Mouse Lewis Lung Carcinoma Research Articles

The influence of an enamine usnic acid derivative (a tyrosyl-DNA phosphodiesterase 1 inhibitor) on the therapeutic effect of topotecan against transplanted tumors in vivo.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a repair enzyme for 3'-end DNA lesions, predominantly stalled DNA-topoisomerase 1 (Top1) cleavage complexes. Tdp1 is a promising target for anticancer therapy based on DNA damage caused by Top1 poisoning. Earlier, we have reported about usnic acid enamine derivatives that are Tdp1 inhibitors sensitizing tumor cells to the action of Top1 poison (Zakharenko in J Nat Prod 79:2961-2967, 2016). In the present work, we showed a sensitizing effect of an enamine derivative of usnic acid (when administered intragastrically) on Lewis lung carcinoma in mice in combination with topotecan (TPT, Top1 poison used in the clinic). In the presence of the usnic acid derivative, both the volume of the primary tumor and the number of metastases significantly diminished. The absence of acute toxicity of this compound was demonstrated, as was the importance of the method of its administration for the manifestation of the sensitizing properties.

Abstract 2588: Oral administration of water extract from Euglena gracilis prevents lung carcinoma growth in mice by alteration of intestinal microbiota

Abstract Euglena gracilis, a single-celled alga, is rich in nutrients and thus, used as a nutritional dietary supplement. This alga can be found in both fresh and saltwater and possesses characteristics of both plants and animals. Euglena gracilis extracts have a wide range of medicinal properties including stimulation of anticancer immunity against multiple types of cancers; however, the anticancer mechanism has not yet been fully elucidated. Therefore, a water extract from Euglena gracilis devoid of water-insoluble mature paramylons was evaluated as an anticancer agent against lung carcinoma. Two different extracts were prepared using dried powder from whole Euglena gracilis. First, partially purified water extract (EWE) was prepared by suspending dry powder in PBS and centrifugation at 10,000g for 20 min followed by a filtration using a 0.22µm pore size membrane. Second, boiled EWE (bEWE) was prepared by immersing unfiltered EWE in boiling water for 12 min followed by centrifugation at 10,000g for 10 min and filtration using 0.22µm pore size membrane. Both EWE and bEWE treatments inhibited the growth of lung carcinoma cells in vitro in a dose- and time-dependent manner. Furthermore, both extracts significantly inhibited the three-dimensional growth of Lewis Lung Carcinoma (LLC) cells. Flow cytometry analysis showed that the EWE treatment attenuates granulocytic myeloid-derived suppressor cells (MDSCs) in bone marrow cell cultures. The in vivo study was conducted using a mouse LLC orthotopic allograft model. Oral administration of EWE and bEWE (100-200 mg/kg/day) three weeks prior to LLC cell inoculation attenuated the tumor growth in the lungs of immunocompetent mice while decreasing the peripheral granulocytes. However, this attenuation was not seen for the extract treatment initiated after LLC cell inoculation. The tumor growth attenuation was more efficient with the bEWE treatment than with EWE treatment. The fecal microbiomes of the mice were analyzed by 16s rRNA gene amplicon sequencing which revealed that alpha diversity in three groups (EWE, bEWE, and PBS control) was similar, however, the microbial compositions of the EWE- and bEWE-treated mouse groups were more diversified than in the PBS group. Specifically, an increase in the ratio of Bacteroidetes to Firmicutes was observed, and a significant increase in Akkermansia and Muribaculum was detected in EWE- and bEWE- treated mice compared to PBS treated mice. These studies suggest that oral administration of partially purified water extracts from Euglena gracilis altered the intestinal microbiome and the alteration may attenuate host MDSCs, thereby preventing lung carcinoma growth. This study was supported by 2016EUGLENA-RC1 (MT), 2017EUGLENA-RC2 (MT and JC), Kansas State University College of Veterinary Medicine SMILE award (MT and JC), and NIH grant P20 RR017686 (MT). Citation Format: Deepa Upreti, Susumu Ishiguro, Mayme Loyd, Nicole Robben, Paige Cote, Morgan Phillips, Ayaka Nakashima, Kengo Suzuki, Jeffrey Comer, Masaaki Tamura. Oral administration of water extract from Euglena gracilis prevents lung carcinoma growth in mice by alteration of intestinal microbiota [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2588.

Tumor burden negatively impacts protein turnover as a proteostatic process in noncancerous liver, heart, and muscle, but not brain.

