Contribution of host immunity to cyclophosphamide therapy of a chemically-induced murine sarcoma

Int. J . Cancer: 11, 358-368 (1973)

CONTRIBUTION OF HOST IMMUNITY TO CYCLOPHOSPHAMIDE THERAPY

OF A CHEMICALLY-INDUCED MURINE SARCOMA

Michael MOORE and Dorothy E. WILLIAMS Charles Salt Research Centre, The Robert Jones and Agnes Hunt Orthopaedic Hospital,

Oswestry, Shropshire, S YIO 7AG, England

The contribution of host immune factors to the therapeutic effect of cyclophosphamide (CPA) on an antigenic chemically-induced sarcoma (MCA-2) transplanted in syngeneic mice with diferent immunological reactivity was investigated. In non-sensitized mice, CPA failed to prevent survival of nascent tumour emboli comprising lo3 cells. By contrast, the enhanced therapeutic efficiency of CPA against clinically detectable neoplasms or nascent tumours developing progressively from small inocula ( lo4 cells) suggested that host sensitization may play a role in cyclophosphamide-mediated regression of this tumour. The hypothesis was investigated by comparison of the effect of CPA treatment on palpable neoplasms growing in immunodepressed mice following whole- body irradiation (400 R ) and specifically immunostimulated mice which had received irradiated (15,000 R ) MCA-2 cells, with their untreated immunocompetent counterparts. In the first group, the antitumour effect of CPA was noticeably weaker compared with that observed in normal mice, while in the second, the eflect was markedly poten- tiated, so that palpable nodules comprising in excess of 5 x JOG cells underwent complete regression. Since the maximum level of resistance evoked by sarcoma MCA-2 per se was of the order of 2 ~ 1 0 ~ cells, the results indicate that pre-immunization significantly augments the antitumour activity of CPA in this system. The specificity of the reaction was demonstrated by the failure of CPA to mediate complete regression of sarcoma MCA-2 growing in mice previously immunized with normal muscle or an antigenically unrelated neoplasm (MCA-7) . Possible mechanisms for the synergistic activity of CPA and host immune factors in this tumour-host system are discussed.

Cyclophosphamide (CPA) is effective in con- trolling the growth of a variety of primary and transplanted rodent neoplasms (Sugiura et al., 1961) and has also been used exten- sively in the treatment of human malignancy (Talley, 1970). The marked effect of this and other drugs o n human neoplasms such as choriocarcinoma (Math6 et al., 1964; Steinfeld, 1967) and Burkitt lymphoma (Ziegler et al., 1970; Burkitt, 1970) has been attributed to concomitant host resistance against these tumours. In these

circumstances it appears that immune mechanisms are not significantly impaired by potentially immunosuppressive agents but may actually increase their therapeutic efficiency.

In animal systems, evidence suggesting that host inimunoreactivity may augment the therapeutic effect of cytostatic drugs has accumulated principally from transplantation experiments involving allogeneic host-tumour systems. This evidence includes (a) the diminished therapeutic effectiveness of drugs on tumours growing in

Received: October 12, 1972. To whom correspondencc should be addressed.

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HOST IMMUNITY IN CYCLOPHOSPHAMIDE THERAPY

hosts with impaired immune reactivity as a result of X-irradiation, cortisone treatment (Mihich, 1962) and neonatal thymectomy (Ferrer and Mihich, 1967a, b) compared with their normal immunocompetent counterparts; (b) the more effective inhibition of sarcoma 180 by cytostatic drugs in random-bred Swiss mice compared with mice with closely matching histocompatibility antigens (Bradner and Pindell, 1965); and (c) the augmentation of therapeutic efficacy of cytostatic agents in hosts previously immunized against tumour antigens (Ferrer and Mihich, 1967~; Sato, 1967). In these experiments which largely utilized long-transplanted tumours, host immunoreactivity was directed against both tumour-specific and histocompatibility antigens. However, experiments undertaken in syngeneic systems using tumours of known tumour-specific antigenicity have tended to confirm the general finding that host immunoreactivity may increase the therapeutic efficiency of cytostatic drugs. Thus, Bremberg (1970) showed that under certain circumstances host factors could potentiate the therapeutic effectiveness of cyclophosphamide against syngeneic transplants of Moloney virus-induced lymphomas, and Lausch et al. (1972) attributed the success of cyclophosphamide therapy of syngeneic transplants of para-adenovirus-7-transformed hamster tumour cells to host resistance mediated by SV40 transplantation antigen carried by the malignant cells. In view of their clinical relevance, it was of interest to know whether these findings might also pertain to other syngeneic tumour-host systems.

