The induction of immunoprotection by mouse ascites tumor cells attenuated in tissue culture

Europ. J. CancerVol. 8, pp. 293-298. Pergamon Press 1972. Printed in Great Britain

The Induction of Immunoprotection by Mouse Ascites Tumor Cells Attenuated in Tissue Culture* j. F. MORGAN and C. P. ENG," Departments of Biochemistry and Cancer Research, University of Saskatchewan, Saskatoon, Canada Abstract-- The loss of transplantability and high degree of immunoprotection shown by 6C3HED-ascites tumor cells in tissue culture have been confirmed with the TA3 ascites tumor. The induction of immunoprotection against solid as well as ascites tumor formation has been demonstrated with 6C3HED non-tumorigenic tissue culture cells. Treatment of virulent 6C3HED ascites cells by chemical or physical agents destroyed completely their immunizing capacity. Similar treatment of non-tumorigenic tissue culture cells reduced but did not destroy their immunizing capacity.

fibroblasts or with virus-transformed hamster cells and have shown loss of transplantability associated with retention of immunoprotective capacity. In the authors' laboratory, a series of tissue culture lines established from the Ehrlich, TA 3, Ehrlich-Lettr6 and 6C3HED mouse ascites tumors were found to have lost their transplantability after from 1 to 5 years of propagation in vitro [9]. The establishment of new and still tumorigenic tissue culture lines from these mouse tumors has made it possible to develop a three-component model system for the study of this phenomenon, based on the tumors in vivo together with their derived tumorigenic and non-tumorigenic cell cultures. The present paper reports data on the immunoprotective capacity of these non-tumorigenic cell cultures and suggests that attenuation of tumor ceils through tissue culture might provide a basis for protection against some types of animal tumors.

INTRODUCTION

ThE PIONEER experiments of Gey in 1941 [1] and of Earle in 1943 [2] demonstrated that normal rat and mouse tissues cultivated in vitro for prolonged periods underwent a progressive series of changes resulting in the development of some malignant characteristics. This observation of a trend towards malignancy in cell cultures derived from normal tissues has been thoroughly proved by m a n y other investigators and has become an accepted concept of tissue culture research. Long-term tissue cultures derived from tumors have also been shown to lose their transplantability. This effect was first demonstrated by De Bruyn and Gey [3] in 1952 with mouse lymphoma cells and has subsequently been confirmed by Hsu and Klatt [4] with rat hepatoma cells, by Foley and Drolet [5] with mouse Sarcoma 180 cells, and by Moore et al. [6] with Ehrlich ascites cells of the mouse. Recently, Harris et al. [7], and Watkins and Chen [8] have hybridized Ehrlich ascites cells with mouse

MATERIAL A N D M E T H O D S Tumors Hypotetraploid lines of the 6C3HED, Ehrlich, TA3, and Ehrlich-Lettr6 ascites tumors were maintained by weekly serial passage with the intraperitoneal inoculation of 2-4 x 106 cells per mouse. The first 3 tumor lines were obtained

Accepted 19 October 1971. *Presented at the IVth International Symposium on the Biological Characterization of H u m a n Tumours, Heidelberg, April 21-23, 1971. tPresent address: Bionetics Research Laboratories, Inc., 5510 Nichotson Lane, Kensington, Md. 20795, U.S.A.

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J. F. Morgan and C. P. Eng

from Dr. G. A. LePage, Stanford Research Institute, Menlo Park, California, and the EhrlichLettr6 tumor line from Dr. M. J. Fraser, University of Manitoba, Winnipeg, Canada. All 4 lines have been carried for many years in the authors' laboratory. These tumors provided cells for the establishment of tissue culture lines and also virulent cells to provide challenge doses in immunoprotection experiments. All studies with the 6 C 3 H E D tumor were conducted in male C 3 H mice, weighing 20 to 25 g, either purchased from the Jackson Laboratory, Bar Harbor, Maine or bred at the University of Saskatchewan from Bar H a r b o r stock. With the other 3 tumors, male Swiss mice weighing 23 to 28 g, from Connaught Medical Research Laboratories, University of Toronto, Canada, were used. These mice were bred under barrier-sustained conditions at the University of Saskatchewan. All mice under experiment were supplied with food and water ad libitum. Mice were inoculated either intraperitoneally or subcutaneously with varying doses of cells established by the Coulter Counter. They were then observed for 90 days and the number dying from tumor development was recorded. At the end of each experiment, any surviving mice were killed and examined for solid tumor formation either in the peritoneal cavity or at the site of inoculation.

