Lung Metastatic Cell Lines Research Articles

Modeling Ewing Sarcoma Lung Metastasis.

Ewing Sarcoma (EwS) is the second most common malignant bone tumor in adolescents and young adults. The single-most powerful predictor of outcome in EwS is presence of metastatic burden at the time of diagnosis. Patients with metastatic Ewing Sarcoma have an abysmal 5-year survival rate of 10%-25%, which has not changed over the past 30-40 years. Thus, unraveling underlying mechanisms of EwS metastasis are imperative for developing effective therapeutic measures. Investigations towards this goal are limited by the lack of reliable genetically engineered mouse models and specialized metastatic models. Using two established cell lines, A673 and TC71, we generated lung specific metastatic cell lines by serial orthotopic intra-tibial injection followed by isolation of cells from lung metastases. The lung metastatic lines generated exhibit distinct differential molecular signatures from the parental cells when analyzed using a multi-omics approach. These signatures overlapped with EwS patient primary bone and metastatic lung specimens supporting the clinical relevance of these preclinical models of EwS. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Intra-Tibial injection in NSG mice Basic Protocol 2: Development and characterization of lung metastatic cell line.

ANTICANCER AND ANTIMICROBIAL POTENTIAL OF BARLERIA PRIONITIS LEAVES ETHANOL EXTRACT

Objective: The present study was focused to screen traditionally used Barleria prionitis for anticancer effects against various cell lines and antimicrobial effect against various pathogenic strains of bacteria and fungi.Methods: Extraction of Barleria prionitis leaves in ethanol was done by the Soxhlet method. After extraction, phytochemical estimation of these seven secondary metabolites like alkaloids, flavonoids, anthraquinones, saponins, terpenoids, tannins, and cardiac glycosides was done as per the protocols of Kokate. Minimum Inhibitory Concentration (MIC) effect of Barleria leaf ethanol (BLE) was done by the dilution method on five bacterial and five fungal strains. Further analysis (anticancer activity) was done with SRB (Sulphorhodamine B) assay. Statistical analysis of antimicrobial and anticancer activity was done by using MS Excel 2007 to±standard deviation and student t-test.Results: Barleria leaf extract with ethanol is a non-polar solvent extract and considered as the best solvent to extract the maximum number of secondary metabolites like alkaloids, saponins, flavonoids, and tannins. BLE extract gave excellent MIC (Minimum Inhibitory Concentration) effects against pathogenic bacteria and pathogenic fungal strains. BLE had highly effective activity against Pseudomonas aeruginosa with 1.25 mg MIC, the OD value of the sample was 0.02±0.0005 (±SD) with 0.0211-0.0245 range. MIC against fungal strains had effective activity against Candida vaginitis with 6.25 mg, the OD value of the sample was 0.02±0.0003 (±SD) with 0.0213-0.0232 range. BLE extract had given more than 70% inhibition against breast cell lines (MCF-7) and 75.16% inhibition of DLD1 cell lines; it was near to Doxorubicin antibiotic (81%). Breast metastatic cell line (MDMAMB-468) was found 60% inhibited with BLE extract and there was a great difference in the results of Doxorubicin. Out of six experimented cell lines, BLE gave very good inhibition for two cell lines, i.e. Breast (MCF-7) and Colon cell lines (DLD-1).Conclusion: BLE extract had shown the best antimicrobial and antifungal effect, against Pseudomonas aeruginosa and Candida vaginitis respectively. BLE also showed an anticancer effect against Lung cell lines (A549), Breast cancer cell line (MCF-7), Breast metastatic cell line (MDMAMB-468), Colon cell line (DLD-1) and lung metastatic cell line (NCIH358) at a statistically significant level (p=<0.05). It did not give any significant results against the colon metastatic cell line (SW620).