Chemically-induced sarcomas in rodents gener- ally possess strong tumour-associated antigens, characteristic for each neoplasm, and constitute a relatively well-studied tumour-host system (Baldwin, 1970). Immune reactivity against these antigens may be demonstrated in pre-immunized hosts by in vivo rejection of transplanted tumour cells, and by passive transfer of immunity to untreated recipients (Klein et al., 1960; Klein and Sjogren, 1960; Old et al., 1962). Colony inhibition tests for the in vitro assay of cell- mediated immunity have revealed significant immune reactivity in lymphoid cells from tumour-bearing as well as from pre-immunized hosts (Hellstrom et al., 1967; Baldwin and Moore, 1971), and humoral antibodies reactive with cell-surface-expressed tumour antigens have

been detected by similar tests and by indirect immunofluorescence using serum from specifically immune donors (Baldwin et al., 1971).

Chemically-induced sarcomas are less susceptible to cyclophosphamide than certain other murine neoplasms and complete tumour regression is rarely achieved (Sugiura et al., 1961). It was therefore of interest to study the effect of this drug on sarcomas growing in mice with various degrees of immune competence. For this purpose we have investigated the effectiveness of cyclophosphamide on a transplanted chemically-induced sarcoma growing in normal mice, mice rendered immunodeficient by non- lethal whole-body irradiation and in mice previously exposed to tumour-associated antigens, in an evaluation of the role of host factors con- tributing to successful cyclophosphamide-induced inhibition of tumour growth.

MATERIAL AND METHODS

Tumour The neoplasm used in this investigation

(designated MCA-2) was a transplantable sarcoma which arose during 1970 in a young adult male C3H mouse 74 days following a single injection (1 .O mg) of 3-methyl-cholanthrene into the right thigh muscle. The histology of the primary lesion was that of a moderately dif- ferentiated fibrosarcoma which grew as a subcutaneous mass on transplantation in syngeneic C3H strain mice, the genetic uniformity of which was routinely confirmed by skin grafting. Experiments were performed with tumour transplants between 16 and 45 generations of in vivo passage. A second chemically-induced sarcoma (MCA-7) used in this investigation arose in the same strain with an approximate latent period of 101 days. Tumour cells used for immunization and cross-challenge in specificity tests were derived from the 18th generation transplant. The growth characteristics of this tumour were comparable with those of MCA-2 but the two neoplasms possessed individually distinctive tumour-associated antigens.

Tumour challenge

For transplantation, tumour was dissociated into a single cell suspension by treatment with 0.25% trypsin in Hank’s balanced salt solution and the viability was assessed by vital dye

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MOORE A N D WILLIAMS

exclusion. Predetermined numbers of cells were inoculated subcutaneously into the right flank where they grew progressively as subcutaneous nodules. An inoculation of lo4 MCA-2 cells to immunocompetent adult mice invariably produced gross tumours at the site of injection which were never observed to spontaneously regress.

The effect of cyclophosphamide on nascent tumour cells was evaluated by recording final tumour incidences for the respective groups. The effectiveness of therapy on clinically established neoplasms was estimated by twice weekly caliper measurements of two tumour diameters at right angles to each other.

Irradiation

Mice were exposed to whole-body irradiation with 400 R delivered at the rate of 35.5 R/min 24 h before tumour grafting.

Iinmimization

Details of the procedure of immunization by implantation of irradiated tumour have been published elsewhere (Moore and Williams, 1972). Briefly, tumour explants were attenuated by exposure to 15,000 R X-irradiation delivered at a rate of 375 R/min. Irradiated pieces were then immediately implanted subcutaneously under the dorsal skin, usually bilaterally so as to stimu- late a larger mass of recipient lymphoid tissue. The usual protocol comprised 3-5 such im- munizations at intervals of 10-14 days.

