Lewis Lung Carcinoma Tumor Cells Research Articles

Exosomal IL-8 derived from Lung Cancer and Colon Cancer cells induced adipocyte atrophy via NF-κB signaling pathway

BackgroundCytokines secreted in the tumor microenvironment function in cancer cachexia (CC), a common clinicopathological syndrome associated with adipocyte wasting and skeletal muscle atrophy. Extracellular vesicles (EVs) secreted by cancer cells actively engage in inter-tissue communication; EVs and enclosed cytokines are largely undefined in CC adipocytes wasting.MethodsEVs derived from Lewis lung carcinoma (LLC) and colorectal cancer C26 cells were extracted and characterized. Conditioned medium and EVs from cancer cells were applied to 3 T3-L1 adipocytes. Recombinant IL-8, IL-8 neutralizing antibody, CXCR2 and NF-κB inhibitor were examined in functional assays. Lipolysis of adipocytes was monitored by Western blots, Oil red O staining and glycerol assays. Furthermore, LLC and C26 cell lines were established as cachexia model to explore the relevance of IL-8 and NF-κB signaling in CC adipose wasting. Adipose tissues were collected for histology analyses.ResultsLLC and C26 cell-derived EVs induced lipolysis of 3 T3-L1 adipocytes. Specially, Dil-labeled EVs were effectively taken up by 3 T3-L1 adipocytes, which were motivated by the delivered IL-8 to elicit the NF-κB pathway. In comparison, special IL-8 neutralizing antibody relieved that lipolysis of 3 T3-L1 adipocytes induced by EVs together with conditioned medium of LLC and C26 cells, respectively. Consistently, both CXCR2 and NF-κB inhibitors would lessen the phenotype of lipolysis in 3 T3-L1 adipocytes. In the in vivo settings, both LLC and C26-tumor bearing mice had higher serum IL-8 levels as compared to the control groups. Two typical lipolysis markers, PGC1α and UCP1, were also up-regulated in the adipose tissues of LLC and C26-tumor mice groups, respectively.ConclusionsEVs secreted by LLC and C26 tumor cells would induce adipocyte wasting via extracellular IL-8-mediated NF-κB signaling. Our study pointed out the physiological and therapeutic values of exosomal IL-8 in CC lipolysis.

Effect of LHS-1269 on morphology and angiogenesis of Lewis lung carcinoma

Background. The new anticancer chemical compound LHS-1269 from the class of indolocarbazoles is undergoing preclinical studies at the N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia. LHS-1269 has a high antitumor activity on transplanted tumors of mice, showed high cytotoxic activity on many tumor cells lines in vitro, and has shown an inhibitory effect on vasculogenic mimicry in tumors in vitro. LHS-1269 does not affect the catalytic activity of human topoisomerases I and IIα. An antiangiogenic mechanism of the drug’s antitumor action is suggested.Aim. To evaluate the effect of LHS-1269 on the morphological features and angiogenesis of transplanted Lewis lung carcinoma (LLC) in BDF1 mice.Materials and methods. In BDF1 mice (n = 20) with transplanted LLC tumor on days 1 and 3 after 5-fold intraperitoneal administration of LHS-1269 in a single dose of 60 mg (total dose – 300 mg/kg), mice (n = 20) on days 5 and 8 after a single intravenous injection of LHS-1269 at a dose of 100 mg/kg, mice (n = 20) with transplanted LLC tumor without administration of LHS-1269 (control). The assessment of the antitumor effect was carried out according to the criterion of inhibition of tumor growth (%) and the study of the morphological features of the tumors. To assess the effect of LHS-1269 on tumor angiogenesis in LLC tumor sections, a visual calculation of the average number (density) of blood vessels and immunohistochemical detection of expression of the CD31+ endothelial marker were performed.Results. The LHS-1269 compound in animal groups with 5-fold and 1-fold use caused tumor growth inhibition – 59–70 and 67–79 %, with pronounced morphological changes and tumor cell death. There is an uneven distribution of blood vessels in tumors and surrounding tissues in all groups. LHS-1269 caused a statistically significant decrease in the average number of blood vessels in the tumor both after 5-fold and after 1-fold administration. The statistically significant decrease in the average number of blood vessels in the surrounding connective tissue tumor was observed only after 5-fold administration of the drug. The decrease in the average number of CD31+ endothelial cells after five times intraperitoneal administration was statistically insignificant compared to control (83.1 ± 8.7 and 59.6 ± 18.9, respectively). The increase in this indicator after a single intravenous injection of LHS-1269 was statistically significant.Conclusion. LHS-1269, when administered five times and once in mice, caused pronounced morphological changes in the form of damage and death of LLC tumor cells. The results of a study of angiogenesis in a tumor do not allow an unambiguous conclusion about the inhibitory effect of LHS-1269 on angiogenesis in LLC tumors in mice.

