Lewis Lung Cancer Cells Research Articles

Antitumor effects obtained by autologous Lewis lung cancer cell vaccine engineered to secrete mouse Interleukin 27 by means of cationic liposome

Interleukin-27 (IL-27), a novel IL-6/IL-12 family cytokine, plays an important role in the early regulation of Th1 responses. The cytokine IL-27 can exert a variety of immune-regulatory functions including cytotoxic T lymphocyte (CTL), CD4+, CD8+ T lymphocytes activation and interferon-γ (IFN-γ) production. In this study, we developed an effective and gene modified tumor cell vaccine. Lewis lung cancer cell LL/2 transfected with the DOTAP:cholesterol cationic liposome could express the mouse IL-27 (mIL-27) gene at a relative high level. The resultant transfectants were then irradiated with X-ray and used as a tumor cell vaccine. This tumor cell vaccine not only contained tumor associated antigen (TAA) of LL/2 cells but also secreted mIL-27 which could induce immune response in mice. The mice vaccinated with LL/2-mIL-27 performed strong tumor inhibiting effect accompanied with a high IFN-γ production. Both CD4+ and CD8+ T lymphocytes were significantly elevated in these mice vaccinated with LL/2-mIL-27 cell vaccine. Moreover, after depletion of CD4+, CD8+ T lymphocytes by injection of antibodies against CD4 and CD8, the vaccinated mice inoculated with autologous LL/2 cells were not protected from tumor challenge. In contrast, vaccinated mice inoculated with autologous LL/2 cells were treated with antibody against natural killer (NK)cells or normal rat IgG still possessed strong antitumor activity. Our data suggested that DOTAP:cholesterol cationic liposome was quite useful in generating an autologous tumor cell vaccine and mIL-27 could be therapeutically used to potentiate the host antitumor immunity.

Animal models of bone cancer pain: Systematic review and meta-analyses

Pain can significantly decrease the quality of life of patients with advanced cancer. Current treatment strategies often provide inadequate analgesia and unacceptable side effects. Animal models of bone cancer pain are used in the development of novel pharmacological approaches. Here we conducted a systematic review and meta-analysis of publications describing in vivo modelling of bone cancer pain in which behavioural, general health, macroscopic, histological, biochemical, or electrophysiological outcomes were reported and compared to appropriate controls. In all, 150 publications met our inclusion criteria, describing 38 different models of bone cancer pain. Reported methodological quality was low; only 31% of publications reported blinded assessment of outcome, and 11% reported random allocation to group. No publication reported a sample size calculation. Studies that reported measures to reduce bias reported smaller differences in behavioural outcomes between tumour-bearing and control animals, and studies that presented a statement regarding a conflict of interest reported larger differences in behavioural outcomes. Larger differences in behavioural outcomes were reported in female animals, when cancer cells were injected into either the tibia or femur, and when MatLyLu prostate or Lewis Lung cancer cells were used. Mechanical-evoked pain behaviours were most commonly reported; however, the largest difference was observed in spontaneous pain behaviours. In the spinal cord astrocyte activation and increased levels of Substance P receptor internalisation, c-Fos, dynorphin, tumor necrosis factor-α and interleukin-1β have been reported in bone cancer pain models, suggesting several potential therapeutic targets. However, the translational impact of animal models on clinical pain research could be enhanced by improving methodological quality.

Simultaneous activation of T helper function can augment the potency of dendritic cell-based cancer immunotherapy

Simultaneous activation of T helper 1 (Th1) cell function has crucial roles in induction of potent cytotoxic T lymphocyte (CTL) responses in cancer immunotherapy. Here, we investigated whether dendritic cell (DC)-based vaccines loaded with both tumor-associated antigen (TAA)-derived MHC class I and pan-MHC class II peptides could elicit more potent CTL responses through simultaneous activation of Th1 function and reduction in CD4(+) regulatory T (Treg) cell proliferation. C57BL/6 mice bearing LLC1, a mouse Lewis lung cancer cell line, were subcutaneously administered DCs loaded with both LLC-derived MHC class I (MUT1&2) and LLC-unrelated pan-MHC class II (PADRE) peptides (DC-MUT1&2-PADRE). In assays using samples from advanced lung cancer patients, peripheral blood mononuclear cells were stimulated with autologous DCs loaded with both MUC1 MHC class I and PADRE peptides (DC-MUC1-PADRE) in vitro. Subsequently, TAA-specific CTL responses and the population of CD4(+) Treg cells were analyzed. The population of spleen CD4(+) PADRE-specific cells producing interferon-gamma (IFNγ) was significantly increased by DC-MUT1&2-PADRE administration. Vaccinations with DC-MUT1&2-PADRE decreased the population of CD4(+) Treg cells in spleen and augmented CTL responses, effectively leading to suppression of tumor growth. In assays with human samples, CD4(+) Treg cells were induced less frequently, and MUC1-specific cytotoxicity was enhanced by stimulation with DC-MUC1-PADRE compared with that by stimulation with DC-MUC1 alone. Simultaneous activation of Th1 function by DCs loaded with both TAA-derived MHC class I and PADRE peptides augments TAA-specific CTL responses while reducing Treg cell proliferation.