Cyclosphospharnide (CPA)

This drug (supplied by WB Pharmaceuticals Ltd., London, UK) was administered as a single intraperitoneal injection in saline, at a concen- tration corresponding to the appropriate CPA dose for a 20-g mouse. Drug concentrations in excess of 200 mg/kg were frequently toxic for young adult C3H mice.

RESULTS

Effect of dosage of cyclophospharnide (CPA) on survival of MCA-2 sarcoma cells

The first experiment was designed to ascertain whether CPA could inhibit the in vivo outgrowth of MCA-2 cells before significant host sensitization to the tumour-associated antigen had occurred. On day 0, groups of mice

were inoculated (SC) with 1 0 cells and one day later injected with CPA in doses ranging from 50 to 200 mg/kg. All mice were thereafter palpated on alternate days to determine the time required for development of clinically detectable tumours in each group.

The results are presented in Figure 1. The majority of mice which received saline only developed palpable tumour nodules by day 7, the proportion rising to 100% by day 10. These tumours grew progressively and ultimately killed the hosts. By contrast there was a marked delay in the appearance of palpable tumours in mice treated with CPA which was a function of the dosage given to each group. However, even at the highest dose (200 mg/kg) complete inhibition of the outgrowth of MCA-2 cells was achieved in only 20% of the mice. These results established that CPA could effectively delay the appearance of MCA-2 tumour but the drug proved inadequate to completely eradicate a population exceeding 1 0 cells in more than a small minority of mice in the presumptive absence of significant host sensitization.

L I A Y S A F T L R I U M V U R - C E l l C H A L l € N G €

FIGURE 1

EFtECT OF DOSAGE OF CYCLOPHOSPHAMIDE (CPA) ON SURVIVAL OF MCA-2 SARCOMA CELLS

Mice received a single injection (IP) of CPA 1 day after inoculation (SC) of lo5 MCA-2 cells. o--o CPA, 200 nig/kg body weight; n-n CPA, 150 rng/kg body weight; A-A CPA, 100rng/kg body weight; m-8 CPA, 50mg/kg body weight; 0-0 Saline control.

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Effect of time of administration of CPA on survival of MCA-2 sarcoma cells

The efficacy of CPA therapy was further evaluated in a series of tests in which mice receiving small viable inocula of MCA-2 cells were treated with the optimal dose of the drug (200 mg/kg) at various time intervals after challenge. In the first experiment, 60 mice received lo5 cells on day 0 and were divided randomly into six groups. Of these, one group received CPA one day before MCA-2 cells while another received saline only simultaneously with tumour cells. The mice in the remaining four groups received a single injection of CPA on days 0, 1, 4 and 7. All mice which received saline alone grew tumours, as did also mice given CPA before the challenge inoculum (Table I). CPA was most effective when administered at the same time as the tumour, four mice out of 10 ultimately yielding palpable tumours from an initial inoculum of lo5 cells. There were no significant differences in tumour incidence re- sulting from CPA therapy administered at varying times after day 0 and only a small minority of mice (<20%) in each group failed to yield tumours which grew progressively to kill the hosts.

TABLE I

EFFECT OF THE TIME OF CYCLOPHOSPHAMIDE (CPA) ADMINISTRATION ON THE SURVIVAL

OF MCA-2 SARCOMA CELLS

No. cells Day CPA treatment inoculated (SC) commenced

1 0 6 -1 0 1 4 7

Saline only

Final tumour incidence

10/10 4/10 9/10 9/10 811 0

loll0

104 - 1 loll0 0 411 0 1 4/10 4 3/10 7 6/10

1 1 5/10 Saline only 10/10

200 mg/kg body weight intraperitoneally.

