Europ. J. Cancer Vol. 11, pp. 1-8. Pergamon Press 1975. Printed in Great Britain

Antitumour Immunity--1 Differential Response of Neuraminidase-Treated and X-Irradiated Tumour Vaccine* P. K. RAY~', V. S. T H A K U R and K. SUNDARAM Medical Division, Cancer Immunobiology, Bhabha Atomic Research Centre, Trombay, Bombay--400 085, India

Abstract---A dimethyl-benz-dithionapthene-induced fibrosarcoma shows reduced transplantability in syngeneic Swiss mice when treated with Vibrio cholerae neuraminidase (VCN). Reduced transplantability of fibrosarcoma has also been observed if they are X-irradiated. Inoculation of VCN treated cells leads to the development of strong antitumour immunity, whereas comparable results are not obtained with Xirradiated cells. However, inoculation of cells treated with VCN foUowed by X-irradiation can also establish lasting antitumour immunity. It is suggested that 'tumour vaccine' produced in this way may be very effective for the immunotherapy of tumour.

INTRODUCTION VARIOUS laboratories [1-10] have reported that Vibrio cholerae neuraminidase (VCN) treated tumour cells have reduced transplantability in an otherwise susceptible host. Animals previously inoculated with VCN treated tumour have been shown to be refractory to normal tumour challenge. Several mechanisms have been proposed as to how VCN treated tumour cells induce tumour specific immunity [1-10]. In a recent review, Weiss [11] has pointed out that absence of tumour or the presence of small, slowlygrowing tumour in animals receiving VCNtreated tumour ceils might be due to the fact that less than LD~o0 doses of undamaged viable cells were given. He mentioned that

Accepted 19 September 1973. *Part of this report was presented at the DAE Symposium on 'Biological approach to problems of medicine, industry and agriculture', Trombay, 12-14 March, 1974. ~'Address reprint requests to: Dr. P. K. Ray, Medical Division, Cancer Immunobiology, Bhabha Atomic Research Centre, Trombay, Bombay-400 085, India.

animals receiving inocula containing similar proportions of living and dead ceils not treated with neuraminidase may also lead to comparable results. Moreover, comparisons of the lethality of inoculated BW 10232 mouse mammary carcinoma cells fail to reveal a significant difference between X-irradiated cells and those incubated with neuraminidase after exposure to 5 0 0 0 R [12]. In this review, Weiss [11] raised the question 'is neuraminidase really necessary?' He stated that X-irradiated cells might exhibit an immunogenic response equal to that shown by those treated with neuraminidase. We tested this phenomenon using VCN treated tumour cells and X-irradiated tumour cells, since X-irradiated vaccines of tumour cells sometimes [13, 14], but not invariably [15], show increased immunogenic activity. This paper reports that VCN-induced increased immunogenicity of tumour cells cannot be achieved with lethally X-irradiated tumour cells. T u m o u r specific immunity, however, can be achieved with V C N treated and X-irradiated tumour cells indicating that VCN modulates the tumour cell surface in a specific way to induce anfitumour immunity unlike the X-irradiated cells.

P. K. Ray, V. S. Thakur and K. Sundaram

MATERIAL AND METHODS Animals Swiss mice originally obtained from Jackson Laboratory have been maintained here by brother-sister mating. Male mice 8-10 weeks old were used. Animals were housed, 4-5 animals/cage; food and water were supplied ad libitum. Turnouts A dimethyl-benz-dithio napthene-induced fibrosarcoma has been obtained from Cancer Research Institute, Pard, by courtesy of Dr. C. V. Bapat and has been maintained here by serial transplantation in syngeneic Swiss mice. This tumour is very weakly immunogenic and never regresses spontaneously.

Neuraminidase Vibrio choterae neuraminidase (VCN) has been obtained from Behrinwerke A.G., Marburg, Lahn, West Germany. It contains 500 units of enzyme/ml. One unit of enzyme activity is equivalent to the release of 1 ~g of N-acetyl neuraminic acid from a glycoprotein substrate at a pH of 5.5, at 37°C. This enzyme has been reported by the manufacturer to be free from aldolases, proteinases, and lecithinase C activity. The enzyme itself is not cytolytic [16, 17] and can release sialic acid from both normal and malignant cell surfaces at physiological pHs [18]. VCN treatment T u m o u r cells were incubated with VCN (25 U / S x 10 v cells) at 37°C for one hour. After incubation, cells were washed ( x 3) in excess of medium 199 (M 199) and finally suspended in M 199. X-irradiation In order to determine if VCN-inhibition of tumour growth was due specifically to VCNtreatments, additional experiments were done using X-irradiated tumour cells. Measured numbers oftumour cells suspended in physiological saline in a petridish have been X-irradiated (100-2000 rads) using an X-ray machine working at a dose rate of 100 rad/min (250 kVp, 15/mA, 0.2 m m Cu, 54 cm targetobject distance). Tumour ceil suspension Tumours were collected in an asceptic way in a petri dish with media containing antibiotics The tumour mass was washed several