BM-26 * CD15 AND E-SELECTIN MEDIATION OF ADHESION OF NON-SMALL CELL LUNG CANCER CELLS TO BRAIN ENDOTHELIUM IN LUNG-BRAIN METASTASIS

BACKGROUND: Lung metastases to the brain are common secondary cancers, representing over 50% of fatal complications of systemic cancer. Metastasis of cancer cells involves multiple steps including intravasation, cancer cell-endothelial rolling and adhesion, extravasation and cerebral colonization. Here, we focused on metastatic Non-Small Cell lung cancer cells adhesion to brain endothelial cells. Specifically, we investigated the role of CD15 in lung cancer and its interaction with E-selectin and brain endothelial cells adhesion. METHODS: Five lung cancer cell lines and one brain endothelial cell line (hCMEC/D3) were used (two primary, COR-L105 and A549; one lymph node lung metastatic cell line, NCI-H1299 and two biopsied secondary lung to brain metastatic cultures). Western blot (WB), immunocytochemistry (ICC) and flow cytometric analysis were used to characterise CD15 expression. To induce E-selectin, endothelial cells were treated with TNF-a. Qualitative and quantitative tumour cell-endothelial adhesion assays and live cell image microscopy were conducted, followed by antibody blocking experiments. RESULTS: All cell lines expressed detectable CD15, as determined by WB, flow cytometry and ICC, with the lung cancer to lymph node cell line expressing the highest, followed by lung to brain cell lines. E-selectin expression was significantly increased in the brain endothelial cell line (p < 0.001) with detectable levels in lung cancer cell lines. There was a significant increase in lung cancer adhesion to brain endothelium exposed to TNFa (P < 0.001); the highest increase seen in the lung-lymph node cells followed by both the lung-brain cells and the primary lung cancer cell line (A549). This adhesion was completely blocked by co-treatment of CD15 antibodies compared to IgM isotype controls. CONCLUSIONS: CD15 may play a significant role in lung cancer cell adhesion to brain endothelium.We postulate that by interfering with CD15 homodimer and heterodimer interactions, adhesion of circulating lung cancer cells to the blood brain barrier would be prevented.

The role of GPC5 in lung metastasis of salivary adenoid cystic carcinoma

ObjectiveProteoglycans play a crucial role in salivary adenoid cystic carcinoma (SACC) tumorigenesis, neurotropic growth and lung metastasis. In this study, we investigated the role of GPC5 in the lung metastasis of SACC. DesignThe expression of heparan sulfate proteoglycans (HSPGs) was detected in SACC-M (high lung-metastatic cell line), SACC-2 (low lung-metastatic cell line) and SACC-83 (low lung-metastatic cell line) cells by relative quantitative Real-Time PCR. The expression of GPC5 was detected by immunofluorescence and Western blot analysis in SACC-M, SACC-2 and SACC-83 cells, and by immunohistochemical analysis in primary tumours from 16 cases of SACC patients with or without lung metastasis. GPC5 expression was silenced in SACC-M cells, cell proliferation and tumour growth was evaluated by MTT assay and nude mice model, respectively. ResultsExpression of most HSPGs was decreased in SACC-M cells, but GPC5 expression increased 3.24-fold and 815.69-fold (more than 3-fold) in SACC-M compared with SACC-2 and SACC-83 cells, respectively. Immunohistochemical analysis showed higher expression of GPC5 in SACC with lung metastasis compared to SACC without lung metastasis. The lung metastasis of SACC-M cells in nude mice was obviously decreased after GPC5 silencing (P<0.05), although GPC5 silencing had no significant effect on SACC-M cell proliferation. ConclusionGPC5 may contribute to lung metastasis of SACC.