Cancer survivors are more susceptible to pathologies such as hypertension, liver disease, depression, and coronary artery disease when compared with individuals who have never been diagnosed with cancer. Therefore, it is important to understand how tumor burden negatively impacts nontumor-bearing tissues that may impact future disease susceptibility. We hypothesized that the energetic costs of a tumor would compromise proteostatic maintenance in other tissues. Therefore, the purpose of this study was to determine if tumor burden changes protein synthesis and proliferation rates in heart, brain, and liver. One million Lewis lung carcinoma (LLC) cells or phosphate-buffered saline (PBS, sham) were injected into the hind flank of female mice at ∼4.5 mo of age, and the tumor developed for 3 wk. Rates of proliferation and protein synthesis were measured in heart, brain, liver, and tumor tissue. Compared with sham, rates of protein synthesis (structural/nuclear, cytosolic, mitochondrial, and collagen) relative to proliferation were lower in the heart and liver of LLC mice, but higher in the brain of LLC mice. In the tumor tissue, the ratio of protein synthesis to DNA synthesis was approximately 1.0 showing that protein synthesis in the tumor was used for proliferation with little proteostatic maintenance. We further provide evidence that the differences in tissue responses may be due to energetic stress. We concluded that the decrease in proteostatic maintenance in liver, heart, and muscle might contribute to the increased risk of disease in cancer survivors.NEW & NOTEWORTHY We present data showing that simultaneously measuring protein synthesis and cell proliferation can help in the understanding of protein turnover as a proteostatic process in response to tumor burden. In some tissues, like hepatic, cardiac, and skeletal muscle, there was a decrease in the protein to DNA synthesis ratio indicating less proteostatic maintenance. In contrast, the brain maintained or even increased this protein to DNA synthesis ratio indicating more proteostatic maintenance.

Effect of Micelle-Incorporated Cisplatin With Sizes Ranging From 8 to 40nm for the Therapy of Lewis Lung Carcinoma.

Insufficient transport of therapeutic cargo into tumor bed is a bottleneck in cancer nanomedicine. Block copolymers are promising carriers with smaller particle size by ratio modification. Here, we constructed cisplatin nanoparticles with sizes ranging from 8 to 40 nm to study the permeability and therapy of Lewis lung carcinoma. We synthesized methoxypoly(ethylene glycol)2000-block poly(L-glutamic acid sodium salt)1979 loading cisplatin through complexation reaction. The cisplatin nanomedicine has high drug loading and encapsulation efficiency. In vitro data demonstrated that cisplatin nanoparticles had equivalent growth-inhibiting effects on Lewis lung carcinoma cells compared to free cisplatin. In vivo evidences showed cisplatin nanoparticles had superior antitumor effects on the Lewis lung carcinoma mouse model with no obvious side effects. All results indicated that optimizing the ratio of block copolymers to obtain smaller sized nanomedicine could act as a promising strategy for overcoming the inadequate accumulation in poorly vascularized tumors.

P53.04 KUgliptin, a Novel and Potent DPP4 Inhibitor, Improves of Combined Therapy with Immune Checkpoint Inhibitor (ICI) in Lung Cancer Immunotherapy

Lung cancer is the most common cause of death rate among all cancer related diseases. Although several drugs including immunotherapeutics (Immune checkpoint inhibitor; ICI) have been developed and used in recent years, the response rate is limited to about 25%. Therefore, many researchers have tried to combine treatment with other drugs to increase the therapeutic efficiency of ICI. Dipeptidyl peptidase 4 (DPP4) is a ubiquitously expressed transmembrane exopeptidase on the cell surface of many hematopoietic cells. According to some reports, DPP4 inhibitors have the ability to suppress tumor proliferation and tumor metastasis. KUgt-123, is a novel and potent DPP4 inhibitor, it is chemical structural based in adamantane structure modified. This study was performed to combined treatment of KUgt-123 for improvement of therapeutic efficacy of immune checkpoint inhibitor for lung cancer. To confirm the cytotoxicity effect of KUgt-123, it was dose dependently treated in human lung carcinoma epithelial cell line (A549) and mouse Lewis lung carcinoma (LLC) cells then using MTT assay. PD-L1 expression level was determined by western blot analysis. TC-1 tumor-bearing mice were treated with either PD-1 antibody treatment group and/or KUgt-123 treatment. KUgt-123 was shown to inhibit the cell proliferation in A549 cells and LLC cells. The result that cell growth was reduced dose dependently of KUgt-123. PD-L1 expression was reduced by KUgt-123 treatment in TC-1 cells. TC-1 tumor bearing C57BL/6 mice, the size of tumors was reduced by alone KUgt-123 treatment and significantly reduced co-treatment with mouse PD-1 antibody. Our data provide evidence that KUgt-123 might be a good co-treatment reagent for improves the efficacy of ICI in lung cancer treatment.