times in media and then cut into very small pieces with scissors, then 0.1% trypsin (1 : 250, DifCo) was added and the tumour pieces were stirred for 10 min using a magnetic stirrer. Clumps were allowed to settle down and supernatant fluid was collected. A fresh volume of media was added and the process repeated several times. The cells were centrifuged (2000 rev/min, 10 rain) and washed three times before finally being suspended in M 199 unless otherwise stated. Cell viability was determined by trypan blue exclusion [19].

RESULTS Effect of VCN on the growth of chemically induced fibrosarcoma in syngeneic Swiss mice Various doses of untreated and VCN treated tumour cells were inoculated subcutaneously in the adult male Swiss mice. Animals were checked every day to detect the tumour nodule and the survival times of the animals from the date of appearance of the tumour nodule were noted. Results are presented in Table 1. The formation of the tumour nodule is much delayed in animals which received VCN treated cells compared to animals which received sham-treated cells. It appears that transplantability of VCN treated cells is very much reduced. This observation confirms the previous findings [ 1-10] which note that at certain concentrations, VCN treated tumour cells cannot grow in an otherwise susceptible host. In the present investigation, it has been established further that VCN-treated tumour cells did not develop tumours, while those animals receiving 1000 sham-treated tumour cells did develop tumours which ultimately killed the host. Although animals inoculated with higher doses of VCN treated turnout cells show tumour development, they survive longer than those receiving the corresponding number of sham-treated cells. The above experiment suggests that though the transplantability of VCN treated tumour cells is very much reduced, their oncogenicity is still retained. VCN treated tumour can grow if inoculated at a high dose. Further, it has been shown that VCN treated tumour cells that do not develop tumours in normal recipients do develop tumours which ultimately kill the recipient when injected in immunosuppressed animals [4, 9]. Thus, the inhibition of growth of VCN treated tumour appears to be mediated through an active immune mechanism in the host. It is highly probable that tumour specific antigens are exposed after

Antitumour Immunity--L Neuraminidase-treated and X-irradiated Tumour Vaccine

3

Table 1. Reduced transplantability of tumour cells treated with Vibrio cholerae neuraminidase ( VCN) Fraction t u m o u r appeared in animals receiving N u m b e r of cells in the initial inoculum U n t r e a t e d cells

106 l0 s 10 * 103 102

V C N treated cells*

6/6 6/6 6/6 6/6 5/6

Average incubation period (days)

U n t r e a t e d cells

6/6 4/6 2/6 0/6 0/6

V C N treated cells

6 11 15 21 23

23 21 24 ---

Survival period (days + S.E.) I"

U n t r e a t e d cells

V C N treated cells

( _+S.E.)

( _+S.E.)

23 29 31 30 35

+ 2.5 _+ 1.5 _+2.0 _+2-5 +_ 3.5

25 + 1.5 36 + 3.0++ 38 +_ 3.5++ Indefinite Indefinite

*Cells were viable by trypan blue exclusion test. i'Standard error of the m e a n (_+ S.E.). ++Animals without turnouts were not included.

V C N treatment. Recognizing them to be of foreign origin, the hosts' immune systems might become fully operative and ultimately reject the tumour cells.

cells. The animals remained tumour-free in perfectly normal health up to 60 days.

Development of antitumour immunity by VCN treated tumour vaccine

Freshly prepared tumour cells were Xirradiated. The irradiated cells were then inoculated into animals and their oncogenic potential were studied (Table 3). A dose of 500 rad can inhibit completely the oncogenic potential of 50,000 tumour cells, while as high as 1500 rad is needed to suppress the tumourogenic ability of 106 tumour cells. It appears from the results that there is a direct relationship between the number of tumour ceils in an inoculum and the amount of X-irradiation to be given to them to suppress their oncogenic potential. In some cases where tumours are seen following inoculation with X-irradiated cells, their appearances are much delayed and

