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Rubio C.A.,Karolinska University Hospital | Delinassios J.G.,International Institute of Anticancer Research
International Journal of Clinical and Experimental Medicine | Year: 2010

Colorectal carcinomas (CRC) usually arise in colorectal adenomas (CRA) displaying high-grade dysplasia (HGD) or carcinoma in situ (CIS). The aim was to assess the frequency of adenomas displaying HGD or CIS in a cohort of consecutive CRA with submucosal invasive carcinoma. Ninety-two consecutive adenomas were investigated. Sub-mucosal invasion was present in the 39 adenomas with HGD (42%) and in 58% (53/92) of the adenomas with CIS (p<0.05). Sections from 49 adenomas were stained with the DNA-specific Feulgen reaction and for the proliferation marker Ki-67. Five consecutive high power fields (HPFs) were evaluated using a x40 objective. Marked Feulgen reaction was recorded in 91.8% or in 101 of the 110 HPFs studied in adenomas with HGD, but in none of the 135 HPFs studied in adenomas with CIS (p<0.05). Intense Ki-67 expression (≥75%) was present in 98.2% or in 108 of the 110 HPFs studied in adenomas with HGD, but only in 1.4% or in 2 of the 135 HPFs in adenomas with CIS (p<0.05). Hence, HGD cells and CIS cells can be differentiated not only morphologically but also chemically by the semi-quantitative appreciation of their DNA content and immunohistochemically by the apparent difference in cell proliferation. Although submucosal invasion occurred significantly more frequently in adenomas with CIS than in those with HGD, as many as 42% of the adenomas with submucosal invasion displayed HGD at histology. Despite morphological, chemical and immunohistochemical dissimilarities, these 2 non-invasive neoplasias might have similar biological behaviour in terms of progression towards submucosal invasion. Source

Delinasios J.G.,International Institute of Anticancer Research | Angeli F.,International Institute of Anticancer Research | Koumakis G.,International Institute of Anticancer Research | Kumar S.,University of Manchester | And 12 more authors.
Anticancer Research | Year: 2015

Aim: to identify biological interactions between proliferating fibroblasts and HeLa cells in vitro. Materials and Methods: Fibroblasts were isolated from both normal and tumour human tissues. Coverslip co-cultures of HeLa and fibroblasts in various ratios with medium replacement every 48 h were studied using fixed cell staining with dyes such as Giemsa and silver staining, with immunochemistry for Ki-67 and E-cadherin, with dihydrofolate reductase (DHFR) enzyme reaction, as well as live cell staining for non-specific esterases and lipids. Other techniques included carmine cell labeling, autoradiography and apoptosis assessment. Results: Under conditions of feeding and cell: cell ratios allowing parallel growth of human fibroblasts and HeLa cells, co-cultured for up to 20 days, a series of phenomena occur consecutively: profound affinity between the two cell types and exchange of small molecules; encircling of the HeLa colonies by the fibroblasts and enhanced growth of both cell types at their contact areas; expression of carbonic anhydrase in both cell types and high expression of non-specific esterases and cytoplasmic argyrophilia in the surrounding fibroblasts; intense production and secretion of lipid droplets by the surrounding fibroblasts; development of a complex net of argyrophilic projections of the fibroblasts; E-cadherin expression in the HeLa cells; from the 10th day onwards, an increasing detachment of batches of HeLa cells at the peripheries of colonies and appearance of areas with many multi-nucleated and apoptotic HeLa cells, and small HeLa fragments; from the 17th day, appearance of fibroblasts blocked at the G2-M phase. Co-cultures at approximately 17-20 days display a cell-cell fight with foci of (a) sparse growth of both cell types, (b) overgrowth of the fibroblasts and (c) regrowth of HeLa in small colonies. These results indicate that during their interaction with HeLa cells in vitro, proliferating fibroblasts can be activated against HeLa. This type of activation is not observed if fibroblast proliferation is blocked by contact inhibition of growth at confluency, or by omitting replacement of the nutrient medium. Conclusion: The present observations show that: (a) interaction between proliferating fibroblasts and HeLa cells in vitro drastically influences each other's protein expression, growth pattern, chromatin features and survival; (b) these functions depend on the fibroblast/HeLa ratio, cell topology (cell-cell contact and the architectural pattern developed during co-culture) and frequent medium change, as prerequisites for fibroblast proliferation; (c) this co-culture model is useful in the study of the complex processes within the tumour microenvironment, as well as the in vitro reproduction and display of several phenomena conventionally seen in tumour cytological sections, such as desmoplasia, apoptosis, nuclear abnormalities; and (d) overgrown fibroblasts adhering to the boundaries of HeLa colonies produce and secrete lipid droplets. © 2015, International Institute of Anticancer Research. All rights reserved. Source

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