Mouse Lewis Lung Carcinoma Research Articles

YAP Involves in Reduced Anabolic Response to Functional Overload in Lewis Lung Carcinoma‐bearing Mice

Cancer‐associated weight loss, also known as cachexia, is connected to poor prognosis, reduced response to chemotherapy, and impaired patient quality of life. While resistance exercise/muscle contraction is a promising therapy, anabolic resistance, the suppressed ability to induce protein synthesis, is present and can’t be solely elucidated by the change of mTOR signaling. Yes‐associated protein (YAP) is a downstream effector of Hippo signaling and is known to control skeletal muscle size in response to mechanical stimulus. However, the role of YAP on anabolic resistance in cancer cachexia needs to be clarified. The purpose of this study was to determine whether the state of YAP activity after functional overload is altered in cachectic mice. Approximately three‐month‐old male C57BL6 mice were divided into two groups: Control group with PBS (100 μl) injection (Con, n=5) and tumor‐implantation group with Lewis lung Carcinoma (LLC) cells injection (2×105 cells in 100 μl PBS, LLC, n=5). Injection was made at the right flank under anesthesia. Four weeks after injection tumor growth was visible, synergist ablation surgery was performed on the left leg (Overload) and the right leg served as an internal control with sham surgery (Sham). Puromycin (0.04 μmol/g body weight, BW) was injected 30 minutes prior to sacrifice and plantaris muscles (PLAN) were removed and snap‐frozen at day 7 following the surgery. After total protein was extracted, routine Western blotting was performed using 60~120 μg of the total protein. Paired t‐test (Sham vs. Overload) and unpaired t‐test (Con vs. LLC) were used for statistical analysis and the levels of significance was set at 0.05. PLAN weights were comparable between Con and LLC with sham surgery (19.8±1.2 mg vs. 17.3±0.5 mg for Con and LLC, respectively, p=0.077). Seven‐day functional overload increased PLAN weights regardless of tumor inoculation, but the degree of hypertrophy was different (42.7±4.0% vs. 27.6±4.6% for Con and LLC, respectively, p<0.05). LLC mice showed reduced levels of phosphorylated p70S6K (p‐p70) with sham surgery (1.0±0.3 vs. 0.4±0.1, for Con and LLC, respectively, p<0.05) and those with SA surgery (4.4±0.7 vs. 1.6±0.7, for Con and LLC, p<0.05). In harmony with these results, the basal levels of muscle protein synthesis in LLC mice were decreased by 51% compared to Con mice and the levels remained lower after functional overload (2.1±0.1 vs. 1.1±0.1 for Con and LLC, respectively, p<0.05). Interestingly, the basal phosphorylated levels of YAP (p‐YAP) were elevated in LLC mice (1.0±0.1 vs. 2.2±0.1, for Con and LLC, respectively, p<0.05). However, functional overload did not alter the levels of p‐YAP in LLC mice while Con mice significantly increased the levels by 55% (p<0.05). In conclusion, the data suggest that YAP plays a role, at least in part, in decreased anabolic response to functional overload in LLC‐bearing mice.Support or Funding InformationSupported by Louisiana Board of Regents Support Fund (LEQSF(2017‐20)‐RD‐A‐22) to SS.

Anti-angiogenic effect of a chemically sulfated polysaccharide from Phellinus ribis by inhibiting VEGF/VEGFR pathway

Previous studies have demonstrated that the sulfated polysaccharide named PRP-S16 could inhibit the proliferation, migration, and tube formation of endothelial cells in vitro. Here, its anti-angiogenic effect and mechanism in vivo were investigated by Lewis lung carcinoma (LLC) mice model. PRP-S16 significantly reduced the microvessel density (MVD) of tumor, exhibiting a high tumor growth inhibitory effect in LLC mice. All designed assays including quantitative real-time PCR, immunohistochemistry, enzyme-linked immunosorbent assay and western blotting showed that PRP-S16 reduced the mRNA and the protein expression of vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2) in serum or tumor tissue of mice. Western blotting also detected decreased phosphorylated (p)-VEGFR-1, p-VEGFR-2, hypoxia-inducible factor-1α (HIF-1α), protein kinase B (Akt), and matrix metalloproteinases-9 (MMP-9). PRP-S16 had no adverse effects on angiogenesis in non-target organs. These findings suggested that the mechanism of anti-angiogenesis of PRP-S16 in vivo was due to inhibition of VEGF/VEGFR signaling pathway and it might be a promising candidate for tumor by anti-angiogenic therapy.

