Mice Bearing Lewis Lung Carcinoma Research Articles

Abstract 4480: Circulating and tumor associated neutrophil subtypes correlate with hyperprogressive disease (HPD) upon immunecheckpoint inhibitors (ICI) in advanced non-small cell lung cancer (aNSCLC) patients (pts) and in vivo models

Abstract Background: HPD was described in ≃10-25% of NSCLC pts upon single-agent ICI and correlated with poor prognosis. High circulating neutrophil count and neutrophils/lymphocytes ratio were associated with shorter survival and HPD in aNSCLC pts. In mouse lung cancer models, interleukin-17 (IL-17) promoted tumor growth upon ICI through increased intratumoral neutrophils. The role of specific circulating and/or tumor-associated neutrophil populations in driving HPD is unknown. Methods: aNSCLC pts treated with ICI and enrolled in a prospective monocentric study were assessed for HPD and circulating neutrophils' phenotype. HPD required 3 tumor measurements (2 before ICI, 1 upon ICI) and was defined as delta tumor growth rate (TGR) TGR (TGR upon ICI - TGR before ICI) >50% and/or TGR ratio (TGR upon ICI/ TGR before ICI) ≥2. Survival curves were estimated by Kaplan Meier and compared by log-rank, correlations with categorical/continuous variables were performed by Fisher/Mann-Whitney tests. The phenotype of circulating low density neutrophils (LDNs) was assessed by flow cytometry (FC) on peripheral blood mononuclear cells from fresh blood samples. Mature and immature LDNs were defined according to the expression of CD10 on CD66b+CD15+ cells, being immature the CD10− neutrophil population. The capacity of immune competent mice to undergo HPD upon anti-PD-1 treatment was further tested in C57BL/6 mice bearing Lewis Lung Carcinoma and treated with anti-murine PD-1. Tumor associated neutrophils' phenotype was assessed by FC analysis. Results: Of 46 aNSCLC, 85% were >65 years, 72% had adenocarcinoma histology, 89% stage IV, 22% had PD-L1 on tumor cells ≥50%, 72% received 1st line ICI. 19 (41%) pts had progression (PD) by RECIST, 4 (9%) pts were HPD by TGR ratio and/or delta-TGR. HPD [4.1 (95% CI 3.1-5) months] correlated with poorer median overall survival compared to non-HPD [6.9 (95% CI 6.1-7.8) months, p=0.03] and to PD pts [4.7 (95% CI 3.2-6.1) months, p=0.8]. Before ICI initiation, the median percentage of circulating immature CD10− CD15+ CD66b+ LDNs was significantly higher in HPD [36.6 (min 29.2; max 64.5)] compared to both non-HPD [8.9 (min 0; max 70.4); p=0.01] and PD non-HPD pts [10.1 (min 0; max 70.4), p=0.04]. HPD defined by TGR ratio and/or delta-TGR occurred in 3 (12.5%) out of 24 immune competent mice treated with anti-murine PD-1. The mean of Gr1highLy6Clow IL-17+ tumor associated neutrophils was significantly higher in HPD compared to non-HPD mice [0.25 (min 0.14; max 0.63) vs 0.06 (min 0.02; max 0.32), p=0.03]. Conclusions: Circulating immature CD10− LDNs and tumor associated IL-17+ neutrophils correlate with HPD upon ICI in aNSCLC pts and in vivo models, respectively. Characterization of neutrophils' functional properties in HPD pts and in vivo models is currently ongoing. Citation Format: Roberto Ferrara, Claudia Proto, Giuseppe Lo Russo, Diego Signorelli, Arsela Prelaj, Giulia Galli, Nicoletta Zilembo, Alessandro De Toma, Giuseppe Viscardi, Riccardo Lobefaro, Marta Brambilla, Monica Ganzinelli, Giuliano Molino, Giovanna Talarico, Marina Chiara Garassino, Mario Colombo, Sabina Sangaletti. Circulating and tumor associated neutrophil subtypes correlate with hyperprogressive disease (HPD) upon immunecheckpoint inhibitors (ICI) in advanced non-small cell lung cancer (aNSCLC) patients (pts) and in vivo models [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 4480.

Circulating and tumor-associated neutrophil subtypes discriminate hyperprogressive disease (HPD) from conventional progression (PD) upon immune checkpoint inhibitors (ICI) in advanced non-small cell lung cancer (NSCLC) patients (pts) and in vivo models.

