Normal Mouse Spleen Research Articles

Low molecular weight polysaccharide of Tremella fuciformis exhibits stronger antioxidant and immunomodulatory activities than high molecular weight polysaccharide

Low molecular weight polysaccharides had higher bio-activity and bioavailability compared to ultra-high molecular weight polysaccharides, this study aimed to obtain low molecular weight polysaccharides from Tremella fuciformis (TFLP) by using high-temperature and high-pressure assisted hydrochloric acid method to degrade Tremella fuciformis polysaccharides (TFP), and the structural characteristics, in vivo antioxidant and immune enhancing activities of TFP and TFLP was explored through Caenorhabditis elegans (C. elegans) and mice model. It was found that TFP and TFLP were acidic polysaccharides with molecular weights of 2238 kDa and 3 kDa, respectively. The glycosidic bonding of TFP and TFLP was mainly composed of different configurations of mannopyranose. TFP and TFLP had excellent in vivo antioxidant activity and stress resistance by regulating the mRNA transcription level and metabolites in C. elegans. Results also showed that TFP and TFLP could enhance the antioxidant capacity and immunity of serum, spleen and small intestine tissues in normal mice and cyclophosphamide-induced immunosuppressive mice through regulating the relative transcription and expression levels of anti-inflammatory related signaling factors, and it has found that TFLP showed better immune enhancement and antioxidant activity than TFP. In addition, Akkermansia, Bacteroides and Alloprevotella were characteristic bacteria at the genus level in immunosuppressed mice intervened with TFLP, with a significant increase in relative abundance. The content of SCFAs significantly increased in immunosuppressed mice by TFLP. These results indicated that TFP and TFLP had potential in vivo antioxidant and immune enhancing activities.

Effects of tumor derived exosomes on T cells markers expression.

Exosomes are 30-120nm bio particles transferred from donor to recipient cells leading to modification in their regulatory mechanisms depending upon the coded message in the form of loaded biomolecule. Cancer cells derived exosomes the true representatives of the parent cells have been found to modify the tumor surrounding/distinct regions and participate in metastasis, angiogenesis and immune suppression. Tis study was aimed to study the effects of tumor mice derived exosomes on the normal mice spleen isolated T cells by using co-culture experiments and flow cytometer analysis. We mainly focused on some of the T cells population and cytokines including IFN-γ, FOXP3+ regulatory T (Treg) cells and KI67 (proliferation marker). Overall results indicated random changes in different set of experiments, where the cancer derived exosomes reduced the IFN-γ expression in both CD4 and CD8 T cells, similarly the Treg cells were also found decreased in the presence of cancer exosomes. No significant changes were observed on the Ki67 marker expression. Such studies are helpful in understanding the role of cancer exosomes in immune cells suppression in tumor microenvironment. Cancer exosomes will need to be validated in vivo and in vitro on a molecular scale in detail for clinical applications.

Discovery of highly immunogenic spleen-resident FCGR3+CD103+ cDC1s differentiated by IL-33-primed ST2+ basophils

Recombinant interleukin-33 (IL-33) inhibits tumor growth, but the detailed immunological mechanism is still unknown. IL-33-mediated tumor suppression did not occur in Batf3−/− mice, indicating that conventional type 1 dendritic cells (cDC1s) play a key role in IL-33-mediated antitumor immunity. A population of CD103+ cDC1s, which were barely detectable in the spleens of normal mice, increased significantly in the spleens of IL-33-treated mice. The newly emerged splenic CD103+ cDC1s were distinct from conventional splenic cDC1s based on their spleen residency, robust effector T-cell priming ability, and surface expression of FCGR3. DCs and DC precursors did not express Suppressor of Tumorigenicity 2 (ST2). However, recombinant IL-33 induced spleen-resident FCGR3+CD103+ cDC1s, which were found to be differentiated from DC precursors by bystander ST2+ immune cells. Through immune cell fractionation and depletion assays, we found that IL-33-primed ST2+ basophils play a crucial role in the development of FCGR3+CD103+ cDC1s by secreting IL-33-driven extrinsic factors. Recombinant GM-CSF also induced the population of CD103+ cDC1s, but the population neither expressed FCGR3 nor induced any discernable antitumor immunity. The population of FCGR3+CD103+ cDC1s was also generated in vitro culture of Flt3L-mediated bone marrow-derived DCs (FL-BMDCs) when IL-33 was added in a pre-DC stage of culture. FL-BMDCs generated in the presence of IL-33 (FL-33-DCs) offered more potent tumor immunotherapy than control Flt3L-BMDCs (FL-DCs). Human monocyte-derived DCs were also more immunogenic when exposed to IL-33-induced factors. Our findings suggest that recombinant IL-33 or an IL-33-mediated DC vaccine could be an attractive protocol for better tumor immunotherapy.