Abstract B7: Hypoxia-inducible factor 1 mediates thrombosis-associated cancer progression

Abstract Background. Cancer patients are up to 50 times more likely to suffer from lethal venous thromboembolism compared with non-cancer patients, but mechanisms that regulate the positive correlation between these conditions are unclear. Our primary aim was to test whether hypoxia within cells contained in the thrombus and adjacent vein leads to upregulation of hypoxia-inducible factor 1 (HIF1), which in turn stimulates the production of factors that mediate tumor growth and metastasis. Methods. Systemic or endothelial and myeloid HIF1 levels were altered (via administration of HIF1 agonist L-mimosine or via cre-Tie2 driven HIF1 deletion respectively) in established mouse models of thrombosis and breast cancer (inferior vena cava stenosis or polyoma middle T mutation driven by mouse mammary tumour virus respectively). Protein arrays were used to quantify the expression of 25 factors that regulate coagulation, thrombosis, and cancer progression. Adhesion and migration assays were used to characterise tumor and endothelial cell behaviour on thrombotic surfaces; while endothelial and tumor cell oxygen consumption rate was measured before and after thrombin treatment. Image analysis was also used to quantify intra-tumor thrombus formation. Results. Thrombus formation or upregulation of HIF1 increased the circulating expression of 10 factors that mediate tumor growth and remodelling including vascular endothelial growth factor (VEGF, 2±1 vs 1±0.2pg/ml in controls, n=6/group, P<0.05), hepatocyte growth factor (HGF, 10±5 vs 7±2% in controls, n=8/group, P<0.05), and stromal cell-derived factor 1 (SDF1, 14±6 vs 2±1% in controls, n=8/group, P<0.05). Hypoxia (1% oxygen) tended to attenuate endothelial cell migration on fibrin (100±13 vs 64±10 cells in controls, n=6/group, P=0.05); while HIF1 deletion in Lewis lung cancer cells abolished their hypoxia-induced increase in adhesion onto fibrin (n=5/group, P<0.05). Thrombin administration reduced the oxygen consumption rate of Lewis lung cancer cells (59±13 vs 79±16 pMol/min in controls, n=5/group, P<0.005) but not endothelial cells (P=0.06). Deposition of fibrin in mammary tumours was reduced following endothelial and myeloid HIF1 deletion (5±1 vs 13±3% in controls, n=7/group, P<0.05) and this was associated with reduced tumour size (n=14, R=0.7, P<0.01). Conclusions. HIF1 regulates the progression of venous thrombosis and cancer, and could also be responsible for the positive association between these conditions. Cell and tissue-specific HIF1 is an attractive therapeutic target for treatments that aim to reduce thrombosis-associated cancer progression. Citation Format: Colin E. Evans, Cristina Branco-Price, Julia Humphries, Ashar Wadoodi, Prakash Saha, Jung-whan Kim, Mattias Belting, Alberto Smith, Randall S. Johnson. Hypoxia-inducible factor 1 mediates thrombosis-associated cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B7.

Xiao-Ai-Ping, a TCM Injection, Enhances the Antigrowth Effects of Cisplatin on Lewis Lung Cancer Cells through Promoting the Infiltration and Function of CD8+T Lymphocytes