9547 Background: HPD occurs in ≃10-25% of NSCLC pts upon single-agent ICI and correlates with poor prognosis. High circulating neutrophil count and neutrophils/lymphocytes ratio have been associated with shorter survival and HPD in NSCLC pts. In mouse lung cancer models, interleukin-17 (IL-17) promoted tumour growth upon ICI increasing intratumoral neutrophils. The role of specific circulating and/or tumour-associated neutrophils in driving HPD is currently unknown. Methods: NSCLC pts treated with single agent ICI were assessed for HPD and circulating neutrophils’ phenotype. Conventional PD was defined by RECIST 1.1. HPD required 3 tumour assessments (2 before ICI, 1 upon ICI) and was defined as delta tumour growth rate (TGR) (TGR upon ICI – TGR before ICI) > 50% and/or TGR ratio (TGR upon ICI/ TGR before ICI) ≥2. Correlations with continuous variables were performed by Mann-Whitney test. Circulating low density neutrophils (LDNs) subtypes were assessed by flow cytometry (FC) on peripheral blood mononuclear cells (PBMCs) from fresh blood samples. LDNs were defined as CD66b+CD15+ cells among CD11b+ PBMCs. Immature subtypes were defined as CD10− and CD10−CD16− LDNs. The occurrence of HPD upon anti-PD-1 treatment was tested in C57BL/6 immune competent mice bearing Lewis Lung Carcinoma and treated with anti-murine PD-1. Tumour associated neutrophils’ phenotype was assessed by FC. Results: Of 52 NSCLC, 65% were > 65 years, 83% had stage IV, 25% PD-L1 on tumour cells ≥50%, 67% received 1st line ICI. PD and HPD occurred in 21 (40%) and 5 (10%) pts, respectively. Before ICI start, HPD pts had higher circulating immature neutrophils measured as median percentage of CD10− LDNs [41.9 (min 26.7; max 83.5) vs 10.1 (min 0.69; max 79.3), p = 0.01] and of CD16− cells among CD10− LDNs [93 (min 89.5; max 98.4) vs 86.3 (min 24.2; max 99), p = 0.03] compared to conventional PD pts. PD and HPD occurred in 17 (71%) and 3 (12.5%) of 24 immune competent mice treated with anti-murine PD-1. The median percentage of IL-17+ tumour associated neutrophils (Gr1highLy6Clow) was significantly higher in HPD compared to PD mice [0.25 (min 0.14; max 0.63) vs 0.06 (min 0.02; max 0.32), p = 0.02]. Conclusions: Circulating immature (CD10− and CD10− CD16−) LDNs and IL-17+ tumour associated neutrophils discriminate HPD from conventional PD upon ICI in NSCLC pts and in vivo models, respectively. Functional characterization of specific neutrophil subsets is ongoing.

Toll-Like Receptor-4 Disruption Suppresses Adipose Tissue Remodeling and Increases Survival in Cancer Cachexia Syndrome

Cancer-induced cachexia, characterized by systemic inflammation, body weight loss, adipose tissue (AT) remodeling and muscle wasting, is a malignant metabolic syndrome with undefined etiology. Here, we show that both genetic ablation and pharmacological inhibition of TLR4 were able to attenuate the main clinical markers of cachexia in mice bearing Lewis lung carcinoma (LLC). AT remodelling was not found in LLC tumor-bearing (TB) TLR4−/− mice due to reduced macrophage infiltration and adipocyte atrophy. TLR4−/− mice were also resistant to cold-induced browning of subcutaneous AT (scAT). Importantly, pharmacological inhibition of TLR4 (Atorvastatin) reproduced the main protective effect against AT remodeling found in TLR4−/− TB mice. Moreover, the treatment was effective in prolonging survival and attenuating tumor mass growth when compared to non-treated-TB animals. Furthermore, tumor-induced elevation of circulating pro-inflammatory cytokines was similarly abolished in both genetic ablation and pharmacological inhibition of TLR4. These data suggest that TLR4 is a critical mediator and a promising target for novel anti-cachexia therapies.

