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A Tumour Cell Aggregation Promoting Substance from Rat Ascites Hepatoma Cells

A substance capable of promoting tumour cell aggregation was released from rat ascites hepatoma cell (possibly from the cell surface) kept in Hanks' balanced salt solution (free of calcium and magnesium) in the cold, and then partially purified by chromatography with DEAE-Sephadex and gel filtration with Bio-gel. The thermostable substance seemed to be a glycoprotein and its molecular weight was about 72,000 when measured by gel filtration on Sephadex G-200. It had no proteolytic activity. The material was clearly effective for rat ascites hepatoma cells as well as SV40 transformed cells, but less effective for Chang's cells and apparently ineffective for normal rat liver cells and red blood cells. The action of this material was more potent than that of Jack bean concanavalin A when assayed for aggregation of SV40 transformed cells. Its effect was not influenced by concanavalin A inhibitors such as alpha-methyl-D-glucopyranoside, N-acetyl-D-glucosamine and D-glucose. ImagesFig. 1Fig. 2Fig. 3


THE MECHANISMS of invasion in cancer
have not yet been established. Various explanations have been given for these mechanisms: (a) a decrease in mutual adhesiveness of tumour cells, by which the cells become free from each other (Coman, 1944;Zeidman, 1947; McCutheon, Coman and Moore, 1948); (b) an increase in ameboid motility and loss of contact inhibition of tumour cells, by which the cells invade the interstitial spaces of the adjacent normal tissues (Hanes and Lambert, 1912;Enterline and Coman, 1950;Hirono, 1958;Abercrombie and Ambrose, 1962;Wood, Robinson and Marzocchi, 1968); (c) invasion by tumour cells of small vessels, by means of which the cells are transported to distant organs (Sato, Suzuki and Kurokawa, 1966); and (d) migration of tumour cells through the vessel wall whereby, under favourable conditions, they lodge and proliferate to form metastatic secondary tumours (Nakamura, 1964;Satoh, 1967). It has been suggested that the invasive character of cancer cells may be in some way associated with an increased locomotion of the cells (Hirono, 1958;Sato, 1967).
As described in previous papers Yoshida et al., 1970), a substance chemotactic for cancer cells has been isolated from some tumour tissues of animal and human origin. After injection, this substance locally induced an extravascular migration of circulating tumour cells and the formation of metastatic tumour (Ozaki et al., 1971). Since the chemotactic factor could only be isolated from tumour tissues but not from the cancer cells themselves, it was supposed that the chemotactic factor was produced outside the cancer cells. It was also demonstrated that local production of the chemotactic factor were associated with the action of a certain neutral protease from cancer cells, e.g. rat ascites hepatoma cells (Koono, Ushijima and Hayashi, 1974) and that activation and release of the neutral protease were associated with a certain thermostable peptide from tumour tissues (Koono, Katsuya and Hayashi, 1974). In a further study (Katsuya, Koono and Hayashi, 1973), it was suggested that dissociation of rat ascites hepatoma cells was related to the action of the neutral protease activated by the peptide.
On the other hand, mechanisms which control cell adhesiveness have been suggested to be intimately related to the surface properties of tumour cells. The introduction of the rotational method of promoting cell aggregation led to the possibility of obtaining reproducible data (Moscona, 1961a). The aggregation of dissociated cells from sponges (Humphreys 1963), chick and mouse embryos (Moscona, 1961b), and tissue culture cells (Moskowitz, 1963) had been investigated using this method, and it has been suggested that specific macromolecular constituents of the cell surface might be involved in the phenomenon (Lilien, 1698). The mechanism of tumour cell adhesiveness is undoubtedly important in the explanation of malignant invasion. The purpose of the present communication is to describe the isolation of an aggregation promoting factor from the surface of the tumour cell and its biological properties.

MATERIALS AND METHODS
Rat ascites hepaton?a. -Rat ascites hepatoma AH136B (Odashima, 1962) has been maintained in our laboratory by routine weekly passage of 1 x 106 AH136B cells injected i.p. into 80-100 g male rats of the Donryu strain. The majority of AH136B cells formed cell islands of varying size in vtvo.