Deficiency of plasminogen activator inhibitor‐2 results in accelerated tumor growth

BackgroundUpregulation of the plasminogen activation system, including urokinase plasminogen activator (uPA), has been observed in many malignancies, suggesting that co‐opting the PA system is a common method by which tumor cells accomplish extracellular matrix proteolysis. PAI‐2, a serine protease inhibitor, produced from the SERPINB2 gene, inhibits circulating and extracellular matrix‐tethered uPA. Decreased SERPINB2 expression has been associated with increased tumor invasiveness and metastasis for several types of cancer. PAI‐2 deficiency has not been reported in humans and PAI‐2‐deficient (SerpinB2−/−) mice exhibit no apparent abnormalities. ObjectivesWe investigated the role of PAI‐2 deficiency on tumor growth and metastasis. MethodsTo explore the long‐term impact of PAI‐2 deficiency, a cohort of SerpinB2−/− mice were aged to >18 months, with spontaneous malignancies observed in 4/9 animals, all of apparently vascular origin. To further investigate the role of PAI‐2 deficiency in malignancy, SerpinB2−/− and wild‐type control mice were injected with either B16 melanoma or Lewis lung carcinoma tumor cells, with markedly accelerated tumor growth observed in SerpinB2−/− mice for both cell lines. To determine the relative contributions of PAI‐2 from hematopoietic or nonhematopoietically derived sources, bone marrow transplants between wild‐type C57BL/6J and SerpinB2−/− mice were performed. Results and ConclusionsOur results suggest that PAI‐2 deficiency increases susceptibility to spontaneous tumorigenesis in the mouse, and demonstrate that SerpinB2 expression derived from a nonhematopoietic compartment is a key host factor in the regulation of tumor growth in both the B16 melanoma and Lewis lung carcinoma models.

Reduced sucrose nonfermenting AMPK-related kinase (SNARK) activity aggravates cancer-induced skeletal muscle wasting

Sucrose nonfermenting AMPK-related kinase (SNARK) is a member of the AMPK family of kinases and has been implicated in the regulation of critical metabolic processes. Recent findings demonstrate that SNARK has an important role in the maintenance of muscle mass with age. Loss of skeletal muscle mass (cachexia) is a key problem for cancer patients. Thus, based on our previous findings with aging, we hypothesized that SNARK would play a role in regulating muscle mass under conditions of cancer cachexia. To test this hypothesis, Lewis Lung Carcinoma tumor cells or vehicle were injected subcutaneously in the right flank of wild type mice, muscle-specific transgenic mice expressing inactive SNARK mutant (SDN) or muscle-specific transgenic mice overexpressing wild-type SNARK (SWT). All tumor-bearing mice presented muscle wasting compared to vehicle-injected mice. However, SDN tumor-bearing mice had more pronounced atrophy compared to wild-type and SWT tumor-bearing mice. Histological analysis confirmed muscle atrophy in tumor-bearing mice, and SDN tumor-bearing mice exhibited a significantly smaller skeletal muscle cross-sectional area than wild-type and SWT tumor-bearing mice. Moreover, SDN tumor-bearing mice had increased skeletal muscle BAX protein expression, a marker of apoptosis, compared to other groups.Thus, lack of SNARK in skeletal muscle aggravates cancer-induced skeletal muscle wasting. These findings uncover a role for SNARK in the maintenance of skeletal muscle mass under cachexia conditions.