Synergic effect of PD-1 blockade and endostar on the PI3K/AKT/mTOR-mediated autophagy and angiogenesis in Lewis lung carcinoma mouse model

BackgroundImmunotherapy has been shown to be effective as a first-line treatment option for non-small cell lung cancer (NSCLC) patients. Unfortunately, it has failed to acquire an anticipant anti-tumour effect for relatively lower clinical benefit rates. It is therefore important to identify novel strategies for improving immunotherapy. Endostar is a novel recombinant human endostatin that exerts its anti-angiogenic effects via vascular endothelial growth factor (VEGF)-related signalling pathways. Anti-programmed death receptor 1 (PD-1) antibody is an immune checkpoint inhibitor that was developed to stimulate the immune system. In this study, the synergy of PD-1 blockade and endostar was assessed in a lung carcinoma mouse model. MethodsLewis lung carcinoma (LLC)-bearing mice were randomly assigned into three groups: controls, anti-PD-1 and anti-PD-1+endostar. The levels of cytokines such as interleukin (IL)-17, transforming growth factor-β1 (TGF-β1) and interferon-γ (IFN-γ) were measured with enzyme-linked immune sorbent assay (ELISA). The expression of VEGF, CD34 and CD31 was assessed with immunohistochemistry (IHC). The proportion of mature dendritic cells (mDC) and myeloid-derived suppressor cells (MDSC) was analysed with flow cytometry. The major proteins in PI3K/AKT/mTOR and autophagy were quantified with Western blot. ResultsAnti-PD-1 combined with endostar dramatically suppressed tumour growth in LLC mouse models. This synergistic effect resulted in decreased pro-inflammatory cytokine IL-17 and immunosuppressive factor TGF-β1 levels, increased IFN-γ secretion, reduced myeloid-derived suppressor cell (MDSC) accumulation, and reversed CD8 + T cell suppression. The expression of VEGF, CD34 and CD31 was significantly down-regulated, while tumour cell apoptosis and PI3K/AKT/mTOR-mediated autophagy was up-regulated. ConclusionThe combination of anti-PD-1 and endostar has a remarkably synergic effect on LLC tumour growth by means of improving the tumour microenvironment and activating autophagy.

Mechanism of Marsdenia tenacissima extract promoting apoptosis of lung cancer by regulating Ca2+/CaM/CaMK signaling

Ethnopharmacological relevanceMarsdenia tenacissima is a traditional Chinese medicine that has been used since the Ming dynasty. It is famous for its apoptotic effects on lung cancer. However, limited information is available about the underlying mechanisms. Aim of the studyWe aimed to determine the mechanisms by which different organic M. tenacissima extracts induce apoptosis in lung cancer cells. Materials and methodsPubMed and CNKI databases were screened for M. tenacissima components that may targets lung cancer; 223 components were selected for drug-like and pharmacokinetic analysis. Moreover, the inhibitory effect of different extracts of M. tenacissima on tumor cells was illustrated using CCK-8 and apoptosis assays, and intracellular [Ca2+] was measured. The in vivo effects were examined by body weight, tumor pathology, and TUNEL staining analysis in a Lewis lung carcinoma mouse model. In vivo levels of the Ca2+ signaling-related proteins Calmodulin, CaMKⅡ, p-CaMKⅡ, MEK1/2, p-MEK1/2, ERK, and p-ERK were measured by Western blot. ResultsPetroleum ether and ethyl acetate extracts of M. tenacissima have stronger inhibitory effects than other extracts on lung cancer cells, with IC50 values of 0.35 ± 0.04 mg/ml and 0.29 ± 0.02 mg/ml for LLC cells and 0.56 ± 0.05 mg/ml and 0.85 ± 0.04 mg/ml for A549 cells, respectively. Compared with the normal control group, A549 and LLC cells of treatment groups exhibited morphological changes typical of apoptosis, and the apoptosis rate was significantly higher, as determined by flow cytometry. Furthermore, the i[Ca2+] changed accordingly, causing a decrease in vivo in Calmodulin, CaMKⅡ, p-CaMKⅡ, p-MEK1/2 and p-ERK levels. ConclusionsM. tenacissima induces apoptosis, both in vitro and in vivo. Hydrophobic extracts are most effective; they increase i[Ca2+], decrease intracellular Calmodulin, CaMKⅡ, p-CaMKⅡ, p-MEK1/2 and p-ERK levels, and activate the apoptotic cascade.

Specific overexpression of 15-lipoxygenase in endothelial cells promotes cancer cell death in an in vivo Lewis lung carcinoma mouse model

PurposeLipoxygenases (LOX) have been implicated in carcinogenesis, however both pro- and anti-carcinogenic effects have been reported in different cancer models. Using transgenic mice, which specifically overexpress human 15-lipoxygenase (ALOX15) in endothelial cells (EC), we previously demonstrated significant inhibition of tumor development. In the Lewis lung carcinoma (LLC) model, the primary tumor developed similarly in both wild type (WT) and ALOX15 overexpressing mice. However, metastases development was significantly inhibited in the transgenic mice. Here, we explored the molecular basis for the anti-metastatic effect of endothelial cell specific ALOX15 overexpression. Materials/methodsWe used ALOX15 overexpressing mice, and in-vitro cell model to evaluate the molecular effect of ALOX15 on EC and LLC cells. ResultsWhen LLC cells were injected in WT and ALOX15 overexpressing mice, we observed a higher degree of apoptosis and necrosis in primary and metastatic tumors of ALOX15 overexpressing animals. These anti-carcinogenic and anti-metastatic effects were paralleled by augmented expression of cyclin-dependent kinase inhibitor 1A (CDKN1A; p21) and of the peroxisome proliferators-activated receptor (PPAR)γ and by downregulation of the steady state concentrations of connexin26 mRNA. Consistent with these in vivo effects, ALOX15 overexpression in LLC and HeLa cancer cells in vitro significantly reduced cell viability in culture. In contrast, similar treatment of non-cancerous B2B epithelial cells did not impact cell viability. ConclusionsTaken together, our data suggests that endothelial cell specific overexpression of ALOX15 promotes apoptosis and necrosis in primary and metastatic tumors in mice, by upregulation of P21 and PPARγ expression in adjacent cancer cells.

