Mouse Lung Carcinoma Research Articles

Human Biofield Therapy Modulates Tumor Microenvironment and Cancer Stemness in Mouse Lung Carcinoma.

Studies have demonstrated that purported biofield therapy emitted from humans can inhibit the proliferation of cancer cells and suppress tumor growth in various cancers. We explored the effects of biofield therapy on tumor growth in the Lewis lung carcinoma and expanded mechanistic outcomes. We found biofield therapy did not inhibit tumor growth. However, the experimental (Ex) condition exposed tumors had a significantly higher percentage of necrosis (24.4 ± 6.8%) compared with that of the Control condition (6.5 ± 2.7%; P < .02) and cleaved caspase-3 positive cells were almost 2.3-fold higher (P < .05). Similarly, tumor-infiltrating lymphocytes profiling showed that CD8+/CD45+ immune cell population was significantly increased by 2.7-fold in Ex condition (P < .01) whereas the number of intratumoral FoxP3+/CD4+ (T-reg cells) was 30.4% lower than that of the Control group (P = .01), leading to a significant 3.1-fold increase in the ratio of CD8+/T-reg cells (P < .01). Additionally, there was a 51% lower level of strongly stained CD68+ cells (P < .01), 57.9% lower level of F4/80high/CD206+ (M2 macrophages; P < .02) and a significant 1.8-fold increase of the ratio of M1/M2 macrophages (P < .02). Furthermore, Ex exposure resulted in a 15% reduction of stem cell marker CD44 and a significant 33% reduction of SOX2 compared with that of the Controls (P < .02). The Ex group also engaged in almost 50% less movement throughout the session than the Controls. These findings suggest that exposure to purported biofields from a human is capable of enhancing cancer cell death, in part mediated through modification of the tumor microenvironment and stemness of tumor cells in mouse Lewis lung carcinoma model. Future research should focus on defining the optimal treatment duration, replication with different biofield therapists, and exploring the mechanisms of action.

Comparative Study of Cytotoxic Activity of Nano Silver Against A549 and L929 Cell Lines

Studies in recent years are focussed on anticancer drugs which can selectively induce cell death with less toxicity to normal cells. The present work therefore aims at exploring the potential of nano silver as selective anticancer drug by comparing its cytotoxic activity against human lung carcinoma cell line (A549) and mouse normal fibroblast cell line (L929) in vitro. Nano silver was synthesized by both chemogenic (AgNP-C) and biogenic (AgNP-B) method and characterized by using PXRD, SEM and TEM. In order to assess the molecular mechanism involved in cytotoxicity, apoptosis inducing effect of nano silver was assessed by Annexin V/PI staining, cell cycle analysis and caspase-3 expression study. From the results, it was confirmed that A549 cells treated with nano silver showed decreased cell viability (AgNP-C: 173.5 ± 2.51 μg/mL, AgNP-B: 29.2 ± 0.22 μg/mL) compared to L929 cells (AgNP-C: 317.2 ± 3.43 μg/mL, AgNP-B: 622.3 ± 1.6 μg/mL), indicating lower toxicity of nano silver towards normal cells. Apoptotic study, cell cycle analysis and caspase-3 studies showed decreased expression of Bcl-2 and increased expression of Bax mitochondrial genes facilitating release of cytochrome c (cyt c) into cytosol by disrupting mitochondrial membrane potential indicating induction of cell death in A549 cells through mitochondrial mediated intrinsic apoptosis pathway. Present investigation provides conclusive evidence for application of biogenic nano silver as a potential candidate for anticancer drug development.

Effects of Photodynamic Therapy with Redaporfin on Tumor Oxygenation and Blood Flow in a Lung Cancer Mouse Model

Three photodynamic therapy (PDT) protocols with 15 min, 3 h and 72 h drug-to-light time intervals (DLIs) were performed using a bacteriochlorin named redaporfin, as a photosensitizer. Blood flow and pO2 changes after applying these protocols were investigated in a Lewis lung carcinoma (LLC) mouse model and correlated with long-term tumor responses. In addition, cellular uptake, cytotoxicity and photocytotoxicity of redaporfin in LLC cells were evaluated. Our in vitro tests revealed negligible cytotoxicity, significant cellular uptake, generation of singlet oxygen, superoxide ion and hydroxyl radicals in the cells and changes in the mechanism of cell death as a function of the light dose. Results of in vivo studies showed that treatment focused on vascular destruction (V-PDT) leads to a highly effective long-term antineoplastic response mediated by a strong deprivation of blood supply. Tumors in 67% of the LLC bearing mice treated with V-PDT regressed completely and did not reappear for over 1 year. This significant efficacy can be attributed to photosensitizer (PS) properties as well as distribution and accurate control of oxygen level and density of vessels before and after PDT. V-PDT has a greater potential for success than treatment based on longer DLIs as usually applied in clinical practice.