Apoptosis of Lewis Lung Carcinoma Cells Induced by Microwave via p53 and Proapoptotic Proteins In vivo.

Background:Microwave therapy is a minimal invasive procedure and has been employed in clinical practice for the treatment of various types of cancers. However, its therapeutic application in non-small-cell lung cancer and the underlying mechanism remains to be investigated. This study aimed to investigate its effect on Lewis lung carcinoma (LLC) tumor in vivo.Methods:Fifty LLC tumor-bearing C57BL/6 mice were adopted to assess the effect of microwave radiation on the growth and apoptosis of LLC tumor in vivo. These mice were randomly assigned to 10 groups with 5 mice in each group. Five groups were treated by single pulse microwave at different doses for different time, and the other five groups were radiated by multiple-pulse treatment of a single dose. Apoptosis of cancer cells was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Western blotting was applied to detect the expression of proteins.Results:Single pulse of microwave radiation for 5 min had little effect on the mice. Only 15-min microwave radiation at 30 mW/cm2 significantly increased the mice body temperature (2.20 ± 0.82)°C as compared with the other groups (0.78 ± 0.29 °C, 1.24 ± 0.52 °C, 0.78 ± 0.42 °C, respectively), but it did not affect the apoptosis of LLC tumor cells significantly. Continous microwave radiation exposure, single dose microwave radiation once per day for up to seven days, inhibited cell division and induced apoptosis of LLC tumor cells in a dose- and duration-dependent manner. It upregulated the protein levels of p53, Caspase 3, Bax and downregulated Bcl-2 protein.Conclusions:Multiple exposures of LLC-bearing mice to microwave radiation effectively induced tumor cell apoptosis at least partly by upregulating proapoptotic proteins and downregulating antiapoptotic proteins. Continuous radiation at low microwave intensity for a short time per day is promising in treating non-small-cell lung cancer.

Systemic platelet‐activating factor‐receptor activation augments experimental lung cancer growth and metastasis (LB496)

Pro‐oxidative stressors including cigarette smoke (CS) generate novel lipids with Platelet‐activated Factor‐receptor (PAF‐R) agonistic activity and mediate systemic immunosuppression, one of the most recognized events in promoting carcinogenesis. Our previous studies have demonstrated that PAF‐R agonists mediated systemic immunosuppression augments the growth of experimental murine B16F10 melanoma tumors in a PAF‐R dependent manner. As CS generates PAF‐R agonists, the current studies sought to determine the impact of PAF‐R‐agonists on lung cancer growth and metastasis using an established experimental murine lewis lung carcinoma (LLC) model. We show that systemic PAF‐R agonist, CPAF augments the growth of subcutaneously implanted LLC tumors in a PAF‐R dependent manner as this effect was not observed in PAF‐R deficient (PAFR‐KO) mice. We next determined the ability of CPAF‐mediated PAF‐R dependent enhanced LLC tumor growth on lung metastasis. We observed increase number of lung mets only in CPAF‐treated WT and not in PAF‐R‐KO mice. In order to investigate the role of tumor or host cell PAF‐R in these effect, we first determined the expression of the PAF‐R in LLC tumor cells. Our results demonstrate that LLC tumor cells lack functional PAF‐R expression as measured by qRT‐PCR and functional Ca2+ mobilization assay and compared with arrays of PAF‐R sufficient and deficient cells. In addition, CPAF treatment failed to induce increase proliferation of LLC cells, indicating the role of host rather tumor cell PAF‐R in these events. This finding has clinical implications as it provides insight into a mechanism through which lung cancer cells evade immune surveillance.Grant Funding Source: NIH R01 HL062996, AICR 09A062, ACSIRG 4185607, Showlater Research Trust Fund 4485602

Role of Trans‐signaling Molecules from Lewis Lung Carcinoma (LLC) Cells on C2C12 Myotube Atrophy and Protein Turnover Regulation

