Elective vital staining of mouse tumors with labelled dyes

Medical Hypotheses 7:

65-75, 1981

ELECTIVE VITAL STAINING OF MOUSE TUMORS WITH LABELLED DYES D. Engel and I. Beneg, Department of Nuclear Medicine and Radiotherapy of the City Hospital Triemli, Zurich. Postal address: D. Engel, Voltastr. 64, 8044 Zurich, Switzerland. Key words:

Vital staining, tumors, labelled dyes ABSTRACT

It was shown by vital staining in previous experiments (1) that carcinoma and sarcoma of mice could be selectively stained by certain dyes of the triphenyl methane . . sulphonic acid group. These findings were nowls51j onfirmed dyes in the present experiments by using labelled ( of the same chemical group. In some mice the tumor tissue was the most radioactive, compared with other tissues. In other mice the tumor tissue was exceeded only by tissues which had a blood supply _5-times to 35-times higher. These were in addition excretory organs the excreta of which was highly radioaciive (kidney and liver). The urine was 50-times more radioactive than the tumor and the bile was most probably not less. After deduction of the radioactivity of these two components, blood and excreta, from the radioactivity of the parenchyma tissue of the kidney and liver, the tumor tissue proved to be the most radioactive of all tissues tested. This indicates that tumor tissue retained the administered labelled dyes in higher concentration than all other tissues. INTRODUCTION It was shown by vital staining (1) that certain tumors such as Ehrlich's carcinoma of mice and mouse sarcoma were electively stained by certain acid dyes of the triphenyl-methane sulphonic acid group (acid fuchsin, rotviolett 5RS, light green and isamine blue). These dyes proved especially active, in contrast to other acidic dyes. The four dyes mentioned turned into a colourless so-called carbinol form (2,3,4) in an alkaline medium, by an intramolecular rearrangement. The carcinoma of rats and the tar carcinoma of mice did not show elective staining with the four dyes tested.

65

It was also shown by Hiiber and Engel (5) that sterile fresh tumor pieces from resected human breasts kept for a few hours at 37OC in an acid fuchsin Ringer solution, discoloured this solution faster than other tissues of similar weight. This observation seems to support the view (1) that it is the living cancer cell and not the necrotic part of the tumor, that has elective staining capacity for the mentioned dye group, as stated by (2) and lately others (6,7,8). In view of these controversies and the practical significance of the problem, some radioactively labelled triphenyl-methane dyes were used for vital staining of tumor and other tissues. This method also offered possibilities for quantitative comparison of the staining capacity of various tumors and organs. METHODS Measurements of Radioactivity Mice of 30 g (LCR/,o) were inoculated S.C. with ascites culture of Ehrlich's mouse carcinoma. After 3-4 weeks, when the tumors were about cherry size, the mice were injected s.c., far away from the tumor, with light green,acid fuchsin or isamine blue. These dyes were labelled with 1251 by the chloramine method of Clark (by Their specific actiDr. Wittings, E.I.R. Wiirl' gen). vity was about 1.04jGi 159I/lg of the dyes. The relatively long half life of the so labelled dyes allowed variations in the intervals between the dye administration and sacrificing of the mice. These intervals varied between 18 end 113 hours. Apart from varying this interval, the dye quantity administered was also varied from 0.25 to 1.5 ml of the 1% dye solution per mouse. Thus the staining capacity of the tissues was compared under varying experimental conditions. The mice were bled under ether anaesthesia by cutting the carotid arteries before being sacrificed. Approximately 0.5 g of tumor, heart, lung, liver, kidney, spleen, muscle, brain, small intestine, testis, skin and fat were then excised for measurement of their radioactivity. For this purpose a Picker's gamma-counter, type Autowell II, was used. The measured radioactivity was compared with a standard solution of 1251 and calculated in $X/g -tissue as a percentage of the applied activity.