CID16020046, a GPR55 antagonist, attenuates sepsis‑induced acute kidney injury.

Acute kidney injury (AKI) is the most common and serious complication of sepsis, and it is also the main cause of mortality in patients with sepsis. The G protein-coupled receptor 55 (GPR55) inhibitor CID16020046 was found to suppress the inflammatory response in sepsis models in mice. The aim of the present study was to investigate the effect of CID16020046 on AKI in sepsis mouse models and elucidate the possible underlying mechanisms. A sepsis model in mice was established by cecal ligation/perforation (CLP). The expression levels of GPR55 in the serum of patients with sepsis and the renal tissues of septic mice were determined via reverse transcription-quantitative PCR and western blot analyses, respectively. The pathological injury of renal tissue was evaluated using H&E and periodic acid-Schiff staining. ELISA was performed to detect the levels of renal injury-related factors, including blood urea nitrogen (BUN), creatinine (Cre), kidney injury molecule 1 (KIM1) and neutrophil gelatinase-associated lipocalin (NGAL) in septic mice. Moreover, the levels of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) were detected via ELISA and western blotting. Apoptosis was determined using TUNEL staining and western blotting. The expression levels of Rho-associated protein kinase (ROCK) pathway-related proteins (Ras homolog family member A, ROCK1 and ROCK2) was measured via western blotting. Finally, H&E staining was used to evaluate the effect of CID16020046 on various organs in mice. Compared with the control subjects, the expression level of GPR55 in the serum of patients with sepsis was significantly increased. Compared with the sham group, CID16020046 (20 mg/kg) significantly decreased the levels of BUN and Cre in the serum, as well as the contents of KIM1 and NGAL in the urine. Furthermore, CID16020046 significantly decreased the contents of TNF-α, IL-6 and IL-1β in the serum and renal tissue of septic mice, and reduced cell apoptosis. In addition, CID16020046 effectively suppressed the expression levels of ROCK pathway-related proteins, and H&E staining revealed that CID16020046 (20 mg/kg) had no toxic effect on the heart, liver, spleen or lung in normal mice. In conclusion, CID16020046 may prove useful for the development of drugs for the treatment of sepsis-induced AKI.

A mixture of Panax ginseng and Scrophularia buergeriana improves immune function in an immunosuppressed murine model

BackgroundImmunomodulatory drugs are currently used for immunosuppressed individuals, but adverse side effects have been reported. Although Panax ginseng and Scrophularia buergeriana are known to have respective pharmacological properties, the potential of a mixture of Panax ginseng and Scrophularia buergeriana (Isam-Tang, IST) as an immunomodulatory drug has not yet been studied. PurposeThe present study was designed to assess the immunomodulatory activity of IST and p-coumaric acid (pCA), an active compound of IST, in the immune system. MethodsThe levels of immunostimulatory cytokines, nitrite, inducible nitric oxide synthase (iNOS), NF-kB activation, and proliferation were examined in RAW264.7 cells, primary splenocytes and splenic NK cells isolated from normal mouse spleen, and in cyclophosphamide-induced immunosuppressed mice using ELISA, quantitative real-time PCR, Western blotting, and immunofluorescence staining. ResultsIST or pCA treatment increased the production of immunostimulatory cytokines and nitrite and the expression of iNOS in RAW264.7 cells and splenocytes. IST or pCA also induced NF-κB signaling activation and promoted the phagocytic activity of RAW264.7 cells. In addition, the splenocyte proliferation and splenic NK activity were enhanced by IST or pCA. IST or pCA increased the levels of immunostimulatory cytokines in immunosuppressed mice and ameliorated splenic tissue damage. ConclusionThese findings suggest that IST supplementation may be used to enhance immune function.