Abstract 1608: Time-restricted feeding attenuates pulmonary metastasis of Lewis lung carcinoma in mice fed a high-fat diet

Abstract Circadian rhythms, which cycle approximately every 24 hours, control patterns of daily functions, e.g. feeding and fasting, rest and activity, and sleeping and waking. Altering the finely tuned circadian rhythms by an erratic lifestyle (e.g. disruption of the daily feeding and fasting pattern) leads to metabolic disorders, including obesity. Obesity is a risk factor for cancer. Obese cancer patients are at a greater risk of recurrence and metastasis than patients with normal body weight. We hypothesized that time-restricted feeding (TRF) attenuates high-fat diet-enhanced metastasis. In a metastasis model of Lewis lung carcinoma (LLC), male C57BL/6 mice were fed the standard AIN93G diet (16% of energy from soybean oil) or a high-fat diet (HFD) containing 45% of energy from soybean oil with or without dark phase restricted feeding (12 hours per day) for 10 weeks. Pulmonary metastases from a primary tumor, established by subcutaneous injection of LLC cells, were quantified. The percent fat body mass of the TRF group was lower than that of the HFD group and remained similar to that of the AIN93G group. There was no difference in energy intake between the TRF and HFD groups. The number and size of lung metastases were significantly higher in the HFD group than in the AIN93G group; however, there were no differences in these measurements between the TRF and the AIN93G groups. Time-restricted feeding prevented HFD-induced increases in plasma concentrations of glucose, insulin, proinflammatory cytokines (leptin, monocyte chemotactic protein-1, and plasminogen activator inhibitor-1), and angiogenic factors (angiopoietin-2, hepatic growth factor, and vascular endothelial growth factor). We conclude that TRF attenuates the HFD-enhanced pulmonary metastasis of LLC. This protection may be through the entrainment of metabolic regulators to the fixed feeding time that results in reductions in body adiposity and associated cancer-promoting inflammatory and angiogenic factors. Citation Format: Lin Yan, Sneha Sundaram. Time-restricted feeding attenuates pulmonary metastasis of Lewis lung carcinoma in mice fed a high-fat diet [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 1608.

Abstract 661: Adipose specific monocyte chemotactic protein-1 knockdown reduces pulmonary metastasis of Lewis lung carcinoma in mice

Abstract Adipose accumulation is a strong indicator of detrimental health outcomes because adipose tissue produces pro-inflammatory adipokines that participate in many pathological processes including cancer. Monocyte chemotactic protein-1 (MCP-1) is a potent adipokine and body adiposity is positively correlated with adipose MCP-1 expression. This study tested the hypothesis that adipose-produced MCP-1 contributes to metastasis. In a metastasis model of Lewis lung carcinoma (LLC), male adipose MCP-1 knockdown (MCP-1-/-) and wild-type (WT) mice were fed the standard AIN93G diet or a high-fat diet (HFD) containing 16% or 45% of energy from soybean oil. The duration of experimental feeding was 11 weeks. The expression of MCP-1 in epididymal adipose tissue was analyzed by quantitative real-time PCR and ELISA. Pulmonary metastases from a primary tumor, established by subcutaneous injection of LLC cells, were the primary endpoints. There was no significant difference in fat body mass between MCP-1-/- and WT mice fed the same diet. There were no differences in energy intake among the four groups. The HFD increased and adipose MCP-1 knockdown decreased MCP-1 mRNA and protein in adipose tissue compared to their respective controls. The HFD increased the number of metastases formed in the lungs in WT mice. The number of metastasis was lower in MCP-1-/- mice fed the HFD than in WT mice fed the HFD but was higher compared to WT mice fed the AIN93G diet. The volume of metastases was smaller in MCP-1-/- mice than in WT mice, regardless of diet. Adipose MCP-1-/- mice, compared to WT mice, exhibited lower concentrations of insulin, pro-inflammatory adipokines (leptin, plasminogen activator inhibitor-1, and resistin), and angiogenic markers (vascular endothelial growth factor, hepatic growth factor, and angiopoietin-2) in plasma. These results indicate that adipose MCP-1 knockdown may lead to the down-regulation of inflammatory and angiogenic pathways during malignant spread. We conclude that adipose MCP-1 deficiency attenuates HFD-enhanced pulmonary metastasis of LLC and supports our hypothesis that adipose-produced MCP-1 contributes to malignant spread. Citation Format: Sneha Sundaram, Lin Yan. Adipose specific monocyte chemotactic protein-1 knockdown reduces pulmonary metastasis of Lewis lung carcinoma in mice [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 661.