Murine cancer cachexia models replicate elevated catabolic pembrolizumab clearance in humans.

BackgroundMonoclonal antibody (mAb) immune checkpoint inhibitor (ICI) therapies have dramatically impacted oncology this past decade. However, only about one‐third of patients respond to treatment, and biomarkers to predict responders are lacking. Recent ICI clinical pharmacology data demonstrate high baseline drug clearance (CL0) significantly associates with shorter overall survival, independent of ICI exposure, in patients receiving ICI mAb therapies. This suggests CL0 may predict outcomes from ICI therapy, and cachectic signalling may link elevated CL0 and poor response. Our aim was to determine if mouse models of cancer cachexia will be useful for studying these phenomena and their underlying mechanisms.MethodsWe evaluated pembrolizumab CL in the C26 and Lewis lung carcinoma mouse models of cancer cachexia. A single treatment of vehicle or pembrolizumab, at a dose of 2 or 10 mg/kg, was administered intravenously by tail vein injection. Pembrolizumab was quantified by an ELISA in serial plasma samples, and FcRn gene (Fcgrt) expression was assessed in liver using real‐time quantitative reverse transcription PCR. Non‐compartmental and mixed‐effects pharmacokinetics analyses were performed.ResultsWe observed higher pembrolizumab CL0 and decreased Fcgrt expression in whole liver tissue from tumour‐bearing vs. tumour‐free mice. In multivariate analysis, presence of tumour, total murine IgG, muscle weight and Fcgrt expression were significant covariates on CL, and total murine IgG was a significant covariate on V1 and Q.ConclusionsThese data demonstrate increases in catabolic clearance of monoclonal antibodies observed in humans can be replicated in cachectic mice, in which Fcgrt expression is also reduced. Notably, FcRn activity is essential for proper antigen presentation and antitumour immunity, which may permit the study of cachexia's impact on FcRn‐mediated clearance and efficacy of ICI therapies.

Z-guggulsterone induces PD-L1 upregulation partly mediated by FXR, Akt and Erk1/2 signaling pathways in non-small cell lung cancer

Programmed death-ligand 1 (PD-L1) is an immune checkpoint molecule, that is overexpressed in non-small cell lung cancer (NSCLC) and has been associated with the response to anti-PD-1/PD-L1 immunotherapy. Z-guggulsterone (Z-GS), an active compound extracted from the gumresin of the Commiphora mukul tree, has been shown to have anti-tumor effects in NSCLC in our previous study. However, whether Z-GS could affect PD-L1 expression levels in tumor cells remains unknown. In this study, we verified the inhibitory effects of Z-GS on NSCLC cell viability and cell cycle progression in vitro, and mouse Lewis lung carcinoma (LLC) tumor growth in vivo. Notably, Z-GS treatment increased PD-L1 surface and mRNA expression levels, and gene transcription in NSCLC cells, in a dose- and time-dependent manner. Mechanistic experiments showed that the upregulation of PD-L1 was mediated, partly by farnesoid X receptor inhibition, and partly by the activation of the Akt and Erk1/2 signaling pathways in Z-GS-treated NSCLC cells. In vivo, Z-GS treatment dose-dependently increased PD-L1 expression levels in mouse LLC tumor models. Overall, our findings demonstrated a promoting role for Z-GS in PD-L1 expression in NSCLC and provided mechanistic insights, that may be used for further investigation into synergistic combined therapies.

AURUM POLYACRYLATE: THE EXPERIMENTAL STUDY OF THE ANTITUMOR ACTIVITY

Introduction. The investigation of metal substituted organic compounds as potential antitumor drugs is one of the promising areas of research in experimental and clinical oncology.Objective. The pre-clinical study of the original antitumor drug aurum polyacrylate (aurumacryl) which belongs to such new for oncology group of compounds as polyacrylates of metals was the aim of this work.Materials and methods. Aurumacryl antitumor activity was determined as the tumor growth inhibitory effect against some of the murine solid tumors (Lewis lung carcinoma, Acatol adenocarcinoma and Ca-755 adenocarcinoma). Drug cytotoxic effect against some of the human tumor cells (Mel-Mo melanoma, A549 lung carcinoma, MCF-7 breast carcinoma, HCT116 colon adenocarcinoma) was evaluated with standard МТТ-test. The aurumacryl pharmacokinetics in tumor bearing mice (Lewis lung carcinoma) was studied. The inductively coupled plasma mass spectrometry method was used for the estimation of the aurum maintenance in the tested tissues (tumor, blood, kidneys, liver, lungs, spleen, brain).Results. The 80–90 % tumor growth inhibitory effect of aurumacryl against some solid tumors in mice had been revealed in vivo as well as the death of the 60–90 % human tumor cells of various origins in vitro. Beside this the strong decrease of the number of proliferating MCF-7 tumor cells had been shown. The distribution of aurumacryl in the body of the mice with the solid tumor had been revealed.Conclusion. On the base of the data obtained the further study of the aurumacryl as a potential antitumor agent seems rather promising.

