Validation of the fluorescent dye Hoechst 33342 as a vascular space marker in tumours

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Abstract
The DNA-binding fluorescent dye Hoechst 33342 (H33342) has been used in a series of investigations of the vascular parameters of two murine tumours. This dye has been shown, to have a short half-life in the circulation (T 1/2 less than 2 min), but is stably bound for at least 2 h after it enters cells. It can be used in morphometric studies on frozen sections to determine the effective vascular volume, the capillary fraction and the size distribution of blood vessels in each tumour. These latter two parameters cannot be deduced from the less labour intensive techniques using radioactive isotopes. The effective vascular volume perfused in 1 min by H33342 was compared with the volume perfused in 30 min with 51Cr labelled erythrocytes. Similar volumes were estimated with the two techniques in a murine carcinoma and in a sarcoma. Both techniques showed that the vascular volume decreased in larger tumours. The H33342 analysis of vessel size showed the decrease in capillary vessels in the carcinomas was even grater, falling from 70% in small tumours to 20% in larger tumours. The deteriorating vascular network in larger tumours sis assocaiated with an increasing fraction of necrotic tissue. Experiments in which the isotopes and dye were co-injected suggest that at 40 mg kg-1 the dye may rapidly lead to a partial shutdown of the tumor vascular bed. This is less marked with 20 mg kg-1. In spite of this effect there is in general a close correlation between the volumes perfused by labelled red blood cells and the fluorescent dye.