Europ. J. Cancer Vol. 8, pp. 267-269. Pergamon Press 1972. Printed in Great Britain

Letter to the Editor

Indication of Hypoxic Areas in Tumours from in vivo NADH Fluorescence*

MARIO GOSALVEZt, RONALD G. THURMAN, BRITTON CHANCE and H. S. REINHOLD:~

Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 19104, U.S.A. and Department of Radiology, Yale University School of Medicine, New Haven, Connecticut, U.S.A.

THE PRESENCE of hypoxic cells in tumours has been demonstrated by different methods [1-4, 7]. The relations between intracellular anoxia and radiobiological anoxia has been questioned both from the theoretical [5] and from the experimental [6] standpoints. It seems of interest to identify hypoxic areas in tumours by direct measurements of the intrace]lular redox state. The fluorescence of N A D H has been shown to be a sensitive and relatively reliable parameter for the measurement of intracellu]ar hypoxia and the study of metabolic control phenomena [8-12].

A biological preparation uniquely suited for studies of localized hypoxia is provided by a tumour growing in a sheet-like fashion (so called "sandwich" tumour) [13] in which an approxi- mately 2-dimensional sheet of tissue could be scanned by N A D H microfluorometry for localized areas of hypoxia.

The recording microfluorometer specially designed for recording N A D H fluorescence from tissue surface has been described else- where [10]. The area seen by the photo- multiplier was restricted to 8 # diam. by placing a 1 mm diaphragm in the image plane. The animals bearing tumour were anesthetized by

Aceepted 15 October 1971. *Supported by GM 12202-05. t T h e work reported in this paper was undertaken

during the tenure of a Research Training Fellowship awarded by the International Agency for Research on Cancer.

++Supported by Public Health Grant USPHS 2 - P O 2 - CA-06519.

urethane. Respiratory gases were controlled by a gas mixer and administered by a mask placed over the animal's snout. The 366 nm excitation beam was focused on the tumour and the scanning of the tumour surface was allowed by displacements of the animal which was placed on the microscope stage.

Figure 1 illustrates a typical reaction to changes in the respiratory gas mixture in an oxygen responsive portion of the sandwich tumour. Time proceeds from left to right in Fig. 1 and fluorescence increase is indicated as an upward defection. The baseline for the

tt~ ¢ -

O) u r " O)

t n

Dark Current .

Fig. 1.

20% o2 20%%

,12 Shutter Open

I I

3 4 5

Time In Minutes

Example of the response of the N A D H fluorescence of the C3H mouse mammary tumour to a decrease of the oxygen concentration of the inspiring air. The fluorescence signal doubles (this is calculated as a 100% increase) when the animal is made hypoxic, and returns rapidly to the normal value when rebreathing air. Exciting wavelength : 366 nm, fluorescence :

450 nm.

267

268 Mario Gosalvez, Ronald G. Thurman, Britton Chance and H. S. Reinhold

unexposed photomuhiplier is indicated in the initial portion of the trace and after 1.2 min of the recording the shutter is opened giving a normoxic value of tissue fluorescence. At 2 min the animal breathes 5 % oxygen and almost immediately the fluorescence increases to the hypoxic level, approximately double the nor- moxic level, which is reached at 3 min and maintained 0.8 min when the animal is allowed to breathe again normal oxygen tension, diminishing the fluorescence to the normoxic level

Figure 2 illustrates an experiment where a continuous scan was performed in normoxia along the line represented by the points. The scan was repeated in anoxia and calculations were made of the fluorescence increase in anoxia, compared to the normoxic conditions, obtained at the particular points which are circled. In the relatively open space between major blood vessels and in the region of small capillary a large fluorescence change (67%) was observed, similar to that obtained in Fig. 1. In other regions even though in the vicinity of blood vessels and branched points in the circu- latory system the fluorescence was smaller, ranging from 20 down to 7 %.

One interpretation of the results is that the areas which showed a smaller fluorescence increase had a greater degree of pyridine nucleotide reduction when the animal was breathing the normal oxygen tension and might

ioo ,_

Fig. 2. Differences in the relative increase of the N A D H fluorescence to a decrease of the oxygen concentration of the inspiring air (Fig. 1) for different locations in a sandwich tumour. The circles represent the measured areas; the figures in the circles give the percentage of.fluorescence increase with 5% oxygen compared to the `fluorescence with 20% oxygen

for that location. The actual measurement of `fluorescence in normoxia and anoxia at each point was performed as in Fig. 1

by continuous scan of each point in normoxia and anoxia.

therefore be considered hypoxic regions of the tumour.

In summary, these experiments show the feasibility of employing a scanning micro- fluorometer technique for identifying in vivo the existence and the location of hypoxic loci and may be of great usefulness in evaluating radiosensitivity in a quantitative relationship to the degree of intracellular hypoxia.

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Tumour N A D H Fluorescence in vivo 269

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