In the second experiment, 70 mice received lo4 cells on day 0 and were randomly distributed into seven groups, of which one received CPA on the day prior to the inoculation of the tumour cells and another received saline only on day 0. Mice in the remaining five groups received CPA on days 0, I , 4, 7 and 1 1 . All mice which received saline only or which were treated with CPA prior to tumour challenge produced tumours. CPA again proved effective when administered concomitantly with the sarcoma cells, four mice out of 10 yielding tumours. However, by contrast with the results obtained with the higher tumour inoculum (lo5 cells), CPA appeared also to be efficacious when administered up to 11 days after challenge. Thus, final tumour incidences following injection of CPA on days 1 , 4 and 7 were 40, 30 and 60 per cent respectively (Table I). Most significant was the 50% tumour incidence in the group given CPA on day 1 1 when palpable tumours were beginning to emerge in the saline-treated controls.

Effect of CPA treatment on growth of MCA-2 sarcoma cells in immunocompetent and immunodeficient mice

These experiments were designed to ascertain whether there were significant differences in the effect of CPA treatment on the outgrowth of MCA-2 sarcoma cells in irradiated (400 R ) and non-irradiated hosts. On day 0, twelve groups of 10 mice, half of which had received 400 R whole- body irradiation 24 h prior to challenge, were inoculated SC with lo4 to lo8 viable cells. When tumours became clinically detectable the mice received CPA at a dosage of 200 mg/kg. All animals were examined for tumour growth at feast twice weekly.

The results are presented in Figure 2. In the case of the higher cell inocula (lo5 and lo6 cells) tumour was palpable in all mice within 6 to 7 days. As anticipated, the rate of tumour outgrowth was most rapid in those mice which received 400 R whole-body irradiation 24 h prior to tumour challenge. Cyclophosphamide significantly impeded the growth of tumour in irradiated mice but the effect of the drug was not so pronounced as in non-irradiated hosts.

This feature was most apparent in mice which received the smallest tumour cell inoculum (lo4 cells). In these groups tumours were not clinically detectable before day 12, after which

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MOORE AND WILLIAMS

FIGURE 2

COMPARISON OF THE EFFECT OF CYCLOPHOSPHAMIDE (CPA) ON GROWTH

AND IMMUNODEFICIENT MICE OF MCA-2 SARCOMA CELLS I N IMMUNOCOMPETENT

Sarcoma MCA-2 was grafted to pre-irradiated (400 R) and non-irradiated mice. Within 2 days of tumours being palpable in all pre-irradiated mice, these mice were divided into two subgroups, of which one received CPA (200mg/kg body weight IP) and the other, saline. Non-irradiated mice were similarly subdivided.

The median tumour diameter in mice which received lo5 and loB cells ranged from 3 to 4 mm and did not differ significantly at the time of CPA treatment in pre-irradiated and non-irradiated groups. In mice receiving the smallest inoculum (lo4 cells) turnours appeared 2-3 days later in non-irradiated compared with pre-irradiated mice. In these circumstances, CPA was administered to tumour-bearing non-irradiated mice at a time when the median tumour diameter (3-4 mm) did not significantly differ from that in pre-irradiated hosts. 0-0 pre- irradiated, no CPA; 0-0 non-irradiated, no CPA; H---H pre-irradiated, CPA-treated; 0-0 non-irradiated, CPA-treated.

the appropriate groups received CPA. Temporary growth inhibition of tumour was again observed in irradiated mice given CPA, but was considerably less marked than CPA-treated non- irradiated hosts, all of which underwent partial remission. In this group tumours ceased to be palable 6 days after CPA treatment and remained clinically undetectable for 12 further days. At day 32 tumours recurred in all mice and grew progressively at a rate comparable with that of the other groups. The pronounced therapeutic effect of CPA was thus shown to be substantially impaired in irradiated (i.e. less immuno- reactive) hosts.

A further observation from these experiments was the influence of the latent period of tumour development on the outcome of CPA therapy. While the size of tumours in all groups was similar at the time of CPA administration, the effect of the drug was considerably more pronounced o n tumours treated 12 to 15 days after challenge (with lo4 cells) than a t 6 or 7 days after challenge (with 1 05-I O6 cells). Thus, tumours with a longer latency period of growth were more susceptible to the inhibitory effect of CPA than those with shorter latency periods.