Self-Assembled Disulfide Bond Bearing Paclitaxel-Camptothecin Prodrug Nanoparticle for Lung Cancer Therapy.

Self-assembled prodrugs (SAPDs), which combine prodrug strategy and the merits of self-assembly, not only represent an appealing type of therapeutics, enabling the spontaneous organization of supramolecular nanocomposites with defined structures in aqueous environments, but also provide a new method to formulate existing drugs for more favorable outcomes. To increase drug loading and combination therapy, we covalently conjugated paclitaxel (PTX) and camptothecin (CPT) through a disulfide linker into a prodrug, designated PTX-S-S-CPT. The successful production of PTX-S-S-CPT prodrug was confirmed by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). This prodrug spontaneously undergoes precipitation in aqueous surroundings. Taking advantage of a flow-focusing microfluidics platform, the prodrug nanoparticles (NPs) have good monodispersity, with good reproducibility and high yield. The as-prepared prodrug NPs were characterized with dynamic light scattering (DLS) and transmission electron microscopy (TEM), demonstrating spherical morphology of around 200 nm in size. In the end, the self-assembled NPs were added to mouse embryonic fibroblast (MEF), mouse lung adenocarcinoma and Lewis lung carcinoma (LLC) cell lines, and human non-small cell lung cancer cell line A549 to evaluate cell viability and toxicity. Due to the redox response with a disulfide bond, the PTX-S-S-CPT prodrug NPs significantly inhibited cancer cell growth, but had no obvious toxicity to healthy cells. This prodrug strategy is promising for co-delivery of PTX and CPT for lung cancer treatment, with reduced side effects on healthy cells.

Toll-like receptor 4 mediates the development of fatigue in the murine Lewis Lung Carcinoma model independently of activation of macrophages and microglia

Cancer-related fatigue at the time of tumor diagnosis is commonly attributed to inflammation associated with the disease process. However, we have previously demonstrated that running wheel deficits occur well before increased expression of proinflammatory cytokines in the liver and brain in a murine model of human papilloma virus-related head and neck cancer (mEER). Further, we have demonstrated that genetic deletion of type I interleukin-1 receptor and MyD88 has no effect. In the current investigation we sought to test the generality of this finding by assessing whether there is a role for toll-like receptor (TLR) 4-dependent inflammation in the fatigue-like behavior observed in mice with Lewis Lung Carcinoma (LLC) or mEER tumors. Genetic deletion of TLR4 attenuated tumor-induced elevations in liver pro-inflammatory cytokine expression in both models. However, it only abrogated wheel running deficits in LLC tumor bearing mice. To determine whether TLR4 signaling in the LLC model involves innate immune cells, mice were treated with the colony stimulating factor (CSF)-1 receptor antagonist PLX-5622 before and throughout tumor development to deplete microglia and peripheral macrophages. Administration of PLX-5622 had no protective effect on wheel running deficits in either mEER or LLC tumor models despite effective depletion of microglia and a down regulation of peripheral proinflammatory cytokine expression. These results indicate that the TLR4 signaling that mediates fatigue-like behavior in LLC mice is not dependent upon microglial or peripheral macrophage activation. Based on the literature and our data demonstrating attenuation of ubiquitin proteasome pathway activation in the gastrocnemius muscle of Tlr4−/− mice implanted with LLC cells, we interpret our current findings as indication that skeletal muscle TLR4 signaling may be involved. These results are important in that they add to the evidence that tumor-induced fatigue develops independently from classical neuroinflammation.

Identification and validation of novel DNA methylation markers for early diagnosis of lung adenocarcinoma.