Cancer cachexia is a metabolic wasting syndrome observed in many cancer patients that is characterized by unintentional body weight loss. Imbalances in rates of protein synthesis and degradation are responsible for cancer‐induced skeletal muscle wasting. Lewis lung carcinoma (LLC) cell implantation is a widely used explant mouse model for examining cancer cachexia and muscle wasting. The purpose of this study is to investigate the influence of trans‐signals induced by LLC tumor cells on C2C12 myotube morphology and protein turnover regulation. C2C12 myotubes were treated with LLC cell culture medium (0%, 15% and 25%) for 4h, 24h and 72h. LLC medium treatment (25% for 72h) significantly decreased myotube diameter. The LLC medium induced both Stat3 and AMPK phosphorylation, while decreasing mTOR (S2448) and 4EBP1 (T37/46) phosphorylation in dose dependent manner. In addition, LLC treatment activated the FOXO3/Atrogin‐1 protein degradation signaling pathway. These results demonstrate that trans‐signaling molecules from LLC tumor cells can result in C2C12 myotube atrophy by both inhibiting mTOR regulation of protein synthesis pathway and activating protein degradation. (RO1CA121249)

Unique expression patterns associated with preferential recruitment of immature myeloid cells into angiogenic versus dormant tumors

Cancer progression from microscopic dormant tumors into disseminated disease involves tumor angiogenesis, and is commonly referred to as the 'angiogenic switch'. CD11b(+)Gr1(+) immature myeloid cells (IMCs) were reported to promote angiogenesis and tumor progression. Here, we studied a model of tumor dormancy, in which Lewis Lung Carcinoma tumor cells were inoculated intra-abdominally into C57Bl/6J mice. Dormancy versus expansive growth was determined by the site of tumor implantation (lower vs upper abdomen). Global gene expression of IMCs was evaluated in different stages of recruitment, starting in the bone marrow, followed by the peripheral blood and finally in the vascular versus dormant tumors. We first demonstrated a ∼3 fold enrichment of IMCs within vascular tumors as compared with dormant tumors, correlating with tumor-infiltrating CD31(+) endothelial cells. Although their migration from the PB into dormant tumors led to differential expression of a relatively small number of genes, recruitment of IMCs into the upper tumors was associated with a profound transcriptional response. Importantly, a large set of proangiogenic genes were significantly upregulated in IMCs derived from vascular tumors compared with those derived from dormant tumors. We therefore, suggest that proangiogenic versus nonangiogenic transcriptional patterns is associated with the ability of IMCs to promote tumor angiogenesis.

Adenosinergic Regulation of the Expansion and Immunosuppressive Activity of CD11b+Gr1+ Cells

Extracellular adenosine and purine nucleotides are elevated in many pathological situations associated with the expansion of CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs). Therefore, we tested whether adenosinergic pathways play a role in MDSC expansion and functions. We found that A(2B) adenosine receptors on hematopoietic cells play an important role in accumulation of intratumoral CD11b(+)Gr1(high) cells in a mouse Lewis lung carcinoma model in vivo and demonstrated that these receptors promote preferential expansion of the granulocytic CD11b(+)Gr1(high) subset of MDSCs in vitro. Flow cytometry analysis of MDSCs generated from mouse hematopoietic progenitor cells revealed that the CD11b(+)Gr-1(high) subset had the highest levels of CD73 (ecto-5'-nucleotidase) expression (Δmean fluorescence intensity [MFI] of 118.5 ± 16.8), followed by CD11b(+)Gr-1(int) (ΔMFI of 57.9 ± 6.8) and CD11b(+)Gr-1(-/low) (ΔMFI of 12.4 ± 1.0) subsets. Even lower levels of CD73 expression were found on Lewis lung carcinoma tumor cells (ΔMFI of 3.2 ± 0.2). The high levels of CD73 expression in granulocytic CD11b(+)Gr-1(high) cells correlated with high levels of ecto-5'-nucleotidase enzymatic activity. We further demonstrated that the ability of granulocytic MDSCs to suppress CD3/CD28-induced T cell proliferation was significantly facilitated in the presence of the ecto-5'-nucleotidase substrate 5'-AMP. We propose that generation of adenosine by CD73 expressed at high levels on granulocytic MDSCs may promote their expansion and facilitate their immunosuppressive activity.