Abstract 137: Pharmacologic activation of integrin CD11b as a novel therapeutic strategy against lung cancer

Abstract Lung cancer is the leading cause of cancer related deaths in the United States. With a 5-year survival rate of 18%, lung cancer patients are in immediate need of new therapeutic options. A major problem facing immunotherapy for cancer is the active immune suppression by the tumor. Tumors show presence of large numbers of tumor associated macrophages (TAMs), which suppress the adaptive immune response, increase neo-vascularization to the tumor, and promote tumor survival. Integrin CD11b is highly expressed on TAMs and is essential for their recruitment and biological functions. Reasoning that CD11b activity is important for controlling tumor growth, we developed a novel small molecule agonist called leuhadherin-1 (LA1), which activates CD11b. We found that CD11b activation by LA1 significantly reduced tumor growth in wild type mice bearing Lewis Lung Carcinoma (LLC) tumors as early as one week post treatment. Conversely, LLC tumors grew at a faster rate in CD11b-/- mice, compared to wild type mice, showing that CD11b is important for controlling tumor growth. Importantly, LA1 treatment in tumor bearing CD11b-/- mice did not show any efficacy, demonstrating the specificity of LA1 to CD11b. These data suggest that CD11b activation via LA1 modulates TAMs in tumors and is a novel therapeutic strategy against cancer. Citation Format: Terese Geraghty, Anugraha Rajagopalan, Samia Khan, Judith Varner, Vineet Gupta. Pharmacologic activation of integrin CD11b as a novel therapeutic strategy against lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 137.

High‐intensity interval training slows down tumor progression in mice bearing Lewis lung carcinoma

AbstractBackgroundWe aimed to determine whether a short‐term high‐intensity interval training (HIIT) protocol could counteract tumor progression in an experimental model of lung cancer.MethodsMice were injected subcutaneously with Lewis Lung Carcinoma (LLC) cells and then randomly assigned into two groups: sedentary mice (LLC group) or mice submitted to HIIT (LLC + HIIT group).ResultsLLC + HIIT group had lower tumor mass than LLC group (‐52% after 18 days), with no differences in glycolytic activity as measured by PET/CT imaging. HIIT increased Cd274 (PD‐L1) mRNA expression by ~6 folds and Vegfa mRNA expression by 2.5 folds, suggesting that HIIT stimulates local inflammation and angiogenesis in LLC tumors. Additionally, HIIT improved running capacity, skeletal muscle contractility and survival rate in LLC tumor‐bearing mice.ConclusionsThese novel findings demonstrate that a short‐term HIIT protocol slows down tumor progression, ultimately increasing survival in LLC tumor‐bearing mice. Thus, this study provides novel pre‐clinical evidence that exercise training may be a beneficial co‐therapy for lung cancer.

Immune regulation effect of lienal polypeptides extract in Lewis lung carcinoma-bearing mice treated with cyclophosphamide.

Polypeptides extracted from animal immune organs have been proved to exert immunomodulatory activities in previous reports. However, relative experimental data regarding the influence of a polypeptide mixture extracted from healthy calf spleen (lienal polypeptide [LP]) on the immune function in tumor therapy are limited, and the components in LP remain unclear. In the present study, the immune regulatory effect of LP was investigated in normal mice and Lewis lung carcinoma (LLC)-bearing mice treated with cyclophosphamide (CTX). The components of LP were identified by liquid chromatography-electrospray ionization-coupled with tandem mass spectrometry (LC-MS/MS) analysis and bioinformatic analysis. In LLC-bearing mice, LP showed a synergic antitumor effect with CTX, whereas LP alone did not present direct antitumor activity. Further, LP was found to enhance immune organ indexes, splenocyte number, and T lymphocyte subsets in normal mice and LLC-bearing mice treated with CTX. The decline of white blood cell and platelet counts, splenocyte proliferation activity, and peritoneal macrophage phagocytic function caused by CTX were also significantly suppressed by LP treatment in LLC-bearing mice. Notably, LP treatment significantly decreased the expression of phagocytosis-related proteins including CD47/signal regulatory protein α/Src homology phosphatase-1 in the tumor tissue of LLC-bearing mice treated with CTX. LC-MS/MS-based peptidomics unraveled the main polypeptides in LP with a length from 8 to 25 amino acids. Bioinformatics analysis further confirmed the possibility of LP to regulate immunity, especially in phagocytosis-related pathway. Our above findings indicated that LP can relieve the immunosuppression induced by chemotherapy and is a beneficial supplement in cancer therapy. Impact statement The immunomodulatory activities of polypeptides extracted from animal immune organs have incurred people's interests since a long time ago. In this study, we investigated the immune regulation effects of a polypeptide mixture extracted from health calf spleen (lienal polypeptide [LP]) in Lewis lung carcinoma-bearing mice treated with cyclophosphamide (CTX). Liquid chromatography-electrospray ionization-coupled with tandem mass spectrometry-based peptidomics and bioinformatics analysis unraveled the main polypeptides in LP and further confirmed that LP is mainly associated with immune regulating pathway, especially in tumor cell phagocytosis-related pathway. Our study for the first time revealed that polypeptides from spleen can relieve the immunosuppression induced by CTX and is a beneficial supplement in cancer therapy.