Tissue culture Cell lines were established as monolayer cultures in Medium 199 [10], supplemented with 5% calf serum [11], by procedures described previously [12]. All ascites cell cultures were treated every 3 months with 500 #g/ml kanamycin (supplied by Bristol Laboratories, Inc., Syracuse, N.Y.) to eliminate any chance of mycoplasma contamination. After prolonged cultivation in vitro, the ascites cell lines failed to produce tumors on back transplantation and were used for immunization studies. For passage, the cells were scraped, aspirated and then transferred to new culture vessels in a 1:2 or 1:3 ratio. This method produced maximum monolayer cultures in 6-8 days. Cell number was determined with a hemocytometer, and cell viability was measured by the dye exclusion test, using erythrosin B. The viability of the non-tumorigenic tissue culture cells ranged between 72 and 81%, while that of the virulent tumor cells taken directly from the mouse always exceeded 96 %.

Immunization Non-tumorigenic tissue culture cells were harvested, washed 3 times in 10 volumes of

Hanks' balanced salt solution (HBSS) [13], and resuspended to the desired concentration in HBSS. Each mouse then received intraperitoneally 5-8 x 10 6 cells in 0.5 ml suspension. The injections were administered once weekly for 3 weeks. One week after the final injection the mice were challenged either intraperitoneally or subcutaneously with varying numbers of virulent tumor cells freshly drawn from a mouse. At the same time, equal numbers of normal nonimmunized mice were given injections of viable tumor cells from the same cell suspension. All mice were then kept for a 90-day observation period. Washed, non-tumorigenic 6 C 3 H E D or TA3 tissue culture cells were suspended in HBSS to a concentration of 8.0 x 10 6 cells per ml and were then treated for 60 sec in an M S E sonic oscillator. Microscopic observation indicated that the cells were completely disrupted after this treatment. This material was then used for immunization purposes as described previously. Similar experiments were conducted with virulent 6 C 3 H E D or TA3 cells freshly harvested from mouse tumors. Virulent ascites tumor cells and their derived non-tumorigenic tissue culture lines were incubated with 10 -3 M sodium iodoacetate at 37°C for 1 hr, according to the procedure of Apffel et al. [14]. These devitalized cells were then used for immunization experiments. Tumor cells and their derived non-tumorigenic tissue cultures received X-irradiation in a Picker Corporation instrument at 200 kVp and 18 mA using a 0-25 N filter. Both types of cells received a total of 4000 rads at an exposure rate of 50 rads/min with a focal distance of 12.1 cm. The dose rate was calibrated with a Baldwin-Farmer dosemeter.

Specificity of immunity Groups of Swiss mice were immunized by weekly intraperitoneal injection for 3 weeks with either spleen cells from C3H mice or with 6 C 3 H E D non-tumorigenic tissue culture cells. Subsequently, both groups of mice were challenged with 1-0x 104 virulent cells of the 6C3HED, TA3, Ehrlich, and Ehrlich-Lettrd ascites tumors. The mice were then held for 90 days and tumor formation was recorded [15]. Chromosome analyses of the ascites tumor cells and of their derived tumorigenic and nontumorigenic tissue cultures were performed as described previously [16]. Mice were subjected to total body X-irradiation in a Picker therapy machine at 200 kVp and 20 mA.

The Induction of Immunoprotection

295

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Generalresearchprogram for study of the attenuation phenomenon. Central portion of the diagram illustrates the three-component tumour model system developed.

RESULTS

Table 1. Important characteristics of tumorigenic and non-tumorigenic ascites cells

Attenuation phenomenon The research program that has been developed to study the phenomenon of attenuation is shown diagrammatically in Fig. 1. The essential part of the system is shown in the central portion of the diagram. It consists of a spectrum of fully virulent ascites tumors maintained by weekly serial passage in mice. From these tumors, fully transplantable tissue culture lines are established which become non-transplantable after long-term cultivation. The system is maintained by the frequent establishment of new tumorigenic tissue culture lines to replace those that have become non-transplantable. The most important features of the tumorigenic and non-tumorigenic ascites cells are summarized in Table 1. The great difference between the tumorigenic and non-tumorigenic cell cultures is shown in their relative transplantability. The tumorigenic culture normally produced 100 % tumors with the injection of 10 z cells, while the non-tumorigenic cell cultures failed to produce any tumors with the intraperitoneal injection of as many as l0 s cells. Similar results were obtained whether normal mice were used or mice which had been subjected to total body X-irradiation to suppress their immune mechanisms.

In vivo

1. 2. 3.