Objectives. To investigate how Xiao-Ai-Ping injection, a traditional Chinese medicine and an ancillary drug in tumor treatment, enhances the antitumor effects of cisplatin on Lewis lung cancer (LLC) cells. Methods. LLC-bearing mice were daily intraperitoneally injected with various doses of cisplatin, Xiao-Ai-Ping, or cisplatin plus Xiao-Ai-Ping, respectively. Body weight and tumor volumes were measured every three days. Results. Combination of Xiao-Ai-Ping and cisplatin yielded significantly better antigrowth and proapoptotic effects on LLC xenografts than sole drug treatment did. In addition, we found that Xiao-Ai-Ping triggered the infiltration of CD8+ T cells, a group of cytotoxic T cells, to LLC xenografts. Furthermore, the mRNA levels of interferon-γ (ifn- γ), perforin-1 (prf-1), and granzyme B (gzmb) in CD8+ T cells were significantly increased after combination treatment of Xiao-Ai-Ping and cisplatin. In vitro studies showed that Xiao-Ai-Ping markedly upregulated the mRNA levels of ifn- γ, prf-1, and gzmb in CD8+ T cells in a concentration-dependent manner, suggesting that Xiao-Ai-Ping augments the function of CD8+ T cells. Conclusions. Xiao-Ai-Ping promotes the infiltration and function of CD8+ T cells and thus enhances the antigrowth effects of cisplatin on LLC xenografts, which provides new evidence for the combination of Xiao-Ai-Ping and cisplatin in clinic in China.

Inhibition of TGF-β1 Signaling With Halofuginone can Enhance the Antitumor Effect of Irradiation in Lung Cancer Xenograft

It is reported that halofuginone can inhibit the transforming growth factor β1 (TGF-β1) signaling pathway. TGF-β1 signaling plays an important role in the invasive and metastatic behavior in many kind of malignancy. Radiation therapy may result in increased expression of TGF--β1 in tumor and surrounding tissues. We hypothesized that halofuginone may enhance the anti-tumor effect of radiation therapy in lung cancer. Lung cancer xenograft was established with Lewis lung cancer cell lines and female C57BL/6J mice. Thirty six animals were randomly divided into 4 groups: control group have no any anti-tumor treatment, halofuginone group received 25μg of halofuginone every day by gavage, radiation group received 30Gy irradiation in 10 daily fractions, and the combined group received daily 25μg of halofuginone 1 hour before the same radiation therapy as in the radiation group. The tumor size in each mouse was evaluated every other day. Three days after the treatment, 3 mice in each group were euthanized and the tumors were weighed and harvested for immunohistochemistry study to evaluate the TGF-β1 expression. The TGF-β1 level in tumor tissue was also measured with enzyme-linked immunosorbent assay (ELISA). Ninety days after the treatment, all the survival mice were euthanized and the lung and liver metastasis lesions were evaluated. Before treatment, there's no significant difference in tumor volume between the groups. From the beginning of the treatment, the tumor growth was significantly delayed in each treatment group than in the control group (P value were all smaller than 0.05 at the 9th day). At the 9th day of the treatment, the tumor volume was 175.2 ± 40.0mm3 in the control group, and it was 118.5 ± 13.2mm3 in the halofuginone group, 106.6 ± 14.1mm3 in the radiation group, and 85.6 ± 17.1mm3 in the combined group. The tumor volume in the combined group was significantly smaller than in every other treatment group. The difference in tumor weight between the groups was similar, with the smallest tumor weight observed in the combined group. Thirty-eight, 3, 9 and 2 metastatic lesions were observed in lung surface in the 4 groups; and 53, 3, 6 and 2 lesions were observed in the liver surface. Immunohistochemistry study revealed decrease level of TGF-β1 expression in the halofuginone group and the combined group than in any other groups. The TGF-β1 level measured with ELISA also showed the similar results. Halofuginone has an anti-tumor effect itself and can enhance the anti-tumor effect of radiation therapy in Lewis lung cancer xenograft. The mechanism most likely due to its inhibition effect to TGF-β1 signaling pathway.

Foxp3 overexpression decreases sensitivity to chemotherapy in mouse Lewis lung cancer cells