Effect of CPA treatment on growth of MCA-2 sarcoma cells in immunostimulated mice

An essential preliminary to this experiment was determination of the maximum number of MCA-2 cells rejected by immunized hosts in the absence of chemotherapeutic intervention. For this purpose syngeneic C3H mice were succes- sively grafted at 10-day intervals with irradiated (1 5,000 R) sarcoma cells and subsequently challenged with graded inocula of viable MCA-2 cells. Alternatively, mice were immunized by surgical extirpation of subcutaneously developing tumour grafts 10-14 days after implantation. The results presented in Table 11 indicate that MCA-2 was a moderately immunogenic sarcoma capable of inducing resistance by the above methods to inocula of approximately 2 x lo5 cells in the majority of immunized recipients. Increased challenge doses of cells progressively surmounted this resistance so that 80% of immune mice challenged with Sx105 MCA-2 cells succumbed to tumour outgrowth while a t loE cells or more tumour incidence was invariably 100%. Immunization with normal muscle or with an antigenically distinct sarcoma

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TABLE I1

IMMUNOGENICITY OF TRANSPLANTABLE METHYLCHOLANTHRENE-INDUCED SARCOMA

MCA-2 IN SYNGENEIC C3H MICE

Tumour challenge

Immunizing r m ~ ~ i Z - Tumour incidence in:

(MCA-2) Treated Untreated procedure ’ tissue

mice controls

MCA-2 Excision 3 x I R 3 xIR 3 xIR 3xIR 3xIR 3xIR

Muscle 3 x lR MCA-1 3 X IR

1 xi05 1 x 105 2x10” 2x10” 5 x lo5 1 x106 2 x 108

1x10” 1 xi05

3/10 2/18 2/13 5/12 8/10

loll0 12/12 loll0 lop0

818 9/10 818 818

10/10 10/10 12/12 loll0 10/10

I IR - Immuniiation by implantation of irradiated (15,000 R) tumour at 10- to 1Cday intervals.

MCA-7 failed to protect against MCA-2 even at the lowest challenge inoculum (lo5 cells).

Having ascertained the maximum level of resistance evoked by immunization with irradiated grafts of MCA-2, we designed experiments to determine whether augmentation of host resistance by this procedure would significantly influence cyclophosphamide therapy of established tumour.

In a series of eight independent experiments performed in syngeneic C3H mice or their F, hybrids, mice were immunized by subcutaneous implantation of irradiated MCA-2 cells and subsequently challenged with cell inocula in excess of the maximum cell number rejected by specifically immunized hosts ( 2 5 x lo5 cells). Controls comprised non-immune mice challenged with identical cell numbers. Tumours were measurable (minimum diameter 4 mm) in all mice of both groups injected between 5 and 7 days (depending on the number of cells grafted) after inoculation. At this time, immune and non- immune groups were sub-divided and treated with saline and CPA respectively. In non- immune hosts, CPA significantly retarded the rate of tumour outgrowth compared with saline- treated controls, but in agreement with previously described experiments (Fig. 2), no complete

regressions were observed in a total of 33 drug- treated hosts (Table HI). In preimmunized mice the effect of CPA treatment was dramatic, the majority of tumours undergoing complete regression. Thus, 29/40 (73%) mice treated with CPA at a time when they had palpable tumours underwent complete tumour regression compared with 1/37 (3%) mice which did not receive CPA. In the remaining 11 CPA-treated mice which ultimately succumbed to tumour outgrowth, remission was only partial. However, in these mice previously palpable tumours were frequently clinically undetectable for periods of up to 21 days after chemotherapy. These experiments thus established that, in combination, CPA and host immunity could ablate the entire cell population of a clinically detectable neoplasm whereas alone they were ineffective.

Further confirmation of this conclusion was derived from four additional transplantation tests in which specifically immunostimulated mice remaining tumour-free for up to 40 days after chemotherapy were rechallenged with various inocula of MCA-2 cells. Resistance to a second challenge could readily be demonstrated but only at inocula (-lo5 cells) comparable with that evoked by this neoplasmper se (Table 11). Thus challenge inocula of 5 ~ 1 0 ~ cells or more grew progressively to kill their hosts (Table IV). That this limited degree of resistance was due to host immunity rather than residual drug is supported by the fact that CPA has a short half-life (less than 1 h) in rodents (Kline et al., 1968).