Lung cancer has the highest mortality of all cancers worldwide. Epigenetic alterations have emerged as potential biomarkers for early diagnosis of various cancer tissue types. To identify methylation markers for early diagnosis of lung adenocarcinoma, we aimed to integrate genome-wide DNA methylation and gene expression data from The Cancer Genome Atlas. To this end, we first examined the global DNA methylation pattern of lung adenocarcinoma and investigated the relationship between DNA methylation subtypes and clinical features. We then extracted differentially methylated and expressed genes, and adopted feature selection techniques to determine the final methylation markers. The performance of the markers in predicting lung adenocarcinoma was evaluated on three independent datasets from Gene Expression Omnibus. Protein levels of marker genes were validated by immunohistochemistry, and their biological function was further verified invivo. We identified three novel methylation markers in lung adenocarcinoma including cg08032924, cg14823851, and cg19161124, mapping to CMTM2, TBX4, and DPP6, respectively. Validating these results on three independent datasets indicated that the three markers can achieve extremely high sensitivity and specificity in distinguishing lung adenocarcinoma from normal samples. Immunohistochemistry quantification results confirmed that markers are weakly expressed in human lung adenocarcinoma, and CMTM2 decreased tumor growth of mouse Lewis lung carcinoma invivo. Overall, our study identified three novel methylation markers in lung adenocarcinoma which may contribute toward an improved diagnosis potentially leading to a better outcome for patients with lung adenocarcinoma.

Mechanisms of Fritillariae Thunbergii Flos in lung cancer treatment from a systems pharmacology perspective

Ethnopharmacological relevanceFritillariae Thunbergii Flos (FTF) included in the Chinese Pharmacopoeia (1977 Edition) is a Chinese medicinal herb traditionally used to treat bronchitis. In recent years, it has been applied in the treatment of lung cancer. However, the molecular mechanism remains largely unknown. MethodsThe screening of bioactive compounds, acquisition of drug targets, network construction, and experimental validation in vivo were combined to explored the mechanism of FTF in the treatment of lung carcinoma with regards to systems pharmacology. ResultsThe network Lung Cancer Pathway consisted of 114 nodes (44 compounds and 70 potential targets) and 361 edges, as well as modules that included inflammatory response, angiogenesis, negative regulation of the apoptotic process, and positive regulation of cell proliferation and migration. It was examined by conducting experiments that involved the administration of ethanol-based extracts of FTF in Lewis lung carcinoma mice. The extracts exerted excellent anti-lung cancer effects in vivo by significantly inhibiting tumor proliferation, thereby extending the survival period of tumor-bearing mice. Moreover, FTF induced the downregulation of PIK3CG, Bcl-2, eNOS, VEGF, p-STAT3, and STAT3 genes in tumor-bearing mice. ConclusionsThe findings of the present study verify the therapeutic effects and mechanism of FTF on lung cancer and provide a theoretical basis to support the comprehensive utilization of FTF resources.

Abstract 3459: Water extract from Euglena gracilis prevents lung carcinoma growth in mice via attenuation of myeloid-derived suppressor cells and granulocytes

Abstract Phytochemicals have been developed into treatments against many diseases, including various cancers. Euglena is a genus of single-cell flagellate eukaryotes having features of both animals and plants. These organisms, which are found in both fresh and salt water, have been used as a dietary supplement for their rich nutrient content. Although recent studies suggest that Euglena extracts have anti-tumor properties, the mechanism is yet to be clarified. Accordingly, the anti-tumor and immunomodulatory effects of partially purified water extract from Euglena gracilis (EWE) was evaluated in cell culture and a mouse orthotopic lung carcinoma allograft model. The EWE was prepared by a PBS extraction at 37°C for 30 min, centrifugation at 2,000 g for 20 min, and filtration through a membrane filter (0.22 μm pore size). Primary components of this extract are water soluble materials and exclude mature paramylons, intact chloroplasts, and oil droplets. In two-dimensional cell culture, the EWE treatment inhibited cell growth of both murine Lewis lung carcinoma (LLC) and human lung carcinoma cells (A549 and H1299) in a dose (1-100 μg/ml) and time (24-72 h)-dependent manner. In contrast, the growth of mouse bone marrow cells (BMCs), but not mouse splenocytes (SPLs) were dose (1-100 μg/ml) and time (24-72 h)-dependently stimulated by the treatment with EWE. In three-dimensional spheroid culture, spheroid growth of LLC cells was significantly attenuated by EWE treatment. Flow cytometry revealed that the EWE treatment attenuated granulocytic myeloid-derived suppressor cells in BMCs. In a mouse LLC orthotopic allograft model, pretreatment with EWE (100-200 mg/kg/day, via drinking water) three weeks prior to LLC cell inoculation significantly attenuated the growth of LLC tumors in the lungs of immunocompetent syngeneic mice. However, this attenuation was not seen for EWE treatment initiated after LLC cell inoculation. The tumor growth attenuation by EWE pretreatment coincided with a significant decrease of myeloid-derived cells, primarily neutrophils. EWE pretreatment appears to inhibit lung carcinoma growth mainly by stimulating host anti-tumor immunity via an attenuation of the host myeloid-derived immune suppressor cells. Hence, the present study suggests that the partially purified extract derived from Euglena gracilis contains bioactive materials that prevent lung carcinoma growth. This study was supported by 2016EUGLENA-RC1 (MT), 2017EUGLENA-RC2 (MT and JC), a Kansas State University College of Veterinary Medicine SMILE award (MT and JC), and NIH grant P20 RR017686 (MT). Citation Format: Susumu Ishiguro, Deepa Upreti, Nicole Robben, Page Cote, Riley Burghart, Damilola Ogun, Tran Le, Jennifer Delzeit, Jayson Carswell, Arashi Nakashima, Ayaka Nakashima, Kengo Suzuki, Jeffrey Comer, Masaaki Tamura. Water extract from Euglena gracilis prevents lung carcinoma growth in mice via attenuation of myeloid-derived suppressor cells and granulocytes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3459.

