|A Vascoactive Intestinal Peptide Antagonist Inhibits Non-small Cell Lung Cancer Growth
|TERRY W. MOODY, FARAH ZIA, MURIEL DRAOUI, DOUGLAS E. BRENNEMAN, MATI FRIDKIN, ARIANE DAVIDSON, AND ILLANA GOZES
|The most prevalent lung cancer, non-small cell lung cancer (NSCLC) has receptors for vasoactive intestinal peptide (VIP). Here the effects of a VIP antagonist (VIPhyb) on NSCLC growth were investigated. In vivo, when VIPhyb (10 µg, s.c.) was daily injected into nude mice, xenograft formation was significantly inhibited by -80%. In vitro, VIP (100 nM) stimulated colony formation -2-fold, whereas 1 µM VIPhyb inhibited colony formation by -50% when adenocarcinoma cell line NCI-H838 was used. The attenuation of tumor proliferation is receptor mediated, as VIPhyb inhibited specific 1251-labeled VIP binding to cell lines NCI-Hl57 and NCI-H838 with an IC50 of 0.7 µM. VIP (10 nM) increased the cAMP levels 5-fold when cell line NCI-H838 was used, and 10 µM VIPhyb inhibited the increase in cAMP caused by VIP. Northern blot analysis and radioimmunoassays have shown VIP mRNA and VIP-like immunoreactivity in NSCLC cells. These data suggest that VIP may be a regulatory peptide in NSCLC and that VIPhyb is a VIP receptor antagonist that inhibits proliferation.
|Use of an Image Analysis System to Count Colonies in Stem Cell Assays of Human Tumors
|BERNHARDT E. KRESSNER, ROGER R. A. MORTON, ALEXANDER E. MARTENS, SYDNEY E. SALMON, DANIEL D. VON HOFF, AND BARBARA SOEHNLEN
|As is well demonstrated elsewhere in this text, the clonogenic assay for tumor colony-forming cells has applicability to a broad scope of human tumors and has proved valuable in studies of biology, clinical course, and clieinosensitivity of human cancers. The development of this promising new area of clinical research, however, has precipitated a substantial new laboratory problem-, namely, the need for automation in counting tumor colonies. This need was not fully apparent until it became clear that the clonogenic assays predicted clinical and biological features of human cancers. In the initial studies, careful qualitative and quantitative evaluations of tumor clusters and colonies in soft agar were conducted by the clinical research laboratory staff of two of the authors (S.E.S., D.D.V.H.). As their studies proceeded, we recognized that there was a major need for a precise automated instrument for selective counting of tumor colonies and therefore initiated a join developmental project with BioLogics Incorporated (formally Bausch & Lomb Incorporated) on the application of image analysis to this task.
|Patterns Of Tumor Colony Development Over Time In Soft-Agar Culture
|WIM J. KIRKELS, OLGA E. PELGRIM, ADRIE M. M. HOOGENBOOM, MATHILDE W. AALDERS,FRANS M. J. DFBRUYNF, G. PETER VOOUS AND CHESTER J. HERMAN
|Human tumors were cultured by the two-layer soft-agar technique and the time course of tumor colony development was evaluated during periods of up to 6 weeks in culture. All colony counting was performed with an automated tumor colony counter (OMNICON; BioLogics Inc., Gainesville, NY, USA - formally Bausch and Lomb, Inc). This instrument provided colony counts per culture plate in six size categories from >60 Mm diameter colonies to > 149 m diameter colonies. Six to 24 culture plates were used for each "growth curve", generally 24. Control (non-drug-treated) cultures were obtained from II 7 tumors, of which 25 also provided enough cells to allow evaluation of the time course of colony development after exposure to cytostatic agents. The development of colonies in non-drug-treated plates usually demonstrated a lag phase, a logarithmic growth phase to maximum colony development and a subsequent deterioration of colonies. In spite of clumps seeded into the agar, real colony growth could be recognized by frequent colony counting of culture dishes, although the temporal patterns of growth were sometimes different if pure single-cell suspensions were compared with suspensions containing clumps from the same tumor. Drug pre-incubation caused changes in the temporal pattern of colony growth as well as in the total number of colonies. Some cultures showed drug sensitivity when evaluated at certain time points while evaluation at later time points showed only borderline drug effect or none at all. The potential utility of tumor colony growth curves in the clinical applications of tumor colony cultures is discussed.