Abstract 5170: Identification of factors secreted by melanoma exhibiting propensity for lung metastases

Abstract Disseminated malignant melanoma is a result of widespread lymphatic and systemic metastases. We hypothesized that melanoma cells secret a cohort of factors that orchestrate tumor microenvironment to enable them to expand and metastasize. A proteomic approach was used to identify factors secreted by a lung metastatic and its parental primary melanoma cell lines as well as four primary cultured melanoma cell strains established from surgical material of melanoma patients. The lead factors identified were matricellular proteins, extracellular matrix proteins, tissue metalloproteinases, proteinase inhibitors, and insulin-like growth factor binding proteins. Several of these factors exhibit differential levels between the primary and the lung metastatic cell lines. To establish a melanoma lung metastasis signature, lung metastatic melanoma tumor cell lines derived from xenograph melanoma were selected. Protein secretion in culture medium by melanoma cells that exhibit a propensity to metastasize to the lung was analyzed using Western blot analysis. Our data show that decreased extracellular level of matrix metalloproteinases (MMP1, MMP2) and increased level of proteinase inhibitors (TIMP1, Glia-derived nexin) are associated with the development of melanoma lung metastases. These results suggest that these proteins can be useful markers in clinical testing for melanoma progression and as potential targets for intervening metastatic melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5170. doi:10.1158/1538-7445.AM2011-5170

Abstract 2024: Over-expression of miR-106b in metastatic hepatocellular carcinoma using an orthotopic metastasis animal model

Abstract Hepatocellular carcinoma (HCC) is one the most fatal cancers worldwide. Early diagnosis is crucial for curing the disease. However, patients are often diagnosis at late-stage with distant metastasis, and the prognosis of metastatic HCC is poor. Molecular pathological studies confirm that gene expression alteration plays a critical role in disease progression. Recently it has been discovered that microRNA (miRNA) can alter the gene expression at post-transcription level. This study aimed to elucidate the role of miRNA in development of metastatic HCC using an orthotopic animal model and the molecular pathway involved. Orthotopic metastasis animal model was established by implanting HCC cell lines onto the liver of the SCID mice. Tumor growth was monitored by in vivo imaging system. Primary tumor and lung metastasis were observed after 12 weeks of inoculation. Tumor were then excised and established into primary tumor cell lines (PT) and lung metastatic cell lines (LM). Functional studies comparing the PT and LM cell lines derived from HCC cell line PLC demonstrated higher cell invasion and migration ability in the metastatic cell line compared with the primary tumor cell line. In addition, the LM cell line possessed more stress fiber than the PT cell line. The PLC-derived cell lines and another metastatic HCC cell line, MHCC97H-derived cell lines were subjected to the miRNA microarray analysis. Fifteen human miRNAs were found to be differentially expressed in the LM cell lines when compared to the PT cell lines. miR-106b was one of the miRNAs which was over-expressed in LM cell lines. We further confirmed our findings in HCC clinical sample and found that miR-106b was over-expressed in HCC tumor compared with the adjacent non-tumor tissue, and its expression was significantly associated with the tumor grade (p=0.018). MicroRNA knock-down study was performed by using the miR-106b LNA knock-down probe. The miR-106b knock-down reverted the cell migration phenotype, and fluorescent staining of stress fiber formation showed that the miR-106b knock-down cells have less stress fiber formation than the parental cell lines and the scramble control. We further studied the expression of E-cadherin in PLC-PT, PLC-LM, the scramble control and the miR-106b knock-down cell lines. Our result demonstrated that miR-106b expression inversely correlated with E-cadherin level. Since decrease of E-cadherin expression is the hallmark for epithelia-mesenchymal transition (EMT), further studies will focus on the role of miR-106b in EMT of HCC cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2024.

Antiproliferative activity of Vietnamese medicinal plants.

