OMNICON Tumor Colony Analyzer
| OMNICON Tumor Colony Analyzer | Published Papers |
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| Article No. | Title |
| TCA1011 | Repeated Urine Cell Culture in Soft Agar: Potential Role in Follow-up of Patients with Transitional Cell Carcinoma. |
| KIRKELS WJ, PELGRIM OK, AALDERS MW, DEBRNYNE FM, VOOYS GP, HERMAN CJ | |
| To evaluate the persistence of cells with clonogenic properties in patients being treated for transitional cell carcinoma, 272 urine samples were collected from 75 patients and cultured in a double layer soft agar "cloning" system. The development of colonies was evaluated with growth curves based on repeated colony counting with an OMNICON automated colony counter at regular time intervals. Forty eight patients had at least one evaluable culture. Comparing the results of colony development in culture with the clinical evaluation of the patients, 9 patients had a histologically proven recurrence preceded or accompanied by tumor colony growth in urine culture. One patient had tumor recurrence with growth negative urine culture (false negative). Fifteen patients have had growth negative urine culture with a negative follow up (mean 19.5 months). Twenty one patients have had growth positive urine cell cultures with no recurrence in their follow up (mean 18.7 months). Although the follow up times are at present relatively short, the present study suggests that repeated soft agar urine culture of patients with low grade, low stage bladder carcinoma may provide a means for identifying those patients at a higher risk for recurrence/progression of their disease. | |
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| TCA1012 | Automated Counting of Human Tumor Colonies in the Courtenay-Mills Assay System. |
| VERHEIJEN RH, WHELAN RD, FEITZ WE, KENEMANS P. VOOYS GP, HERMAN CJ, HILL BT | |
| A procedure for using the OMNICON automated image analysis system for counting colonies grown from a human tumour cell line (COLO 205) in the Courtenay-Mills assay is described. This involves the transfer of the agar medium from culture tubes into petri dishes. Comparisons of observer and instrument counts were done on a blinded basis. Run-to-run correlation coefficient was 0.996 for automated counting and the inter-observer correlation coefficient was 0.984. Both assessments showed a linear relationship between the number of cells plated and the number of colonies grown. Automated colony counting is fast, reliable and provides additional information on colony size distribution, not obtainable with manual counting. This automated procedure will greatly facilitate in vitro drug sensitivity evaluation. | |
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| TCA1013 | Evaluation of an Automated Image Analysis System for Counting Human Tumor Colonies. |
| SALMON SE, YOUNG L, LEBOWITZ J. THOMSON S. EINSPHAR J. TONG T. MOON TE | |
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The OMNICON FAS II image analysis system was applied to counting tumor colonies grown in a soft agar human tumor clonogenic assay with a detailed protocol designed to assess the instrument's sensitivity, specificity, precision, and accuracy. Comparisons of technician and instrument counts were done on a blinded basis. Sensitivity studies (which used metal microspheres) yielded a correlation coefficient (r) of 0.999 between technicians and the counter. A field by field analysis of the instrument's specificity for identifying individual objects correctly as tumor colonies rather than artifacts (as identified by the technician) was excellent (r = 0.95). In the precision studies (determined with repeated automated counting of the same samples for five days), the median coefficient of variation was less than 7%. Accuracy was evaluated on cultures of fresh biopsies from 30 human cancers obtained for drug sensitivity testing as well as on a series of tumor cell lines. The correlation between the mean number of colonies counted by the technicians and by the colony counter was greater than 0.91. Similar comparisons of mean percent survival of tumor colony forming cells after drug exposure between technician and machine were also quite acceptable (r = 0.85). We conclude that the colony counter provided sufficient reliability to be applied to counting human tumor colonies grown in vitro. In addition, the colony counter performed the Petri dish counts ten times faster than experienced technicians and without associated operator fatigue. |
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| TCA1014 | Blockade of the epidermal growth factor receptor tyrosine kinase suppresses tumorigenesis in MMTVyNeu 1 MMTVyTGF-a bigenic mice |
