Detection of genomically-tagged cancer cells in different tissues at different stages of tumor development: lack of correlation with the formation of metastasis
Introduction
The presence of a very small number of viable tumor cells in an otherwise healthy tissue is, from a clinical point of view, a significant finding with important consequences for the evaluation of therapeutic strategies and prognostic of patients [1], [2], [3], [4], [5], [6]. This is a reflection of the widely accepted assumption that the presence of tumor cells in distant parenchymas correlates with the subsequent development of metastases with clinical relevance. For this reason, many studies have focussed on the development of high sensitivity methods for the detection of tumor cells in distant parenchymas during early stages of tumor progression.
Standard cytology has been the most widely used method for the detection of cell dissemination. However, this method can only detect tumor cells with a limited degree of sensitivity (one tumor cell per 100 normal cells), and is notoriously inaccurate. Immunocytochemistry, although more sensitive (detecting one cell per 105 normal cells), is dependent on the availability of specific antibodies to tumor-associated cell surface antigens, and may be prone to false-positive results due to cross-reactivity with antigens present in normal cells.
In the case of circulating cancer cells, techniques based on molecular technology such as polymerase chain reaction (PCR) provide a more sensitive method for detection. Reverse transcription coupled with amplification by polymerase chain reaction (RT-PCR) of relatively small amounts of mRNAs encoding tumor cell proteins allows the detection of as few as one tumor cell per 107 normal cells [1], [2], [5], [6], [7], [8]. In patients with colorectal cancer, amplification of tumor markers including carcinoembryonic antigen (CEA) [5], [6], [7], CD44 [1] and cytokeratin [8] with this technology has been used to detect the presence of tumor cells in the bloodstream. Unfortunately, these mRNAs encode epithelial markers that are not specifically expressed by tumor cells, and therefore this technology cannot be applied to the detection of tumor cells in parenchymatous tissues.
In the present study we describe the development of an experimental model of cancer using a recombinant DNA-based technology that allows the detection of small numbers of cells in apparently healthy organs and blood, even during very early stages of the disease. Using this technology we have evaluated whether there is a correlation between the presence of cancer cells in any target organ and the subsequent appearance of metastases in that organ.
Section snippets
Cell culture
The cell line DHD/K12-PROb (also called DHD/K12-TRb; referred to as DHD cells herein) was used. This clonal cell line was initially established by Martin et al. [9] in 1983 from a colon adenocarcinoma that had been induced in syngenic BD-IX rats by administration of 1,2-dimethylhydrazine. Cells were cultured as monolayers in a mixture of DMEM and Ham's F10 (1:1, v/v; GibcoBRL, Life Technologies, Paisley, Scotland), supplemented with 10% fetal bovine serum (GibcoBRL) and gentamycin (0.005%;
Growth of tumors
To determine whether the stable insertion of pcDNA3.1CAT sequences into the genome of DHD cells might alter their capacity to form tumors or their aggressiveness, we monitored the growth of tumors generated after injections of DHD-CAT cells by measuring the external diameter of tumors weekly, and compared these measurements with those taken from tumors generated by injection of wild-type cells (‘un-tagged’ cells). As indicated in Fig. 1, no significant difference (Student t-test analysis) in
Discussion
We have developed an experimental model of cancer based on genomic tagging of tumor cells using a recombinant DNA-based technology that allows one to detect relatively small numbers of cells in all organs and the blood without any effect on the cancer aggressiveness. This model allowed us to establish whether there is a direct correlation between the presence of cells in a given organ during relatively early stages of the disease, and the subsequent development of metastases in that organ.
Acknowledgements
This study was supported in part by two grants from ‘Cultural Albacete’ (Spain) and ‘Plan Nacional de I’ (Spain), respectively.
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