Grow very slowly in tissue culture 75-78 chromosomes 100% tumours produced by 102 cells

In vitro Tumorigenic 1. Grow rapidly in tissue culture 2. Average 123 chromosomes, with metacentric 3. 100% tumors produced by I02 cells

1

Non-tumorigenic I. Grow rapidly in tissue culture 2. Average 117 chromosomes, with 3 or 4 metacentrics 3. O tumors with as many as 108 ceils

Immunoprotection The extent of the tumor-specific immunity induced by the non-tumorigenic tissue culture cells was studied by immunizing groups of mice with 6C3HED or TA3 cells and challenging with the homologous ascites cells, as summarized in Tables 2 and 3. A very high degree of immunoprotection is evident, with 95% protection against challenge of 106 6C3HED cells and 70% protection at this dose with TA3 cells. When 107 6C3HED cells were used for the challenge dose, the immunoprotection was decreased to 60%.

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J. F. Morgan and C. P. Eng

Table 2. Immunoprotection induced by 6C3HED nontumorigenic tissue culture cells against challenge with virulent 6C3HED ascites cells at different dosages* Challenge dose (no. of ceils inoculated per mouse)

Normal mice

Immunized mice

Survivors~

Survivorst Protection

Two mice which received intraperitoneal injections of 106 virulent TA3 ascites cells are shown in Fig. 2. The normal control mouse (right) has died from an ascites tumor while the pre-immunized mouse (left) shows no sign whatever of tumor development. Similar results were obtained with 6C3HED cells.

(%) 10 3 10 4 10 s 10 6 10 7

0/20 0/20 0/20

10/10 20/20 20]20 19/20 12/20

0/20 0/20

100 100 100 95 60

*Immunized mice received intraperitoneal injections of 5-8 × 106 non-tumorigenic cells once a week for 3 weeks and were challenged with virulent cells 1 week later. ~Expressed as number of mice surviving for 90 days/ number of mice challenged.

Table 3. Immunoprotection induced by TA3 nontumorigenic tissue culture cells against challenge with virulent TA3 ascites cells at different dosages* Challenge dose (no. of cells inoculated per mouse)

Normal mice

Immunized mice

Survivorst

Survivors1" Protection

(%) 102 10 a 10" 105 106

0/20 0/20 0•20 0/20 0/20

20/20 20/20 20/20 20/20 14/20

100 100 100 100 70

*Immunized mice received intraperitoneal injections of 5-8 × 106 non-tumorigenic cells once a week for 3 weeks and were challenged with virulent cells 1 week later. tExpressed as number of mice surviving for 60 days/ number of mice challenged.

Table 4.

Effect of chemical and physical treatments Previous studies from this laboratory [ 15] had established that maximum efficiency of immunization was obtained when more than 105 nontumorigenic cells were administered at each injection and that two or more immunizing doses were most effective. It was considered of interest to determine the effect of chemical and physical treatment of both tumorigenic and non-tumorigenic cells upon their immunizing capacity, as summarized in Table 4. It is evident that the physical and chemical treatments used have completely devitalized the virulent ascites cells since no tumors were elicited by injection of the treated cells. These treatments, however, have also destroyed the immunizing capacity of the cells since no protection whatever is afforded against subsequent challenge with virulent cells. In contrast, the attenuated tissue culture cells still retain a high proportion of their immunizing capacity after the chemical and physical treatments. X-irradiation and iodoacetate appear to be less damaging in their effects than are lyophilization, sonication or freezing and thawing.

Passive immunity and cross-protection Our previous studies [15] have shown that the immunoprotection induced by non-tumorigenic 6C3HED tissue culture cells can be demonstrated by both the serum and the spleen

Effect of chemical and physical treatments on immunoprotective capacity of virulent and attenuated 6C3HED cells*

Treatment of cells before immunization

Treatment controls (virulent cells) Survivors

No treatment Iodoacetate Lyophilization Freezing and thawing Sonication X-irradiation

0/20 20/20 20]20 20]20 20/20 20]20

Immunization with Virulent cells Survivors -0/20 0/20 0/20 0/20 0/20

Attenuated cells Survivors 20/20 15/20 11/20 13/20 12/20 16120

Protection (%) 100 75 55 65 60 80

*Results are expressed as number of mice surviving after 60 days/number of mice inoculated. Immunizations were carried out with 5 x 10 e ceils intraperitoneally once a week for 3 weeks, followed by challenge with 105 untreated virulent cells 1 week later.

Fig. 2. Two mice inoculated intraperitoneally with 10 6 virulent TA3 ascites cells. Mouse on left had been pre-immunized with non-tumorigenic TA3 tissue culture cells. Non-immunized control mouse is ol~ right.