| ANNE E. G. LENFERINK*, JEAN F. SIMPSON†‡, LAURA K. SHAWVER§, ROBERT J. COFFEY*‡, JAMES T. FORBES*†, AND CARLOS L. ARTEAGA*‡I** | |
| Overexpression of ErbB-2yNeu has been causally associated with mammary epithelial transformation. Here we report that blockade of the epidermal growth factor receptor (EGFR) kinase with AG-1478 markedly delays breast tumor formation in mouse mammary tumor virus (MMTV)/ + Neu 1 MMTV transforming growth factor a bigenic mice. This delay was associated with inhibition of EGFR and Neu signaling, reduction of cyclin-dependent kinase 2 (Cdk2) and mitogenactivated protein kinase (MAPK) activities and cyclin D1, and an increase in the levels of the Cdk inhibitor p27Kip1. In addition, BrdUrd incorporation into tumor cell nuclei was prevented with no signs of tumor cell apoptosis. These observations prompted us to investigate the stability of p27. Recombinant p27 was degraded rapidly in vitro by untreated but not by AG-1478-treated tumor lysates. Proteasome depletion of the tumor lysates, addition of the specific MEK1/2 inhibitor U-0126, or a T187A mutation in recombinant p27 all prevented p27 degradation. Cdk2 andMAPK precipitates from untreated tumor lysates phosphorylated recombinant wild-type p27 but not the T187A mutant in vitro. Cdk2 and MAPK precipitates from AG-1478-treated tumors were unable to phosphorylate p27 in vitro. These data suggest that increased signaling by ErbB receptors up-regulates MAPK activity, which, in turn, phosphorylates and destabilizes p27, thus contributing to dysregulated cell cycle progression. | |
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| TCA1015 | The Functional Reserve of Corneal Endothelium. |
| SHAW EL, RAO GN, ARTHUR EJ, AQUAVELLA JV | |
| With recent advances in our knowledge of corneal physiology, coupled with the development and increasing availability of the specular microscope as a clinical instrument, valid observations relating the morphologic appearance of the corneal endothelium to its functional capacity are within our reach. Manual methods of data analysis are cumbersome, time consuming, and associated with human error and investigator bias. The OMNICON pattern analysis system lends itself to objective analysis of morphologic features, offers the possibility of quantifying the data obtained and, hopefully, will lead to a better understanding of the many aspects of endothelial cell morphology which, in total, relate to the functional reserve of a given cornea. | |
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| TCA1016 | Characterization of Nerve Growth Factor Precursor Protein Expression by Human Prostate Stromal Cells: A Role in Selective Neurotrophin Stimulation of Prostate Epithelial Cell Growth |
| ROBERT DELSITE and DANIEL DJAKIEW | |
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BACKGROUND. Nerve growth factor (NGF) immunoreactive proteins derived from human prostatic stromal cells (hPS) have been implicated in the paracrine regulation of prostate epithelial cell growth. However, mature NGFß does not appear to be expressed by these cells. In order to determine whether NGF precursors are expressed by these cells, we investigated the potential processing and expression of precursor forms of NGF by human prostatic stromal cells, and examined the effects of NGF precursor moieties along with the other members of the neurotrophin family of gene products on soft agar colony formation of prostate epithelial cells. METHODS. Specific antibodies to the peptide domains defined as N4 and L38, and the NGFß moiety of prepro-NGF, were used in immunoblot assays to characterize the molecular weight forms of precursor NGF secreted by human prostatic stromal cells. The potential processing of NGF precursors with two enzymes, NGFy and trypsin, was performed by incubation with stromal cell secretory protein containing precursor NGF. The selective effects of the N4, L38, and NGF13 peptide domains of precursor NGF, along with the remaining members of the neurotrophin family, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), were examined for their ability to stimulate growth of prostate tumor epithelial cells in an assay of soft agar colony formation. |
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| TCA1017 | Constitutive expression of Heregulin induces apoptosis in an erbB-2 overexpressing breast cancer cell line SKBr-3 |
| F.K. GUERRA-VLADUSIC, G. SCOTT, V. WEAVER, E.A. VLADISIC, M.S. TSAI, C.C. BENZ and R. LUPU | |