Modulation of CXCR1 and CXCR3 expression on NK cells via Tim-3 in a murine model of primary biliary cholangitis

Tim-3, which is expressed on a variety of innate immune cells including NK cells, plays a key role in many autoimmune diseases. However, the immunomodulatory actions of Tim-3 on NK cells in primary biliary cholangitis (PBC) remain uncertain. Using a murine model of PBC we evaluated the expression of Tim-3 and its ligand Gal-9 in peripheral blood, liver, and spleen. Additionally, we studied Tim-3 regulation of chemokine receptors (CXCR1 and CXCR3) in vitro. Flow cytometric analysis indicated large numbers of infiltrating NK cells in the liver which exhibited high expression of Tim-3 and CXCR3. Moreover, we found overexpression of CXCR1 in liver tissue and liver-derived NK cells in PBC mice. We also observed lower levels of soluble Tim-3 in the serum of PBC mice. In vitro experiments with liver-derived NK cells from PBC mice indicated that CXCR3 was up-regulated by treatment with recombinant mouse TIM-3 Fc (rmTim-3 Fc) to activate the Tim-3 pathway. Furthermore, stimulating normal mouse spleen NK cells with poly I:C resulted in elevated expression of CXCR1 and interferon-γ release. Nonetheless, adding rmTim-3 Fc or rmGal-9 significantly down-regulated CXCR1 expression and IFN-γ release in NK cells activated by poly I:C, proposing a means to exploit the Tim-3 pathway to reverse responses in NK cells. In conclusion, our data demonstrate that dysregulation of Tim-3/Gal-9 is involved in modulating the local immune microenvironment in PBC mice. Our findings highlight the potential of Tim-3 pathway to modulate chemokine responses in NK cells during autoimmunity.

Abraxane-induced bone marrow CD11b+ myeloid cell depletion in tumor-bearing mice is visualized by μPET-CT with 64Cu-labeled anti-CD11b and prevented by anti-CSF-1.

To investigate the utility of noninvasive µPET-CT with 64Cu-DOTA-anti-CD11b (64Cu-αCD11b) in assessing bone marrow status after anticancer therapies, and the protective role of anti-CSF-1 (αCSF-1) against bone marrow suppression induced by Abraxane.Methods: MDA-MB-435 tumor-bearing mice were treated with Abraxane, αCSF-1, or αCSF-1 plus Abraxane. µPET-CT and biodistribution of 64Cu-αCD11b were performed after intravenous injection of the radiotracer. Cells from mouse bone marrow and MDA-MB-435 tumor were analyzed by flow cytometry. A humanized αCSF-1 was investigated for its role in protecting bone marrow cells, using a transgenic mouse model that expresses functional human CSF-1.Results: μPET-CT showed that 64Cu-αCD11b had high uptake in the bone marrow and spleen of both normal and tumor-bearing mice. Abraxane significantly reduced 64Cu-αCD11b uptake in the bone marrow and spleen of treated mice compared to untreated mice. Interestingly, 64Cu-αCD11b μPET-CT revealed that αCSF-1 alleviated the depletion of bone marrow cells by Abraxane. These changes in the bone marrow population of CD11b+ myeloid cells were confirmed by flow cytometry. Moreover, αCSF-1 potently enhanced tolerance of bone marrow granulocytic myeloid cells to Abraxane, decreased cell migration, and suppressed recruitment of myeloid cells to the tumor microenvironment. The humanized αCSF-1 also alleviated the effects of Abraxane on bone marrow cells in transgenic mice expressing human CSF-1, suggesting clinical relevance of αCSF-1 in prevention of bone marrow suppression in addition to its role in reducing tumor-infiltrating myeloid cells.Conclusions: Abraxane-induced bone marrow CD11b+ myeloid cell depletion in tumor-bearing mice could be noninvasively assessed by μPET-CT with 64Cu-αCD11b and prevented by αCSF-1.

Structural and Functional Abnormalities in the Spleen of anmFtz-F1 Gene–Disrupted Mouse