Targeting macrophages for cancer therapy disrupts bone homeostasis and impairs bone marrow erythropoiesis in mice bearing Lewis lung carcinoma tumors

Macrophages are represented in all tissues by phenotypically distinct resident populations that show great functional diversity. Macrophages generally play a protumoral role, and they are attractive targets for cancer therapy. In this study, we found that CD169+ macrophages depletion inhibited the growth of established Lewis lung carcinoma tumors in mice. Benefits must be weighed against potential adverse effects in cancer therapy. Here, we investigated the adverse effects of CD169+ macrophages depletion on bone and bone marrow in mice bearing Lewis lung carcinoma tumors. Our studies showed that depletion of CD169+ macrophages in LLC tumor-bearing mice disrupted bone homeostasis, including bone weight loss and bone mineral density decrease. Further studies revealed that bone marrow erythropoiesis was severely impaired after depletion of CD169+ macrophages in LLC tumor-bearing mice. Our findings suggest that depletion of macrophages for cancer therapy may be associated with potential adverse effects that need to be recognized, prevented, and optimally managed.

Montelukast Induces Apoptosis-Inducing Factor-Mediated Cell Death of Lung Cancer Cells.

Developing novel chemo-prevention techniques and advancing treatment are key elements to beating lung cancer, the most common cause of cancer mortality worldwide. Our previous cohort study showed that cysteinyl leukotriene receptor antagonists, mainly montelukast, decreased the lung cancer risk in asthma patients. In the current study, we conducted in vivo and in vitro experiments to demonstrate the inhibiting effect of montelukast on lung cancer and to investigate the underlying mechanisms. Using Lewis lung carcinoma-bearing mice, we showed that feeding montelukast significantly delayed the tumor growth in mice (p < 0.0001). Montelukast inhibited cell proliferation and colony formation and induced the cell death of lung cancer cells. Further investigation showed the down-regulation of B-cell lymphoma 2 (Bcl-2), up-regulation of Bcl-2 homologous antagonist/killer (Bak), and nuclear translocation of apoptosis-inducing factor (AIF) in montelukast-treated lung cancer cells. Montelukast also markedly decreased the phosphorylation of several proteins, such as with no lysine 1 (WNK1), protein kinase B (Akt), extracellular signal-regulated kinase 1/2 (Erk1/2), MAPK/Erk kinase (MEK), and proline-rich Akt substrate of 40-kDa (PRAS40), which might contribute to cell death. In conclusion, montelukast induced lung cancer cell death via the nuclear translocation of AIF. This study confirmed the chemo-preventive effect of montelukast shown in our previous cohort study. The utility of montelukast in cancer prevention and treatment thus deserves further studies.

Toll-like receptor 4 mediates Lewis lung carcinoma-induced muscle wasting via coordinate activation of protein degradation pathways

Cancer-induced cachexia, characterized by muscle wasting, is a lethal metabolic syndrome with undefined etiology. Current consensus is that multiple factors contribute to cancer-induced muscle wasting, and therefore therapy requires combinational strategies. Here, we show that Toll-like receptor 4 (TLR4) mediates cancer-induced muscle wasting by directly activating muscle catabolism as well as stimulating an innate immune response in mice bearing Lewis lung carcinoma (LLC), and targeting TLR4 alone effectively abrogate muscle wasting. Utilizing specific siRNAs we observed that LLC cell-conditioned medium (LCM)-treated C2C12 myotubes underwent a rapid catabolic response in a TLR4-dependent manner, including activation of the p38 MAPK−C/EBPβ signaling pathway as well as the ubiquitin-proteasome and autophagy-lysosome pathways, resulting in myotube atrophy. Utilizing a reporter cell-line it was confirmed that LCM activated TLR4. These results suggest that LLC-released cachexins directly activate muscle catabolism via activating TLR4 on muscle cells independent of immune responses. Critically, LLC tumor-bearing TLR4−/− mice were spared from muscle wasting due to a blockade in muscle catabolic pathways. Further, tumor-induced elevation of circulating TNFα and interleukin-6 (IL-6) was abolished in TLR4−/− mice. These data suggest that TLR4 is a central mediator and therapeutic target of cancer-induced muscle wasting.