The specificity of CPA-induced regression of MCA-2 was investigated in a further series of tests with an antigenically distinctive sarcoma, MCA-7. Mice were immunized with normal muscle, MCA-2 and MCA-7 and subsequently challenged with identical inocula of both tumours. As previously, when tumours were measurable ( 2 4 mm) in all injected mice, each group was subdivided and treated with saline and CPA respectively. CPA therapy failed to induce regression of either MCA-2 or MCA-7 growing in mice preimmunized with the respective antigenically unrelated tumours, or regression of MCA-2 in mice previously exposed to irradiated normal tissues (Table V). By contrast, palpable tumours of 13/18 (72%) mice pre- immunized and challenged with MCA-2 cells underwent complete regression on treatment with

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MOORE AND WILLIAMS

TABLE 111

CYCLOPHOSPHAMIDE-INDUCED REGRESSION OF SARCOMA MCA-2 IN SPECIFICALLY IMMUNOSTIMULATED HOSTS

Complete tumour regressions

mice mice

Recipient mice Expt. No. tumour No. cells grafted Pretreatment I Saline-!reated CPA-treated Strain

1 5 x lo6 C3H

2 1 x 1 0 6 C3 H

3 5 x 105 (C3H X CBA)Fl - 014 014 4xIR 014 314

4 1 x106 (C3H x CBA)Fl - 014 014 4 x l R 013 214

5 2x106 C3H - 012 012 5xIR 014 415

6 2 x 106 C3H

7 2 x 106 C3 H

8 5 X lo6 C3H

' The remaining three mice in this group went into prolonged partial remisaion (>21 days) but subsequently grew tumours. * IR = Immunization by implantation of irradiated (15,000 R) tumour at 10- to 14-day intervals. The median tumour diameter

ranged from 4 to 8 mm. Diameters of immunized and non-immunized groups differed by 2 mm or less. Sarcoma MCA-2 was grafted to pre-immunized and non-immunized syngeneic mice. Within 2 days of turnours being palpable in all pre-immunized mice, these mice were divided into two subgroups, of which one received CPA (200 mg/kg body weiqht intraperitoneally) and the other. saline. Non- immuniied controls were similarly sub-divided.

TABLE IV

EVALUATION OF HOST IMMUNE STATUS FOLLOWING CYCLOPHOSPHAMIDE (CPAbINDUCED

REGRESSION O F SARCOMA MCA-2 IN SPECIFICALLY IMMUNOSTIMULATED HOSTS

Tumour rechallenged

Tumour incidence in '

(MCA-2) treated untreated Cell dose Host status

mice mice

CPA treated tumour-free 1 x lo5 014 4/4 (Expt. 1 ) CPA treated tumour-free 5 x lo5 314 414 (Expt. 2) CPA treated tumour-free 1 x lo5 016 7/7 (Expt. 7) CPA treated tumour-free 1 x lo8 313 414 (Expt. 8)

' Mice were challenged 40 days after a single treatment with

Experiment numbers refer to those documented in Table 111. CPA (200 mg/kg) when still clinically tumour-free.

CPA. These experiments established that the therapeutic efficacy of CPA is significantly augmented by specific immunoreactivity directed against tumour-associated antigens and is unlikely to be attributable to non-specific host factors.

DISCUSS I 0 N

Tumour cells may be destroyed both by treatment with cytostatic drugs and by host immune reactivity directed against tumour-associated antigens. In the chemotherapy of antigenic neoplasms the immune response might potentiate the efficacy of a drug provided that its potency to destroy is not insignificant in comparison with the anti-tumour effect of the agent. Such a relative limitation of the host response might either be inherent or due to the immunosuppressive activity of the drug. The primary aim of this investigation was to determine whether the therapeutic outcome of CPA treatment of a

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TABLE V

SPECJFICITY OF CYCLOPHOSPHAMIDE (CPA)-INDUCED REGRESSION OF SARCOMA MCA-2

IN IMMUNOSTIMULATED HOSTS

Immunizing tissue '