Abstract 3457: Plasma metabolomic changes in mice with time restricted feeding-attenuated pulmonary metastasis of Lewis lung carcinoma

Abstract Erratic eating behavior disrupts diurnal rhythms of metabolism and may contribute to obesity. Time-restricted feeding (TRF), in which food is available in the active period of the day (the dark phase for nocturnal animals), restores metabolic homeostasis in mice. In a mouse model of Lewis lung carcinoma (LLC), we found that TRF (12 hours, dark phase) attenuates obesity-enhanced spontaneous metastasis to the lungs in a study that compared differences in the extent of metastasis among four dietary groups: non-LLC-bearing mice fed the standard AIN93G diet and LLC-bearing mice fed the AIN93G, an obesogenic high-fat diet (HFD) or TRF with the HFD. With the goal of determining potential plasma fingerprints underlying TRF efficacy, we performed untargeted metabolomic analysis upon the plasma samples from this study. Using gas chromatography time-of-flight mass spectrometry, we identified 152 compounds of which 24 differed among the four groups. Ten analytes that were the most influential in treatment separation were alpha-tocopherol, phenylalanine, isoleucine, leucine, pyruvic acid, dihydrocholestrol, arachidonic acid (ARA), docosahexaenoic acid (DHA), lactic acid, and cholesterol. In mice fed the AIN93G diet, compared to non-LLC-bearing mice LLC elevated alpha-tocopherol, ARA, isoleucine, phenylalanine, cholesterol, lauric acid, and 4-hydroxybenzoate and decreased lactic acid, lactamide, and pyruvic acid. In LLC-bearing mice, compared to the AIN93G diet the HFD elevated asparagine, leucine, phenylalanine, dehydrocholesterol, p-cresol sulfate, and hexuronic acid and decreased 1,5-anhydroglucitol, palmitoleic acid, myristic acid, lauric acid, and 4-hydrobenzoate. Compared to the HFD, TRF elevated linoleic acid and 2-hydroxyhexanoic acid and decreased alpha-tocopherol, asparagine, isoleucine, leucine, phenylalanine, dihydrocholesterol, p-cresol sulfate, hexuronic acid, and DHA. This study showed that LLC metastatic growth and an increase in dietary fat consumption disrupted metabolic homeostasis, as reflected in plasma analyte changes compared to their respective controls. TRF, through its temporal regulation of food intake, restored HFD-mediated metabolic disruption and may have contributed to its inhibition of obesity-enhanced LLC metastasis. Citation Format: Lin Yan, Bret M. Rust, Matthew J. Picklo. Plasma metabolomic changes in mice with time restricted feeding-attenuated pulmonary metastasis of Lewis lung carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3457.

BMP2 signalling activation enhances bone metastases of non-small cell lung cancer.