Carboxyamidotriazole Synergizes with Sorafenib to Combat Non-Small Cell Lung Cancer through Inhibition of NANOG and Aggravation of Apoptosis.

Lung cancer is currently the leading cause of cancer-related deaths worldwide. In this study, we investigated the combination of carboxyamidotriazole (CAI) and sorafenib in non-small cell lung cancer (NSCLC) in vitro and in vivo to test whether CAI enhances the antitumor effects of sorafenib and reduces its side effects. The combination index (CI) showed that coadministration of CAI and sorafenib synergistically inhibited the proliferation of NSCLC cells (Lewis lung carcinoma, A549, and NCI-H1975 cells). Cell death as a result of the combination treatment was attributed to apoptosis, which was accompanied by activation of caspase-3 and poly(ADP-ribose) polymerase. In addition, combination therapy induced the accumulation of mitochondrial-associated reactive oxygen species, as well as depolarization of mitochondrial and reduced NANOG (homeobox protein NANOG) mRNA and protein expression. Basic fibroblast growth factor, a stimulator of NANOG, was applied to identify the possible mechanism. The addition of basic fibroblast growth factor followed by combined treatment may stimulate NANOG expression and synchronously rescue the accumulation of reactive oxygen species. C57BL/6J mice bearing Lewis lung carcinoma were randomized to receive vehicle (polyethylene glycol 400), CAI (30 mg/kg), low-dose sorafenib (SFB-L; 10 mg/kg), high-dose sorafenib (SFB-H; 30 mg/kg), or a CAI and SFB-L combination. Tumor growth was significantly suppressed in the combination group, and the efficacy of combination treatment was equivalent to that of the SFB-H monotherapy group. Furthermore, the combination group had reduced side effects compared with the SFB-H group, as indicated by weight preservation in mice. Our study illustrates that CAI enhances the antitumor activity of sorafenib in NSCLC and provides a novel strategy for NSCLC treatment.

Antitumor and anti-metastatic effects of photolon-mediated Sono-Photodynamic therapy on mice bearing Lewis lung Carcinoma

Antitumor and anti-metastatic effects of photolon-mediated Sono-Photodynamic therapy on mice bearing Lewis lung Carcinoma

Theabrownin Inhibits Cell Cycle Progression and Tumor Growth of Lung Carcinoma through c-myc-Related Mechanism.

Green tea, the fresh leaves of Camellia sinensis, is not only a health-promoting beverage but also a traditional Chinese medicine used for prevention or treatment of cancer, such as lung cancer. Theabrownin (TB) is the main fraction responsible for the medicinal effects of green tea, but whether it possesses anti-cancer effect is unknown yet. This study aimed to determine the in vitro and in vivo anti-lung cancer effect of TB and explore the underlying molecular mechanism, by using A549 cell line and Lewis lung carcinoma-bearing mice. In cellular experiment, MTT assay was performed to evaluate the inhibitory effect and IC50 values of TB, and flow cytometry was conducted to analyze the cell cycle progression affected by TB. In animal experiment, mice body mass, tumor incidence, tumor size and tumor weight were measured, and histopathological analysis on tumor was performed with Transferase dUTP nick-end labeling staining. Real time PCR and western blot assays were adopted to detect the expression of C-MYC associated genes and proteins for mechanism clarification. TB was found to inhibit A549 cell viability in a dose- and time-dependent manner and block A549 cell cycle at G0/G1 phase. Down-regulation of c-myc, cyclin A, cyclin D, cdk2, cdk4, proliferation of cell nuclear antigen and up-regulation of p21, p27, and phosphate and tension homolog in both gene and protein levels were observed with TB treatment. A c-myc-related mechanism was thereby proposed, since c-myc could transcriptionally regulate all other genes in its downstream region for G1/S transitions of cell cycle and proliferation of cancer cells. This is the first report regarding the anti-NSCLC effect and the underlying mechanism of TB on cell cycle progression and proliferation of A549 cells. The in vivo data verified the in vitro result that TB could significantly inhibit the lung cancer growth in mice and induce apoptosis on tumors in a dose-dependent manner. It provides a promising candidate of natural products for lung cancer therapy and new development of anti-cancer agent.