|In-Use Evaluation of the OMNICON Automated Tumor Colony Counter
|CHESTER J. HERMAN, OLGA E. PELGRIM, WIM J. KIRKELS, RENE VERHEIJEN, FRANS M. J. DEBRUYNE,PETER KENEMANS, AND G. PETER VOOIJS
|The reproducibility and accuracy of the OMNICON (BioLogics, Inc., Gainesville, VA - formally Bausch and Lomb Inc.) automated tumor colony counter for counting tumor colonies growing in double layer soft agar is evaluated and the reproducibility is compared with manual tumor colony counting. Replicate within day run-to-run colony counts of the OMNICON show a median correlation coefficient (r) of >0.985, and day-to-day median r of >0.980. In contrast, for manual colony counting, the best intra-observer reproducibility achieved is a r of 0.943 and the best inter-observer reproducibility is a r of 0.831. Analysis of results from individual culture plates counted by the OMNICON on 5 separate days shows a median coefficient of variation of 10% with 77% of the culture dishes showing coefficients of variation of colony counts over 5 days of less than 20%. Counting of culture plates during incubation shows that the OMNICON is counting tumor colonies developing after plating of a single cell suspension.
|Improved Detection Of Drug Cytotoxicity In The Soft Agar Colony Formation Assay Through Use Of A Metabolizable Tetrazolium Salt
|MICHAEL C. ALLEY, CINDY B. UHL, AND MICHAEL M. LIEBER
|Use of a metabolizable tetrazolium salt was observed to facilitate assessments of tumor cell drug sensitivity in the soft-agar colony formation assay. Enzyme-mediated staining permits discrimination between viable and non-viable groups of cells so that drug-induced cytotoxicity is clearly identifiable by visual inspection as well as by computerized image analysis. The technique appears to be especially useful in the evaluation of primary tumor cell cultures which often contain substantial numbers of non-viable cellular aggregates.
|Feasibility of Drug Screening with Panels of Human Tumor Cell Lines Using a Microculture Tetrazolium Assay
|MICHAEL C. ALLEY, DOMINIC A. SCUDIERO, ANNE MONKS, MIRIAM L. I-LURSEY, MACIEJ J. CZERWINSKI, DONALD L. FINE, BETTY J. ABBOTT, JOSEPH G. MAYO, ROBERT H. SHOEMAKER, AND MICHAEL R. BOYD
|For the past 30 years strategies for the preclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. The feasibility of implementing an alternate approach, namely combined in vitro/in vivo screening for selective cytotoxicity among panels of human tumor cell lines derived from a broad spectrum of human solid tumors is under investigation. A group of 30 cell lines acquired from a variety of sources and representing 8 lung cancer pathologies as well as 76 cell lines representing 10 other categories of human cancer (carcinomas of colon, breast, kidney, prostate, ovary, head and neck, glioma; leukemia; melanoma; and sarcoma) have exhibited acceptable growth characteristics and suitable calorimetric profiles in a single, standard culture medium. Measurements of in vitro growth in microculture wells by cell-mediated reduction of tetrazolium showed excellent correlation (0.89 < r' < 0.98) with measurements of cellular protein in adherent cell line cultures as well as viable cell count in suspension cell line cultures (0.94 < r' < 0.99). Since the microculture tetrazolium assay provides sensitive and reproducible indices of growth as well as drug sensitivity in individual cell lines over the course of multiple passages and several months' cultivation, it appears suitable for initial-stage in vitro drug screening.