Enhanced Tumor Metastasis in Response to Blockade of the Chemokine Receptor CXCR6 Is Overcome by NKT Cell Activation

Invariant NKT (iNKT) cells can induce potent antitumor responses in vivo. However, the mechanisms that regulate the effects of iNKT cells are unclear. The chemokine receptor CXCR6, and its ligand CXCL16, have been shown to play critical roles in iNKT cell homeostasis and activation. Thus we investigated the role of CXCR6 in protection against experimental metastasis of B16-F10 melanoma (B16) and Lewis lung carcinoma (LLC) cells to the liver and lungs. Wild-type and CXCR6(-/-) mice exhibited no differences in tumor cell metastasis to the lungs. However, metastasis of LLC and B16 tumor cells to the liver was enhanced in CXCR6(-/-) mice. Liver metastasis was also increased in wild-type mice treated with a CXCL16 neutralizing Ab. As Ab treatments did not alter iNKT cell numbers, this implicates a direct role for CXCR6/CXCL16 in regulating antitumor immunity. Cytokine induction was significantly attenuated in CXCR6(-/-) mice upon systemic iNKT cell activation with the glycolipid Ags alpha-galactosylceramide (alpha-GalCer), alpha-C-GalCer (a Th1 polarizing derivative), or OCH (a Th2 polarizing derivative). Despite differences in the levels of cytokine production, liver and lung metastasis were inhibited significantly in both wild-type and CXCR6(-/-) mice treated with glycolipids. Single doses of alpha-GalCer, alpha-C-GalCer, or OCH were sufficient to prevent liver metastasis and subsequent doses failed to elicit optimal cytokine responses. Our findings implicate a role for CXCR6 in natural immunosurveillance against liver metastasis. However, CXCR6 deficiency could be overcome by systemic iNKT cell activation, demonstrating that even suboptimal iNKT cell activation can protect against metastasis.

Whole tumor cell vaccine with irradiated S180 cells as adjuvant

Whole tumor cell vaccines have been widely studied and remain promising cancer immunotherapies. In the present study, we discovered that vaccination with irradiated mouse sarcoma S180 tumor cells stimulated robust antitumor immunity to autologous tumor cells in both syngenic and allogenic mice. The antitumor activity requires both T and B cells, but not NK cells. When a mouse lung carcinoma (TC-1) whole tumor cell vaccine was combined with the S180 vaccine, the antitumor immunity against live TC-1 tumor cells is significantly enhanced compared to a TC-1 whole cell vaccine alone. This antitumor immunity not only prevents live tumor challenge but also eradicates existing tumor cells. A similar phenomenon was also observed when S180 vaccine was combined with LL2 Lewis lung carcinoma tumor cells. Therefore, S180 vaccine may serve as an adjuvant for other whole tumor cell vaccines.