Abstract 3735: An anti-tumor immune response is evoked by partial-volume single dose radiation

Abstract Purpose: To study tumor growth delay resulting from partial irradiation in preclinical mouse models. Methods and Materials: We investigated 67NR murine orthotopic breast tumors in both immunocompetent and nude mice. Treatment was delivered to 50% or 100% of the tumor using a 2x2 cm collimator on a micro-irradiator. Radiation response was modulated by treating with anti-CD8 and anti-ICAM antibodies. Similar experiments were performed using the less immunogenic Lewis Lung Carcinoma (LLC) mouse model. Tumor growth delay and γH2AX phosphorylation were measured and immune response was assessed by immunofluorescence and flow cytometry at 1 and 7 days post-radiotherapy (RT). Tumor expression of cellular adhesion molecules was also measured at different times post-RT. Results: Partial irradiation led to tumor responses similar to fully exposed tumors in immunocompetent mice, but not in nude mice. After a single dose of 10Gy, infiltration of CD8+ T cells was observed, along with increased expression of ICAM. The response to 10Gy in hemi-irradiated tumors was abrogated by treatment with either anti-CD8 or anti-ICAM antibodies. Similar responses were obtained in the less immunogenic LLC mouse model delivering 15Gy to half the tumor volume. Treatment with FTY720, a compound that inhibits T cell egress from lymph nodes, did not affect tumor response at the time of CD8+ T cells infiltration in the non-irradiated area of the tumor, indicating that the most likely source of these cells is the irradiated portion of the hemi-irradiated tumors. In addition, a significant abscopal effect was observed after partial irradiation with a single dose of 10Gy in the 67NR model. Conclusions: In these models, radiation controls tumor growth both directly through cell killing and indirectly through immune activation. This raises the possibility that this effect could be induced in the clinic. Citation Format: Adriana Haimovitz-Friedman, Johnn Humm, Joseph O. Deasy, Jedd Wolchok, Taha Merghoub, James Russell, Hongyan Li, Robert Samstein, Sadna Budhu, Simon Powell, Chloe Bodden, Ela Markovsky, Qing Chen. An anti-tumor immune response is evoked by partial-volume single dose radiation [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 3735.

Lipidomic Composition of Lewis Lung Carcinoma Metastases from Mice Fed an Obesogenic, High-fat Diet (P08-126-19)

ObjectivesMetastasis is a hallmark of cancer. Diet-induced obesity enhances lung metastasis from the primary tumor of Lewis lung carcinoma (LLC) in mice and the lipidomic profile of LLC primary tumor reflects the fatty acid composition of the diet. The objective of this study was to evaluate the impact of feeding an obesogenic, high-fat diet (HFD) on the lipidomic composition of resulting LLC metastases in mice. MethodsLung metastases were obtained from LLC primary tumor-bearing mice fed the AIN93G diet or a HFD providing 16% or 45% of energy from soybean oil. Triacylglycerols (TAG) in the metastases were analyzed by using a mass spectrometry method wherein the ion source was calibrated with an equimolar TAG standard. Neutral mass losses were used to determine the relative content of selected fatty acids across Brutto structures. Lipidomic data were analyzed by using MetaboAnalyst software (Version 4.0) with a false discovery rate of 0.05. ResultsAnalysis of TAG composition in metastases identified 51 separate TAGs by Brutto structure. Six TAGs were significantly different between the two diet groups. Concentrations of TAGs containing 48 and 50 acyl carbons were lower and those containing 54 acyl carbons were higher in the HFD-fed mice. In mice fed the HFD, TAG 54:6 was higher whereas TAGs 48:1 and 50:1 were lower in the metastases than in mice-fed the AIN93G diet. The volcano plot analysis of TAGs showed that differences in 3 TAGs between the two groups were at least two-fold. In neutral loss analysis, the acyl chain C and saturation values of 36:4 and 36:5 corresponding to 18:2_18:2 and 18:2_18:3, respectively, were significantly higher in the HFD group than in the AIN93G group. Increases in these linoleic and linolenic acid-containing species reflected the higher concentrations of these fatty acids in the HFD. ConclusionsThe lipidomic profile of LLC metastases reflects the fatty acid composition of the diet. It suggests that lipidomic alteration of the metastases by an obesogenic diet may contribute to the aggressiveness of metastatic growth in the lungs. Funding SourcesUSDA ARS 3062-51000-050-00D.