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

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

Water-soluble extract of Saxifraga stolonifera has anti-tumor effects on Lewis lung carcinoma-bearing mice

Saxifraga stolonifera is an evergreen and herbaceous plant well known in Korea, Japan and western China, which has great potential applications in gardening and pharmacology. The aim of this study is to evaluate the anti-tumor effects of S. stolonifera extraction on lung tumors of Lewis mice. By the measurement of MS/MS, we found that there were four main bioactive components in methanol extract of S. stolonifera, including gallic acid, norbergenin, protocatechuic acid and bergenin, and the results of quantitative analysis showed that the contents of gallic acid, protocatechuic acid and bergenin in methanol extract of S. stolonifera were 5.150, 1.492, 24.559mg/g, respectively. Animal experiment showed that the mean tumor weight of Lewis lung carcinoma-bearing mice treated with water-soluble extract of S. stolonifera was obviously smaller than model group (cis-DDP), and its inhibition rate was 49.2%. In addition, histopathological evaluation and immunohistochemical assay confirmed the anti-tumor effects of S. stolonifera. Investigation of four haematological parameters revealed that the Lewis mice fed with S. stolonifera showed good resilience in the level of leukocyte, haemoglobin, blood platelets and red blood cell compared with the model group. In addition, RT-PCR suggested that the relative expression of pro-apoptosis gene p53, Sox and Bax was enhanced, while the relative expression of anti-apoptosis gene Bcl2 was diminished in comparison with model group. These results suggested that water-soluble extract of S. stolonifera has anti-tumor effects on Lewis lung tumors.

MicroPET imaging with 2-[18F]fluoropropionic acid in Lewis lung carcinoma-bearing mice

Objective To prepare 2-[18F]fluoropropionic acid (18F-FPA) and evaluate its biodistribution and imaging capacity in Lewis lung carcinoma-bearing mice. Methods 18F-FPA was prepared by nucleophilic substitution reaction, and its hydrophilicity was analyzed. 18F-FPA (7.4-11.1 MBq) was injected into Lewis lung carcinoma-bearing mice via tail vein. MicroPET imaging was performed at 20, 80 min after the injection. The biodistribution of 18F-FPA in organs was analyzed. The blocking effects of sodium propionate and dichloroacetate to 18F-FPA were tested in vivo. Data were analyzed by two-sample t test using GraphPad Prism software. Results The synthesis of 18F-FPA took 40 min. 18F-FPA had high radiochemical purity (>99%) and hydrophilicity. 18F-FPA was mainly distributed in the carcinoma, the urinary bladder and the caecum. The radioactive uptakes in muscles, brown fat and bones were relatively low. Quantitative analysis showed that the uptake of 18F-FPA in Lewis lung carcinoma from 20 min to 80 min was slightly increased ((17.03±2.87) %ID/g vs (19.33±2.45) %ID/g) without significant difference (t=1.100, P>0.05). Neither sodium propionate nor dichloroacetate could block the uptake of 18F-FPA in Lewis lung carcinoma (t=1.544, 0.894; both P>0.05). Conclusions 18F-FPA can be quickly synthesized and has good physicochemical properties. It can be used as a tracer to visualize Lewis lung carcinoma in mice, and its tumor uptaking can not be blocked by propionate and dichloacetate. 18F-FPA PET has the potential to detect lung cancer noninvasively in clinic. Key words: Propionic acids; Isotope labeling; Fluorine radioisotopes; Tomography, emission-computed; Tomography, X-ray computed; Lung neoplasms; Mice

Abstract A195: CB-1158 inhibits the immuno-oncology target arginase and causes an immune mediated anti-tumor response