66

The tissue pieces were enclosed in a testtube immediately after excision, to prevent exsication, and weighed before activity measurement. The tumors were mostly fleshy non-necrotic. In some cases a central and a peripheral section was chosen for comparison. Examination of blood content of the tumor and some organs Since the radioactivity of tissues depended not only on their parenchyma but also on their blood content, the latter was examined in two mice. The heart blood of two normal mice was taken under anaesthesia and diluted with normal saline 1 : 1. A drop of liquemin (anticoagulant) was added to this dilution and the erythrocytes were labelled with 51chromium. 1 ml of the labelled blood was injected into the caudal vein of an adult mouse which had an Ehrlich's carcinoma of hazelnut size previously s.c.ly implanted. Another tumor mouse injected subcutaneously with the same quantity of labelled blood, was used as a control. The two mice were sacrificed 30 minutes after the injection of labelled blood. The tumor and 5 organs, the brain, kidney, liver, spleen and the lung were minced with scissors and their homogenates' radioactivity per gram was measured by a Picker's gamma courter type Autowell II, adapted for 51chromium measurement. The results are given in Table III. Comparing radioactivity of tumor with that of urine and bile Since the dyes used in our experiments are excreted by the kidney and liver, the urine and bile were examined for their radioactivity after a tumor-mouse was injected with 0.5 ml of a 1% light green solution, labelled with 1251 and sacrificed 22 hours later. The radioactivity of the tumor and of the urine was then measured. Unfortunately, owing to technical difficulties, no bile could be obtained from the minute gallbladder. Instead? the activity of the duodenum was tested in the assumption that it might contain some bile. Comparing the staining capacity (radioactivity) of the centre and periphery of the tumor Two mice with a subcutaneous tumor, 3 weeks old, 2 x 2 x 1 cm in size each, were injected S.C. one A) with 1 ml 0" a 1% light green solution labelled with l25J and sacrificed

67

75 hours after the injection; the other mouse (B) was injected with 0.5 ml of the same solution and sacrificed 22 hours after injection. On the cut surface all the tumors looked fleshy and free of necrosis. Two pieces were taken from the centre and two from the periphery of each tumor. The radioactivity of the tumors was examined by the method above described. RESULTS The dye - of tissues, of Table

degree of radioactivity - indicating uptake of the tumor tissue, compared with that of other is shown in Table I: -it is based on the figures II (Appendix7

From the two Tables it is evident that the tissues of the kidney, the tumor and the liver, in this sequence, showed the highest radioactivity of all the tissues; the lung was next in intensity and the spleen occupied the fifth place. The brain and the muscle were the most inactive of all tissues. The other tissues tested, especially fat, testis and intestine proved much less active than the first five tissues. For this reason the testing of their activity was not pursued beyond two to three trials. It was also noticed that in those mice in which the tumor was the most active tissue of all, it was 11-times (in mouse 4) and 5-times (in mouse 10) more active than its "riv~l'~,the kidney. Such marked differences were not seen between other tissues of our series. In view of the very high blood content of the kidney, the liver and the spleen and the lung, on one hand, and the very poor blood supply of the tumor, on the other, an observation which is even macroscopically self-evident, it was considered of interest to determine quantitatively the blood content of these tissues. It seemed most likely that the radioactivity tested in our experiments was caused not only by the parenchyma but also by the activity of the blood carrying radioactive dyes not yet absorbed by the tissues. The figures given in Table III fully confirmed our expectations: According to this the lung contained 36.18-times, the spleen 34.97-times, the liver 12.58times, the kidney 5 24-times more blood than the tumor. ??le tissues of the dontrol mouse, which had the labelled blood injected subcutaneously, showed no radioactivity at testing at all. Judging from the radioactivity of the kidney and liver, which ranked higher than that of the lung and

66

spleen, as shown in Tables I and II, one would have expected that the kidney and liver would rank in the first place with regard to their blood content. Why this was not the case is explained by the high radioactivity of the urine and the bile found in our experiments. The radioactivity of the tumor was 0.055~.$i/~in~d that of 1 ml urine was 2.533 )ICi./g. Thus 1 g was about 50-times more radioactive, due to its dye content, than 1 g of tumor. It is, therefore, most likely that the strong radioactivity of the kidney tissue in our experiments was, to a great extent, due to its urine- and blood content. Because of the small gallbladder of the mouse it was not possible to obtain sufficient bile for examination. As second best, the radioactivity of the duodenum was examined in the expectation that at least some small quantities of bile might be found in the duodenum. In fact the activity of the duodenum was nearly as high as that of the spleen, tumor or liver (in mice No. 13, 14, 16 and 18). The bile was most likely a major contributory factor to the high radioactivity of the liver, just as urine was to the activity of the kidney. Both are excretory organs known to excrete triphenyl methane dyes with the urine and bile respectively. Thus the urine and bile content. in addition to a far superior blood supply, explain why these two organs showed a higher radioactivity in some mice than did the anemic tumor. The central and peripheral parts of the tumor were compared because some authors believe that the necrotic and not the living tumor cells are electively stained. Our present experiments confirm previous findings (1) that this is not the case with our tumors. The radioactivity readings of the two tumors were the following: Periphery Mouse A: Mouse B:

Centre

0.007 pCi/g 0.055 yCi/g

O.O08yCi/g 0.056 ,uCi/g

Thus the peripheral and central sections of both tumors showed practically equal activity despite different dosage of dye and different interval between administration of dye and sacrificing the mice. These results could be repeated in any of our tumors since in our experiments only the fleshy, non-necrotic parts were selected for examination.