Fig. 3. Two mice inoculated subcutaneously in left thigh with 10 6 virulent TA3 ascites cells. Mouse on right had been pre-immunized with nontumorigenic T A 3 tissue culture cells. Non-immunized control mouse is on left and shows development of large solid tumour.

(to f a c e p. 296)

The Induction of Immunoprotection cells of the immunized mice. Protection rates of 73% and 70% were obtained, respectively, against challenge with 10" virulent 6C3HED cells. Mice immunized with 6C3HED non-tumorigenic tissue culture cells showed a considerable degree of crossprotection when challenged with virulent TA3, Ehrlich or Ehrlich-Lettr6 cells [15]. The degree of crossprotection achieved was 40% for TA3 cells, 60% for Ehrlich-Lettr6 cells, and 70% for Ehrlich cells, while 100% protection was obtained against the homologous 6C3HED cells. The high degree of crossprotection obtained suggests that some common antigens have been developed during the long-term cultivation in vitro. Immunoprotection against solid tumors Ascites cells inoculated subcutaneously rather than intraperitoneally produce large solid tumors at the site of injection. Since the nontumorigenic tissue culture lines had been shown to induce a strong immunoprotection against challenge with virulent cells administered intraperitoneally, it was considered of interest to determine whether such immunity would extend to the solid tumor system. Accordingly, groups of C3H mice were immunized with non-tumorigenic 6C3HED tissue culture ceils and were then challenged with graded doses of virulent 6C3HED cells injected subcutaneously in the dorsal region of the right thigh. The results summarized in Table 5 show that a high degree of immunoprotection, totaling 90% at a challenge dose of 10 v cells, has been attained. This degree ofimmunoprotection is somewhat higher than that recorded previously for intraperitoneal challenge in the same cell system [15]. Table 5. Immunoprotectioninduced against solid tumor formation with attenuated 6C3HED cells* Challenge dose

Normal mice

103 10s 107

10/20 19/20 20/20

Immunized mice 0/20 0/20 2/20

Protection (%) 100 100 90

*Results expressed as number of mice developing solid tumours/number of mice challenged with virulent 6C3HED cells subcutaneously. Two mice which received subcutaneous injections of 106 virulent TA3 cells in the left thigh are shown in Fig. 3. The normal control mouse (left) has developed a very large solid tumor while the pre-immunized mouse (right) shows no sign whatever of tumor development.

297

DISCUSSION The previous observation [9, 15] that 6C3HED ascites tumor cells become nontumorigenic after prolonged cultivation in vitro, but retain a high immunoprotective capacity has now been confirmed with TA3 ascites tumor cells. With both cell lines nearly complete protection has been achieved against a challenge dose of 10 6 fully virulent ascites cells. Extension of these studies has shown that immunoprotection can be achieved against solid tumor as well as ascites tumor formation at a similar high challenge dose of virulent cells. This observation is in accord with our previous report [15] that either spleen cells or sera from immunized mice could neutralize the transplantability of virulent tumor cells in vivo. It would appear that the immunoprotection elicited by non-tumorigenic tissue culture cells is a whole body immunity response. When virulent ascites cells were devitalized by chemical or physical treatments they failed to induce immunoprotection in mice. Following similar treatments, the non-tumorigenic tissue culture cells still retained most of their antigenic capacity, although such treatments as lyophilization and sonication reduced this capacity to a considerable extent. It would appear from these results that untreated nontumorigenic cells are far more effective as immunizing agents than are devitalized virulent ascites cells. The concept of the loss of transplantability by tumor cells during long-term tissue culture is not a new one since the idea of attenuation was expressed clearly by Hsu and Klatt [4] in 1959 and by Sanford et al. [17] in 1955. The present studies appear to be unusual in the complete loss of transplantability obtained, coupled with an extremely high antigenic capacity. The fact that a moderate degree of cross-protection against other ascites tumors can be developed [15] and that the immunity can be transferred by spleen cells or serum suggest that the phenomenon of attenuation deserves intensive study. The mechanism of the loss of transplantability has not yet been established. Selection of nontumorigenic cells from a heterogeneous population, some form of regression from the malignant state, or some form of cell hybridization appear to be obvious possibilities. The attenuation process appears to occur spontaneously and on a different time basis for each ascites tumor line studied. If the mechanism of attenuation could be elucidated it might become possible to expedite the process and control it experimentally, steps that would be crucial in the development of attenuated cells for therapeutic purposes.

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Acknowledgements--The financial support of the Medical Research Council of Canada is gratefully

acknowledged. We wish to express our appreciation to Mrs. M. D. Heuchert for skilled technical assistance.

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