Farnesoid X Receptor Constructs an Immunosuppressive Microenvironment and Sensitizes FXRhighPD-L1low NSCLC to Anti-PD-1 Immunotherapy.

The farnesoid X receptor (FXR) regulates inflammation and immune responses in a subset of immune-mediated diseases. We previously reported that FXR expression promotes tumor cell proliferation in non-small cell lung cancer (NSCLC). Here we study the relevance of FXR to the immune microenvironment of NSCLC. We found an inverse correlation between FXR and PD-L1 expression in a cohort of 408 NSCLC specimens; from this, we identified a subgroup of FXRhighPD-L1low patients. We showed that FXR downregulates PD-L1 via transrepression and other mechanisms in NSCLC. Cocultured with FXRhighPD-L1low NSCLC cell lines, effector function and proliferation of CD8+ T cell in vitro are repressed. We also detected downregulation of PD-L1 in FXR-overexpressing Lewis lung carcinoma (LLC) mouse syngeneic models, indicating an FXRhighPD-L1low subtype in which FXR suppresses tumor-infiltrating immune cells. Anti-PD-1 therapy was effective against FXRhighPD-L1low mouse LLC tumors. Altogether, our findings demonstrate an immunosuppressive role for FXR in the FXRhighPD-L1low NSCLC subtype and provide translational insights into therapeutic response in PD-L1low NSCLC patients treated with anti-PD-1. We recommend FXRhighPD-L1low as a biomarker to predict responsiveness to anti-PD-1 immunotherapy.

Biological and anti-vascular activity evaluation of ethoxy-erianin phosphate as a vascular disrupting agent.

The effects of ethoxy-erianin phosphate (EBTP) on cellproliferation, mitotic cell arrest, migration, infiltration, and endothelial tubular structures were evaluated in this study. The antiproliferative activity of EBTP and combretastatin A-4P (CA4P) was analyzed on several tumor cells (including MCF-7, HeLa, 2LL, and 2LL-IDO) and on an endothelial cell (human umbilical vein endothelial cells [HUVECs]) as well as a human normal liver cell (L02). The results showed that EBTP possessed antiproliferative activity in the micromole range and was relatively less toxic than CA4P. Treating HUVECs with EBTP caused cell accumulation in the G2/M phase, and wound-healing assays indicated that EBTP inhibited cell migration. Furthermore, EBTP and CA4P destroyed the vasculature in endothelial cells and showed vascular disrupting activity of the chorioallantoic membrane in fertilized chicken eggs. In addition, we found that EBTP suppressed the expression of indoleamine 2,3-dioxygenase (IDO) and significantly inhibited IDO-induced migration and infiltration of 2LL-IDO cells. Administration of EBTP blocked vasculogenic mimicry in 2LL-IDO cells, which was typically observed in tube formation assays of 2LL-IDO cells. Moreover, the results of Lewis lung carcinoma in mice showed a high inhibition rate of EBTP. EBTP is an effective vascular disrupting agent that is superior to CA4P and may prevent and treat malignancy by inhibiting the expression of IDO.

Cytotoxic efficacy of Nannochloropsis Extracts on Lung Carcinoma in Mice

Cytotoxic efficacy of Nannochloropsis Extracts on Lung Carcinoma in Mice

Time-restricted Feeding Attenuates High-fat Diet-enhanced Spontaneous Metastasis of Lewis Lung Carcinoma in Mice.

