion and infiltration of MDSCs in multiple organs and subsequent inflammation. In the LAL-deficient (lal(-/-)) mouse model, melanoma metastasized massively in allogeneic lal(-/-) mice, which was suppressed in allogeneic lal(+/+) mice owing to immune rejection. Here we report for the first time that MDSCs from lal(-/-) mice directly stimulated B16 melanoma cell in vitro proliferation and in vivo growth and metastasis. Cytokines, that is, interleukin-1β and tumor necrosis factor-α from MDSCs are required for B16 melanoma cell proliferation in vitro. Myeloid-specific expression of human LAL (hLAL) in lal(-/-) mice rescues these malignant phenotypes in vitro and in vivo. The tumor-promoting function of lal(-/-) MDSCs is mediated, at least in part, through overactivation of the mammalian target of rapamycin (mTOR) pathway. Knockdown of mTOR, Raptor or Rictor in lal(-/-) MDSCs suppressed their stimulation on proliferation of cancer cells, including B16 melanoma, Lewis lung carcinoma and transgenic mouse prostate cancer-C2 cancer cells. Our results indicate that LAL has a critical role in regulating MDSCs' ability to directly stimulate cancer cell proliferation and overcome immune rejection of cancer metastasis in allogeneic mice through modulation of the mTOR pathway, which provides a mechanistic basis for targeting MDSCs to reduce the risk of cancer metastasis. Therefore MDSCs possess dual functions to facilitate cancer metastasis: suppress immune surveillance and stimulate cancer cell proliferation and growth.

Myeloid-derived suppressive cells (MDSCs) are inflammatory cells that play critical roles in promoting cancer growth and metastasis. In order to facilitate characterization of biochemical and cellular mechanisms of MDSCs, it is urgent to establish an “MDSC-like” cell line for pharmacological and immunotherapeutic applications. Lysosomal acid lipase (LAL) is a critical lipid enzyme in the metabolic signaling pathway that hydrolyzes cholesteryl esters (CE) and triglycerides (TG) in lysosomes. In mice, lack of LAL in genetically ablated knockout mice (lal-/-) shows systemic expansion of MDSCs, which directly stimulate cancer cell proliferation, and suppress T cell proliferation and impair T cell function. The Affymetrix Genechip microarray assay reveals over-activation of the mTOR signaling pathway in lal-/- MDSCs. By cross breeding of immortomouse (simian virus 40 large T antigen transgenic mice) with wild type and lal-/- mice, we have established wild type (HD1A) and lal-/- (HD1B) myeloid cell lines. Compared with HD1A cells, HD1B cells demonstrated many characteristics similar to lal-/- MDSCs. HD1B cells exhibited increased lysosomes around perinuclear areas. HD1B cells showed increased glycolytic metabolism during blockage of fatty acid metabolism to fuel the energy need. Compared with HD1A cells, HD1B cells showed increased glucose concentration, suggesting the enhanced glycolytic metabolic pathway, in which glucose converts into pyruvate. Indeed, the pyruvate concentration was increased in HD1B cells compared with that in HD1A cells. Glycolysis occurs in the cytosol of the cell. Pyruvic acid supplies energy to living cells through the citric acid cycle (TCA) in the mitochondria, which generates NADH for the oxidative phosphorylation (OXPHOS, electron transport pathway) to produce ATP. Aconitase is the rate-limiting enzyme in the TCA cycle. Its activity was doubled in HD1B cells compared with HD1A cells. GLUT (SLC2) family members are the major membrane transporters. Among them, GLUT 1-5 have been well characterized as glucose and/or fructose transporters in various tissues and cell types. Thirteen GLUT proteins have been reported to be expressed in mice (14 in humans). Using the Real-time PCR method, expression of all GLUT members was assessed in HD1A and HD1B cells, in which GLUT3, GLUT6, GLUT8, GLUT12, and CLUT13 were upregulated, while GLUT 5 and GLUT 9 were downregulated in HD1B cells. This supports a concept that the LAL metabolic pathway controls the balance of glucose transportation to fuel the energy need in HD1B cells. In addition, dysfunction of mitochondria in HD1B cells skews toward fission structure, damaged membrane potential, and increased ROS production. Similar to lal-/- MDSCs, the mTOR signal pathway in HD1B cells is overly activated. Rapamycin treatment of HD1B cells reduced ROS production and restored the mitochondrial membrane potential. HD1B cells showed much stronger immunosuppression on CD4+ T cell proliferation and function in vitro, and enhanced cancer cell