Chemotherapy is the main strategy for the treatment of lung cancer. However, sensitivity to chemotherapy, one of the main factors affecting the survival rate of patients with lung cancer, is extremely poor. Forkhead box P3 (Foxp3) is the key regulatory gene for the development and function of CD4+CD25+ regulatory T cells (Tregs). Increased levels of Tregs and Foxp3 expression in the peripheral blood and tumour specimens of cancer patients are associated with tumour progression and poor prognosis. In addition, certain studies have suggested that Tregs may be resistant to conventional chemotherapy and thus, enhance tumour immune evasion. Previous studies have demonstrated that Foxp3 is also expressed within tumour cells and that it may mimic the function of Tregs. Currently, the correlation between the tumour cell expression of Foxp3 and sensitivity to chemotherapy is unclear. Therefore, it was hypothesised that Foxp3 causes resistance to chemotherapeutic agents in lung cancer cells and that it may consequently promote the progression of lung cancer. In the current study, the expression of Foxp3 in mouse Lewis lung cancer (LLC) cells was detected using RT-PCR and immunocytochemistry. The overexpression of Foxp3, which was accomplished by the transient transfection of recombinant pcDNA3.1-Foxp3 or empty plasmids into LLC cells, was confirmed by RT-PCR and western blot analysis. The inhibition of cell proliferation was measured using MTT assay. The expression of multidrug resistance protein1 (mdr1) mRNA and its protein product, P-glycoprotein (P-gp), were detected by RT-PCR and flow cytometry, respectively. The results revealed that Foxp3 was expressed by LLC cells. The inhibitory rate of cell proliferation in Foxp3-overexpressing LLC cells compared with those transfected with an empty plasmid was significantly decreased following adriamycin (ADM) and mitomycinC (MMC) treatment. The IC50 values of ADM and MMC in Foxp3-overexpressing LLC cells were increased. The expression levels of mdr1 mRNA and P-gp were significantly upregulated in Foxp3 overexpressing LLC cells. These results suggest that Foxp3 reduces the sensitivity of LLC cells to ADM and MMC, thus promoting tumour progression, by upregulating the expression of mdr1 mRNA and P-gp.

TGF-β of lung cancer microenvironment upregulates B7H1 and GITRL expression in dendritic cells and is associated with regulatory T cell generation

The effects of TGF-β on dendritic cells (DCs) on the tumor microenvironment are not well understood. We report, here, the establishment of an in vitro lung cancer microenvironment by co-incubation of seminaphtharhodafluor (SNARF) labeled Lewis lung cancer (LLC) cells, carboxyfluorescein succinimidyl ester (CFSE) labeled fibroblasts and 4-chloromethyl-7-hydroxycoumarin (CMHC) labeled DCs. Raw 264.7, EL4 and NCI-H446 cells were able to synthesize TGF-β which was determined by flow cyto-metry and western blotting, respectively. Furthermore, TGF-β efficiently increased regulatory T-cell (Treg) expansion and upregulated DC B7H1 and GITRL expression. TGF-β and the co-incubation of LLC cells, fibroblasts with DCs could augment the expression of B7H1 and GITRL molecules of DCs. The data presented here indicate that the B7H1 and GITRL molecules may play an important role in TGF-β-induced Treg expansion of lung cancer microenvironment.

Effects of Chinese herbal medicine Feiyanning Decoction on the ratio of CD4+CD25+ regulatory T cells and expression of transcription factor Foxp3 in mice bearing Lewis lung carcinoma

To study the effects of Feiyanning Decoction, a compound traditional Chinese herbal medicine, on the ratio of CD4(+)CD25(+) regulatory T cells and expression of transcription factor Foxp3 in mice with Lewis lung cancer. Thirty-two male wild-type C57BL/6 mice aging from 6 to 8 weeks were inoculated with Lewis lung cancer cells to establish the tumor-bearing model of Lewis lung carcinoma and were randomly divided into model group, Chinese medicine group, chemotherapy group and Chinese medicine combined with chemotherapy group. After intervention for 14 d with corresponding drugs, behaviors, physical signs and changes of feed consumption of the mice were observed. All mice were sacrificed after drug treatment, and tumors and organs were removed to weigh and calculate organ indexes (lung index, spleen index and thymus index). The percentages of CD4(+)CD25(+) regulatory T cells in the thymus, spleen and tumor were determined by flow cytometry. The expression of Foxp3 mRNA in the thymus, spleen and tumor tissues was detected by quantitative real-time polymerase chain reaction. Compared with those in the model group, the mice in the Chinese medicine group showed significant reductions in spleen and thymus indexes and tumor weight, and elevation in the body weight without tumor (P<0.05). The numbers of CD4(+)CD25(+) regulatory T cells in spleen, thymus and tumor were lower in the Chinese medicine group than in the model group (P<0.05). The expression of Foxp3 mRNA in spleen, thymus and tumor was significantly down-regulated in the Chinese medicine group compared with the model group (P<0.05). There were no significant differences in CD4(+)CD25(+) regulatory T cell ratio and Foxp3 mRNA expression between the Chinese medicine combined with chemotherapy group and the chemotherapy group. Feiyanning Decoction can enhance the antitumor immune response and thus play a role in antitumor therapy by reducing the ratio of CD4(+)CD25(+) regulatory T cells and down-regulating the expression of Foxp3 mRNA.