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COMMENT In 2 mice out of 18 the cancer tissue stained with 12%-labelled dyes was the most radioactive, in 3 mice it was second most active, in 6 it was third, in another 6 in the fourth, and in 1 in the fifth place, compared with the other tissues. Only the kidney exceeded the tumor in respect of radioactivity. The liver was practically equal and all other tissues were less active than the tumor. The lungs varied according to their blood content or possibly oedema. In comparing the radioactivity of kidney, liver and lung with that of the tumor, two facts must be borne in mind: firstly, that the kidney, liver and lung contain many times more radioactive blood than does the tumor (see Table III>; secondly, that 1 g of urine (which fills the kidney tubules) proved to be 50-times more radioactive than 1 g of tumor tissue. This suggests that, in making a valid comparison between kidney and tumor, the radioactivity of the urine and blood must be deducted from the total radioactivity of the kidney. Similarly, in the case of the liver, the radioactivity of the blood and the bile must be deducted from the total activity of the liver. After such an adjustment the tumor tissue would prove to be the most radioactive of all the tissues tested. The radioactivity figures obtained in all our present experiments are in direct relation to the staining capacity of the tissues. Thus the results obtained with radioactive dyes confirm previous findings (1) according to which certain mouse-tumors have an elective staining capacity for a group of triphenyl methane dyes. DISCUSSION An important question concerning the problem of vital staining of cancer is whether it is the necrotic or the living tissue that is stained by certain dyes. In previous experiments it was shown (1) that it was the living and not the necrotic cancer tissue that is electively stained by a group of triphenyl methane dyes. In the present experiments chiefly young, fleshy tumors were selected for radioactive testing. Since the above publication several papers have appeared (6,7,8,9 and others) on vital staining of animal tumors with lissamine green. This dye was chosen by these authors because it does not lose its color in alkaline medium and because it does not penetrate the cell membrane of living cells. For the

70

purpose of comparing the staining capacity of living cancer cells with that of normal tissues, lissamine green would appear most unsuitable. In our previous and present experiments dyes were used which do lose color in alkaline medium and do penetrate the cell membrane of living cells. On the advice of (lo), (5) produced membranes by adding protein and lecithin to collodion sacks. The colourless carbinol derivates of the triphenyl methane dyes passed through the membranes so prepared more easily than did the coloured dyes. It seems that the cancer cell membranes are likewise more permeable to these carbinols Hijber and than to the original coloured form w&5) Wankel also proved that the colourless derivates (carbinols) penetrate the cell membrane of living opalina ranarum more easily than does the coloured form of the dye. l

In previous experiments (1) distribution of living and dead cells was ascertained by carefully comparing frozen vital-stained sections with corresponding H.E.-stained paraffin sections. In the experiments of Goldacre & Sylven dye distribution in the tumor followed a certain pattern. In my (E) previous, as well as in our present, experiments, no such pattern was discernible, irrespective of whether the tumor was examined 22 or 75 hours after the injection of the labelled dye. On the contrary, the dye distribution was practically even; this was shown by the equal radioactivity of the central and peripheral parts of the tumors. The statement (7,s) is not correct that the tumor cells, in my experiment (E) could not be stained because of a low dye concentration in the blood. 1 ml of a 1% to 2% dye solution was injected S.C. three times, 18, 7 and 2 hours before sacrificing the animals, and not, as stated, once only. The triphenyl methane dyes are not the only substances that penetrate tumor cells more readily than normal cells. 18 times more bismuth is absorbed by tumor tissue than by normal tissue of similar weight, while less thorium nitrate is absorbed per/g by tumor tissue than by any other tissue (11). The different behaviour of tumor tissue towards certain dyes and other substances, compared with that of normal tissues, is probably due to the different metabolism and different physico-chemical qualities of the two. The

71

differences apply chiefly, if not exclusively, to the living tumor cells. From a therapeutic standpoint it is important to emphasize this fact because the elective staining of tumors with the triphenyl methane dye group was perhaps the first proof that cancer tissue csn retain an administered chemical substance in higher concentration than other tissues. Since the triphenyl methane dyes are not toxic substances and have special affinities to cancer tissue, it is conceivable that such dyes might possibly be used as vehicles to bring to the cancer cell therapeutic agents (cytostatica or radionucleids) attached as sidechains, in higher concentration than could be achieved with the free agent alone. Acid fuchsin is certainly non-toxic to man. 20 ml of a 5% solution of it was given subcutaneously to 20 adult men (12) without the slightest ill-effect. The labelled triphenyl methane dyes may also have diagnostic possibilities. If these dyes should accumulate in human csncer, as they do in some animal tumors, it might be possible to visualize their presence by scintillation technique. , K I-

1

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Li

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n n n n

Tu . . . . ,== . . . . . .