AbstractThe spleen has two main functions. The first is to provide a proper microenvironment to lymphoid and myeloid cells, whereas the second involves clearance of abnormal erythrocytes. Ad4BP/SF-1, a product of the mammalian FTZ-F1 gene (mFTZ-F1), was originally identified as a steroidogenic, tissue-specific transcription factor. Immunohistochemical examination of the mammalian spleens confirmed the expression of Ad4BP/SF-1 in endothelial cells of the splenic venous sinuses and pulp vein. In mFtz-F1 gene–disrupted (KO) mice, several structural abnormalities were detected in the spleen, including underdevelopment and nonuniform distribution of erythrocytes. Examination of the spleen of KO fetuses showed failure of development of certain tubular structures during embryogenesis. These structures are normally assembled by Ad4BP/SF-1 immunoreactive cells, and most likely form the vascular system during later stages of development. Other structural abnormalities in the spleen of the KO mice included defects in the tissue distribution of type-IV collagen, laminin, c-kit, and vimentin. These morphologic defects in the vascular system were associated with a decrease in the proportion of hematopoietic cells, although differentiation of these cells was not affected significantly. A high number of abnormal red blood cells containing Howell-Jolly bodies were noted in the KO mice, indicating impaired clearance by the splenic vascular system. We also detected the presence of an mRNA-encoding cholesterol side-chain cleavage P450 in the spleen, resembling the findings in steroidogenic tissues such as the gonads and adrenal cortex. The mRNA transcript was not involved in splenic structural defects as it was detected in the spleens of both normal and KO mice, indicating that the regulatory mechanism of theP450 gene in the spleen is different from that in steroidogenic tissues. Our results indicate that a lack of the mFtz-F1 gene in mice is associated with structural and functional abnormalities of the splenic vascular system.

게르마늄 수경재배 미나리 추출물 처리에 의한 In Vitro/In Vivo에서의 사이토카인 감소 및 면역 활성에 대한 효과

RAW 264.7 cells were used to test if the extracts of water dropwort (Oenanthe javanica), cultivated in either tap water (OJ) or water containing germanium (GO) affect the secretion of nitric oxide and pro-inflammatory cytokines. Normal male ICR mice were administered OJ or GO extracts orally to evaluate the production of nitric oxide/pro-inflammatory cytokines and activities of natural killer (NK) cells in spleen and abdominal cavity. ICP-AES determined the germanium contents of the GO extracts with 116.68 mg germanium being found in 1 kg of the GO extracts. The chemical compositions of the GO extracts were analyzed using LC-Q-orbitrap and chlorogenic acid, neochlorogenic acid, caffeic acid, quercetin, isorhamnetin were detected in the GO extracts. In lipopolysaccharide-stimulated RAW 264.7 cells, the levels of nitric oxide and pro-inflammatory cytokines were decreased by treatments with the OJ and GO extracts, but the interferon-gamma (IFN-γ) levels were increased. The nitric oxide levels were reduced by both OJ and GO extracts in the spleen of normal male ICR mice fed 400 mg/kg of OJ or GO extracts. The levels of pro-inflammatory cytokines were also decreased by both OJ and GO extracts, but the IFN-γ levels were increased. Both OJ and GO extracts elevated the additional NK cell activities in the spleen and abdominal cavity. Overall, these results provide evidence that GO extracts are better than OJ extracts in terms of inhibiting the inflammatory responses. Therefore, GO extracts may be used as novel and natural functional food source to produce anti-inflammatory foods.