In vitro assay for cell aggregation.-This was performed essentially by a modification of the method of Moscona (1961a). One ml of the test sample at the same concentration (absorbancy 0 5 at 280 nm/ml) was mixed with 1 ml of cell suspension in a Falcon tube (1.5 x 9-5 cm) and incubated at 37°C in a roller tube culture apparatus, model Te-Her (Hirasawa Co., Tokyo, Japan) of one rotation/ 8 min. At intervals of 5, 15, 30 and 60 min after incubation, cell aggregation in both gross and microscopic features was recorded.
The grading of the induced cell aggregation was achieved by counting the aggregating cells and floating cells respectively in the fluid at 30 min of incubation, at which macroscopic cell aggegation became satisfactory. The cell aggregates formed were carefully removed from the tube with a pipette, suspended in 2 ml of Hanks' balanced salt solution and dissociated mechanically by pipetting; total numbers of dissociated cells w-ere counted by utilizing the microscopic apparatus with blood corpuscle counting chamber (Erma Optical Co., Tokyo, Japan). Total numbers of floating cells were also counted. Thus, the intensity of cell aggregation was roughly graded as follows: + +, over 70 + 500 of originally suspended cells w ere aggregated; +, 50 ± 5 % aggregated; 4, 30 ± 5 % aggregated; and -, below 20% aggregated.
Preparation of dissociated cell s8uspension.-AH136B cell suspension wvas prepared as follows: the ascitic fluid was collected by i.p. saline, the cells w-ere meclhanically dissociated by pipetting and sedimented by centrifugation at 55 g for 10 min. The cell suspension was finally prepared wtithl Hanks' balanced salt solution at concentration of 2 x 105 cells/ml; the majority of the cells in the suspension wAas found to be free.
Chang's cell suspension was prepared as follows: The cells were grown in Eagle's MEM containing 200 horse serum. The cells in 12-day-old culture w-ere wAashed writh Hank's balanced salt solution (free of calcium and magnesium), dissociated meclhanically by pipetting and sedimented by centrifugation at 55 g for 10 inin. The cell suspension was finally prepared with Hanks' balanced salt solution at concentrations of 5 x 105 or 25 x 106 cells/ml; the majority of the cells in the suspension wNere found to be free.
Normal rat liver cell suspension wN-as prepared as follows: liver cells of healthly male Donryu rats (80-100 g) wAere dissociated and collected according to the iiethod of Anderson (1953). After perfusion w ith 20 ml of 01 mmol/l EDTA in physiological saline through the portal vein, the liver wias excised and cut into small cubes or slices wNith a razor in 01 mmol/l EDTA solution. The cell suspension wNas filtered through 3 sheets of gauze and the liver cells wrere sedimenited by centrifugation at 55 g for 10 min and washed. The cell suspension containing 5 x 105, 1 x 106 or 3 x 106 cells/ml was finally prepared with Hanks' balanced salt solution; most of the cells wNrere found to be free. Red blood cells of healthy male Donryu rats (80-100 g) wN-ere collected and the cell suspension containing 5 x 105 or 1 x 107 cells/ml was prepared with Hanks' balanced salt solution.
Chromatography.-This was performed on columns of DEAE-Sephadex A-50 (3-5 mEq/g, Pharmacia, Uppsala, Sweden) prepared by the method of Porath and Lindner (1961) and Bio-gel A-5m (Bio-Road Laboratories, Richmond, California, U.S.A.) prepared by the method of Hjerten (1964). Protein concentrations Awere determined by the method of Wrarburg and Christian (1941) after measurement of the absorbancies at 280 nm and 260 nmn of the test samples. Desired concentrations on protein solutions were obtained by vacuum pressure dialysis using the Visking cellulose tubing.
Aggregation promoting factor in cell-free supernatant (Step 1). All procedures were performed in the cold (0°C). The ascitic fluid (200 ml), withdrawn 10 days after i.p. inoculation of AH136B cells, was diluted 1: 10 Awith 04450 NaCl and kept at room temperature for 60 min to allow red blood cells to separate. Tumour cell islands were collected by centrifugation at 55 g for 10 min, washed with 3 changes of 0-4500 NaCl and resuspended in 200 ml of Hanks' balanced salt solution (free of calcium and magnesium). The cell suspension received 50 gentle pipettings and was allowed to stand for 3 h. The supernatant fluid, obtained after centrifugation at 300 g for 10 min, was further centrifuged at 10,000 g for 30 min for removing visible cellular components. Before assay the supernatant fluid wN-as filtered through Millipore filters (pore size 0 3 em). Tumour cells sedimented wvere stained wNith 0500 trypan blue in Hanks' balanced salt solution; the numbers of damaged cells, as shown by diffuse staining with the dye, w-ere only 2-5% of the suspended cells, indicating that the above procedures induced very little cell damage.