Inhibition of the oncogenic potential of tumour cells by X-irradiation

In the above experiment, animals which received V C N treated tumour cells and did not develop any tumour up to 60 days were challenged with double the initial doses of untreated tumour cells. Results are presented in Table 2. These animals were found to be resistant against the challenging inoculum. It is quite significant that these immune animals can resist such high doses of tumour cells, while cohort mice all died. After 60 days, these resistant animals received a second challenge with 10 times the initial inoculum and could withstand even this high dose of viable tumour

Table 2. Developmentof strong antitumour immunity by VCN-treated tumour vaccine

G r o u p of animals I

Initial inoculum 10 s

II

104

III

10 3

IV

102

Cell treatment VCN None VCN None VCN None VCN None

Fraction 1° t u m o u r appearance 4/6 6/6 2/6 6/6 0/6 6/6 0/6 5/6

Challenging inoculum* 1st 2x . 2× . 2x . 2x .

Fraction t u m o u r appeared

2nd

105 . 104 . 10 s . 102 .

106 . 10 s . 10 4 . 103 .

Ist

2nd

0/2

0/2

0/4

0/4

0/6

0/6

0/6

0/6

. . . .

Survival time (_+ S.E.) Indefinite'~ 29 A- 1.5 Indefinite 31 _+2"0 Indefinite 30 + 2.5 Indefinite 35 + 3.5

*Animals which did not show primary tumours were only challenged with untreated viable t u m o u r cells after 60 days. Second challenge was given 60 days after the first challenge. I f the initial challenge is given with 10 times the initial inoculum, in m a n y cases t u m o u r developed in the beginning. 1"Only the t u m o u r free animals were considered.

4

P . K . Ray, V. S. Thakur and K. Sundaram

they usually survive longer than the control groups of animals.

Development of antitumour immunity by X-irradiated tumour cells Animals which did not develop any t u m o u r following the inoculation of X - i r r a d i a t e d t u m o u r cells in the above e x p e r i m e n t (Table 3), were challenged with viable u n t r e a t e d t u m o u r cells (double the initial inoculum). Results are presented in T a b l e 4. Most of these animals were not resistant to the challenging inoculum indicating that the i m m u n o genicity of the t u m o u r cells was not increased

with X - i r r a d i a t i o n to p r e v e n t t u m o u r growth. T o examine the i m m u n o g e n i c potential of this c o m b i n a t i o n the following experiments were performed.

Development of antitumour immunity by VCN treated and X-irradiated tumour vaccine T h r e e groups o f animals were inoculated with either 10 s V C N treated cells, X - i r r a d i a t e d (1500 rads) t u m o u r cells, or V C N treated and X - i r r a d i a t e d (1500 rads) t u m o u r cells. Eight out of ten animals receiving V C N treated cells developed tumours while none of the animals receiving irradiated or V C N treated and

Table 3. Reducedtransplantability of X-irradiated tumour cells

Group of animals

Number of cells in tile intial inoculum 2x 1x 5x 5x 5x 5x 5x

II

Dose of irradiation (X-rays, rads)

Fraction tumour appearance

Average incubation period (days + S.E.)

500 500 500 500 500 500 500

6/6 1/6 0/6 0/6 0/6 0/6

18 + 2"5 21 + 3.0 19_+2-5 -----

32++2.5 33+*2-5 32+3-0 Indefinite Indefinite Indefinite Indefinite

100 200 300 400 500 750 1000 1500 2000

4/4 4/4 6/6 6/6 4/6 2/3 2/6 0/3 0/3

11 _+1.5 14 + 1.5 14 + 2"0 15 + 1.5 30 + 2.5 21 _+3-5 19 + 2.5 ---

26 + 1.5 27 + 2"5 32 _+3.0 34 + 2.5 33 + 3-0 36 + 3-5 43 + 4.0 Indefinite Indefinite

106 106 l0 s 104 10 a 102 10

1 x 106

5/5

Average period of survival of tumour bearing animals*

*Animals with tumours were only included. following X-irradiation, at least to the extent that could inhibit the growth of the challenging inoculum. T h u s it appears that inoculation of X-irradiated t u m o u r cells does not lead to the establishment of c o m p a r a b l e a n t i t u m o u r i m m u nity obtained with V C N treated t u m o u r cells. Inhibition o f oncogenicity o f t u m o u r cells by X - i r r a d i a t i o n m a y be due to the killing of the t u m o u r cells. O n the other hand, the inhibition of the growth of V C N treated t u m o u r cells, which are viable, appears to be due to the heightened t u m o u r specific i m m u n i t y developed in those animals (Table 2). F r o m the point o f view o f actual t h e r a p y using V C N treated t u m o u r cells, which are otherwise viable and can show t u m o u r developm e n t at higher doses (Table 1), it was necessary to combine V C N t r e a t m e n t o f t u m o u r cells