Abstract 5265: Spleen metabolism altered by human pancreatic cancer xenografts

Abstract Cancer-induced cachexia accounts for approximately 20% of all cancer deaths [1] and affects nearly 80% of pancreatic cancer patients [2, 3]. Cachectic patients experience a wide range of symptoms affecting several organ functions such as muscle, liver, brain, and heart that decrease quality of life and worsen prognosis. A major characteristic of cachexia is the accelerated skeletal muscle and fat storage wasting causing nutrient mobilization both directly as lipid and amino acids, and indirectly as glucose derived from the exploitation of liver gluconeogenesis that reaches the tumor through the bloodstream [4]. Here, for the first time, we have performed high-resolution quantitative 1H magnetic resonance spectroscopy (MRS) of spleen tissue obtained from normal mice and mice bearing PDAC that are cachectic (Pa04C) and non-cachectic (Panc1). The Panc1 (non-cachectic), and Pa04C (cachectic) PDAC cell lines were used [5]. Eight-week-old male severe combined immunodeficient mice were inoculated in the right flank with cancer cells (5×106). Mice were euthanized once tumors were ~ 300 mm3. Control, cachectic and non-cachectic groups consisted of 9, 10 and 9 mice per group respectively. Spleen water soluble metabolites were extracted, freeze dried, reconstituted in D2O PBS and transferred to an NMR tube for spectral acquisition with a 750 MHz (17.6T) MR spectrometer [6]. As anticipated, mice with cachexia-inducing Pa04C tumors showed significant weight loss with time. We observed, for the first time, that spleens from cachectic Pa04C tumor bearing mice showed a profound weight loss compared to normal mice and mice with Panc1 tumors. However, an increase of liver and spleen size has been previously reported in terminal cachectic human patients from colorectal cancer measured by CT scan 2-11 months prior to death [7]. A significant decrease in almost all amino acids was observed in cachectic (Pa04C) mouse spleens compared to normal and non-cachectic (Panc1) mouse spleens. Differences in choline metabolites, creatine, glutamine, glutamate, glutathione and aspartate were observed in cachectic mouse spleens compared to non-cachectic mouse spleens and spleens from healthy control mice. The significant decrease of amino acids in the cachectic spleens may reflect increased utilization of amino acids by the tumor or other organs during the cachexia muscle/protein wasting [4]. These results provide new insights into changes in spleen metabolism during cachexia, and support investigating metabolic targets to reduce cachexia associated morbidity. Supported by NIH R01CA193365 and R35CA209960. Reference: 1. Argiles et al. Nat. Rev. Cancer 2014, 14 (11), 754; 2. Fearon et al. HPB (Oxford) 2010, 12 (5), 323; 3. Ozola et al. Pancreatology 2015, 15 (1), 19; 4. Porporato et al. Oncogenesis 2016, 5, e200; 5. Winnard et al. Can. Res. 2016, 76(6): 1441; 6. Penet et al. Clin. Can. Res. 2015, 21 (2), 386; 7. Lieffers et al. Am J Clin Nutr. 2009, 89 (4), 1173 Citation Format: Santosh Kumar Bharti, Paul T. Winnard, Raj Kumar Sharma, Yelena Mironchik, Marie-France Penet, Zaver M. Bhujwalla. Spleen metabolism altered by human pancreatic cancer xenografts [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 5265.

Adipocytes: A Novel Target for IL-15/IL-15Rα Cancer Gene Therapy.

IL-15 is a proinflammatory cytokine that plays an essential role in the development and activation of natural killer (NK) cells. Adipose tissue acts as an endocrine organ that secretes cytokines and is an important reservoir for lymphocytes. We hypothesized that activation of the IL-15 signaling in adipose tissue will activate and expand the NK cell population and control tumor growth. We recently developed an adipocyte-targeting recombinant adeno-associated viral (rAAV) vector with minimal off-target transgene expression in the liver. Here, we used this rAAV system to deliver an IL-15/IL-15Rα complex to the abdominal fat by intraperitoneal (i.p.) injection. Adipose IL-15/IL-15Rα complex gene transfer led to the expansion of NK cells in the adipose tissue and spleen in normal mice without notable side effects. The i.p. injection of rAAV-IL-15/IL-15Rα complex significantly suppressed the growth of Lewis lung carcinoma implanted subcutaneously and exerted a significant survival advantage in a B16-F10 melanoma metastasis model. The antitumor effects were associated with the expansion of the NK cells in the blood, spleen, abdominal fat, and tumor, as well as the enhancement of NK cell maturity. Our proof-of-concept preclinical studies demonstrate the safety and efficacy of the adipocyte-specific IL-15/IL-15Rα complex vector as a novel cancer immune gene therapy.

Anti-Toxoplasma gondii antibodies attach to mouse cancer cell lines but not normal mouse lymphocytes.

Toxoplasma gondii (T. gondii) is prevalent intracellular parasite and a cause of worldwide infection in the human population. An inhibitory effect of this parasite on cancer growth has been demonstrated in cell culture and animal models. To determine whether the anticancer activities of T. gondii are associated with host immune response, in the current study the reactivity of anti-T. gondii antiserum with the surface of cancer cell lines was investigated. Anti-T. gondii antibodies were raised in rabbit and the reaction of this antiserum in comparison with other anti-parasite antisera (anti-T. vaginalis, anti-hydatid cyst fluid, anti-protoscolices antigens) with mouse melanoma or breast cancer cells lines was investigated using flow cytometry. Anti-T. gondii antiserum reacted markedly with the surface of mouse melanoma and breast cancer cells, and less so with the normal mouse spleen lymphocytes. Meanwhile, the other anti-parasite antisera did not react strongly with the surface of cancer cells compared with normal mouse spleen lymphocytes. In summary, it has been demonstrated herein that anti-T. gondii antiserum may selectively react with the surface of mouse cancer cells but not with normal mouse spleen lymphocytes. Therefore, further study on anti-Toxoplasma antibodies may be useful for directing the application of selective drug delivery in cancer treatment.

