GLYCOGEN AND FAT IN MALIGNANT NEW

GROWTHS OF THE MOUSE.

BY M. HAALAND.

IN the transplantable mouse-tumours we meet with a rapidity of growth which is hardly surpassed by any other known form of cell proliferation. A few centigrams of tissue when introduced into a mouse are in many cases capable of producing 1-2 grms. of solid tumour mass in 10 days, and 4-5 grms. in 2-3 weeks, in mice of 15-20 grms. weight. An exact comparison with the growth of foetal tissues is hardly possible, because we have no means of ascertaining how many cells form the starting point of proliferation in the transplanted material. The interest of this cell proliferation is not limited to its bearing on the problems of cancer alone, but it is also of the greatest importance for a general biological study of cell-life and cell-metabolism in higher animals.

Of the phases of cell-metabolism that can be studied with histological methods, those characterised by the appearance of glycogen and fat have attracted greatest interest, partly because of the ease with which these chemically sharply-defined substances can be demonstrated. The great amount of glycogen in foetal tissue suggested long ago its relation to rapid cell growth. Brault brought forward the hypothesis that the presence of glycogen in tumours is in proportion to their rate of growth, and that the estimation of its amount can be used to form a prognosis as to the malignancy of tumours. His results, however, have not been confirmed by Lubarsch, Best, and Gierke. The conclusions of these authors as to the significance of the appearance of glycogen differ. While Lubarsch ascribes great importance to inherited qualities of the cells, Best has shown that tissues normally containing no glycogen, may under inflammatory conditions, contain a great amount. He therefore considers the appearance of glycogen as an inflammatory reaction. Gierke concluded from his observations that it could be

explained as a result of circulatory conditions. Lubarsch has pointed out that the presence of glycogen in different kinds of cells can hardly be explained from a single standpoint.

In this connection we have thought it of some interest to examine the rapidly growing mouse-tumours for the presence of glycogen. In Best's method of staining glycogen in sections by a special carmine solution we have an easy and elegant method which, with the employment of different Iodine methods † as a control, offers every surety, so far as histological reactions can give them.

• Best's Method for staining Glycogen (latest modification). (Zeitschrift für Mikroskopie, Bd. 23, 1906, p. 319.)

Fix in absolute alcohol and imbed in celloidin.

Prepare the following stock solution :—

The solution is then ready for staining. Keep in the dark. The solution deteriorates after about 2 months in winter, after a few weeks in summer. Filter before use.

(1) Stain the celloidin-sections in Delafield's, Böhmer's, or other hæmatoxylin till nuclei are dark (differentiate in acid 70% alcohol if necessary). Wash in tap water. (2) Transfer to the following, prepared immediately before use:

(3) Transfer the sections directly into following differentiating solution :

Differentiate for 1-3-5 minutes till no more red colour is given off.

(4) 80°, Alcohol.

(5) Absolute Alcohol.

(6) Xylol or Toluol.

(7) Canada Balsam.

Nuclei and protoplasm blue; glycogen granules red.

+ Ehrlich's, Barfurth's, and Langhans's method. See Encyklopädie der mikroskopischen Technik 1903, paragraph Glykogen.

The tumours we have had the opportunity of examining in this way

are:

(1) A great many spontaneous mammary adeno-carcinomata of the mouse, collected in the laboratory of the Imperial Cancer Research Fund and described by Murray on previous pages. In all these we failed to find glycogen in the parenchyma.

(2) Numerous transplanted tumours of mammary origin of different strains, mentioned in various papers in this Report (strain 27, 37, 39, 50, 46, and Jensen's tumour). Of these the first four have an adenocarcinomatous structure, 39 and 50 are hæmorrhagic tumours as described by Gierke in this Report, 46 and Jensen's tumours are prototypes of alveolar carcinomata.

In none of these tumours, examined when they are used for transplantation, have we been able to find glycogen in the specific tumour parenchyma with certainty, even in the most rapidly growing tumours. We frequently find granules of glycogen in the leucocytes penetrating between the cancer-cells, and in the surrounding tissue, fatty tissue, neighbouring skin, and hair follicles. A positive result by chemical examination therefore need not necessarily imply that this glycogen is present in the specific tumour parenchyma, and only Best's method, with its distinct staining of both glycogen and nuclei, can show us exactly in which cellular elements it is present.

(3) In the transplanted tumours of a squamous-cell carcinoma (tumour 32), described in detail by Murray in an earlier paper in this Report, glycogen is found in the alveoli showing distinct keratinisation, and there only in the middle layer of cells, corresponding to the upper part of the stratum mucosum in the normal skin (fig. 1). On the other hand, we do not find glycogen in the great bulk of the tumour, i. e. in the cells of the undifferentiated alveoli. In this tumour the appearance of glycogen seems to be limited to a certain phase in the differentiation of the squamous epithelium, such as is already known to be exhibited normally by the same layers in skin.

(4) The mixed tumours we have described in the paper on sarcoma development occurring during continued transplantation of carcinoma have been examined. In them we occasionally meet with granules of glycogen in some of the sarcomatous cells of the stroma.

(5) The pure sarcomatous tumours developing from the mixed tumours, and two other transplantable sarcomata of spontaneous origin have been examined, one of mouse (tumour 92, described by Murray on pp. 78-81)

J. R. Ford, del. Transplantable squamous cell carcinoma (20 days old). To show in the middie layer of stratified squamous epithelium in a keratinised Glycogen could not be demonstrated in the tumours when keratinisation

J. R. Ford, del. FIG. 2.-37/7Q-8,G. Margin of graft of mammary adenocarcinoma, preserved 48 hours after inoculation. Granules of glycogen in the parenchyma-cells and in the cells of the reaction tissue. Adipose tissue of the host at the lower part of the figure. XI.

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