Distant metastases occur when non‐small cell lung cancer (NSCLC) is at late stages. Bone metastasis is one of the most frequent metastases of NSCLC and leads to poor prognosis. It has been reported that high expression of BMP2 in NSCLC correlates with poor survival, but whether BMP2 contributes to NSCLC bone metastasis remains largely unknown. The activation of BMP signalling is found in metastatic bone tumours of mice Lewis lung carcinoma and predicts poor survival in human NSCLC. BMP2 signalling activation can enhance bone metastasis of Lewis lung carcinoma. Moreover, BMP2 secreted by stroma fibroblasts can promote the migration and invasion of NSCLC cells. Besides, in combination with pre‐osteoblast and LLCs, BMP2 could enhance the differentiation of macrophages into osteoclasts to play roles in the osteolytic mechanism of NSCLC bone metastasis. Interestingly, NSCLC cells can also enrich BMP2 to pre‐osteoblasts to function in the osteoblastic mechanism. Our results firstly demonstrate the detailed mechanisms about what roles BMP2 signalling play in enhancing NSCLC bone metastases. These findings provide a new potential therapy choice for preventing bone metastases of NSCLC via the inhibition of BMP2 signalling.

Knockdown of IKKβ Inhibits Tumor Development in a Leptomeningeal Metastasis Mouse Model and Proliferation of Lung Cancer Cells.

ObjectiveThis study will explore the role of IKKβ in the leptomeningeal metastasis (LM) of lung cancer cells.MethodsIn vitro studies were conducted in mouse Lewis lung carcinoma (LLC) cells with IKKβ knockdown. Cell proliferation was explored using CCK-8 and tumor colony-forming assays. The expression of the extracellular signal-regulated kinase (ERK) signaling pathway was detected by Western blot. Tumor cell apoptosis was identified through Western blot detection of Bax and Bcl-2. The migration of tumor cells was identified by a wound healing assay. In vivo studies used an LM mouse model developed by injecting LLC cells with or without IKKβ knockdown into the cisterna magna. Mouse brain and spinal samples were sectioned for hematoxylin and eosin staining.ResultsIn vitro: IKKβ knockdown inhibits tumor cell proliferation, initiates apoptosis, and attenuates cell migration. In vivo: IKKβ knockdown inhibits tumor growth in the LM mouse model. In addition, the in vitro results showed that IKKβ knockdown attenuated the expression of ERK phosphorylation in LLC cells.ConclusionBlocking the NF-κB signaling pathway by IKKβ knockdown in LLC cells inhibited tumor growth in the LM mouse model. IKKβ supports leptomeningeal tumor progression by promoting cancer cell proliferation and migration and inhibiting cancer cell apoptosis, and these actions may be correlated to ERK signaling.

Gemcitabine and checkpoint blockade exhibit synergistic anti-tumor effects in a model of murine lung carcinoma

Lung cancer is leading cause of cancer death in the world. Chemotherapy is currently one of the standard treatments for lung cancer. Gemcitabine is a pyrimidine nucleoside drug which has been approved by FDA to treat lung cancer. However, acquired resistance inevitable develops after Gemcitabine treatment, limiting clinical efficacy. Lewis lung carcinoma (LLC) cells were treated with Gemcitabine and cell apoptosis and programmed cell death-ligand 1 (PD-L1) expression were analyzed by flow cytometry. LLC mouse model was established to analysis the proportion and programmed cell death-1 (PD-1) expression of CD8 + T cells. Anti-tumor effect by treating with Gemcitabine and anti-PD-1 antibody was measured through in vivo LLC mouse model. Gemcitabine treatment induces tumor cell apoptosis and PD-L1 expression. Further study showed that Gemcitabine treatment also increases CD8+ and CD4+ T cells proportion, PD-1 and PD-L1 expression in LLC mouse model. Combination therapy with Gemcitabine and αPD-1 not only has strong anti-tumor effect, but also could inhibit postsurgical recurrence of LLC. Our findings demonstrated that the combination therapy of Gemcitabine and αPD-1 is an effective therapeutic strategy for lung cancer.

A UPLC-Q-TOF/MS-based plasma metabolomics approach reveals the mechanism of Compound Kushen Injection-based intervention against non-small cell lung cancer in Lewis tumor-bearing mice