MCA-2 Normal muscle MCA-2 MCA-7

~

Complete tumour Tumour challenge regressions

Tumour No. cells Saline- CPA- treated treated mice

MCA-2 2x10' 0116 13/18

MCA-2 2X106 018 018 MCA-7 2 x lofi Oil0 0/9 MCA-2 2X lo6 019 011 1

~~ ~~~ ~ ~~ ~ ~

' Immunization by implantation of irradiated (15,000 R)

The median tumour diameter ranged from 4 to 8 mm. Within 2 days of tumours being palpable in all pre-immunized

mice, they were subdivided into two groups, of which one received CPA (200 mg/kg body weight intraperitoneally) and the other, saline.

grafts at 10- to 14-day intervals.

transplantable antigenic neoplasm could be significantly influenced by host factors. Reliable effective assessment of cytostatic activity was critical for disclosure of any significant host influences, and the solid tumour system used in this study was considered to fulfil this require- ment.

The ability of CPA to inhibit tumour growth was found to be critically dependent on the size of the tumour-cell population and on the time of drug administration. CPA was most effective when given concomitantly with challenges of lo4 and lo5 cells, presumably exerting its action by an immediate and direct cytocidal effect, since, consistent with its short biological half-life (Kline er a[., 1968), drug given 1 day prior to tumour-cell inocula failed to suppress tumour growth. A sequence of events more complicated than this was, however, suggested by experiments in which CPA was administered at various time intervals after challenge with the lower inoculum (lo4 cells). Here the therapeutic efficiency of CPA when administered concurrently and between 4 and 1 1 days after challenge was comparable. During this period the tumour cell population had progressively increased, so that by 11 days after challenge palpable tumours were beginning to emerge. Since it was unlikely that the restricted regimen of CPA could eradicate the entire population of cells present at this

stage of tumour development, these observations suggested that host immune factors might contribute to the outcome of CPA therapy.

This hypothesis was subjected to more defini- tive investigation in experiments where the effect of CPA therapy on established tumours growing in normal and immuno-depressed mice was compared. The effect of CPA on palpable neoplasms in preirradiated mice was noticeably weaker than in normal non-irradiated hosts. This phenomenon was reproducibly demonstrated on clinically detectable tumours emerging at different time intervals after challenge with three different tumour-cell inocula ranging over two logarithmic units, and strongly suggested that in the normal immunocompetent host, immune factors contribute substantially to chemotherapy.

Tumours with the longest latency period of development were more susceptible to CPA therapy than those with shorter latencies, again emphasizing the role of concomitant host sensitization in determination of the outcome of chemotherapy. This interpretation is in line with what is known of the kinetics of the immune response to tumour antigens and the ineffective- ness of CPA therapy against nascent tumours consisting of the order of lo5 cells in the absence of significant host sensitization.

Augmentation of host sensitization with irradiated tumour cells profoundly influenced the outcome of CPA therapy. Palpable nodules growing in previously immunized hosts comprising, in some instances, more than 5 x lo6 cells, underwent complete regression in the majority of cases. While CPA proved incapable of eradicating a population of lo5 cells in non- sensitized mice, and the maximum level of resistance evoked by sarcoma MCA-2 per se was of the order of 2 x LO5 cells, host immunity and CPA treatment in combination were between 50 and 100 times more effective in this tumour- host system than either immune reactivity or chemotherapy alone. The effect was moreover highly specific since pre-immunization with normal tissues and an antigenically unrelated neoplasm failed to promote cyclophosphamide- induced regression of clinically established tumours. These studies thus confirmed that palpable tumours may completely regress as a result of the synergistic action of CPA and specific immunoreactivity directed against antigenic tumour cells.

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Whether this synergism operates sequentially or concurrently was not resolved in this study. Brernberg (1970) has suggested that the effect of the drug is to decrease the tumour-cell population to a certain minimum level characteristic for each tumour-host system, in order to allow any substantial influence of immune factors on the therapeutic outcome. A concurrent effect, however, must also be considered, particularly in view of the fact that CPA possesses the properties of a powerful immunosuppressant (Berenbaum and Brown, 1963) and of an anti-neoplastic agent. At dosages comparable to those used in this study CPA was shown to abrogate the immunologically-mediated regression of Moloney sarcoma virus-induced sarcomas in mice (Fefer, 1969, 1970). On the other hand, in several tumour- host systems where specific (Glynn et al., 1969; Fefer, 1969; Vadlamudi et al., 1971) and non- specific (Currie and Bagshawe, 1970; Kal- paktsoglou and Good, 1970) immunotherapy has been used in combination with chemotherapy, the immunosuppressive activity of CPA has apparently not dominated the therapeutic outcome.