Obesity is a risk factor for cancer. Disruption of the daily feeding and fasting rhythm can contribute to obesity. This study tested the hypothesis that time-restricted feeding (TRF) attenuates obesity-enhanced metastasis. In a spontaneous metastasis model of Lewis lung carcinoma (LLC), male C57BL/6 mice were fed the standard AIN93G diet or a high-fat diet (HFD) with or without dark-phase restricted feeding (12 h per day) for 10 weeks. Pulmonary metastases from a subcutaneous tumor were quantified. The number and size of lung metastases were greater in the HFD group than in the AIN93G group, but did not differ between the TRF and AIN93G groups. TRF prevented HFD-induced increases in plasma concentrations of glucose, insulin, proinflammatory cytokines (leptin, monocyte chemotactic protein-1, plasminogen activator inhibitor-1), and angiogenic factors (angiopoietin-2, hepatic growth factor, vascular endothelial growth factor). TRF attenuates the HFD-enhanced spontaneous metastasis of LLC in mice.

IDO1 Inhibition Overcomes Radiation-Induced “Rebound Immune Suppression” by Reducing Numbers of IDO1-Expressing Myeloid-Derived Suppressor Cells in the Tumor Microenvironment

The limitation of hypofractionated radiation efficacy is due partly to the immunosuppressive tumor microenvironment. Indoleamine 2,3-dioxygenase 1 (IDO1) is an important regulator of tumor immune suppression. We evaluated the effects of IDO1 in hypofractionated radiation using a Lewis lung carcinoma (LLC) mouse model and tested whether IDO1 inhibition could sensitize those tumors to hypofractionated radiation. Bilateral LLC tumors were established in C57BL/6 mice. Primary tumors were treated with 3 fractions of either 12Gy or 6Gy, and the IDO1 inhibitor INCB023843 was given starting on the first day of radiation. Plasma tryptophan and kynurenine levels were quantified by liquid chromatography and tandem mass spectrometry. Tumor-infiltrating immune cells were isolated from the tumors, stained, and quantified by flow cytometry. The combination of INCB023843 and three 12-Gy fractions led to better tumor control and survival than radiation alone; INCB023843 plus three 6-Gy fractions had no benefit. IDO1 expression by tumor-infiltrating immune cells was increased by three 12-Gy doses and inhibited by the addition of INCB023843. Nearly all IDO1+ immune cells were also F4/80+. Percentages of IDO1+F4/80+ immune cells were drastically increased by three 12-Gy fractions and by three 6-Gy fractions, but only INCB023843 combined with three 12-Gy fractions reduced those percentages. IDO1+F4/80+ immune cells were further found to be CD11b+, Gr1-intermediate-expressing, CD206-, and CD11c- (ie, myeloid-derived suppressor cells). Three 12-Gy fractions also increased the percentages of tumor-infiltrating T regulatory cells and CD8+ T cells, but adding INCB023843 did not affect those percentages. In addition to its immune activation effects, hypofractionated radiation induced "rebound immune suppression" in the tumor microenvironment by activating and recruiting IDO1-expressing myeloid-derived suppressor cells in a dose-dependent manner. Adding an IDO1 inhibitor to hypofractionated radiation reduced the percentages of these cells, overcame the immune suppression, and sensitized LLC tumors to hypofractionated radiation.

Synthetic Phenolic Antioxidant TS-13 Suppresses the Growth of Lewis Lung Carcinoma and Potentiates Oncolytic Effect of Doxorubicin.

ROS are important intracellular messengers; their ambiguous role in malignant processes was demonstrated in many studies. The effects of a synthetic phenolic antioxidant sodium 3-(3'-tert-butyl-4'-hydroxyphenyl)propyl thiosulfonate sodium (TS-13) on the tumor growth and oncolytic properties of doxorubicin were studied in the experimental model of Lewis lung carcinoma in mice. In mice receiving TS-13 with drinking water (100 mg/kg), suppression of tumor growth by 32.3% was observed on day 21 after inoculation of Lewis lung carcinoma cells. Two-fold intraperitoneal injections of doxorubicin in a cumulative dose of 8 mg/kg were followed by inhibition of tumor growth by 49.5%. Combined treatment with TS-13 and doxorubicin suppressed the tumor growth by 55.4%. In contrast to doxorubicin, TS-13 inhibited NO generation by peritoneal macrophages. The results show the prospect of studying TS-13 in the context of overcoming drug-resistance of tumors.

Yifei Tongluo, a Chinese Herbal Formula, Suppresses Tumor Growth and Metastasis and Exerts Immunomodulatory Effect in Lewis Lung Carcinoma Mice.