proliferation. Knockdown of mTOR with siRNA reduced the HD1B cell ability to immunosuppress T cells and stimulate cancer cell proliferation. Therefore, the HD1B myeloid cell line is an “MDSC-like” cell line. HD1B cells can be used as an alternative in vitro system to study how the lipid metabolic signaling pathway regulates myeloid cell functions. Both HD1A and HD1B cell lines will be useful for identifying pharmacological drugs to suppress MDSCs expansion in multiple inflammation-induced diseases that involve MDSCs. Grant support: This work was supported by National Institutes of Health Grants HL087001 (to H. D.), and CA138759, CA152099 (to C. Y.). Citation Format: Cong Yan, Xinchun Ding, Lingyan Wu, Hong Du. Establishment of myeloid lineage cell line that resembles myeloid-derived suppressive cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A12.

Inflammation critically contributes to cancer metastasis, in which myeloid-derived suppressor cells (MDSCs) are an important participant. Although MDSCs are known to suppress immune surveillance, their roles in directly stimulating cancer cell proliferation and metastasis currently remain unclear. MDSCs development and homeostasis is controlled by lysosomal acid lipase (LAL), a critical enzyme in the metabolic signaling pathway that hydrolyzes cholesteryl esters (CE) and triglycerides (TG) in lysosomes. Lysosomal acid lipase (LAL) deficiency causes systemic expansion and infiltration of MDSCs in multiple organs. LAL-deficient (lal-/-) MDSCs arise from dysregulated production of progenitor cells in the bone marrow. In humans, increased CD14+CD16+ and CD14+CD33+ MDSC subsets have been reported with heterozygote carriers of LAL mutations. Patients with mutations in the LAL gene has been reported to be associated with carcinogenesis. In the LAL-deficient (lal-/-) mouse model, melanoma metastasized massively in allogeneic lal-/- mice, which was suppressed in allogeneic lal+/+ mice due to immune rejection. Here we report for the first time that MDSCs isolated from lal-/- mice directly stimulated B16 melanoma cell proliferation in vitro, and growth and metastasis in vivo. Cytokines i.e., IL-1β and TNFα from MDSCs are required for B16 melanoma cell proliferative function in vitro. Myeloid-specific expression of human LAL (hLAL) in lal-/- mice rescues these malignant phenotypes in vitro and in vivo. The tumor-promoting function of lal-/- MDSCs is mediated, at least in part, through over-activation of the mammalian target of rapamycin (mTOR) pathway. mTOR is a serine/threonine protein kinase that regulates cell growth, proliferation, migration, survival, protein synthesis, and transcription in response to growth factors and mitogens. mTOR is the catalytic subunit of two distinctive complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Unique accessory proteins, regulatory-associated protein of mTOR (RAPTOR), and rapamycin-insensitive companion of mTOR (RICTOR) define the TORC1 and TORC2 complexes, respectively. Affymetrix GeneChip microarray analysis and Ingenuity Pathway Analysis of gene transcripts revealed up-regulation of multiple genes in the mTOR signaling pathway in lal-/- MDSCs. We have shown that inhibition of mTOR in lal-/- MDSCs increased apoptosis, decreased ATP synthesis, decreased ROS production, and recovered impairment of mitochondrial membrane potential. As a result, lal-/- MDSCs reversed the increased cell proliferation, corrected enhanced lal-/- MDSCs development from lineage negative progenitor cells, and reduced systemic MDSC expansion. More importantly, knockdown of mTOR, Raptor or Rictor in lal-/- MDSCs reversed the immune suppression on T cell proliferation and function, as well as suppressed their stimulation on proliferation of cancer cells (including B16 melanoma, LLC and Tramp-C2 cancer cells). Our results indicate that LAL plays a critical role in regulating MDSCs through modulation of the mTOR pathway. MDSCs possess dual functions to overcome immune rejection and facilitate cancer metastasis: 1) suppress immune surveillance, and 2) stimulate cancer cell proliferation, growth, and metastasis. Grant support: This work was supported by National Institutes of Health Grants CA138759, CA152099 (to C. Y.) and HL087001 (to H. D.). Citation Format: Hong Du, Ting Zhao, Xinchun Ding, Katlin Walls, Cong Yan. Myeloid-derived suppressor cells suppress immune surveillance and stimulate cancer cell proliferation/metastasis through activation of mTOR pathway in lal-/- mice. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A11.