A number of recent studies have emphasized the selective action of CPA on the lymphoid system of the mouse, producing changes in lymphoid cell populations expressing different immunological functions, and cell-to-cell interactions (Turk and Poulter, 1972; Marbrook and

Baguley, 1971). Of particular relevance is the study of Poulter and Turk (1972) who reported a proportional increase in thymus-dependent (T) lymphocytes in peripheral lymphoid tissue following treatment with CPA. Conceivably, one effect of CPA treatment may be the preferential suppression of B lymphocyte function compared with T lymphocyte function, thereby diminishing the effect of “blocking” or enhancing antibodies. In a skin allograft system Griswold et al. (1972) reported that cytosine arabinoside (ara-C), depending on the dose and time of administration, could exhibit a selective suppression of humoral immunity without a concomitant inhibition of cellular immunity.

Elucidation of the nature of the interaction of cyclophosphamide with both tumour and the lymphoid system of the sensitized host would appear to be warranted on the basis of this and other studies (Bremberg, 1970; Lausch et al., 1972) in syngeneic tumour-host systems.

ACKNOWLEDGEMENTS

We thank Dr. N. W. Nisbet, Director of Research, for his criticism of this manuscript and Miss Janet Hotchkiss for secretarial assis- tance.

This study was supported by grants from the Cancer Research Campaign and the Medical Research Council.

CONTRIBUTION DE L’IMMUNITE DE L’H6TE AU TRAITEMENT A U CYCLOPHOSPHAMIDE D’UN SARCOME MURIN CHIMIO-INDUIT

Nous avons ttudit la contribution des facteurs immuns de I’hdte a I’effet thtra- peutique du cyclophosphamide (CPA ) sur un sarcome antigtnique chimio-induit ( MCA-2) greflt sur des souris synginiques ayant des rkactions immunologiques difftrentes. Chez les souris non sensibilistes, le CPA n’a pas empichi la survie des emboles de tumeurs naissantes comportant 106 cellules. Par contre, I’eficacitt thtra- peutique accrue du CPA a I’tgard des ntoplasmes cliniquement decelables ou des tumeurs naissantes se dtveloppant progressivement a partir de petits inoculums ( lo4 cellules) donne ir penser que la sensibilisation de I’hbte peut jouer un rble dans la rtgression de la tumeur mtdite par le cyclophosphamide. Cette hypothtse a t t t ttudite sous I’angle de I’effet du traitement au CPA sur des ntoplasmespalpables se developpant chez des souris immunodtprimtes aprPs irradiation du corps entier (400 rad) ou chez des souris spicifiyuement immunostimultes qui avaient recu des cellules MCA-2 irradites (15,000 rad), que I’on a compartes avec leurs hornologues imniunocompttentes non traities. Dans le premier groupe, I’eflet antitumoral du CPA itait sensiblement plus faible que chez les souris normales, alors que dans le secondgroupe, I‘effet itait nettement plus fort, a tel point que les nodules palpables comportant plus de 5 x log cellules avaient

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totalement regress&. Etant donnk que le niveau maximum de rksistance suscitke par le sarconie MCA-2 est de I’ordre de 2 x lo5 cellules, les resultats indiquent que la put- itnmunisation augtnente considkrablement I’activitk antitumorale du CPA dans ce systkme. La spkcificiti de la reaction a kte dkmontree par le fait que le CPA ne peut mtdier une regression complkte du sarcome MCA-2 qui se dkveloppe chez des souris prealablement immunistes avec du muscle normal ou un nkoplasme tout a fait di’irent du point de vue antiginique (MCA-7). Les auteurs analysent les mkcanismes possibles de I’activitk synergistique du CPA et des facteurs immuns de I’hdte dans ce systkme hdte-tumeur.

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Contribution of host immunity to cyclophosphamide therapy of a chemically-induced murine sarcoma