This study was aimed to investigate the anti-tumor, anti-metastasis and immunomodulatory effects of Yifei Tongluo (YFTL), a Chinese herbal formula, in Lewis lung carcinoma mice and to explore the underlying mechanisms. LLC cells were inoculated subcutaneously in C57BL/6 mice to establish the Lewis lung carcinoma model. We observed that YFTL effectively inhibited tumor growth and prolonged the overall survival of tumor-bearing mice. Additionally, YFTL treatment resulted in a significantly decreased number of surface lung metastatic lesions compared with the model control group. Meanwhile, TUNEL staining confirmed that the tumors from YFTL-treated mice exhibited a markedly higher apoptotic index. The results suggest that Akt and mitogen-activated protein kinase (MAPKs) pathways may be involved in YFTL-induced apoptosis. The results show that YFTL also inhibited the vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMP)-2, MMP-9, N-cadherin, and Vimentin expression, but increased E-cadherin expression. Mechanistic studies indicated that YFTL could suppress the angiogenesis and the epithelial-mesenchymal transition (EMT) of the tumor through Akt/ERK1/2 and TGFβ1/Smad2 pathways. In addition, YFTL also showed immunomodulatory activities in improving the immunosuppressive state of tumor-bearing mice. Therefore, our findings could support the development of YFTL as a potential antineoplastic agent and a potentially useful anti-metastatic agent for lung carcinoma therapy.

Interaction of aminophylline with photoilluminated riboflavin leads to ROS mediated macromolecular damage and cell death in benzopyrene induced mice lung carcinoma

Riboflavin (Rf) or vitamin B₂ is a known photosensitizer whose photophysical and photochemical properties are well established. Aminophylline (Am) is a phosphodiesterase inhibitor and is currently used as a bronchodilator. Although there are several reports of haemolytic and proteolytic interaction of photoilluminated riboflavin with aminophylline, the cytotoxicity of this system against malignant tissue is not well defined and fully unravelled. Here, we are evaluating anticancer activity of this system against B(a)P induced lung carcinoma in swiss albino mice. We observed marked increment in the level of cellular redox scavengers as well as oxidative stress markers. A significant DNA damage was observed using comet assay. Histopathological studies further confirmed induction of apoptosis in lung tissues of Am-Rf treated animals. Scanning electron microscopy revealed altered surface morphology of the malignant tissue, which characteristically improved in the treatment group. Since malignancy is characterised by compromised redox status, therefore, further increment in ROS due to the action of this system derives cellular system towards extensive macromolecular damage and consequent ROS mediated apoptosis. We anticipate the usage of this system in developing efficient photodynamic therapy against lung cancer that can be clinically realised.

Apoptotic and antimetastatic activities of betulin isolated from Quercus incana against non-small cell lung cancer cells.

BackgroundGlobally, the prevalence and mortality rates of lung cancer have been escalated with the increasing trend of tobacco smoking. The toxicity and irresponsive nature of the available drugs for lung cancer treatment demands an alternative approach.MethodsIn this study, four known compounds namely, cirsimaritin (4′,5, -dihydroxy-6,7-di-methoxyflavone) (1), eupatorin (5,3′-dihydroxy-6,7,4′-trimethoxyflavone) (2), betulin (Lup-20 (29)-ene-3, 28-diol) (3), and β-amyrin acetate (12-Oleanen-3yl acetate) (4) have been isolated from the leaves extract of Quercus incana. Preliminary screening of these natural compounds (1–4) was performed against non-small cell lung carcinoma (NCI-H460) and normal mouse fibroblast (NIH-3T3) cell lines.ResultsThe compounds were found to be antiproliferative against cancer cells with wide therapeutic index in comparison to the normal cells. Effects of betulin (3) on cell migration, invasion, apoptosis, and expression of important apoptosis- and metastasis-related markers were observed at different concentrations. The results showed significant dose-dependent induction of apoptosis after the treatment with betulin (3) followed by increased expression of the caspases family (ie, caspase-3, -6, and -9), proapoptotic genes (BAX and BAK), and inhibiting anti-apoptotic genes (BCL-2L1 and p53). Furthermore, wound healing and transwell invasion assays suggested that betulin (3) could also regulate metastasis by inhibiting MMP-2/-9. Osteopontin, a central regulator of apoptosis and metastasis was also inhibited in a dose-dependent manner.ConclusionThe present findings suggest that betulin (3) can be an attractive chemotherapeutic target for treating resistant lung cancers.

