Automatische Zytofluorometrie solider Malignome nach Zellenvereinzelung durch intraperitoneale Anzucht von Zellkomplexen in jungen Ratten

Abstract Introduction The use of isolated cells is a basic requirement besides data processing techniques to obtain optimum results with the automated flow cytofluorometry. Several techniques have been employed in order to get cell dispersal. But the claim for reproducible and representative cell samples cannot exactly be fulfilled: gross parts of cells are damaged by any dispersal method applied on solid tissues. Therefore we want to introduce a special method for cell isolation, which avoids mechanical and chemical damage of the treated cells. For this the intraperitoneal tolerance of growing tumor cells in young animals was used, in the species of which solid growth of such tumor cells is established. The investigations reported here were carried out with the Walker-tumor 256 on the rat as a model for solid tumor growth. Our interest was focussed on the determination of cellular CNA distribution in tumor cells and contaminating somatic cells grown in the ascitic fluids. Material and Methods Walker-tumors are permanently cultivated on Sprague-Dawley rats. The solid tumors (1 cm diameter) were removed under sterile conditions, cutted with scissors, and resuspended in 0.9% saline. Ten rats (37 days old, body weight 90–120 g) had received an intraperitoneal injection of 1 ml of such prepared tumor cell suspensions. Four or five days after inoculation of tumor cells 2–8 ml of ascitic fluid were punctuated and supplemented with 0.1 ml Liquemin (5000 Roche). One million tumor cells could be obtained by such a procedure. After spontaneous sedimentation of cells in order to reduce the number of erythrocytes, the cells were processed and fixed according to Trujillo and Van Dilla (1972) , digested with heated RNase (1 mg/ml tris-buffer; 0.18 M and pH 7.5), pepsin treated, and then stained with ethidium bromide (40 μg/ml tris-buffer) for 30 min in the cold. After rinsing with distilled water several times the cells were resuspended in buffer and measured within an hour immediately after passing a 10 μm filter to avoid cell aggregates. Results and Discussion The cell suspensions obtained from ascitic fluids were free from cellular debris and revealed a correct DNA-analysis by automated flow cytofluorometric techniques. Cells, transferred in such a manner, showed no deviations in stem line and variability of their DNA-content. It is demonstrated that also cell material derived from small metastases can be amplified during intraperitoneal growth. By means of this specific “biological amplification” new advantages in the field of flow-through fluorometry might be possible as for example (1) investigation onto compact tumors which can hardly be suspended into single cells or (2) determinations of the DNA stem line and DNA-variability in very small pieces of neoplastic tissues and metastases. The problem of selection of cell clones during metastasis might be more open to quantitative analyses. (3) Studies on different responses of metastases to cytostatic drugs with respect to the presence of clonal cell lines in tumors and their manifestation during metastatic processes.