Background Lysosomal acid lipase (LAL) controls development and homeostasis of myeloid lineage cells. Loss of the lysosomal acid lipase (LAL) function leads to expansion of myeloid-derived suppressive cells (MDSCs) that cause myeloproliferative neoplasm. Methodology/principal findings Affymetrix GeneChip microarray analysis identified detailed intrinsic defects in Ly6G(+) myeloid lineage cells of LAL knock-out (lal-/-) mice. Ingenuity Pathway Analysis revealed activation of the mammalian target of rapamycin (mTOR) signaling, which functions as a nutrient/energy/redox sensor, and controls cell growth, cell cycle entry, cell survival, and cell motility. Loss of the LAL function led to major alteration of large GTPase and small GTPase signal transduction pathways. lal-/- Ly6G(+) myeloid cells in the bone marrow showed substantial increase of cell proliferation in association with up-regulation of cyclin and cyclin-dependent kinase (cdk) genes. The epigenetic microenvironment was significantly changed due to the increased expression of multiple histone cluster genes, centromere protein genes and chromosome modification genes. Gene expression of bioenergetic pathways, including glycolysis, aerobic glycolysis, mitochondrial oxidative phosphorylation, and respiratory chain proteins, was also increased, while the mitochondrial function was impaired in lal-/- Ly6G(+) myeloid cells. The concentration of reactive oxygen species (ROS) was significantly increased accompanied by up-regulation of nitric oxide/ROS production genes in these cells. Conclusions/significance This comprehensive gene profile study for the first time identifies and defines important gene pathways involved in the myeloid lineage cells towards MDSCs using lal-/- mouse model.

Homeostasis of platelet number in human and other mammals is well maintained for prevention of minor bleeding and for other immunological functions, but the exact molecular mechanism responsible for immune thrombocytopenic purpura (ITP) has not been fully understood. In an effort to identify genetic factors involved in initiation of platelet production in response to bleeding injury or platelet destruction, we have successfully generated an animal model of human ITP via intraperitoneal injection of anti-platelet antibody into the Balb/c mouse. Platelet counts were dropped dramatically in animals that received antibody injection within 4 h, maintained at the minimum level for a period of 44 h, started to rebound after 48 h, and reached to the maximum at 144 h (6 days). Final homeostasis reached at approximately 408 h (17 days), following a minor cycle of platelet number fluctuation. Using semi-quantitative RT-PCR, we assessed and compared mRNA level of CD41, c-myb, c-mpl, caspase-3, caspase-9, GATA-1, and Bcl-xl in bone marrow and spleen. Alteration of mRNA expression was correlated with the change of platelet level, and an inverse relationship was found for expression of the genes between bone marrow and spleen. No transcription was detectable for any of the seven genes in bone marrow at the time when platelet number reached the maximum (144 h). In contrast, mRNA transcripts of the seven genes were found to be at the highest level in spleen tissue. This is the first study of simultaneous detection of multiple platelet related genes in a highly reproducible ITP animal model. Our results provided the supportive evidence that expression of the above seven genes are more related to negative regulation of platelet number in spleen tissue, at least in the model animals.