Ribavirin induces widespread accumulation of IMP dehydrogenase into rods/rings structures in multiple major mouse organs

Ribavirin (RBV) is a guanosine analogue triazole most commonly used in the treatment of chronic hepatitis C (HCV) infection. Although its mechanism of action is a matter of debate, several possibilities have been proposed, including depletion of guanine nucleotides through inhibition of inosine monophosphate dehydrogenase (IMPDH). IMPDH has been shown to assemble into micron-scale rod- and ring-shaped structures (rods/rings or RR), also called “IMPDH filaments,” both in vitro and in vivo. Formation of RR structures can occur naturally, potentially to influence IMPDH activity, or when de novo guanosine monophosphate biosynthesis or IMPDH itself are inhibited by nutrient deprivation or drugs like RBV. Numerous studies have also reported the occurrence of autoantibodies targeting RR structures (anti-RR) in HCV patients previously treated or under treatment with interferon-α and ribavirin (IFN/RBV) combination therapy. For this brief study, we considered the strong association between RR autoantibodies and IFN/RBV treatment, and the lack of data assessing how RBV affects RR formation in a variety of tissues in vivo. First, RR structures formed in the spleen and pancreas of normal mice without any treatment. Then, in RBV-treated mice, we detected RR structures in a number of tissues, including stomach, liver, spleen, kidney, brain, skin, and cardiac and skeletal muscle. We made several intriguing observations: predominance of RR structures in the mucosa and submucosa layers of the stomach wall; a high proportion of RR-positive cells in the cerebral cortex, suggesting that RBV actually crosses the blood-brain barrier; and a higher ratio of rings to rods in the epidermis compared to the dermis layer of the skin. Screening for RR structures appears to be a useful method to track tissue penetration of RBV and the many RR-inducing drugs previously identified.

A polysaccharide from Antrodia cinnamomea mycelia exerts antitumor activity through blocking of TOP1/TDP1-mediated DNA repair pathway

A polysaccharide (termed ACPS-1) from mycelia of Antrodia cinnamomea under submerged culture was purified by hot water extraction and successive DEAE-52 cellulose and Sephadex G-100 column chromatography, and structurally characterized by FTIR, NMR, periodate oxidation, Smith degradation, and GC–MS. ACPS-1 (MW 2.296 × 104 Da) was composed primarily of Man, Xyl, Ara, Fuc and Rha with a molar ratio of 31.27:1.77:1.44:1.34:1.00, and its backbone consisted of repeating α-(1 → 3), α-(1 → 6), α-(1 → 2), and α-(1 → 4) glycosidic linkages. ACPS-1 displayed strong in vitro growth-inhibitory effects on several human and mouse cancer cell lines (HeLa, A431, H22 and S180), and were not cytotoxic to normal mouse spleen cells. Studies of the inhibitory mechanism revealed that ACPS-1 induced apoptosis and cell cycle arrest (cells remained in G2/M phase) through blocking of topoisomerase I/tyrosyl-DNA phosphodiesterase I (TOP1/TDP1)-mediated DNA repair pathway. Our findings suggest that ACPS-1 has strong potential applications in pharmaceutical and food industries, and as a novel anticancer agent based on its dual TOP1/TDP1 inhibitory effect.

Abstract 1445: Metastasis-resistant fatty liver induced by a choline-deficient diet

Abstract Background: Fatty liver disease is increasing worldwide. The differences of the liver metastasis between fatty liver and normal liver remain unclear. We established the EL4-RFP lymphoma models in mice with fatty liver induced by a choline-deficient-diet (CDD) comparing the frequency of liver-metastasis of this tumor in mice with fatty and normal livers. Material and method: C57BL/6-GFP transgenic mice were fed with a CDD in order to establish a fatty liver model. EL4-RFP malignant lymphoma cells were injected in the spleen of fatty-liver mice and normal mice. Using the Olympus SZX7 microscope and the Olympus FV1000 confocal microscope, metastases in mice with fatty liver or normal liver were imaged. Results: EL4-RFP tumors were observed to metastasize to the liver and bone marrow as well as form ascites. Fewer metastases in the fatty liver were observed than in the normal liver. Similarly, fewer cancer-associated fibroblasts (CAFs) were observed in the fatty liver. Conclusion: The relative metastatic resistance of the fatty liver may be due to the reduced number of CAFs in the fatty liver. The fatty liver may not be a suitable environment for CAFs which may be due to re-programmed methionine metabolism which may have occurred during the time the animals were on the CDD. Citation Format: Miki Nakamura, Atsushi Suetsugu, Tomoyuki Satake, Kosuke Hasegawa, Masahito Shimizu, Shigetoyo Saji, Hisataka Moriwaki, Robert M. Hoffman. Metastasis-resistant fatty liver induced by a choline-deficient diet [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1445.