Antitumor effect of SCV-07 in murine lung and renal cancer models

14012 Background: SCV-07 (γ-Glu-Trp) is an immunomodulatory peptide with demonstrated antitumor effects in the murine B16 melanoma model. Herein, the antitumor effects of SCV-07 were evaluated in mice bearing Lewis lung carcinoma (LLC) or renal carcinoma (RenCa) cells. The effect on growth of cultured LLC and RenCa cells was also evaluated to address whether the antitumor effect of SCV-07 is mediated by cytotoxicity. Methods: LLC and RenCa cells were implanted subcutaneously (sc) into the right axillary area of C57BL/6 and Balb/C mice, respectively. SCV-07 was sc administered (at a site different from tumor implantation) once daily at doses of 10 or 20 mg/kg (for LLC) and 5 or 10 mg/kg (for RenCa) for 14 consecutive days. Cisplatin (CDDP, given at 6 mg/kg on days 1, 6, and 12) and cyclophosphamide (CTX, given at 40 mg/kg every other day) were positive controls for LLC and RenCa tumors, respectively. The animals were euthanized on the termination day (day 16 in LLC model, day 17 in RenCa model) and tumors excised and weighed. For the evaluation of cytotoxic effect, LLC or RenCa cells were cultured in 96-well plates with SCV-07 at various concentrations, ranging up to 100 μg/mL, representing the plasma concentrations after effective doses in the mouse models. After 48- (LLC) or 72- (RenCa) hour incubation, inhibitory effects on cell proliferation were determined by the MTT method. Results: In both LLC and RenCa cancer models, SCV-07 treatment led to significant inhibition of tumor growth compared to vehicle control. On the day of study termination, decreases of 16.84% (p<0.05) and 37.45% (p<0.01) in LLC tumor weight were seen after SCV-07 treatment at 10 and 20 mg/kg, respectively, and decreases of 30.02% (p<0.01) and 28.33% (p<0.01) in RenCa tumor weight seen at 5 and 10 mg/kg, respectively. Growth inhibition in the controls were 77.35% (p<0.01) for LLC and 90.12% (p<0.01) for RenCa. In the cultured LLC or RenCa cells, SCV-07 did not inhibit cell proliferation at any tested concentration. Conclusions: Administration of SCV-07 once daily for 14 consecutive days via the sc route is effective against growth of LLC and RenCa tumors. SCV-07 does not induce in vitro cytotoxic effects in cultured LLC and RenCa tumor cells, indicating the antitumor effect of SCV-07 is related to its immunomodulatory actions, rather than cytotoxicity. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration SciClone Pharmaceuticals, Inc. SciClone Pharmaceuticals, Inc SciClone Pharmaceuticals, Inc.

Effect of expansile paclitaxel-loaded nanoparticles on growth of lung cancer in vivo

7593 Background: Surgical resection remains the most effective treatment for patients with non-small cell lung cancer; however, even in early stage diagnoses the presence of occult microscopic disease at the surgical resection margin significantly impacts long-term patient survival. Thus, the focus of this study is to establish proof of concept that locoregional growth of microscopic tumor burden can be prevented through the application of a novel “expansile” nanoparticle drug delivery system. Methods: Biocompatible polymer nanoparticles that enter tumor cells and swell 300 fold in response to pH were loaded with paclitaxel (Pax-NP) and assessed for efficacy against murine Lewis lung carcinoma (LLC) cells using cell viability assays in vitro and tumor implantation in vivo. Paclitaxel-loaded non-expansile nanoparticles (Pax-neNP) that are not sensitive to pH variation were also produced. For the in vivo studies, 7.5 x 105 LLC tumor cells were co-injected with 0.25 mg unloaded control (expansile) NP, Pax-NP, Pax-neNP or paclitaxel alone in 100μL Cremophor EL/ethanol (each equivalent to a 2.5 μg dose of paclitaxel) subcutaneously in C57Bl/6 mice. Results: In vitro assays demonstrated that co-culture of LLC cells and Pax-NP results in significant inhibition of tumor growth (p<0.01, t-test vs control). In contrast, LLC incubated with unloaded NP did not inhibit tumor growth. Moreover, Pax-NP prevented tumor growth in vivo. Whereas LLC alone (n=11) or LLC with 0.25mg unloaded NP (n=14) or Pax-neNP (n=12) resulted in the development of tumors with volumes of 388±86, 382±82 and 268±63 mm3, respectively (mean ± SE), only one mouse (n=15) developed a small tumor (2±2 mm3) following the injection of LLC and Pax-NP (p<0.05 via ANOVA). Further, injection with an equivalent dose of paclitaxel did not significantly reduce tumor growth (225±95, n=6). Conclusions: These results establish that locoregional growth of microscopic tumor burden can be inhibited by intracellular nanoparticle-mediated drug delivery. These early results suggest that Pax-NP, which undergo cellular uptake with resultant drug release, may afford enhanced local drug delivery aimed at preventing the growth of occult microscopic disease present at the resection margin following parenchyma-sparing surgery for lung cancer. No significant financial relationships to disclose.

Functional endogenous cytotoxic T lymphocytes are generated to multiple antigens co-expressed by progressing tumors; after intra-tumoral IL-2 therapy these effector cells eradicate established tumors.