The G protein-coupled receptor mFPR2 promotes anti-tumor host defense (161.7)

Abstract Abstract mFPR2 is a G protein-coupled receptor interacting with bacterial and host-derived chemotactic agonists. In vivo, mFPR2 plays a major role in innate and adaptive immune responses as shown by reduced severity of allergic airway inflammation in mFPR2-KO mice due to impaired trafficking of antigen presenting dendritic cells (DCs). The aim of this study is to examine the role of mFPR2 in anti-tumor host responses. We found that mFPR2-KO mice bearing subcutaneously implanted Lewis lung cancer (LLC) cells exhibited significantly shortened survival due to more rapidly growing tumors. In contrast, in mFPR2-transgenic (Tg) mice, subcutaneously implanted LLC tumors grew more slowly than those in wild type (WT) littermates. Mechanistic studies revealed an increased number of macrophages associated with tumors grown in mFPR2 KO mice and macrophages derived from mFPR2 KO mice showed a more potent chemotactic response to LLC-derived supernatant (LLC Sup), which could be neutralized by an anti-CCL2 antibody. In addition, macrophages from mFPR2 KO mice were more prone to assume an M2 phenotype after incubation with LLC Sup. These results suggest that mFPR2 plays an important role in host defense against tumor by sustaining macrophages in an M1 phenotype.

Liposomal curcumin inhibits Lewis lung cancer growth primarily through inhibition of angiogenesis

Curcumin has been proven to effectively inhibit tumor growth by both targeting tumor cells and angiogenesis; however, poor water solubility limits further clinical application. In the present study, we prepared water-soluble liposomal curcumin to investigate its anti-tumor effects and the underlying mechanism. The MTT assay was used to test the anti-proliferative activities for the MS1 murine endothelial and LL/2 Lewis lung cancer cell lines. Apoptosis and cell cycle arrest induced by liposomal curcumin were analysed by flow cytometry. Anti-angiogenic agents and the resulting anti-tumor effects were investigated in a murine lung cancer model. Zebrafish were used to investigate the anti-angiogenic effect of liposomal curcumin in the development of embryos. In vitro, liposomal curcumin inhibited the proliferation of MS1 cells and induced cell cycle arrest and apoptosis. Notably, LL/2 cells showed less sensitivity to the liposomal curcumin in vitro. In vivo, the systemic administration of liposomal curcumin resulted in significant inhibition of tumor growth. CD31 immunohistochemical analysis and alginate encapsulation assay revealed that angiogenesis was decreased by liposomal curcumin treatment. Angiogenesis was also suppressed in the development of zebrafish. Liposomal curcumin showed potent inhibitory activity against murine endothelial cells but not lung cancer cells. Liposomal curcumin treatment is capable of significantly inhibiting tumor growth in vivo, a process that may depend primarily on its anti-angiogenic effects. Our study also indicates that liposomal curcumin may be developed not only for cancer therapy, but also for the treatment of other angiogenesis-related diseases.

Abstract 5546: UVC irradiation inhibits superficial tumor growth in the brain of nude mice

Abstract Temozolomide and UVC light were used for induction of cancer cell apoptosis of cancer cells which was imaged in real time at the subcellular level in the brain. Temozolomide inhibited brain tumor growth (p &amp;lt; 0.001). UVC irradiation inhibited superficial brain tumor growth (4.7-fold) (p &amp;lt; 0.0001) and improved survival (39.0%) (p = 0.002). This model can be used to evaluate new treatment modalities of brain metastasis or brain cancer at the subcellular level. In order to visualize single cancer cell nuclear-cytoplasmic dynamics of cancer cells in the brain in real time, including mitosis and apoptosis, Lewis lung cancer cells or human U87 glioma cells were injected into the brain either stereotactically or via the carotid artery of nude mice. The cancer cells were labeled with GFP in the nucleus and RFP in the cytoplasm, such that mitosis and apoptosis could be clearly imaged. A craniotomy open window was made for real-time fluorescence imaging of the cancer cells growing in the brain. Mitosis as well as treatment-induced apoptosis of the individual cancer cells were imaged in real time. UVC irradiation was initiated 1 day after the cancer cell inoculation and tumors were irradiated for 120 seconds on days 1, 4, 7, and 10. The tumor volume and survival were compared with untreated mice. UVC irradiation inhibited 4.7-fold changes in the brain tumor growth (p &amp;lt; 0.0001) and improved 39% higher survival (p = 0.002). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5546. doi:1538-7445.AM2012-5546

