Vol. Lll, No. 4 (August, 1971) was issued 2.9.71. THE BRITISH JOURN OF EXPERIMENTAL PATHOLOGY VOL. LII OCTOBER, 1971 NO. 5 A PHENOMENON RESEMBLING OPSONIC ADHERENCE SHOWN BY DISAGGREGATED CELLS OF THE TRANSMISSIBLE VENEREAL TUMOUR OF THE DOG D. COHEN, B. W. GURNER AND R. R. A. COOMBS From the Department of Animal Pathology and Immunology Division, Department of Pathology, University of Cambridge Received for publication February 5, 1971 SUMMARY.-Twenty to 30 per cent of the cells in a suspension of trypsindisaggregated tumour cells of the Transmissible Venereal Tumour of the dog exhibit a property resembling opsonic adherence. In the particular technique used this shows as rosette-formation with antibody-sensitized sheep red cells surrounding the reacting tumour cells. This phenomenon, which is a well known immunological activity of phagocytic cells, is also displayed by a considerable percentage of cells in other tumours. It is considered that the reacting cells are tumour cells and not phago - cytic host cells. OPSONIc adherence, as usually understood, is the first stage of phagocytosis where phagocytic cells make contact and adhere to antigenic molecules, particles or cells after these have been sensitized by antibody (often in this context called an opsonin). Opsonization due to antibody is often confined to IgG although complement adsorbed on IgM antibody leads to complement-mediated opsonic adherence (Coombs and Franks, 1969). Phagocytic cells, both macrophages and polymorphonuclear leucocytes have 2 distinct membrane receptors, one of which reacts with a site on antibody activated following combination with antigen and one which reacts with a site on fixed C3. The cell receptor reactive with fixed C3 is susceptible to the action of trypsin. The present paper is concerned with a phenomenon resembling antibody opsonic adherence. The Transmissible Venereal Tumour (TVT) of the dog is a naturally occurring, coitally transmitted and experimentally transplantable neoplasm (Karlson and Mann, 1952). Its histogenesis and aetiology are uncertain (Bloom, Paff and Noback, 1951; Dozza and Torlone, 1960; Makino, 1963). The phenomenon to be described, namely the strong adherence of sensitized red cells to a percentage of TVT cells was observed while investigating the use of 41
448 D. COHEN, B. W. GURNER AND R. R. A. COOMBS the mixed antiglobulin reaction (Mori and Coombs, 1969) for the detection of antibodies in dogs against the tumour cells. In this reaction, serum-treated TVT cells are further treated with anti-dog immunoglobulin serum and centrifuged together with antibody-sensitized indicator red cells. In these tests besides the mixed agglutination due to anti-tumour antibodies, another complicating phenomenon was observed. This showed as extremely strong rosette-formation of red cells around certain tumour cells, even in the absence of tumour specific antibody, or of antiglobulin serum. On analysis it was clear that this phenomenon resembled opsonic adherence and although this was a decided complication in the tests we were doing, it was, in itself, of potential interest. The present paper records some of the experimental findings on this phenomenon exhibited by the tumour cells. MATERIALS AND METHODS Tumour.-The Transmissible Venereal Tumour was obtained from a 3-yr-old male Labrador dog and was serially transplanted by s.c. injection of trypsinized cell suspensions in Hanks' Balanced Salt Solution (BSS). Preparation of cell 8swpen8ions.-Tumours were removed from dogs 20-30 days after transplantation. Single cell suspensions were prepared by stirring chopped tumour in 0-25 per cent trypsin containing a few drops of 0-02 per cent deoxyribonuclease at 37. Cell counts were performed in an improved Neubauer counting chamber and viability assessed by the trypan blue exclusion test. Diluent.-A 0-2 per cent Bovine Serum Albumin (BSA) solution in BSS was used as diluent. Preparation of 8ens'itized red cell8.-equal volumes of 2 per cent sheep red cells in Phosphate Buffered Saline (PBS) and dog anti-sheep red cell serum at a dilution of 1: 400 were incubated for 1 hr at room temperature. Following incubation, the red cells were washed x 3 in PBS and resuspended to a 0 4 per cent suspension in BSS containing 0-2 per cent BSA. Te8ting the tumour cell 8uspen8ion for ro8etting.-to one drop of the tumour cell suspension (3 X 106 cells/ml.) in a narrow tube (5 x 50 mm.) was added one drop of a 0 4 per cent suspension of sensitized red cells. The tubes were centrifuged at 1500 rpm for 2 min. The deposited cells were resuspended and examined under the microscope, on a siliconed slide with siliconed cover slip overlaid and waxed at the edges. In some tests toluidine blue dye was added to stain the tumour cells. Fixation and 8taining of ro8etted tumour cells.