Summary In previous pharmacologic studies, the native fluorescent properties of doxorubicin (DOX) have been utilised to visualise tissue and cellular drug distribution. Such distribution studies provide valuable additional information to that obtained by measuring tissue drug concentration alone. An alternative immunocytochemical method of drug localisation using a rabbit immunoadsorbed antiserum to DOX and silver-enhanced gold-labelled second antibodies has been used to achieve visualisation of DOX Danesi et al., 1988;Hindenburg et al., 1989) and this characteristic provides a means of identifying treatment regimens or drug analogues which may improve drug delivery to tumour, of correlating drug distributions with specific organ toxicities, or of comparing intracellular distribution of drug in anthracycline-sensitive and -resistant tumours and cells. Phenotypic variations in the intracellular localisation of DOX have also been demonstrated by fluorescence (Aghai & Tokes, 1990).We have previously demonstrated that the distribution of the anti-cancer drug VP16-213 in normal and malignant tissues can be visualised using immunocytochemical methods employing a specific VP16-213 antiserum and enzymelabelled second antibodies (Henneberry et al., 1987
Materials and methods
Primary antiserumThe polyclonal DOX antiserum was raised in a rabbit (GR 52) against DOX conjugated to bovine serum albumin (BSA) (Piall et al., 1982). The antiserum was immunoadsorbed on a high-capacity aldehyde activated silica (100 nm diameter; IOOA pore size; Clifmar Associates, Guildford) column, to which was coupled 100 mg BSA g-' silica beads. Before application of the crude antiserum (1 ml), the column (0.7 x 7 cm) was washed with 10 ml 0.1 M glycine/HCI pH2, followed by 20 ml 0.2 M phosphate buffered saline pH 7.4. After elution of the purified antiserum with 5 ml of the buffer, the column was washed with glycine/HCI followed by 0.2 M phosphate buffered saline containing 0.1% thiomersal. The sealed column was then stored at 4'C until required again.For immunocytochemistry, the purified antiserum was diluted 1 in 100 in 0.01 M phosphate buffered saline (PBS) containing 0.1% BSA, 0.5% sodium azide, pH 7.2.Tissues and controls Tissues (liver, kidney, heart, small intestine, sarcoma) were obtained at the end of infusion from rats treated with 10 mg DOX kg-', infused intravenously over 1-2 h. EMT6 mammary tumours were obtained from mice 1 h after administration of 10 mg DOX kg' i.p. Human primary breast tumour biopsy material was obtained from patients during surgery 1 h following the administration of DOX (25 mg i.v.) as previously described (Stallard et al., 1990). Controls were either tissues from untreated animals or sections from drugtreated subjects, which were incubated without specific antibody.Cytological samples A metastatic human breast cancer cell line, ZR75, was grown in RPMI-1640 medium (Northumbria Biologicals Ltd) until the cells were almost confluent and then treated with DOX (0-1,000 ng ml-' (0-1.84 pM)) at 37°C for 24 h. ...