Abstract The role of myeloid derived suppressor cells (MDSCs) has become increasingly recognized as an important mechanism of tumor immune evasion. However, to date there are no effective means to antagonize the immunosuppressive activity of these cells in patients. MDSCs suppress cytotoxic T-cells and Natural Killer cells through the secretion of the enzyme arginase which depletes local arginine concentrations. The depletion of arginine in the tumor microenvironment renders cytotoxic T-cells unable to proliferate and therefore unable to effectively mount an anti-tumor attack. Similarly, M2 macrophages and polymorphonuclear cells (PMNs) express high levels of arginase and may contribute to the local suppression of immune responses. Restoration of arginine levels in the tumor microenvironment via arginase inhibition would be expected to allow T-cell activation and proliferation to occur and result in T-cell mediated anti-tumor responses. We have developed novel, potent, and specific inhibitors of arginase. Our clinical candidate CB-1158 has an IC50 of less than 100 nM in a recombinant human arginase assay. As expected, inhibition of arginase in cell culture does not have a direct anti-proliferative effect on any cell type tested. However, in Peripheral Blood Mononuclear Cells (PBMCs) from a patient with renal cell carcinoma containing both suppressive MDSCs and cytotoxic T-cells, the addition of an arginase inhibitor to the MDSC/T-cell co-culture resulted in a dose-dependent increase in T-cell proliferation relative to vehicle controls. CB-1158 has high oral bioavailability in mice and rats. In mice bearing Lewis Lung Carcinoma (LLC) syngeneic tumors, treatment with CB-1158 results in a 3-4 fold increase in tumor arginine levels with a clear pharmacokinetic/pharmacodynamic relationship. The pharmacodynamic effect of arginase inhibition in tumors was sustained throughout a 24-hour period using a twice-daily oral dosing schedule and has been observed in multiple syngeneic models. Moreover, systemic plasma arginine levels are significantly increased in mice following dosing with CB-1158. Importantly, oral dosing with CB-1158 results in single agent anti-tumor efficacy in the LLC model in C57.Bl/6 mice. In contrast, treatment of immunocompromised C57/scid mice bearing LLC tumors with CB-1158 had no effect on tumor growth. This finding is consistent with the observed anti-tumor efficacy in immune competent mice being mediated through an immune mechanism. Evaluation of tumors treated with arginase inhibitors revealed an increase in CD3+ T-cell infiltrates further supporting an immune-based mechanism of action. CB-1158 has been very well tolerated in rodents with no impact on body weights or serum chemistry enzymes following multi-week dosing schedules. CB-1158 is a first-in-class arginase inhibitor that targets the immunosuppressive effects of myeloid cells in the tumor microenvironment and is currently in development as a novel immuno-oncology strategy. Based on this novel mechanism of action there is also a potential for enhanced therapeutic benefit by combining CB-1158 with other immune checkpoint inhibitors. Citation Format: Matthew Gross, Jason Chen, Ethan Emberley, Julie Janes, Weiqun Li, Andy Mackinnon, Alison Pan, Francesco Parlati, Mirna Rodriguez, Susanne Steggerda, Tracy Wang, Melissa Works, Jing Zhang, Winter Zhang, Mark Bennett. CB-1158 inhibits the immuno-oncology target arginase and causes an immune mediated anti-tumor response. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A195.

Fas Ligand Deficiency Impairs Tumor Immunity by Promoting an Accumulation of Monocytic Myeloid-Derived Suppressor Cells.

The Fas receptor ligand FasL regulates immune cell levels by inducing apoptosis of Fas receptor-positive cells. Here, we studied the impact of host FasL on tumor development in mice. Genetically targeting FasL in naïve mice increased myeloid cell populations, but, in marked contrast, it reduced the levels of myeloid-derived suppressor cells (MDSC) in mice bearing Lewis lung carcinoma tumors. Analysis of the MDSC subset distribution revealed that FasL deficiency skewed cell populations toward the M-MDSC subset, which displays a highly immunosuppressive activity. Furthermore, tumor-bearing mice that were FasL-deficient displayed an enhanced proportion of tumor-associated macrophages and regulatory T cells. Overall, the immunosuppressive environment produced by FasL targeting correlated with reduced survival of tumor-bearing mice. These results disclose a new role for FasL in modulating immunosuppressive cells.

Eritoran inhibits S100A8-mediated TLR4/MD-2 activation and tumor growth by changing the immune microenvironment.