Abstract 5715: Modulation of Radiation Response after TGFβ signaling Blockade in Non-small Cell Lung Cancer (NSCLC) Cell Carcinomas

Abstract Lung cancer remains one of the most prevalent and deadliest malignancies worldwide. Radiotherapy (RT) is a major therapeutic modality for inoperable non-small cell lung cancer (NSCLC). There is substantial evidence that ionizing radiation triggers activation of TGFβ especially in lung, breast and liver tumor. TGFβ inhibition prior to IR inhibits the DNA damage response in epithelial cells via blockade of ATM kinase activity (Cancer Res 62:5627, 2002; Cancer Res 66:10861, 2006) and sensitizes radiation responses in breast cancer cell lines and tumors (Clin Cancer Res 2011;17:6754-6765.). The current studies test whether TGFβ blockade prior to irradiation modulates radiation response of NSCLC cell lines, NCI-H1299, NCI-H292, H460 and Lewis Lung Cancer (LLC), assessed in vitro and in vivo. All NSCLC cell lines responded to TGFβ by phosphorylation of Smad2, which was blocked by LY364947, a small molecule TGFβ type 1 receptor inhibitor. NCI-H1299 and LLC were insensitive to TGFβ growth inhibition in vitro. LY364947 increased radiosensitivity in NCI-H1299 and H460 in clonogenic assays. Consistent with increased clonogenic cell death, TGFβ inhibition also compromised radiation-induced phosphorylation of H2AX and ATM in NCI-H1299. Treatment of cultured LLC cells with either small molecule TGFβ type 1 receptor inhibitor or TGFβ neutralizing antibody, 1D11, significantly increased LLC radiosensitivity as measured by clonogenic assay. Furthermore, C57BL mice bearing subcutaneous LLC tumors treated before and after RT with 1D11 or 13C4 isotype control antibody (20 mg/kg i.p.) resulted in greater tumor growth delay compared to RT and control antibody. Together, these data indicate that TGFβ inhibition prior to radiation attenuates DNA damage responses, enhances clonogenic cell killing, and promotes tumor growth delay. Given the recognized role of radiation-induced TGFβ in lung fibrosis, the therapeutic index for RT could be significantly increased if circumstances are defined in which TGFβ inhibition during RT both enhances lung cancer radiosensitivity and protects normal lung. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5715. doi:1538-7445.AM2012-5715

Identification of a Cancer Stem-like Population in the Lewis Lung Cancer Cell Line

Although various human cancer stem cells (CSCs) have been defined, their applications are restricted to immunocompromised models. Developing a novel CSC model which could be used in immunocompetent or transgenic mice is essential for further understanding of the biomolecular characteristics of tumor stem cells. Therefore, in this study, we analyzed murine lung cancer cells for the presence of CSCs. Side population (SP) cells were isolated by fluorescence activated cell sorting, followed by serum-free medium (SFM) culture, using Lewis lung carcinoma cell (LLC) line. The self-renewal, differentiated progeny, chemosensitivity, and tumorigenic properties in SP and non-SP cells were investigated through in vitro culture and in vivo serial transplantation. Differential expression profiles of stem cell markers were examined by RT-PCR. The SP cell fraction comprised 1.1% of the total LLC population. SP cells were available to grow in SFM, and had significantly enhanced capacity for cell proliferation and colony formation. They were also more resistant to cisplatin in comparison to non-SP cells, and displayed increased tumorigenic ability. Moreover, SP cells showed higher mRNA expression of Oct-4, ABCG2, and CD44. We identified SP cells from a murine lung carcinoma, which possess well-known characteristics of CSCs. Our study established a useful model that should allow investigation of the biological features and pharmacosensitivity of lung CSCs, both in vitro and in syngeneic immunocompetent or transgenic/knockout mice.