Gel filtration of aggregation promoting factot on Bio-gel (Step 3).-All procedures were performed in the cold (0°C). The second peak (eluted in 0 02 mol/l phosphate buffer plus 0-3 mol/l NaCl on DEAE-Sephadex) from Step 2 was concentrated under vacuum pressure dialysis. The second peak (5 ml, 5r-51 absorbancy 4-6 at 280 nm/ml) was placed on a Bio-gel column (2-0 x 90 cm) equilibrated with Hanks' balanced salt solution. Filtration was performed at a rate of 5 drops/ min and 4 g effluent fractions were collected. Before assay, each effluent fraction was dialysed against Hanks' balanced salt solution for 12 h and then filtered through Millipore filters (pore size 0-3 ,um). Estimation of molecular size.-According to the method of Andrews (1965), estimation of molecular size of an aggregation promoting factor was performed by gel filtration on a column (2.4 x 50 cm) of Sephadex G-200 (Pharmacia, Uppsala, Sweden) equilibrated with 0-05 mol/l Tris-RCl buffer (pH 7-4). The flow rate was 25 ml/h. Cytochrome c (12,400 mol. wt; Sigma, St Louis, Missouri, U.S.A.), bovine serum albumin (67,000 monomer mol. wt; 134,000 in dimer mol. wt; Armour, Kankakee, Illinois, U.S.A.), and urease (480,000 mol. wt; Merck, Darmstadt, Germany) were used as standard substances. The elution volumes for each substance were plotted against the logarithmic scale of molecular weight, and the linear relationship between the elution volume and logarithmic value of molecular weight was recorded.

RESULTS
I. Isolation and partial purification of aggregation promoting factor (a) Aggregation promoting factor in cell-free supernatant.-Equal volumes (1 ml) of the cell-free supernatant fluid (absorbancy 0-5 at 280 nm/ml) from Step 1 and of AH136B cell suspension (5 x 105 cells/ml) were mixed and incubated. Formation of macroscopic cell aggregates became visible as early as 5 min after the start of incubation. With increasing incubation time, the cell aggregates became larger, fused with each other and sedimented as a mass on the bottom of the culture tube (Fig. 1), the aggregated cells showing a tendency to arrange in a concentric pattern (Fig. 2). The activity of the factor (graded +) was found still present when assayed in 4-fold dilutions with Hank's balanced salt solution. On the other hand, AH136B cells were not aggregated in the absence of the supernatant fluid even after 2 h of incubation (Fig. 3). No difference in the potency of the supernatant fluid was revealed before and after dialysis against Ranks' balanced salt solution for 12 h, indicating that the active substance of the supernatant fluid was non-dialysable.
(b) Aggregation promoting factor on DEAE-Sephadex.-After elution of the cell-free supernatant (10-20 ml, absorbancy 5-7 at 280 nm/ml) from Step 1 on DEAE-Sephadex, 5 chromatographicpeaks were obtained (Fig. 4) and the total yield, measured as absorbance at 280 nm, was about 70% of the proteins applied. The first peak contained 2-0%, the second 26-0%, the third 14.4%, the fourth 18.5% and the fifth 9.2%. However, the second peak (absorbancy 0-5 at 280 nm/ml) only caused a strong activity for AH136B cell aggregation under the same conditions as described above. Its affinity (graded +) was still present even when tested in 32-fold dilutions with Hanks' balanced salt solution, indicating an increase in the potency. No activity was revealed with the same concentration (absorbancy 0-5 at 280 nm/ml) of other peaks.