Deep Profiling of Mouse Splenic Architecture with CODEX Multiplexed Imaging

A cytometric imaging approach, called CO-Detection by indEXing (CODEX), that enables high parameter multiplexing of antibody-tagged target epitopes is used here to create high parameter imaging datasets of normal mouse and lupus (MRL/lpr) spleens. In this procedure, antibody binding events are rendered iteratively using DNA barcodes, fluorescent dNTP analogs, and an in-situ polymerization-based indexing procedure. Fluorescent signals from multiple rounds of indexing are computationally combined into a multi-channel image stack and subjected to image segmentation and quantification. A segmentation and linear model algorithm was developed to accurately quantify membrane antigen levels on dissociated cells as well as tissue sections. Leveraging the spatially resolved nature of CODEX multiplexed single-cell imaging data, quantitative de novo characterization of lymphoid tissue architecture was enabled and overlaid onto previously described morphological features. We observed an unexpected, profound impact of the cellular neighborhood on the expression of protein receptors on immune cells. By comparing normal murine spleen to spleens from animals with systemic autoimmune disease (MRL/lpr), extensive and previously uncharacterized splenic cell interaction dynamics in the healthy versus diseased state was observed. The fidelity of multiplexed imaging data analysis demonstrated here will allow deep proteomic analysis and systematic characterization of complex tissue architecture in normal and clinically aberrant samples.

Induction of apoptosis by Fe(salen)Cl through caspase-dependent pathway specifically in tumor cells

Iron-based compounds possess the capability of inducing cell death due to their reactivity with oxidant molecules, but their specificity towards cancer cells and the mechanism of action are hitherto less investigated. A Fe(salen)Cl derivative has been synthesized that remains active in monomer form. The efficacy of this compound as an anti-tumor agent has been investigated in mouse and human leukemia cell lines. Fe(salen)Cl induces cell death specifically in tumor cells and not in primary cells. Mouse and human T-cell leukemia cell lines, EL4 and Jurkat cells are found to be susceptible to Fe(salen)Cl and undergo apoptosis, but normal mouse spleen cells and human peripheral blood mononuclear cells (PBMC) remain largely unaffected by Fe(salen)Cl. Fe(salen)Cl treated tumor cells show significantly higher expression level of cytochrome c that might have triggered the cascade of reactions leading to apoptosis in cancer cells. A significant loss of mitochondrial membrane potential upon Fe(salen)Cl treatment suggests that Fe(salen)Cl induces apoptosis by disrupting mitochondrial membrane potential and homeostasis, leading to cytotoxity. We also established that apoptosis in the Fe(salen)Cl-treated tumor cells is mediated through caspase-dependent pathway. This is the first report demonstrating that Fe(salen)Cl can specifically target the tumor cells, leaving the primary cells least affected, indicating an excellent potential for this compound to emerge as a next-generation anti-tumor drug.

Escherichia coli maltose-binding protein (MBP) activates mouse Th1 through TLR2-mediated MyD88-dependent pathway and TLR4-mediated TRIF-dependent pathway