irradiated cells developed turnouts ( T a b l e 5). Animals which r e m a i n e d t u m o u r free were t h e n challenged with u n t r e a t e d t u m o u r cells (2 x 10s). All animals which received irradiated t u m o u r cells as a p r i m a r y i n o c u l u m developed t u r n o u t without any change in their i n c u b a t i o n time or in the period of survival of the animals ( T a b l e 5). Most o f the animals which received V C N treated and X - i r r a d i a t e d cells as a p r i m a r y inoculum were i m m u n e to the challenging inoculum. It is i m p o r t a n t to note that while X-irradiation inhibits the growth potential of 10 s V C N treated t u m o u r cells, it does not alter the i m m u n o g e n i c properties of V C N treated cells. F r o m the therapeutic point o f view, this m e t h o d o f p r e p a r a t i o n o f t u m o u r vaccine will have significance for a variety of reasons: (a) there will be no d a n g e r of inducing a fresh t u m o u r growth by the inoculating cells,

Antitumour Immunity--L Neuraminidase-treated and X-irradiated Tumour Vaccine (b) t u m o u r vaccine containing large n u m b e r o f cells can be inoculated at a time (which is necessary for effective i m m u n o t h e r a p y ) to i n d u c e t u m o u r specific i m m u n i t y , and (c) a syngeneic t u m o u r or even a portion of the t u m o u r to be treated can serve as the source of cells for the p r e p a r a t i o n of the vaccine, t h e r e b y diminishing a n y c h a n c e o f graft-versushost reaction.

Table 4. Initial inocutum 1 x 106 1 x 106 1 x 106 1 X 106 5 x l0 s 1 x 10 s 5x 104 5 X 103

5

transplantability o f t u m o u r cells, it is not associated with the establishment o f c o m p a r a b l e a n t i t u m o u r i m m u n i t y . I n some cases, some degrees o f i m m u n i t y (lower t h a n t h a t given b y V C N treated cells) could be established. But the degree of i m m u n i t y is always lower c o m p a r e d to w h a t has been observed with V C N treated t u m o u r inoculation. H o w e v e r , V C N treated and X - i r r a d i a t e d t u m o u r cells

Weak antitumour immunity as developed by X-irradiated tumour cells

Cell treatment Fraction 1° tumour (X-rays, rads) appearance 2000 1500 1000 500 500 500 500 500

0/6 0/6 2/6 5/6 1/6 0/6 0/6 0/6

Challenging inoculum 2 × 106 2 x 106 2 × 106 2 X 106 1 x 106 2 x l0 s 1 x l0 s 1 X 104

Fraction turnout appeared 6/6 6/6 4/4 0/1 5/5 6/6 6/6 3/6++

Mean day of death ( +_S.E.)* 23+2.0 25 + 2.5 22 + 1.5 Indefinitet 31 +_3.0 29 + 3-5 26+2"5 27 + 2"5

*Only those animals with tumours were included. tThis animal could resist a dose up to 4 x 10 6, but not 107 cells. ++These animals could resist a dose up to 2 x 104, but not IOs cells.

Table 5.

Development of antitumour immunity by VCN-treated and X-irradiated tumour vaccine

Initial inoculum

Cell treatment

Fraction 1o tumour appearance

Challenging inoculum*

Fraction tumour appearance

1 x lO s

VCN 1500 rad VCN+ 1500 rad

8/10 0/10 0/10

2 x 105 2 × 105 2 x l0 s

0/2 10/10 2/10

Mean day of death ( +_S.E.)t Indefinite~: 24 + 3 Indefinite

*Animals which did not show p r i m a r y t u m o u r were only challenged with untreated viable t u m o u r cells. ~'Standard Error of the m e a n (S.E.).

++Animals without tumours were included.