-preparations of rosetted tumour cells were suspended in 1: 3 v/v heated normal dog serum in BSS. A drop of this suspension was placed on a microscope slide for 1 min., smeared and dried at 37. The dried smear was fixed with ethanol for 10 min. and stained with Giemsa or Leishman. EXPERIMENTAL STUDY Percentage of cells in tumour suspension rosetted by sensitized sheep red cells When a suspension of trypsinized TVT cells was centrifuged with sheep red cells sensitized with a subagglutinating dose of dog anti-sheep red cell serum (diluted 1/400) 20 per cent of cells in the tumour suspension formed tight rosettes (Figs. 8 and 9). The rosettes appear as spheric aggregations of red blood cells around a central tumour cell. The tumour cells may be clearly visualized by the addition of toluidine blue (Fig. 8). If trypan blue is added, instead of toluidine blue, most of the tumour cells remained unstained indicating that they were living. It was clear that most of the rosettes formed around living cells. Cells of the tumour suspension were either completely free of adhering red cells or extremely firmly rosetted; there were very few intermediate or weakly rosetting
OPSONIC ADHERENCE AND TUMOUR CELLS 449 cells. No rosettes formed if unsensitized sheep red cells were used instead of sensitized red cells. If the tumour cell suspension (and not the red cells) was treated with the same dog anti-sheep red cell serum at the same dilution and subsequently washed before adding unsensitized sheep red cells, the rosetting reaction did not occur; this excluded, in this particular test, both cytophilic antibody and mixed agglutination from being involved. A similar rosetting phenomenon was obtained if the sheep red cells were sensitized with a subagglutinating dose of sheep cell antibody made in the horse, guinea-pig, rabbit, rat or mouse although the strongest reactions were obtained with dog antibody. As the characteristics of the reacting tumour cells were undefined, cells were prepared from tumours grafted for 30, 40, 63 and 200 days. The percentage rosetting cells in the 4 preparations was 23, 23, 14 and 14 per cent respectively. There was no difference if tests were performed on cells liberated early in the trypsin-treatment or at the end of the incubation. With another TVT tumour 30 per cent of cells in the suspension rosetted. A suspension of TVT cells disaggregated by mechanical means without any treatment with trypsin gave the reaction just as strongly. Tumour cells could also be frozen in 20 per cent glycerol-in-bss down to -180 over liquid nitrogen and the reaction demonstrated again on thawing and reconstitution. Rosetting with cell suspensions from other tumours and cells from normal tissues Opsonic adherence is a reaction expected of all phagocytic cells in normal tissues. A test performed on peritoneal " macrophages " washed out of a peritoneal cavity and not treated with trypsin gave dog over 90 per cent rosettes. Pieces of tissue (buccal mucosa, bladder epithelium, liver and kidney) were taken from a dog, immediately after death. The tissues were treated with trypsin and Of the buccal suspension, the disaggregated cells tested, as were the tumour cells. 10 per cent formed rosettes but epithelial cells were not involved; of the bladder suspension 10 per cent formed rosettes but again no epithelial cells were involved. Five per cent of cells in the kidney suspension reacted but no epithelial cells; while in the liver 60 per cent of cells reacted,but these did not include hepatic cells. Many of the hepatic cells in this preparation were broken down and so a special preparation of hepatic cells was made following disaggregation of the cells inm/200 ethylamine-diamine-tetra-acetate in buffered saline. This gave an excellent preparation of hepatic cells and none of these gave the rosetting reaction. In the 4 preparations of normal cells, the reacting cells were in all probability macrophages or cells of the reticulo-endothelial system. Transplantable tumours of rats, hamsters and mice were then tested to see whether this activity was to be found in cells of other tumours. Cell preparations were obtained following treatment with trypsin. A solid methyl cholanthreneinduced rat fibrosarcoma (MC2) exhibited a very strong reaction (approximately 20 per cent reacting cells); likewise a solid spontaneous rat fibrosarcoma (SP7). A hamster fibrosarcoma (induced by murine sarcoma virus) and growing for many passages in suspension tissue culture, gave a weak reaction (< 1 per cent reacting cells). With these cells there was no difference whether the cells were treated with trypsin or not. A suspension of BP8 mouse ascitic tumour cells stored at 180 over liquid nitrogen after removal from the peritonealcavity of the