|Morphometric and Colorimetric Analyses of Human Tumor Cell Line Growth and Drug Sensitivity in Soft Agar Culture
|M. C. ALLEY, C. M. PACULA-COX, M. L. HURSEY, L. R. RUBINSTEIN, AND M. R. BOYD
|Previous studies have demonstrated the suitability of image analysis of tetrazolium-stained colonies to assess growth and drug sensitivity of human tumor cells cultivated in soft agar culture. In the present study, the potential utility of calorimetric analysis to expedite experimental drug evaluations using human tumor cell lines was investigated. The same culture dishes were assessed by image analysis and by formazan colorimetry for purposes of comparing multiple methods of measuring growth as well as growth inhibition. Replicate cultures treated with 2-(p-iodonitrophenyl) -3-p-nitropben), 1-5-plienyltetrazolium chloride or 3-(4,5-dimethyltbiazol-2-yi)-2,5-diphenyltetrazolium bromide exhibited nearly identical colony count and volume indices as well as excellent correlation in calorimetric end points. Colony-forming unit volume analysis versus calorimetric assessment of the same cultures following dimetityl sulfoxide extraction of prolamine sulfate-rinsed, dried soft agar cultures exhibited excellent linear correlation for both growth (Pearson r ranging from 0.95 to 1.00) and drug sensitivity (Pearson r ranging from 0.90 to 0.99, and Spearman r ranging from 0.82 to 0.97) and similar drug sensitivity profiles. Results of the current investigation indicate that end Points of soft agar culture remain stable for a period of at least 2 weeks following assay termination. In addition, a calorimetric detection range of 1.3-2.2 log units permits determinations of survival levels ranging from 100 to 5% of respective control levels. Colorimetric analysis is anticipated to expedite soft agar colony formation assay evaluations (a) by reducing tile need to use the more rigorous and (time-consuming image analysis procedures to measure activity in preliminary drug sensitivity assays and (b) by permitting the determination of effective concentration ranges of new experimental agents for subsequent, more detailed investigations.
|Morphometric Analysis of Lymphocyte Nuclei in Chronic Lymphocytic Leukemia.
|OSTAPENKO VA, KRUCHINSKII NG, SMIRNOVA LA, CHEREDNIK AB, NESTEROV VN, TEPLIAKOV AI
|This work is dedicated to the study of use of quantitative analysis of cell nucleus structure for the analysis of peripheral blood lymphocytes in patients with chronic lymphocytic leukaemia. The structure of lymphocytic nuclei of healthy donors was evaluated by means of staining by toluidine blue purified cell suspensions smears. The preparations were analysed on the television measuring system OMNICON with measurements of the following parameters: square of the nucleus, euchromatin, heterochromatin, and the ratio of heterochromatin and euchromatin squares. Actuarial analysis and nuclei classification of the previously mentioned parameters showed, that in peripheral blood of patients with chronic lymphocytic leukemia a large amount of atypical lymphocytes is present with reduced nucleus sizes. Atypical cells retain the ratio of structural components of chromatine, characteristic to normal cells, which show their low proliferative activity.
|Quantitative Analysis of Nuclear Area Variation in Benign and Malignant Breast Fine Needle Aspirates.
|KAUSHIK N. SARDANA S. DAS DK, LUTHRA UK
|The measurement of nuclear area was carried out in 30 benign and 32 malignant breast lumps using OMNICON Alpha 500 Image Analyzer. The mean nuclear area of duct cells in malignant group was greater (157.6 +/ 58.64 sq.microns with a peak around 140 sq.microns) and more heterogenous within and amongst cases than observed in duct cells from most of the cases of fibroadenoma (85.05 ae 14.2 sq.microns with a peak around 80 sq.microns). Taking into consideration 110 sq.microns as a differentiating limit, a significant difference was observed between benign and malignant conditions (p). Similarly taking 118 sq.microns as differentiating limit duct cell carcinomas could be divided into two groups i.e. 9(28.1 %) cases of small nuclear type with a range of 80 118 sq.microns and 23(71.9%) cases of large nuclear type with a range of 118 320 sq microns .6(18.8%) cases with small nuclei had an overlap with fibroadenoma. Although 13(72.2%) cases of large nuclear type carcinomas had lymph node metastasis as against 4(44.4%) in small nuclear group, the difference was not statistically significant.
|Automation of Data Acquisition and Processing in Assays for Anchorage-Independent Growth: Application to the Purification of Epithelial Transforming Growth Factor.
|DUNNINGTON DJ, PINSKY S. MATTES D, PRICHETT W. EARL CQ, GREIG R. ANZANO MA
|We have developed a method for automated data collection from anchorage-independent growth assays by direct interfacing of an OMNICON image analysis system with a VAX mainframe computer network. By use of this interface, data generated with the OMNICON can be acquired and manipulated by the VAX, providing several advantages including high throughput, elimination of operator error, flexibility and speed, and capacity of mainframe data processing. We have applied these techniques to aid in the purification of a novel growth factor for human epithelial cells. Both column elusion profiles and dose-response data were processed to graphic formats, and ED50 values for the individual purification steps were obtained by Hill transformation of the dose-response curves. The assay for anchorage-independent growth is widely used for purification of growth factors and testing of chemotherapeutic agents against human tumor cells. The present technique should be useful in facilitating these labor-intensive studies.
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