Dendritic cells pulsed with placental gp96 promote tumor-reactive immune responses

Defining and loading of immunogenic and safe cancer antigens remain a major challenge for designing dendritic cell (DC)-based cancer vaccines. In this study, we defined a prototype strategy of using DC-based vaccines pulsed with placenta-derived heat shock protein gp96 to induces anti-tumor T cell responses. Placental gp96 was efficiently taken up by CD11c+ bone marrow-derived DCs (BMDCs) and resulted in moderate BMDC maturation. Splenocytes and cytotoxic T cells (CTLs) generated with mouse BMDCs pulsed with placental gp96 specifically lysed B16 melanoma and LLC lung carcinoma cells. In both transplantable melanoma and lung carcinoma mice models, immunization with placental gp96-stimulated BMDCs led to a significant decrease in tumor growth and mouse mortality with respect to mice treated with liver gp96-pulsed BMDCs or placental gp96 alone. This vaccine induced strong cross-reactive tumor-specific T cell responses. Our results revealed that DCs pulsed with placenta-derived gp96 represent an effective immunotherapy to induce tumor-reactive immune responses, possibly via loading DCs with its associated carcinoembryonic antigens.

Multiwalled Carbon Nanotubes Prevent Tumor Metastasis Through Switching M2-Polarized Macrophages to M1 via TLR4 Activation.

Targeting tumor-associated macrophages (TAMs) has emerged as a novel therapeutic strategy for cancer metastasis. Here, we investigated whether carboxylated multiwalled carbon nanotubes (MWCNTs-COOH) prevent tumor metastasis through regulating macrophage polarization. In Lewis lung carcinoma (LLC) or B16F10 melanoma-bearing mice, intratracheal instillation of MWCNTs-COOH significantly reduced metastatic burden in the lungs. MWCNTs-COOH promoted the expression of M1 markers (iNOS and CXCR10) and inhibited the expression of M2 markers (CD206 and Arg-1) along with an increased expression of Th1 cytokines (TNF-α and IL-12) and decreased expression of Th2 cytokines (TGF-β and IL-10). Such changes were accompanied with TLR4 mRNA and protein elevation. Importantly, macrophage depletion in mice lungs reversed the anti-metastatic effects of MWCNTs-COOH. In vitro, MWCNTs-COOH switched IL-4/13-treated macrophages to the M1 phenotype and thus prevented the migration and invasion of LLC cells, accompanied by the upregulation of toll-like receptor (TLR)-4/NF-κB p65 signaling. Moreover, TAK-242 (resatorvid), a specific TLR4 inhibitor, reversed the effects of MWCNTs-COOH on macrophage polarization. In summary, MWCNTs-COOH effectively prevent tumor metastasis through skewing M2-polarized macrophages to M1 via activating TLR4/NF-κB signaling. Thus, targeting TAMs by MWCNTs-COOH is a potential therapeutic approach against tumor metastasis.

Evaluation of Oncolytic Viral Therapy Combined with or without Artificial Immunoadjuvant Cell Therapy in Immunocompetent Mouse Tumor Models