450 D. COHEN, B. W. GURNER AND R. R. A. COOMBS mouse gave 2 per cent reacting cells; but these could well have been host macrophages. Identity of the cells rn the tumour suspensions showing rosettes Opsonic adherence is a property of phagocytic macrophages, polymorphonuclear leucocytes and other cells of the reticuloendothelial system. One might first suspect that the reacting cells were invading reticuloendothelial cells of the host, but their number-up to 30 per cent is against this when the homogeneity of the tumour, seen either in section or on imprint (Figs. 1-3) is considered. If the tumour itself were of reticuloendothelial origin, one might again expect nearly all the cells to react in this manner-while only 20-30 per cent did. When the suspension of trypsinized TVT cells was examined cytologically (after gently smearing in 30 per cent dog serum, fixing and staining with Leishman) at least 3 cell types could be distinguished on the basis of their morphological appearance (Fig. 4). However, rosette formation was not restricted to any one of the 3 cell types but occurred on all of them (Figs. 5, 6, 7). Attempts to remove the potentially rosetting cells by first treating the cell suspension with carbonyl iron at 370 and passing the suspension through a magnet were unsuccessful. Cells which phagocytosed iron as well as cells which did not were involved in rosette formation. DISCUSSION Should the phenomenon described be that of opsonic adherence and should the reacting cell in the tumour cell suspension be tumour, as seems most likely, it is of great interest. The findings point to the phenomenon being one of antibody mediated opsonic adherence. It can only be reproduced when sheep red cell indicator cells are sensitized with subagglutinating doses of antibody; the antibody can be from many species. That the red cells have to be sensitized with antibody would seem to rule out the possibility that the reaction is caused by virus haemadsorption. A cytophilic antibody reaction and mixed agglutination were also excluded by the fact that no rosette formation followed if the tumour cells, instead of the sheep red cells, were treated with the sheep red cell antibody. Of normal host cells, polymorphonuclear leucocytes, macrophages or other reticuloendothelial cells would give the same reaction but a 20-30 per cent content EXPLANATION OF PLATES FIa. 1.-A section of the Transmissible Venereal Tumour (TVT) 4 weeks after transplantation. The cells are densely packed and supported by very little stroma. H. and E. x 75. FIG. 2.-A section of the tumour showing the homogeneous appearance of the cells. H. and E. x 75. FIa. 3.-Impression smear of the TVT showing the homogeneous appearance of the cells. Giemsa. x 675. FIa. 4.-A smear of a trypsin disaggregated TVT cell suspension. The cells are rather heterogeneous in appearance. Giemsa. x 675. FIGS. 5, 6 and 7.-Smears of rosetted TVT cells. Three morphologically distinct TVT cell types surrounded by a rosette of sensitized sheep red cells. Giemsa. x 675. FIGs. 8 and 9.-Photographs of rosetted TVT cells in suspension. Fig. 8: wet preparation with toluidine blue-bright field illumination. X 185. Fig. 9: wet preparation with phase contrast microscopy. x 185.
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OPSONIC ADHERENCE AND TUMOUR CELLS 451 of such cells in prepared TVT cell suspension would seem to be very unlikely and could be excluded cytologically. This tumour is very cellular and contains little stroma (Figs 1 and 2). Serological studies on this tumour by D.C. using the indirect membrane immunofluorescence test and host serum antibodies has shown that over 90 per cent of the cells in the TVT suspensions used in the present studies were, in fact, tumour cells. On cytological grounds alone (Figs 4, 5, 6, 7) it may be seen that many of the rosetted cells resemble other tumour cells in the smears. More pains would have been taken to identify more surely the precise nature of the reacting cells but investigations presently in progress on human autochthonous tumours show the same phenomenon and again many of the reacting cells have the appearance of tumour cells. In the human tumours the phenomenon is found not only with sarcomata as examined in the present report but also with carcinomata where any reticuloendothelial origin is ruled out. Tumours vary in the proportion of cells showing the capacity to adhere to sensitized red cells and we intend to investigate the significance of this variable population of tumour cells which have a membrane property giving a phenomenon similar to opsonic adherence. REFERENCES BLOOM, F., PAFF, G. H. AND NOBACK, C. R.-(1951) Am. J. Path., 27, 119. COOMBS, R. R. A. AND FRANKS, D.-(1969) Prog. Allergy, 13, 174. DOZZA, G. AND TORLONE, V.-(1960) Veterinaria ital., 11, 647. KARLSON, A. G. AND MANN, F. C.-(1952) Ann. N.Y. Acad. Sci., 54, 1197. MAXINo, S.-(1963) Ann. N.Y. Acad. Sci., 108, 1106. MORI, W. AND COOMBS, R. R. A.-(1969) Br. J. Cancer, 23, 622.