:::

L.IS

M

S

/

D

sk

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(p

T.

Tissues of , turnour %

F

organs

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Table I shows the radioactivity (= dye retention) of 14 %issues of 18 mice. It is based on the data of Table II and shows diagrammatically the frequency with which the various tested tissues fall into categories 1 to 9 (1 = highest radioactivity, 9 = lowest radioactivity).

72

2

1

4

3

5

G. 0.5cc,24h

G. 0.5cc.52h

G. 0.75cc,42h

G. 0.5cc,18h

G. 0.25cc,41h-

Lu K Li Tu s M

K Tu z Lu M s

K LU Li Tu M S

z K LU L1 s M

K Tu Ll LU S M

0.274 0.034 0.018 0.017 0.007 0.006

0.021 0.13 0.11 0.11 0.008 0.005

7

6

0.041 0.039 0.033 0.030 0.020 0.016

0.549 0.046 0.039 0.037 0.014 0.009

9

8

10

G. 0.25cc,23h

G. 0.25cc.66h

G. 0.25cc,U3h

G. 0.25cc,2U)hG.

K Lu Li Tu s M

K Li E Lu s M

K 0.0096 LU 0.0071 ~1 0.0068 0.0049 " S 0.0029 B 0.0013

K 0.0291 LU 0.0053 Tu 0.0046 L10.0031 s 0.0015 B Cl.0009 F 0.0006

0.035 0.022 0.020 O.Ol& 0.011 0.010

0.009 0.005 0.004 0.003 0.002 0.0008

12

11

14

13

0.012 0.077 0.006 0.004 0.002 0.001 ___--

Tu Li K VE S SI LU

lcc,??h 0.526 O.lOf! 0.107 0.024 ‘0.02~. 0.018 0.00s _____~__

15

G. 0.5cc,47h

G. O.?5cc,67h

G. lcc,Z?h

G. lcc,22h

Is 0.5cc,lEih

K 0.215 Li 0.147 s Tu 0.08 0.086

Lu K w Li Te LI S B

1.23 0.28 0.22 O.lG 0.074 0.024 0.024 0.012

LU K Li Du Tu ov S B

0.82 0.538 0.43 0.255 0.23 0.107 0.089 0.024

K

0.41 0.33 0 0.21 Tu 0.16 DU 0.153 LU c.141 S 0.089 B 0.02

K 0.132 L1 0.0"" Sk O.O:t' Tu 0.03', 1.u 0.027 s 0.02; TC 0.01E! 0.01: B 0.003

Is 1.5cc,102h

F

0.5cc,lPh

F

1.5cc,lD?h

Li K Tu s Du F Lu M B

K Tu Sk Li LU S Te M B

0.351 0.057 0.053 0.049 0.024 0.020 0.016 0.010 0.003

K Li TU s Du LU M F B

0.437 0.134 0.106 0.047 0.040 0.037 0.017 0.010 0.004

SK Ve Te B

0.055 0.05 0.05 0.012

17

16 1.057 0.397 0.182 0.118 0.106 0.101 0.060 0.026 0.011

Ll

1%

Table II Giving radioactivity of tumor and or an tissues of 18 mice injected with dyes labelled with 12% , expressed in @i/g and calculated on the basis of the specific activity of the dye injected. The figure of each of the 18 columns pertain to one mouse. At the head of each box F = fuchsin-S; G = light green, Is = isamine blue, followed by the quantity of dye (all 1%) injected (in ml), and the interval between the injection and killing (in h). The figures within the columns indicate the radioactivity for the following tissues: B = brain, D = duodenum, F = fat, I = intestine (small), K = kidney, Li = liver, Lu = lung, M = muscle, 0 = ovary, S = spleen, Sk = skin, Te = testis, Tu = tumor, Ve = vesicula semin. The tissues within each column are listed in descending order of ra~U-;p",$vi-~i~,;zr instance: The radioactivity of the lung 4 ranks 3.and will appear in the third horizontal column of Table I. 73

Table III

Relation of bloodcontent compared with tumor

Radioactivity of blood in organs CPM/g Lung Spleen Liver Kidney Brain Tumor

36.18-fold 34.97-fold 12.38-fold 5.24-fold 2.80-fold

25.472 24.623 8.716

/

Showin a) the radioactivity of the blood content (labelled with 5 B-chromium) of 5 organs and tumor;b) the relative blood content of the 5 organs compared with the blood content of the tumor. REl?E.RENCES -1.

Engel

2.

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3.

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11.

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