Regulation of Phenotypic Expressions in Lung Cancer Models In Vitro and In Vivo

Khan, Mohammad Zareen (1990) Regulation of Phenotypic Expressions in Lung Cancer Models In Vitro and In Vivo. PhD thesis, University of Glasgow.

Abstract

The objectives were to investigate the relationship between lung cancers and to determine the effects of micro-environmental and genetic manipulation on regulation of phenotypic expression. A small cell lung carcinoma (SCLC) cell line, NCI-H69 was selected as a model for the study of heterogeneity. Two adherent sublines (H69V and H69VZ) have been isolated, and characterized, for species and lineage confirmation, and for expression of neuroendocrine markers. They have also been characterized for their growth, morphological properties, and invasiveness in vitro, radiation- and chemo-sensitivity. The cell lines have also been investigated for tumorigenicity, invasion and metastases formation in vivo. The cell lines were also investigated for c-myc expression by immunostaining. Significant morphological differences have been found in culture, between the parental and the derivative lines. While the parental line grew as densely packed free floating cellular aggregates, the sublines grew as a monolayer of epithelioid cells. Both the derivative lines either expressed very low levels or did not express neuroendocrine cell markers including L-dopa-decarboxylase, creatine kinase-BB isoenzyme, bombesin-like immunore activity, neuron specific enolase, and neurosecretory type granules, compared to the parental line. All the lines stained positively for epithelial markers. Karyotypic analysis of the derivative lines showed features of SCLC. Differences have also been observed in their growth characteristics, both in vitro and in vivo. The growth rates of both the derivative lines are faster than the parental line, with doubling times closer to non-small cell lung carcinoma (NSCLC) cell lines. In vitro invasion studies of the cell lines showed early, single cell invasion in the parental cells, while solitary invasion was absent in both the derivative lines. Both the parental and the derivative lines were tumorigenic in nude mice. The parental line was both invasive locally, and formed multiple metastatic lesions in visceral organs, while local invasion was not found in either of the derivative lines, and they were non-metastatic. Xenograft histology of all the lines revealed an anaplastic, pleomorphic and rather large cell type tumours. However, the derivative lines showed focal spindle cell morphology, and spaces filled with an amorphous material, which was alcianophilic, and some of the cells contained glycogen. These features were not found in the parental line. Therefore, it seems likely that H69 cell line contains cells of both small and non-small cell like phenotypes, suggesting a common origin for these two cell types. Further studies showed that H69 clones were able to give rise to a heterogeneous cell population similar to the parental line. These data suggest a common origin for both derivative and parental lines. Having established that phenotypic changes can occur spontaneously in H69 cells, they were also investigated further to establish whether similar phenotypic changes could be induced in these cells by micro-environmental manipulation. The cells were exposed to different chemical inducers, and investigated for various parameters as above. The specific aim was to see whether malignant cells still respond to changes in microenvironment, and if they do, whether it was possible to alter the malignant behaviour of cells by micro-environmental manipulation. Cells treated with hexamethylene bisacitamide (HMBA), dibutyryl cyclic adenosine monophosphate (dbcAMP), and sodium butyrate (Na-But) showed cytostatic effects on cell growth in culture, changes in culture morphology, inhibition of invasion in vitro, decrease in final tumour volume in vivo, changes in tumour histology, and reduction in expression of some neuroendocrine markers e.g. L-dopa decarboxylase, suggesting that the H69 cell phenotype can be regulated be microenvironmental alterations. A NSCLC cell line (WIL) derived from an adenocarcinoma of lung was selected for the study of phenotypic expression in this group of lung cancer, caused by varying the micro-environmental influences. Growth of cells in conventional monolayer, with and without stromal interactions, was compared with the growth in a histotypic tissue culture model and xenografts in vivo. The malignant cells grown in optimum conditions (histotypic culture) also were exposed to various chemical agents, and effects on morphology both at light and electronmicroscopic level, and on the production of mucin-like glycoproteins, and immunohistochemical markers were analyzed. Cells grown on collagen in histotypic culture showed increased production of mucin-like glycoproteins and formation of surface microvilli, and intracellular mucin-like inclusions.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Medicine, Oncology
• Date of Award: 1990
• Depositing User: Enlighten Team
• Unique ID: glathesis:1990-78180
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 28 Feb 2020 12:09
• Last Modified: 28 Feb 2020 12:09
• URI: https://theses.gla.ac.uk/id/eprint/78180