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.

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.

Controlling the Phenotype of Tumor-Infiltrating Macrophages via the PHD-HIF Axis Inhibits Tumor Growth in a Mouse Model.

SummaryThe tumor microenvironment (TME) polarizes tumor-infiltrating macrophages toward tumor support. Macrophage-abundant tumors are highly malignant and are the cause of poor prognosis and therapeutic resistance. In this study, we show that the prolyl hydroxylase (PHD) inhibitor FG-4592 (FG) inhibits tumor growth of macrophage-abundant tumors and prolongs mouse survival. FG not only normalizes tumor vessels and improves tumor oxygenation but also directly affects macrophages and activates phagocytosis through the PHD-hypoxia-inducible factor (HIF) axis. Remarkably, FG can promote phagocytic ability of the Ly6Clo subset of tumor-infiltrating macrophages, leading to tumor growth inhibition. Moreover, Ly6Cneg macrophages contributed to blood vessel normalization. Using a malignant tumor mouse model, we characterized macrophage function and subsets. Altogether, our findings suggest that the PHD inhibitor can promote the anti-tumor potential of macrophages to improve cancer therapy.

PGC-1α activator-induced fatty acid oxidation in tumor-infiltrating CTLs enhances effects of PD-1 blockade therapy in lung cancer.

The present study aims to investigate the efficacy and mechanisms of peroxisome proliferator-activated receptor γ coactivator 1-α agonist, as adjuvant to programmed death-1 (PD-1) blockade in hyporesponsive lung cancer cells-derived in vivo tumor model, using bezafibrate. Mouse Lewis lung carcinoma (LLC) xenograft models were established and treated with programmed death-ligand 1 (PD-L1) monoclonal antibodies with or without bezafibrate. Tumors or peripheral blood of mice were harvested to investigate the quality, quantity, and function as well as energetic metabolism of cytotoxic T lymphocytes (CTLs) by flow cytometry or quantitative real-time polymerase chain reaction. The combination of bezafibrate plus anti-PD-L1 reached synergistic tumoricidal effect in LLC xenograft mouse models, even though bezafibrate alone had no effect on tumor growth. Bezafibrate significantly facilitated CD8+ T cells infiltrating into tumor tissues by enhancing the expression of CXCL9 and CXCL10 within tumors as well as the receptor CXCR3 in infiltrating CTLs. Activated CTLs within tumors were also significantly upregulated by bezafibrate. Further data demonstrated that bezafibrate treatment could maintain the survival and functional capacity of tumor-infiltrating CTLs. Moreover, cellular reactive oxygen species in infiltrating CTLs and fatty acid oxidation (FAO)-related genes (PGC-1α, Cpt1a, and LCAD) expression within tumors were significantly increased after treatment with bezafibrate. Bezafibrate synergized the tumoricidal effect of PD-1 blockade in hyporesponsive lung cancer by expansion of effector CTLs within tumor microenvironment. The potential mechanism may relate to the capacity of bezafibrate in regulating FAO of tumor-infiltrating CTLs.

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.

Cryptotanshinone has curative dual anti-proliferative and immunotherapeutic effects on mouse Lewis lung carcinoma

Lung cancer is currently the leading cause of cancer-related mortality with very limited effective therapy. Screening of a variety of traditional Chinese medicines (TCMs) for their capacity to inhibit the proliferation of human lung cancer A549 cells and to induce the in vitro maturation of human DCs led to the identification of cryptotanshinone (CT), a compound purified from the TCM Salvia miltiorrhiza Bunge. Here, CT was shown to inhibit the proliferation of mouse Lewis lung carcinoma (LLC) cells by upregulating p53, downregulating cyclin B1 and Cdc2, and, consequently, inducing G2/M cell-cycle arrest of LLC cells. In addition, CT promoted maturation of mouse and human DCs with upregulation of costimulatory and MHC molecules and stimulated DCs to produce TNFα, IL-1β, and IL-12p70, but not IL-10 in vitro. CT-induced maturation of DCs depended on MyD88 and also involved the activation of NF-κB, p38, and JNK. CT was effective in the treatment of LLC tumors and, when used in combination with low doses of anti-PD-L1, cured LLC-bearing mice with the induction of subsequent anti-LLC long-term specific immunity. CT treatment promoted T-cell infiltration and elevated the expression of genes typical of Th1 polarization in LLC tumor tissue. The therapeutic effect of CT and low doses of anti-PD-L1 was reduced by depletion of CD4 and CD8 T cells. This paper provides the first report that CT induces immunological antitumor activities and may provide a new promising antitumor immunotherapeutic.

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.