MBP (maltose-binding protein) is a component of Escherichia coli. Our previous study found that MBP directly induces the activation of Th1 (T helper type 1), but the molecular mechanism remains unclear. In the present study, CD4+T cells were purified from the spleens of normal mice using antibody-coated immunomagnetic beads by negative selection. CD4+T cells activated with a CD3/CD28 antibody were stimulated with MBP. The results indicated that MBP elevated IFN-γ mRNA levels in activated CD4+T cells and promoted IFN-γ production from activated CD4+T cells. To explore TLR2/TLR4 signaling involved in the mechanism of MBP-induced activation of Th1, we further detected downstream molecules of TLR2/TLR4 signaling. We found that MBP increased the mRNA levels of MyD88, TRAF6, TRIF and TRAF3 expressed in CD4+T cells. The results suggested that downstream molecules of TLR2/TLR4 signaling may be involved in MBP-induced activation of CD4+T cells. Furthermore, MyD88, TRIF, TRAF3 and TRAF6 expressed in activated CD4+T cells blocked with anti-TLR2 antibody or anti-TLR4 antibody followed by treatment with MBP were detected via RT-PCR and western blotting, respectively. MBP decreased the production of IFN-γ in CD4+T cells in the presence of anti-TLR2, accompanied by the down-regulated expression of MyD88 and TRAF6. However, MBP increased the production of IFN-γ in CD4+T cells in the presence of anti-TLR4 antibody accompanied by the up-regulated expression of MyD88 and the down-regulated expression of TRIF, TRAF6 and TRAF3. The results suggested that the MyD88-dependent pathway of TLR2 and TRIF-dependent pathway are involved in the mechanism of Th1 activation induced by MBP. Our study has contributed to the clarification of the molecular mechanism of MBP-induced activation of CD4+T cells.

Choline-Deficient-Diet-Induced Fatty Liver Is A Metastasis-Resistant Microenvironment

Fatty liver disease is increasing in the developed and developing world. Liver metastasis from malignant lymphoma in the fatty liver is poorly understood. In a previous report, we developed color-coded imaging of the tumor microenvironment (TME) of the murine EL4-RFP malignant lymphoma during metastasis, including the lung. In the present report, we investigated the potential and microenvironment of the fatty liver induced by a choline-deficient diet as a metastatic site in this mouse lymphoma model. C57BL/6-GFP transgenic mice were fed with a choline-deficient diet in order to establish a fatty liver model. EL4-RFP cells were injected in the spleen of normal mice and fatty-liver mice. Metastases in mice with fatty liver or normal liver were imaged with the Olympus SZX7 microscope and the Olympus FV1000 confocal microscope. Metastases of EL4-RFP were observed in the liver, ascites and bone marrow. Primary tumors were imaged in the spleen at the injection site. The fewest metastases were observed in the fatty liver. In addition, the fewest cancer-associated fibroblasts (CAFs) were observed in the fatty liver. The relative metastatic resistance of the fatty liver may be due to the reduced number of CAFs in the fatty livers. The mechanism of the effect of the choline-deficient diet is discussed.

MicroRNA expression profiles of granulocytic myeloid‑derived suppressor cells from mice bearing Lewis lung carcinoma.

Myeloid-derived suppressor cells (MDSCs) are a group of heterogeneous myeloid cells that can suppress antitumor immunity. MDSCs are divided into granulocytic (G-MDSCs) and monocytic subsets. In the present study, the microRNA profiles of the G-MDSCs were determined and the differential expression of microRNAs between G-MDSCs from tumor-bearing mice and tumor-free mice was examined. The number of G-MDSCs in spleens of Lewis lung carcinoma (LLC)-bearing mice was ~6-fold higher than in spleens of normal mice (13.54±1.74% vs. 2.14±1.44%; P<0.01) and G-MDSCs account for about 72.9% of all MDSCs. The microRNA (miRNA) profiles of the G-MDSCs from spleen of LLC-bearing mice were obtained using a microRNA microarray and compared with their counterparts from spleens of tumor-free mice. A total of 43 miRNAs with >1.3-fold increased or decreased change were differentially expressed between the experimental and control group mice. The levels of nine of these differentially expressed miRNAs, miRNA-468 (miR-486), miR-192, miR-128, miR-125a, miR-149, miR-27a, miR-125b, miR-350 and miR-328, were also analyzed by RT-qPCR to validate the microarray data. The concordance rate between the results tested by the two methods was 88.9%. Bioinformatics analyses revealed that these miRNAs may act on various target genes, including Adar, Pik3r1, Rybp and Rabgap1, to regulate the survival, differentiation and the function of tumor-induced granulocytic MDSCs. The results revealed microRNAs and potential targets that may be vital for regulating survival, differentiation and function of G-MDSCs induced by LLC. Further investigation should be performed to clarify the roles of these microRNAs in regulating LLC-induced granulocytic MDSCs and the target genes that mediate their functions.