DISCUSSION

T h e above results suggest t h a t V C N treatm e n t o f t u m o u r cells reduces the transplantability of these cells, thus confirming the earlier observations from a n u m b e r of laboratories [1-10]. S a m e doses of s h a m - t r e a t e d t u m o u r cells (10 3) as g r o w n in 100% o f animals, do not show a n y t u m o u r at all if previously t r e a t e d with V C N . A t higher doses (104-105), a fraction o f the animals receiving V C N t r e a t e d t u m o u r cells show t u m o u r development. Animals which did not show a n y t u m o u r following inoculation with V C N t r e a t e d t u m o u r cells, have been found to be r e f r a c t o r y to n o r m a l t u m o u r challenge. T h e y could inhibit the g r o w t h o f ten times the doses o f initial inoculum. O n the other h a n d , although X - i r r a d i a t i o n o f t u m o u r cells could reduce the

established c o m p a r a b l e a n t i t u m o u r i m m u n i t y as observed with V C N treated t u m o u r cells with one extra benefit t h a t higher doses c a n be inoculated. This ' t u m o u r vaccine' perhaps can be successfully used for the i m m u n o t h e r a p y of tumours. I t should be m e n t i o n e d t h a t different tumours and different laboratories yield different results [1-10, 20, 21], with V C N - i n d u c e d modification o f t u m o u r cells. While most o f the laboratories [1-10] confirmed the ability o f V C N to increase the i m m u n o g e n i c i t y o f t u m o u r cells, c o n t r a d i c t o r y reports [20, 21] are also available. H o w e v e r , it appears from the present r e p o r t that V C N treated t u m o u r cells, as also the V C N t r e a t e d and X - i r r a d i a t e d t u r n o u t cells, lead to the establishment o f strong a n t i t u m o u r i m m u n i t y , unlike the i r r a d i a t e d t u m o u r cells.

P. K. Ray, V. S. Thakur and K. Sundaram

The above results point out that VCN perhaps acts in a specific way, increasing the availability of immunogens while the effect of irradiation appears to be non specific. The exact mechanism as to how VCN treated cells help the host to establish antitumour immunity is not known. However, there are a number of possible explanations. Sanford [1], and Currie and Bagshawe [2] have suggested that the mechanism of increased immunogenicity of VCN treated tumour cells could be the result of "unmasking" of histocompatibility antigens on the tumour cells. This has been contradicted by Ray and Simmons [16, 22] and subsequently by others [20, 23, 24]. However, while not unmasking H-2 antigens, VCN could unmask other antigens. Kassulke et al. [25] demonstrated an increased number of blood group isoantigens on VCN treated human leukemic cells. Ray et al. [26, 27] have demonstrated unmasking of xenogeneic neo-antigens on mouse lymphoid cells. Further, Ray and associates [16, 17, 26] have demonstrated that VCN treated normal lymph node cells become extremely susceptible to cytolysis by alloantibody and complement. Even autologous serum could show cytolysis of VCN treated autochthonous lymphoid cells [28]. Recently, R a y and Sundaram [29] have suggested the presence of autoantibody directed against VCN-exposed antigenic determinants on the autochthonous lymphoid cells. VCN treated autochthonous red blood cells have been found to form rosettes with autologous T cells [30]. In MLC, VCN treated stimulatory cells could give stimulation to the syngeneic human lymphocytes [31]. VCN treated tumour cells could stimulate the autologous human lymphocytes in culture [32]. Whatever may be the mechanism responsible for the suppression of growth of VCN treated tumour, it

becomes apparent that VCN treated cells are more easily reacted upon by the immune components of the body. We have reported earlier that VCN treated normal [33], and foetal cells [34] become increasingly immunogenic. It has also been observed that VCN treated sheep erythrocytes are more easily phagocytized [35], and phagocytosis is directly related to antigen handling and/or processing. Thus, the antigen processing may be facilitated by the above mechanisms. Further, VCN results in the decrease in the cell surface negative charges [36] and hence may facilitate the binding between a VCN treated cell and a negatively charged antigen responsive cell. VCN also results in an increased deformability of the cell membrane [36]. Increased deformability would facilitate a larger area of cell-to-cell contact and allow for an increased number of contacts between the antigenic determinants and the antigenreceptor sites on the antigen responsive cell. However, it is difficult to establish precisely as to how the tumour specific immunity could be achieved following inoculations of VCN treated tumour vaccine. It is not known if either one or the other, or a summation of the events as discussed above might give rise to antitumour immunity. Whatever the mechanism, the present method provides an effective way for producing a "tumour vaccine". The possible use of such a vaccine in the immunotherapy of cancer is obvious. Using tumour vaccine produced in this manner, experimental trials with Yoshida sarcoma in rat and a chemically induced fibrosarcoma in mice have achieved significant growth inhibition of these tumours resulting in the prolongation of life span of the treated animals (unpublished observation).

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P. K. Ray, V. S. Thakur and K. Sundaram

,8 29. 30.

31.

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