S100A8/A9 is a major component of the acute phase of inflammation, and appears to regulate cell proliferation, redox regulation and chemotaxis. We previously reported that S100A8/S100A9 are upregulated in the premetastatic lung. However, the detailed mechanisms by which S100A8 contributes to tumor progression have not been elucidated. In this study, we investigated the TLR4/MD-2 dependency by S100A8 on tumor progression. We found that S100A8 (2-89) peptide stimulated cell migration in a manner dependent on TLR4, MD-2 and MyD88. The S100A8 (2-89) peptide also activated p38 and NF-κB in TLR4-dependent manner. The peptide induced the upregulation of both IL-6 and Ccl2 in peritoneal macrophages obtained from wild-type mice, but not TLR4-deficient mice. We then investigated the responsible region of S100A8 for TLR4/MD-2 binding by a binding assay, and found that C-terminal region of S100A8 binds to TLR4/MD-2 complex. To further evaluate the TLR4 dependency on tumor microenvironment, Lewis lung carcinoma-bearing mice were treated with Eritoran, an antagonist of TLR4/MD-2 complex. We found that both tumor volume and pulmonary recruitment of myeloid-derived suppressor cells were reduced with the treatment of Eritoran for five consecutive days. Eritoran reduced the development of tumor vasculature, and increased tumor-infiltration of CD8(+) T-cells. Taken together, S100A8 appears to play a crucial role in the activation of the TLR4/MD-2 pathway and the promotion of a tumor growth-enhancing immune microenvironment.

MicroR-545 enhanced radiosensitivity via suppressing Ku70 expression in Lewis lung carcinoma xenograft model

ObjectiveRadiotherapy is an important therapeutic method for lung cancer. However, in clinical situations, cellular resistance to radiotherapy is a significant component of tumor treatment failure. Thus, clarification in cellular mechanism underlying radiosensitivity of cancer cell is urgently needed. In this study, we established a radiation model of Lewis lung carcinoma in C57BL/6 mice and investigated the possible signaling molecule involved in this process.MethodsC57BL/6 mice were subcutaneously transplanted with Lewis lung carcinoma cells and locally irradiated followed by measurement in tumor volume. Levels of miR-545 and Ku70 mRNA expression were determined by using Quantitative Real-Time PCR. Expression of Ku70 was determined by using western blot assay. Cell viability was analyzed by MTT assay. Cell apoptosis was examined by using TUNEL assay.ResultsIn mice bearing Lewis lung carcinoma tumor, local radiotherapy suppressed tumor growth as well as enhanced expression of miR-545 and downregulated Ku70 level. Inhibition of miR-545 expression reduced radiosensitivity of Lewis tumor. In vitro Lewis lung carcinoma cells experiment, we observed that miR-545 regulated Ku70 expression by targeting Ku70 3′UTR and this process was involved in radiotherapy. This was demonstrated by result of cell proliferation assay in which irradiation reduced apoptosis of cells was mediated by miR-545 inactivation which was reversed by Ku70 silence.ConclusionmiR-545 increased radiosensitivity of Lewis lung carcinoma via inhibiting Ku70 expression.

Effects of Feijining Decoction on vascular endothelial growth factor protein expression and changes of T cell subsets in Lewis lung carcinoma-bearing mice.

Angiogenesis is crucial for cancer growth and metastasis. T cells are also key members of the adaptive immunity against tumorigenesis. The aim of the present study was to observe the effects of Feijining Decoction (FJND) on vascular endothelial growth factor (VEGF) protein expression and T cell subsets [cluster of differentiation 4+(CD4+) and CD8+ T lymphocyte] in Lewis lung carcinoma (LLC)-bearing mice. C57BL/6J mice were subcutaneously implanted with LLC cells. Forty carcinoma-bearing mice were randomly assigned to four groups (10 animals/group). The control group (CG) were the untreated group, the cisplatinum (DDP) group (DG) mice were treated with DDP, the FJND group (FG) were treated with FJND and the FJND + DDP group (FDG) were treated with FJND and DDP. Western blot and flow cytometry were used to evaluate the VEGF protein expression of tumor tissue and T cell subsets of the spleen. Spontaneous activity in 5 min was observed by the photoelectric counting method. DDP + FJND (FDG group) markedly inhibited tumor growth compared to the DG mice. The protein expression of VEGF was significantly downregulated in the carcinoma of FG mice compared to CG mice. VEGF protein expression was significantly reduced in FDG compared to DG mice. In the FG mice, the splenic CD4+ and CD4+/CD8+ cells were significantly increased compared to the CG mice, and the splenic CD4+ cells in the FDG mice were significantly increased compared to the DG group. In conclusion, FJND can inhibit tumor growth by downregulating VEGF protein expression and improving the immune function.