Tumors contain many antigens that may be recognized by the immune system. It is not known whether these antigens, and the epitopes within these antigens, can all be recognized by the anti-tumor immune response or if such responses are restricted to a few dominant epitopes. Effector function of endogenous cytotoxic T lymphocytes (CTL) generated during tumor progression has previously been assessed by indirect, ex vivo assays, which often focused on a single antigen. Therefore, we evaluated the endogenous in vivo CTL response to multiple neo tumor antigens using murine Lewis lung carcinoma tumor cells transfected with ovalbumin or a polyepitope construct. Both express multiple MHC class I-restricted epitopes. Ovalbumin contains a known hierarchy of epitopes for given MHC molecules, whilst the polyepitope expresses a number of dominant epitopes. We show that as tumors progress, potent effector CTL are generated in vivo that are restricted to dominant epitopes; we did not see the responses to subdominant or cryptic epitopes. Our data show that the CTL recognizing tumor antigens vary in their lytic capacity, as the CTL responding to two of the four epitopes were particularly potent killers. The presence of these effector CTLs did not prevent tumor growth. However, intra-tumoral IL-2 treatment altered the potency, but not the hierarchy, of these CTL such that they mediated tumor regression. These results have implications for immunotherapy protocols.

The Serum Glycoprotein Fetuin-A Promotes Lewis Lung Carcinoma Tumorigenesis via Adhesive-Dependent and Adhesive-Independent Mechanisms

Fetuin-A is a serum glycoprotein in the cystatin family associated with the regulation of soft tissue calcification. We tested the role of systemic fetuin in tumor cell growth and metastasis by injecting Lewis lung carcinoma (LLC) cells into fetuin-A null and their wild-type (WT) littermate control C57BL/6 mice via the tail vein, s.c., and intrasplenic routes. In the experimental metastasis assay, the lungs of the WT mice were filled with metastatic nodules, whereas the lungs of the fetuin-A null mutant mice were virtually free of colonies at the end of 2 weeks. Lung colonization responded to the levels of serum fetuin-A in a dose-dependent manner, as observed by the formation of half as many colonies in mice heterozygous for the fetuin-A locus compared with homozygous WT mice and restoration of lung colonization by the administration of purified fetuin-A to fetuin-A-null mice. Serum fetuin-A also influenced the growth of LLC cells injected s.c.: fetuin-A-null mice developed small s.c. tumors only after a substantial delay. Similarly, intrasplenic injection of LLC cells resulted in rapid colonization of the liver with metastasis to the lungs within 2 weeks in the WT but not fetuin-A null mice. To examine the mechanism by which fetuin-A influences LLC colonization and growth, we showed that LLC tumor cells adhere to fetuin-A in a Ca(2+)-dependent fashion, resulting in growth of the tumor cells. These studies support the role of fetuin-A as a major growth promoter in serum that can influence tumor establishment and growth.

New Triterpene Aldehydes, Lucialdehydes A—C, from Ganoderma lucidum and Their Cytotoxicity Against Murine and Human Tumor Cells.

AbstractFor Abstract see ChemInform Abstract in Full Text.

New triterpene aldehydes, lucialdehydes A-C, from Ganoderma lucidum and their cytotoxicity against murine and human tumor cells.

Three new lanostante-type triterpene aldehydes, named lucialdehydes A-C (1-3), were isolated from the fruiting bodies of Ganoderma lucidum, together with ganodermanonol (4), ganodermadiol (5), ganodermanondiol (6), ganodermanontriol (7), ganoderic acid A (8), ganoderic acid B8 (9), and ganoderic acid C1 (10). The structures of the new triterpenes were determined as (24E)-3 beta-hydroxy-5 alpha-lanosta-7,9(11),24-trien-26-al (1), (24E)-3,7-dioxo-5 alpha-lanosta-8,24-dien-26-al (2), and (24E)-3 beta-hydroxy-7-oxo-5 alpha-lanosta-8,24-dien-26-al (3), respectively, by spectroscopic means. The cytotoxicity of the compounds isolated from the ganoderma mushroom was tested in vitro against Lewis lung carcinoma (LLC), T-47D, Sarcoma 180, and Meth-A tumor cell lines. Lucialdehydes B, C (2, 3), ganodermanonol (4) and ganodermanondiol (6) showed cytotoxic effects on tested tumor cells. Of the compounds, lucialdehyde C (3) exhibited the most potent cytotoxicity against LLC, T-47D, Sarcoma 180, and Meth-A tumor cells with ED(50) values of 10.7, 4.7, 7.1, and 3.8 microg/ml, respectively.