Oncolytic viruses have a great potential to provide clinical improvement for cancer patients who do not respond to conventional therapies. The mechanisms of action include direct infection, immune-mediated cell death, and vascular collapse. We previously reported that a miRNA-regulated, thymidine kinase-deleted vaccinia virus (MDVV) specifically infects multiple myeloma (MM) cells and showed that the intravenous injection of MDVV significantly prolonged survival of severe combined immunodeficiency mice harboring a subcutaneous human RPMI8226 MM tumor (Blood 2014:124:2082, Mol Ther Oncolytics 2017;6:57). Although it was highly effective, there remained to be confirmed whether the MDVV would be similarly effective in the immunocompetent setting. To address this question, we performed several in vivo infection using immunocompetent C57BL/6 (B6) models.A vaccinia virus (VV) strain LC16, which was derived from the Lister strain, was widely used in 1970s for the smallpox eradication program with no severe adverse effect. Because its safety is robust, we used the LC16 for the backbone of the MDVV. To improve its safety and tumor specificity, we deleted viral thymidine kinase gene, and introduced miRNA (let-7a)-target sequences into the critical viral B5R gene. Because let-7a is abundant in normal differentiated tissues, and the endogenous let-7a down-regulates viral B5R gene, the MDVV becomes far more tumor specific (Mol Ther Oncolytics 2017;6:57).To observe whether the MDVV protects immunocompetent mice from MM, we generated a mouse MM model by transplanting 1x10^6 Vk*Myc tumor cells into syngeneic B6 mice. After confirming M-proteins in the serum, 1x10^7 pfu of MDVV was injected intravenously. Contrary to our expectation, we could not observe a clearly infected region, nor a prolongation of survival of these mice; probably due to difference in infectivity between species. Although mouse derived cell lines are generally insensitive to vaccinia virus as compared with human cell lines, a mouse lung carcinoma cell line 3LL showed a relatively high infectivity to VV in our screening in vitro infection experiment (Figure), therefore we used the B6/3LL subcutaneous tumor model instead of B6/Vk*Myc model.The B6/3LL mice were injected with mock or 1x10^7 pfu MDVV intravenously when the subcutaneous tumor became more than 5 mm in diameter. Although viral signal (determined by in vivo imaging using luciferase) disappeared 3 days after infection, the therapeutic benefit had remained, and prolongation of survival was observed Because natural killer (NK) cells were thought to play in the clearance of VV in the B6 mice, we attempted to extend the duration of infection by depleting NK cells using anti-asialo GM1 antibody, which, we thought, enhances viral oncolytic activity. As expected, we observed a persistent viral signal for up to 2 weeks in anti-asialo GM1 antibody treated B6/3LL mice. However, the anti-tumor effect seen with anti-asialo GM1 antibody was rather milder than that of without anti-asialo GM1 antibody, indicating the importance of NK activities in anti-tumor immunity.Based on this finding, we, in reverse, tried to stimulate the anti-tumor immunity using artificial immunoadjuvant cells co-expressing CD1d and tumor antigen/GM-CSF fusion protein. The CD1d is one of non-classical MHC molecules and is involved in the presentation of lipid antigens. When α-GlaCer is loaded on the CD1d, it activates natural killer T (NKT) cells through the interaction with invariant T cell receptor on NKT cells. When activated, NKT cells produce Th1 and Th2 cytokines, and activate NK cells. GM-CSF stimulates antigen-presenting cells (APCs), and fusion proteins of tumor antigen and GM-CSF are more efficiently taken up by APCs than tumor antigen alone (Nature 1993, 362:755). We generated the artificial immunoadjuvant cells by transducing 3LL tumor cells with lentiviral vectors expressing CD1d and ovalbumin (OVA)/GM-CSF fusion protein. After loading with α-GlaCer, the immunoadjuvant cells were irradiated with 50Gy, and injected intravenously into B6/3LL-OVA mice. We observed a significant reduction in tumor mass and prolongation of survival as compared with mock-injected mice. Currently, we are exploring the optimal setting of combination with MDVV virotherapy and immunoadjuvant cell therapy. In this meeting, we will present these results. DisclosuresNo relevant conflicts of interest to declare.

142 - Antitumor effect of synthetic phenolic antioxidant TS-13 in Lewis lung carcinoma mouse model

Reactive oxygen and nitrogen species are important intracellular messengers that are strongly implicated in tumor processes, yet their precise role in cancerogenesis is still largely unclear and often debatable. Plant and synthetic phenols and polyphenols are known to have antitumor effect through multiple mechanisms most importantly the one that activates Keap1/Nrf2/ARE pathway. We have previously demonstrated that water-soluble synthetic phenolic antioxidant sodium 3-(3′-tert-butyl-4′-hydroxyphenyl) propyl thiosulfonate (TS-13) increased cell susceptibility to doxorubicin in cell culture. Antitumor action of TS-13 was attributed to its effect on mitochondria followed by Keap1/Nrf2/ARE activation and subsequent autophagy and apoptosis. The effect of TS-13 on tumor growth and the oncolytic effect of doxorubicin was studied in experimental model of transplantable tumor (Lewis lung carcinoma, LLC) in mice. On day 21 after inoculation of LLC cells, tumor growth was inhibited by 32.3% in mice that received TS-13 with drinking water (100 mg/kg). Two intraperitoneal injections of doxorubicin in a cumulative dose of 8 mg/kg body weight reduced tumor growth by 49.5%, while co-administration of TS-13 and doxorubicin resulted in tumor growth inhibition by 55.4%. Animals with inoculated tumor (+ or - doxorubicin) had significantly higher rate (by 27 and 39%, respectively, p