Abstract A58: Galbanic acid isolated from Ferula assafoetida exerts in vivo antitumor activity in association with antiangiogenesis and antiproliferation

Abstract To investigate whether galbanic acid (GBA) exerts antiangiogenic and anticancer activities. Using human umbilical vein endothelial cell (HUVEC) model, we analyzed effects of GBA on cellular and molecular events related to angiogenesis. We tested its direct antiproliferative action on mouse Lewis lung cancer (LLC) cells and established its in vivo antiangiogenic and antitumor efficacy using LLC model. GBA significantly decreased vascular endothelial growth factor (VEGF)-induced proliferation and inhibited VEGF-induced migration and tube formation of HUVECs. These effects were accompanied by decreased phosphorylation of p38-mitogen-activated protein kinase (MAPK), c-jun N-terminal kinase (JNK), and AKT, and decreased expression of VEGFR targets endothelial nitric oxide synthase (eNOS) and cyclin D1 in VEGF-treated HUVECs. GBA also decreased LLC proliferation with an apparent G2/M arrest, but did not induce apoptosis. In vivo, inclusion of GBA in Matrigel plugs reduced VEGF-induced angiogenesis in mice. Galbanic acid given by daily i.p. injection (1 mg/kg) inhibited LLC-induced angiogenesis in an intradermal inoculation model and inhibited the growth of s.c. inoculated LLC allograft in syngenic mice. Immunohistochemistry revealed decreased CD34 microvessel density index and Ki-67 proliferative index in GBA-treated tumors. Overall, our findings suggest that GBA exerts anticancer activity in association with antiangiogenic and antiproliferative actions. Citation Format: Sunghoon Kim, Kwanhyun Kim, Soojin Jeong, Hyojeong Lee, Junxuan Lu. Galbanic acid isolated from Ferula assafoetida exerts in vivo antitumor activity in association with antiangiogenesis and antiproliferation [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A58.

Oral administration of Qing-Shu-Yi-Qi-Tang reduce lung cancer-induced cachexia in mice

This study is to investigate anti-inflammatory and anti-cachectic effect of Qing-Shu-Yi-Qi-Tang (QSYQT) in RAW 264.7 cells and Lewis lung cancer cells bearing mice. We examined the effect of QSYQT on LPS-induced inflammatory activity in a murine macrophage cell line, Raw 264.7. We evaluated cachectic parameters, such as weight loss, food intake and tumor size in tumor-bearing and non-tumor-bearing mice treated with QSYQT decoction or a normal diet. Cytokine production in cell culture and in cachectic mice was quantified by ELISA. NF-kB expression was measured using real-time polymerase chain reaction (PCR). QSYQT significantly reduced the IL-1β, IL-6 and TNF-α production in LPS-stimulated RAW 264.7 cells. Treatment of QSYQT prevented weight loss in tumor-bearing mice without affecting food intake or tumor growth. Furthermore, the level of proinflammatory cytokines, IL-1β, IL-6 and TNF-α in sera were significantly reduced in tumor-bearing mice treated with QSYQT. NF-kB expression in spleen of LLC-derived mice was decreased in the presence of QSYQT. Our results revealed that QSYQT exerts an anti-cachectic effect on LLC-induced cachectic mice. The effect is conclusively associated with modulation of IL-6 production through NF-kB. Key words: Chinese traditional medicine, cachexia, anti-inflammation, IL-6, NF-kB, palliative medicine.

Effect of Endostatin on Preventing Postoperative Progression of Distant Metastasis in a Murine Lung Cancer Model

The relapse and metastasis of cancer remain a predominant cause of death after surgical removal of the primary tumor. There is a positive linkage between the postoperative upregulation of systemic angiogenic activity and distant tumor metastasis. In the present study, we established a spontaneous metastasis model and investigated whether antiangiogenic therapy using endostatin could prevent the progression of distant metastasis after removal of the primary tumor. Female C57BL/6 mice were implanted subcutaneously with 1 × 106 Lewis lung cancer cells. Twenty days after implantation of the cancer cells, the primary tumor was removed and the mice were randomly divided into three groups. The NS group received normal saline, the L-ES group received 3 mg/kg endostatin, and the H-ES group received 20 mg/kg endostatin intravenously daily for 10 days. The effect of endostatin on lung metastases and the survival time of the mice were observed. Flow cytometry and immunohistochemistry were carried out to assess the angiogenic activity. The serum endostatin levels in peripheral blood were measured using an enzyme-linked immunosorbent assay. The mean number of metastatic pulmonary nodules and the mean net lung weight in the NS, L-ES and H-ES groups was 10.2, 2.8 and 4.0, and 0.55 g, 0.31 g and 0.36 g, respectively. The difference between the NS group and the endostatin-treated groups was statistically significant (P <0.05). The endostatin-treated mice showed prolonged overall survival (P <0.05). Compared with the NS group, the endostatin-treated groups had lower levels of circulating endothelial cells in peripheral blood and showed a decrease in microvessel density in the metastatic tumors, with a more marked reduction in the L-ES group (P <0.05). The systemic presence of endostatin was gradually increased with the continued administration of endostatin to the mice. Antiangiogenic therapy with endostatin is effective in inhibiting the postoperative progression of distant metastasis.