Annonaceous acetogenins from the Leaves of Annona montana

A novel Annonaceous acetogenin, montanacin F, with a new type of terminal lactone unit, was isolated from the leaves of Annona montana. Its structure was determined on the basis of spectral evidences and chemical methods, and a possible biosynthetic pathway was discussed. In addition, the cytotoxicity of montanacin F was evaluated in vitro against Lewis lung carcinoma (LLC) tumor cell lines. Furthermore, the previously isolated cytotoxic acetogenin annonacin against LLC was examined for in vivo antitumor activity with LLC tumor cells.

Chemoattraction of femoral CD34+ progenitor cells by tumor-derived vascular endothelial cell growth factor.

Patients and animals with GM-CSF-producing tumors have an increased number of mobilized CD34+ progenitor cells within their peripheral blood and tumor tissue. These CD34+ cells are inhibitory to the activity of intratumoral T-cells. The present study used the murine Lewis lung carcinoma (LLC) model to assess mechanisms that could lead to the accumulation of CD34+ cells within the tumor tissue. In vitro analyses showed that LLC tumor explants released chemoattractants for normal femoral CD34+ cells. The LLC tumor cells contributed to the production of this activity since CD34+ cell chemoattractants were also released by cultured LLC cells. Antibody neutralization studies showed that most, although not all, of the chemotactic activity that was produced by LLC cells could be attributed to VEGF. In vivo studies with fluorescent-tagged CD34+ cells showed their accumulation within the tumor tissue, but not within the lungs, spleen or bone marrow, suggesting a selective accumulation within the tumor. Whether or not VEGF could chemoattract CD34+ cells in vivo was measured with a VEGF-containing Matrigel plug assay. Infusion of fluorescent-tagged CD34+ cells into mice after the plugs became vascularized revealed the accumulation of fluorescent-tagged cells within the plugs. However, these CD34+ cells failed to accumulate within the VEGF-containing Matrigel plugs when they were infused together with neutralizing anti-VEGF antibody. Through a combination of in vitro and in vivo analyses, the LLC cells were shown to be capable of chemoattracting CD34+ cells, with most of the tumor-derived chemotactic activity being due to tumor release of VEGF.

A simple cell labeling technique by means of lectins linked to fluorochromes for the detection of cells on tissue sections

We designed a protocol for cell labeling with the lectin wheat germ agglutinin (WGA) linked to the fluorochrome tetramethyl-rhodamine isothiocyanate (TRITC) for effective detection of the B16F10 melanoma and Lewis lung carcinoma (LLc) cells on pulmonary histological sections from C57BL 6 mice. We have also determined a suitable concentration of WGA-TRITC (10 μg/ml), which leads to a very intense and homogeneous labeling of the cells, as it avoids cell clumping due to the presence of the lectin WGA. In order to determine to what extent the method affects these tumor cells, we have studied some important aspects related to their metastatic behavior, taking into account three parameters: a) viability and rate of proliferation of the cells cultured in vitro; b) percentage of animals ( C57BL 6 mice) bearing metastasis 15 days after intravenous inoculation with 10 5 B16F10 or LLc cells; and c) pattern of distribution of tumor foci in lung. There were no significant differences in these three parameters between the WGA-TRITC labeled-cells compared to the cultures of non-labeled cells in either of the cell lines (B16F10, LLc). Thus, we conclude that B16F10 and LLc tumor cells can be labeled following the protocol set-up in our study, as it allows these cells to be neatly identified on tissue sections and it causes no important physiological changes in the cells, with regard to metastatic behavior. These points make this technique very suitable for the detection of B16F10 and LLc cells on histological sections in studying their behavior during the first stages of the metastatic process.