| We have previously reported that Heregulin (HRG)/neu differentiation factor (NDF) induced growth arrest and cellular differentiation in breast cancer cells overexpressing erbB-2 receptor To elucidate the cellular mechanisms underlying the growth inhibition by HRG, we developed un in vitro model by transfection of HRG eDNA into the erbB-2 overexpressing breast cancer cell line, SKBr-3. We showed that the enforced expression of HRG in SKBr-3 cells induces dramatic morphological changes and pronounced inhibition of anchorage-independent and independent growth. Most SKBr-3/HRG transfected (SK/HRG) cells exhibited about 15-fold increase in size and persisted 'giant' multinucleated cells with extended flattened vacuole-filled cytoplasm with reduced cell attachment. The growth suppression of SK/HRG cells was accompanied by a reduction in S phase, the presence of a G2-M cell cycle delay, and an increase in DNA aneuploid components. In addition, DNA fragmentation assays showed that HRG induced apoptosis of SKBr-3 cells. In contrast, while HRG treatment of other erbB-2 overexpressing breast cancer cell lines led to growth arrest and cell detachment, it did not induce apoptotic features. Thus, this study demonstrates that while growth arrest and cell detachment are general effects of HRG towards erbB-2 overexpressing cells, the ability of HRG to induce apoptosis is a phenomenon confined to selective cells including SKBr-3 cells. | |
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| TCA1018 | Improved optical detection of colony enlargement and drug cytotoxicity in primary soft agar cultures of human solid tumour cells |
| M.C. ALLEY AND M.M. LIEBER | |
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The presence of cellular aggregates in cell suspensions derived from human solid tumours often complicates subsequent evaluation of colony formation in primary soft agar cultures (Agrez et al., 1982b). In the present study, performance of a conventional colony formation assay was observed to lack sufficient sensitivity to identify growth and active chemotherapeutic agents in the majority of specimen cultures. Modification of conventional methodologies to include filtration of cell suspensions, use of "proliferation control" and "cytotoxicity control" cultures as well as vital staining were found to be essential fot the valid assessment of primary soft agar cultures in our laboratory. In addition, application of drugs to culture surface in place of culture incorporation appeared to facilitate culture performance and drug sensitivity testing. |
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| TCA1019 | Measurement of human tumour cell growth in soft-agar cultures using computer-assisted volume analysis |
| M.C. ALLEY & M.M. LIEBER | |
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Growth in soft-agar bilayer cultures of human tumour cells derived from 4 in vitro continuous cell lines, from 21 xenofrafts carried in athymic mice, and from 197 samples of fresh human solid tumours of various histologic types was analyzed by computer-assisted image analysis. Replicate cultures for each specimen were assessed on successive days of incubation for the number and volume of growth units within multiple size categories. Our results confirm the recent finding of others that there is an upper limit ~10 +9 um +3 to the cumulative growth unit bolume obtainable in a 2 ml bilayer soft agar culture system. Since this upper limit to the carrying capacity of the closed culture system exists, the extent of growth within the cultures is determined in a fundamental way by the cumulative volume of growth units initially inoculated into cultures. A growth index of >= 16-fold was only seen when initial comulative growth unit bolume was <10 +7 um +3 per culture dish. Computer-assisted volume analysis (CAVA) appears to be a useful quantitative method to study the growth of human tumour cells in soft agar cultures. |
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| TCA1020 | Drug application to the surface of soft-agarose cell cultures |
| M.C. ALLEY & M.M. LIEBER | |
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Evaluation of human tumor cell chemosensitivity using soft-agar colony formation assays generally have utilized "One-hour exposure" followed by washing and resuspension of cells prior to inoculation in culture or 2) "continuous exposure" by incorporation of drug into soft-agar cell suspensions prior to inoculation (Alberts et al, 1980; Soehnlen et al, 1980). The present study was designed to assess possible advantages of an alternate method of drug exposure: that is, drug application to the surfaces of soft-agarose cultures following inoculation. In contrast to other techniques, surface application was viewed as a means to eliminate mechanical manipulation of cells in the presence of drug and to permit all cultures to be set up from a single, "bulk" cell suspension with minimal handling. |
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