Methanol, methanol-water (1:1) and water extracts were prepared from seventy-seven Vietnamese medicinal plants and tested for their antiproliferative activities against human HT-1080 fibrosarcoma cells. Among them, fifteen extracts including seven methanol extracts of Caesalpinia sappan, Catharanthus roseus, Coscinium fenestratum, Eurycoma longifolia, Hydnophytum formicarum and Streptocaulon juventas (collected at two areas), six methanol-water (1:1) extracts of Cae. sappan, Cat. roseus, Co. fenestratum, H. formicarum and S. juventas (at two areas), and two water extracts of Cae. sappan and S. juventas exhibited antiproliferative activities in a concentration-dependent manner. Their antiproliferative activities against human cervix HeLa adenocarcinoma, human lung A549 adenocarcinoma, murine colon 26-L5 carcinoma, murine Lewis lung carcinoma (LLC) and murine B16-BL6 melanoma cells were then examined. Co. fenestratum showed selective activity against lung carcinoma and/or lung metastatic cell lines, A549, LLC and B16-BL6, while H. formicarum and S. juventas showed selective activity against human tumor cell lines, HeLa and A549. Characteristic morphological change and DNA fragmentation indicated the antiproliferative activity to be due to the induction of apoptosis.

Highly metastatic variant of a mouse colon carcinoma cell line, LM17 and its response to GM-CSF gene therapy.

In order to establish a highly metastatic variant of a mouse colon carcinoma cell line (CT26), BALB/c mice were first subcutaneously injected with CT26 cells. Several weeks later, metastatic tumors in lungs were resected, mechanically dispersed into a single cell suspension and cultured in vitro until cells reached confluency. These tumor cells were then subcutaneously injected into new mice. After repeating this procedure five times, a highly lung metastatic cell line, denoted as LM17, has been established. The LM17 cells grow in vitro with or without serum, whereas parental CT26 cells require serum for their growth. The LM17 cells adhere to type I collagen or fibronectin stronger than CT26 cells do. The LM17 cells invade through Matrigel-coated basement membrane in greater number than CT26 cells. By gelatin zymography, LM17 cells showed higher proteinase activity than CT26. Furthermore, subcutaneous injection of irradiated LM17 cells infected with adenovirus harboring mouse GM-CSF gene prevents the growth and lung metastasis of pre-existing subcutaneous tumor. The injection of irradiated GM-CSF-producing LM17 cells after the surgical removal of pre-existing tumor also protected the occurrence of lung metastasis. These results suggest that this highly metastatic LM17 cell line could be useful for analysis of the lung metastatic mechanism and as the mouse GM-CSF gene therapy model.

Expression of human choriogonadotropin-like material correlates with metastatic phenotype of R3230 AC rat adenocarcinoma.

Human choriogonadotropin (hCG)-like material has been found in variable amounts on the surface of cells of human and animal tumors. Intravenous injection of R3230 AC rat adenocarcinoma cells, one of the models investigated, results in multiple lung foci seeding. We analyzed the phenotypic diversity of this tumor by cloning and culturing two distinct cell subpopulations from a cell culture of this tumor, hereafter called OR or original cell culture. One was obtained after repeated exposure of the OR to increasing concentrations of concanavalin A and wheat germ agglutinin. A single clone was isolated and was named lectin-resistant (LR) cell line. The LR cells did not metastasize but maintained stable tumorigenicity and morphology over at least 10 passages. A second cell line was obtained by repeated passage and injection of cells from a single metastatic node. After repeating the process five times, a single clone of cells was selected from the final variant and was called lung metastatic (LM) cell line. The LM cultured cells maintained stable tumorigenicity, morphology, and metastatic properties for no more than 10 passages. OR, LR, and LM cells were assessed by their doubling time (DT), chromosome counts, and hCG immunocytochemistry. The results demonstrated that the LM cell line had a higher chromosome count than the LR and the OR cell lines, and its DT was the shortest. Immunocytochemistry of the transplanted OR neoplasm showed scattered expression of the hCG-like material. By the same techniques a complete lack of reactivity of the LR cells was found. However, almost all cells of the LM line were strongly positive for hCG-like material. After a few passages, the great majority of the LM cells also became unreactive. Our data demonstrate: (i) the existence of marked heterogeneity of the expression of hCG-like material in the primary tumor cell population; (ii) that the expression of hCG-like material correlates with the metastasizing capacity of the cells; and (iii) that there is a phenotypic instability for the expression of hCG-like material by tumor cells when maintained in vitro.