Compound Kushen Injection (CKI), a well-known Chinese Medicine preparation, has been used to treat non-small cell lung cancer (NSCLC) for more than 15 years, and its clinical curative effect is considered to be beneficial. This study was designed to evaluate the effects and underlying mechanisms of CKI against NSCLC using an ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS)-based plasma metabolomics approach. 4',6-diamidino-2-phenylindole (DAPI) staining and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) dye reduction assay were employed to assess apoptosis and the viability of A549 cells with and without CKI treatment. The weight/volume of Lewis lung carcinoma (LLC) sarcomas and histopathological examinations were used to evaluate the anti-tumor effects of CKI against NSCLC. A UPLC-Q-TOF/MS method combined with multivariate data analysis was developed to characterize metabolomic fingerprinting and to screen functional biomarkers that are linked to the CKI treatment of LLC mice, and then metabolic pathway analysis was used to investigate the therapeutic mechanism of CKI. DAPI staining and MTT dye reduction assays indicated that CKI-induced apoptosis and inhibited the proliferation of A549 cells, respectively, in a concentration-dependent manner. The sarcoma volumes and weights in LLC tumor-bearing mice in CKI-dosed groups were significantly lower than those in a model group, which was treated with physiological saline. Histopathological analysis of sections of sarcomas and left pulmonary lobes indicated that CKI exerts an ameliorative effect against LLC. Fourteen functional biomarkers that are related to the therapeutic effects of CKI on LLC were screened and identified using a metabolomics study. Analysis of metabolic pathways revealed that the therapeutic effects of CKI on LLC mainly involved glycerophospholipid metabolism, amino acid metabolism and sphingolipid metabolism. As glycerophospholipid metabolism is a crucial feature of cancer-specific metabolism, the enzymes that are involved in 1-acyl-sn-glycero-3-phosphoinositol biosynthesis were further evaluated. Western blotting results indicated that CKI modulated the abnormal biosynthesis pathway of 1-acyl-sn-glycero-3-phosphoinositol by activation of cytidine diphosphate-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) and cytosolic phospholipase A2 (cPLA2), and by inhibition of lysophosphatidic acid acyltransferase gamma (AGPAT3). This study demonstrated that CKI has a favorable anti-tumor effect and that a UPLC-Q-TOF/MS-based metabolomics method in conjunction with further verifications at the biochemical level is a promising approach for investigating its underlying mechanisms.

Antitumor immunity targeting fibroblast activation protein-α in a mouse Lewis lung carcinoma model.

The tumor stromal microenvironment is an integral part of the occurrence and development of tumor. Cancer-associated fibroblasts (CAFs) are a key component of most tumor stromal microenvironments. The present study aimed to investigate the use of CAFs-targeted immunotherapy to fibroblast activation protein-α (FAP-α) expressed in CAFs. Recombinant adenoviral vectors containing the mouse FAP-α cDNA (rAd-FAP-α) were constructed. C57BL/6 mice were immunized with rAd-FAP-α infected dendritic cells (DCs) against FAP-α, which is overexpress in CAFs. The results demonstrated that mice vaccinated with rAd-FAP-α DCs gave rise to potent FAP-α-specific cytotoxic T lymphocytes capable of lysing Lewis lung cancer (LLC) CAFs. Furthermore, mice vaccinated with rAd-FAP-α-transduced DCs induced an effective therapeutic or protective antitumor immunity to LLC in a subcutaneous model, and prolonged overall survival time compared with mice vaccinated with the control recombinant adenovirus-transduced DCs (rAd-c DCs) or DCs alone. The results of the present study suggested that FAP-α, which is preferentially expressed in CAFs, may be considered as a potential target for killing or destroying CAFs within the tumor stromal microenvironment, and may be exploited to develop immunogenic tumor vaccines.

Optimization and Characterization of Protein Nanoparticles for the Targeted and Smart Delivery of Cytochrome c to Non-Small Cell Lung Carcinoma.

The delivery of Cytochrome c (Cyt c) to the cytosol stimulates apoptosis in cells where its release from mitochondria and apoptotic induction is inhibited. We developed a drug delivery system consisting of Cyt c nanoparticles decorated with folate-poly(ethylene glycol)-poly(lactic-co-glycolic acid)-thiol (FA-PEG-PLGA-SH) to deliver Cyt c into cancer cells and tested their targeting in the Lewis Lung Carcinoma (LLC) mouse model. Cyt c-PLGA-PEG-FA nanoparticles (NPs) of 253 ± 55 and 354 ± 11 nm were obtained by Cyt c nanoprecipitation, followed by surface decoration with the co-polymer SH-PLGA-PEG-FA. The internalization of Cyt c-PLGA-PEG-FA nanoparticles (NPs) in LLC cells was confirmed by confocal microscopy. NP caspase activation was more efficient than the NP-free formulation. Caspase activity assays showed NPs retained 88–96% Cyt c activity. The NP formulations were more effective in decreasing LLC cell viability than NP-free formulation, with IC50 49.2 to 70.1 μg/mL versus 129.5 μg/mL, respectively. Our NP system proved to be thrice as selective towards cancerous than normal cells. In vivo studies using near infrared-tagged nanoparticles show accumulation in mouse LLC tumor 5 min post-injection. In conclusion, our NP delivery system for Cyt c shows superiority over the NP-free formulation and reaches a folic acid-overexpressing tumor in an immune-competent animal model.