(c) Aggregation promoting factor on Bio-gel.-After elution of the second peak (5 ml, absorbancy 4-6 at 280 nm/ml) from Step 2 on Bio-gel, 2 chromatographic peaks were obtained (Fig. 5). The total yield, measured as absorbance at 280 nm, was about 95% of the proteins applied; the first peak contained 25% and the second 70%. The second peak (absorbancy 0-5 at 280 nm/ml) showed a strong activity for AH136B cell aggregation under the same conditions as described above; its activity (graded +) was still present even when tested in 42-fold dilutions with Hanks' balanced salt solution, indicating an increased potency. No activity was demonstrated with the -first peak (absorbancy 0-5 at 280 nm/ml) under the same conditions. The poltncy of the aggregation promoting factQr in each step of purification is summarized (Table I). 18553 ml for bovine serum albumin monomer; 136*2 ml for bovine serum albumin dimer; and 177X7 ml for aggregation promoting factor. The linear relationship between the elution volume and logarithmic value of molecular weight was recorded and the molecular weight of this aggregation promoting factor was estimated at 72,000 ± 7200 (Fig. 6). (b) Effect of heat on aggregation promoting factor. The active substance from Step 3 was found to be thermostable because most of the potency of this substance remained unchanged when heated to to 60°C for 30 min. The activity of the substance was kept satisfactorily at 0°C or in the frozen state. Aggre( mixture (2.0 ml), an equal volume of the active substance from Step 3 was added for assay of cell aggregation. The effect of the aggregation promoting factor was not, however, influenced in the presence of these sugar preparations even at highest concentration (100 mmol/l).
(d) Reactions of aggregation promoting factor with various reagents.-The active substance from Step 3 seemed to be a protein because positive results were obtained in the biuret, ninhydrin and Folin reactions. The reaction with phenol sulphate for sugar was also positive. On the other hand, the reactions with indol and orcinol for nucleic acid were all negative.
(e) Effect of aggregation promoting factor on other cells. Following the same method as described above, the effect of the active substance (absorbancy 0.5 at 280 nm/ml) from Step 2 was assayed on SV40-transformed cells, Chang's cells, normal liver cells and red blood cells.
The active substance was clearly effective in aggregating SV40-transformed cells (2 x 105 cells/ml) and its potency was  Step 3 was tested by a modification (Hayashi et al., 1962(Hayashi et al., , 1965 of the casein digestion method of Kunitz (1947) and by a modification (Sliwinski, Doty and Landmann, 1959) of the haemoglobin digestion method of Anson (1939). The assay was performed at various pH ranges (3.0-6.0 against haemoglobin and 6*0-1.0-0 against casein) and concentrations (absorbancy 1*0-3*0 at 280 mn/ml) of the factor. It had no proteolytic activity.

DISCUSSION
The observations described here demonstrated that a tumour cell aggregation promoting factor was released possibly from the cell surface from rat ascites hepatoma AH I36B cells forming cell islands, when kept in cold Hanks' balanced salt solution (free of calcium and magnesium). It could be partially purified by chromatography using DEAE-Sephadex and by gel filtration using Bio-gel. The substance was assumed to be a thermostable glycoprotein with a molecular weight of about 72,000. This is of some interest in view of the well known evidence that sugar containing molecules of animal cell membranes may play a part in mediating cell adhesions, perhaps as " recognition sites ". The substance had no proteolytic activity when tested against casein and haemoglobin.
The action of this material was characterized by lack of inhibition of AH136B cell aggregation by sugar preparations which inhibit the effect of aggregation promoting factors such as Jack bean concanavalin A (Inbar and Sachs, 1969), wheat germ glycoprotein (Burger and Goldberg, 1967) and plant phytoagglutinins (Tomita et al., 1970). This strongly suggests the presence of a binding site for the factor on the cell surface, different from the sites for aggregation promoting factors of plant origin mentioned above.
The factor also differed from concanavalin A in its higher aggregating activity when compared on SV40-transformed cells. The potency of 11l,tg of the aggregation promoting factor seemed to correspond to that of 250,ug ofJack bean concanavalin A.
The substance was clearly effective for aggregating dissociated AH136B cells as well as SV40 transformed cells, but less effective for Chang' cells. On the other hand, it was apparently ineffective for normal liver cells and red blood cells of rats. This may be due to functional differences on the surface of hepatoma cells and liver cells which need further investigation.
The power to induce tumour cell aggregation has been seen in the serum (Tal, Dishon and Gross, 1964) and ascitic fluid of cancer patients (Mori, Akedo and Tanigaki, 1970) and of tumour bearing mice (Oppenheimer and Humphreys, 1971). However, the problem of whether such potency may be related to an aggregation promoting factor of cancer cell origin has not yet been established. We have recently demonstrated that a substance similar to the present aggregation promoting factor can be separated from the sera and ascitic fluid of rat ascites hepatoma AH136B or AH109A transplanted rats (Kudo and Hayashi, 1972) and this suggested that the factor was released from these cancer cells.