Non-cell metastasis of malignant tumor: A preliminary experiment

The present study aimed to investigate the non-cell of malignant tumor (metastasis at the molecular level). Primers for the specific SPY gene of male mouse were designed and used to detect the SPY gene in the Lewis lung cancer (LLC) cells by PCR; LLC cells were inoculated into females C57BL/6 mice. H and E staining was performed to detect the subcutaneous primary tumor and metastatic tumors in the lungs and liver, and polymerase chain reaction (PCR) and In situ hybridization (ISH) were employed to detect the sex determining region of the Y (SRY) gene in the primary and metastatic tumors. After inoculation of LLC cells carrying SRY gene into female C57BL/6 mice, the SRY gene was detectable in the metastatic tumor in these female mice, which suggests the metastatic cancer cells originate from the primary tumor. Our results demonstrated the of cancer cells at the cellular level but not the non-cell metastasis. It is very difficult to verify the of non-cell metastasis by animal experiment for the limit of itself. Further studies with new strategies are required to elucidate our hypothesis due to the presence of limitations in the present study. Key words: Cancer, metastasis, sex determining region of the Y (SRY) gene.

Development of New Spontaneous Metastatic Heterotopic Model of Lewis Lung Carcinoma Imaged by GFP Expression

Many studies have demonstrated the importance of spontaneous metastases in cancer research. Until now, we still had only a few spontaneous metastatic models with high occurrence rate of metastasis in distant lymph and visceral tissues. We report a syngeneic heterotopic metastatic model using the Lewis lung cancer cell line with high metastatic ratio in C57BL/6 mice after transplantation by injection of cancer cells and without surgical intervention. Metastatic process was declared for each mouse in two groups –sacrificed 3 or 5 weeks after subcutaneous (s.c.) injection of the tumor cells into the dorsal side of the tail. The total number of metastases was counted as the sum of observed macrometastases. Our model produced produced a 100% rate of spontaneous lymphatic and visceral metastases after a simple injection transplantation into the heterotopic site. In mice with large primary tumors which are non-lethal, visceral and lymph macrometastases were observed. Tumor volume correlated linearly not only with the tumor growth time, but also with the number of metastases in lymph nodes and organs. This new metastatic model could be useful for studying the metastasis mechanism and for developing therapy for lymph and visceral metastases.

Experimental Research on Effect of Medicated Serum contained with Yiqiyangyinjiedu Recipe on Proliferation and Apoptosis of Lewis Lung Cancer Cells in vitro

Objective To observe the effect of medicated serum contained with yiqiyangyinjiedu recipe on proliferation and apoptosis of Lewis lung cancer cells(LLC) in vitro.Methods A total of 30 male rats waere divided into Low,medium,high dose Yiqiyangyinjiedu recipe,Cisplatin(DDP),Yiqiyangyin recipe combined with DDP and normal saline(NS) group.The medicated serum was prepared by the serum pharmacology method.Then the serum was added to LLC in vitro.The proliferation of LLC was measured by methyl thiazolyl tetrazolium(MTT) method.The cell apoptosis were measured by flow cytometry(FCM) and Annexin V-FITC/PI.Immunohistochemistry(SP method) measured the expression levels of Caspase-3.Results Yiqiyangyin detoxification recipe containing drug serum had a remarkable growth-inhibition on LLC in vitro,these effects showed characters being time-dependent and concentration-dependent.Effect of high concentrations on LLC in 72h,the growth inhibiting rate(IR) was 50%.Inhibiting rate of yiqiyangyin detoxification recipe joint DDP containing drug serum up to 94.67%.Compared with the control group,each group containing serum can induce apoptosis of LLC,and showed characters being time-dependent and concentration-dependent.Yiqiyangyin detoxification recipe joint DDP containing drug serum on LLC for 72h,the apoptosis rate declined and the necrosis increase.Expression of Caspase-3 was being dose an time dependent manner(P0.05).Compared with other groups,expression of Caspase-3 of chinese combined DDP was highest(P0.05).Conclusion Yiqiyangyin detoxification recipe can inhibit proliferation and induce apoptosis of LLC.The promotion of Caspase-3 protein expression may be one of the mechanisms of inducing apoptosis of LLC.