Myeloid-derived suppressor cells (MDSCs) in mouse are inflammatory cells that play critical roles in promoting cancer growth and metastasis by directly stimulating cancer cell proliferation and suppressing immune surveillance. In order to facilitate characterization of biochemical and cellular mechanisms of MDSCs, it is urgent to establish an "MDSC-like" cell line. By cross breeding of immortomouse (simian virus 40 large T antigen transgenic mice) with wild type and lysosomal acid lipase (LAL) knock-out (lal-/-) mice, we have established a wild type (HD1A) and a lal-/- (HD1B) myeloid cell lines. Compared with HD1A cells, HD1B cells demonstrated many characteristics similar to lal-/- MDSCs. HD1B cells exhibited increased lysosomes around perinuclear areas, dysfunction of mitochondria skewing toward fission structure, damaged membrane potential, and increased ROS production. HD1B cells showed increased glycolytic metabolism during blockage of fatty acid metabolism to fuel the energy need. Similar to lal-/- MDSCs, the mTOR signal pathway in HD1B cells is overly activated. Rapamycin treatment of HD1B cells reduced ROS production and restored the mitochondrial membrane potential. HD1B cells showed much stronger immunosuppression on CD4+ T cell proliferation and function in vitro, and enhanced cancer cells proliferation. Knockdown of mTOR with siRNA reduced the HD1B cell ability to immunosuppress T cells and stimulate cancer cell proliferation. Therefore, the HD1B myeloid cell line is an "MDSC-like" cell line that can be used as an alternative in vitro system to study how LAL controls various myeloid cell functions.

Inflammation plays crucial roles at all stages of tumor development, from tumor initiation to metastatic progression, during which myeloid-derived suppressor cells (MDSCs) are an important participant. Although MDSCs are known to suppress immune surveillance, their roles in directly stimulating cancer cell proliferation and metastasis currently remains unclear. Lysosomal acid lipase (LAL) deficiency causes systemic expansion and infiltration of MDSCs in multiple organs and subsequent inflammation. In the LAL-deficient (lal-/-) mouse model, we found that melanoma metastasized massively in allogeneic lal-/- mice, which was suppressed in allogeneic lal+/+ mice due to immune rejection. Therefore, we hypothesized that MDSCs with LAL deficiency directly stimulate cancer cell proliferation and metastasis. Bone marrow-derived MDSCs from lal-/- mice directly stimulated B16 melanoma cell proliferation in vitro by co-culture analysis and in vivo by co-implantation in the Matrigel plugs. These tumor cell-stimulatory effects were diminished when myeloid-specific human LAL (hLAL) was expressed in myeloid cells in lal-/- mice. In addition, lal-/- MDSCs facilitated B16 melanoma cell metastasis in the lungs of recipient lal+/+ mice via tail vein injection. Furthermore, the mammalian target of rapamycin (mTOR) and its downstream gene products were significantly up-regulated in lal-/- MDSCs. Knockdown of mTOR in lal-/- MDSCs suppressed their stimulation on B16 melanoma cell proliferation, growth and metastasis, indicating the tumor-promoting function of lal-/- MDSCs is mediated, at least in part, through over-activation of the mTOR pathway. Finally, lal-/- MDSCs stimulated proliferation and growth of Lewis lung carcinoma (LLC), and transgenic mouse prostate cancer (TRAMP-C2) cells, and these effects were impaired after mTOR inhibition. Our results indicate that LAL plays a critical role in regulating MDSCs ability to directly stimulate cancer cell proliferation, and overcome immune rejection of cancer metastasis in allogeneic mice through modulation of the mTOR pathway, which provides a mechanistic basis for targeting MDSCs to reduce the risk of cancer metastasis. Therefore, MDSCs possess dual functions to facilitate cancer metastasis: suppress immune surveillance, and stimulate cancer cell proliferation and growth. Citation Format: Ting Zhao, Hong Du, Xinchun Ding, Katlin Walls, Cong Yan. Activation of mTOR pathway in myeloid-derived suppressor cells with lysosomal acid lipase deficiency stimulates cancer cell proliferation and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 161. doi:10.1158/1538-7445.AM2014-161