Attenuation in Antitumor Resistance Acid against ...

Protection by S-2-(3-Aminopropylamino)ethylphosphorothioic Acid against Radiation- and Cyclophosphamide-induced Attenuation in Antitumor Resistance Luka Milas, William H. McBride, Nancy Hunter, et al. Cancer Res 1984;44:2382-2386. Published online June 1, 1984.

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[CANCER RESEARCH 44, 2382-2386,

June 1984]

Protection by S-2-(3-Aminopropylamino)ethylphosphorothioic Radiation- and Cyclophosphamide-induced Resistance1

Acid against

Attenuation in Antitumor

Luka Milas,2 William H. McBride, Nancy Hunter, and Hisao Ito Department ol Experimental Radiotherapy. The University of Texas M. D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77030 Â¡L.M., N. H., H. I.], and Department ol Bacteriology, University of Edinburgh, University Medical School, Edinburgh, Scotland [W. H. M.]

ABSTRACT Studies were performed to investigate whether S-2-(3-aminopropylaminojethylphosphorothioic acid (WR-2721) can protect antitumor immune rejection responses against the damaging effects of whole-body irradiation (WBI) and cyclophosphamide (CY). Among these damaging effects were radiation-induced enhancement of s.c. tumor take and radiation- and CY-induced enhancement of lung colonization by tumor cells injected i.v. The ability of WR-2721 to protect against WBI-induced decreased radioresponse of solitary tumors was also investigated. All ex periments were performed with an immunogenic fibrosarcoma syngeneÃ-cto C3Hf/Kam mice. WR-2721 was given i.p. at a dose of 400 mg/kg 30 min before WBI with -y-rays or CY injection. WBI with 650 rads reduced the number of tumor cells needed for tumor take in 50% of animals from 5.1 x 104 cells in normal mice to 2.0 x 102. WR-2721 given before WBI almost entirely abolished the effect of WBI: the number of tumor cells needed for tumor take in 50% of animals was 1.4 x 104. Treatment of mice with WBI or CY increased the number of tumor nodules in the lung generated by fibrosarcoma cells injected i.v. 5 days later, in a linear dose response. WR-2721 greatly reduced this metastasis enhancement effect of WBI and CY with protection factors of 2.5 for WBI and 1.8 for CY. Fibrosarcomas of 8 mm in diameter exhibited a decreased radiocurability when growing in WBI mice: the dose of irradiation yielding local tumor control in 50% of animals in these mice was 5950 compared to a dose of irradiation yielding local tumor control in 50% of animals of 4160 rads in normal mice. WR-2721 given before WBI inhibited this effect of WBI: the dose of irradiation yielding local tumor control in 50% of animals was 5210 rads. The proportion of macrophages in tumors growing in WBI mice was significantly reduced, but not when WR-2721 was first given. WR-2721 greatly reduced the damaging effects of WBI and CY on natural killer cell activity. Therefore, WR-2721 was capable of protecting the immune mechanisms involved in antitumor resistance against WBI and CY. This might be of therapeutic benefit when WR2721 is combined with radio- or chemotherapy. INTRODUCTION Growth characteristics and response to therapy of experimen tal animal tumors can be influenced by the immune system. For

example, immunosuppression can facilitate tumor take, growth, and metastatic spread (2, 9, 18, 26), and make tumors less responsive to treatments with ionizing radiation (31, 32) or chemotherapeutic drugs (5). The opposite effect can be achieved by stimulating antitumor immune responses of the tumor host (3,21,23). Under certain circumstances, both tumor chemother apy and radiotherapy, especially large-field irradiation, suppress the immune system of the tumor host (14,30), which can reduce their therapeutic efficacy. Therefore, it might be beneficial to protect against development of immunosuppression. WR-27213 is a phosphorothioate compound that protects tissues against ionizing radiation (27, 36) and alkylating agents (33,36). It has been the subject of much recent research because of its ability to preferentially spare normal tissues rather than tumors (reviewed in Refs. 27 and 36). Although a wide range in the degree of radioprotection of normal tissues by WR-2721 has been reported, it is not uncommon to find that 2 to 3 times higher doses of irradiation are required to produce the same level of damage in certain tissues, in particular hematopoietic tissue, if animals are given WR-2721 prior to irradiation (27, 36). Several immune functions have also been reported to be well protected by WR-2721 against the damaging effects of irradiation. These are hemolytic plaque formation by spleen cells (34), cytolytic activity of T-lymphocytes (12), and delayed-type hypersensitivity reaction (29). Here we report the ability of WR-2721 to protect antitumor resistance of mice against damage by whole-body ionizing radia tion and CY. Specifically, WR-2721 was capable of a significant protection against radiation-induced enhancement of s.c. tumor take, radiation- and CY-induced enhancement of lung coloniza tion by i.v.-injected tumor cells, and WBI-induced decrease in tumor radiocurability. In addition, we describe protection against radiation- and CY-induced suppression of NK cell activity. MATERIALS AND METHODS Mice Inbred C3Hf/Kam mice of both sexes bred and maintained in our own specific pathogen-free mouse colony were used. Mice were 11 to 13 weeks old at the beginning of the experiments. Within each experiment, the mice were of the same sex, and they were housed 4 to 6/cage. Tumor

' This investigation was supported in part by Grants CA-06294 and CA-16672, awarded by the National Cancer Institute, Department of Health and Human Services. Animals used in this study were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accord ance with current regulations and standards of the United States Department of Agriculture and Department of Health and Human Services, NIH. 2To whom requests for reprints should be addressed. Received November 21,1983;

accepted March, 6,1984.

A methylcholanthrene-induced 3 The abbreviations

FSA syngeneic to C3Hf/Kam mtee was

used are: WR-2721, S-2-(3-aminopropylamino)ethylphos-

phorothioic acid; FSA, fibrosarcoma; T Dw, number of tumor cells needed for tumor take in 50% of animate; TCDÂ», dose of irradiation yielding local tumor control in 50% of animals; WBI, whole-body irradiation (irradiated); CY, cyclophosphamide; NK, natural killer.

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VOL. 44

WR-2721 and Antitumor Resistance used in all experiments

involving tumor cells in this study. Single-cell

lation spectrometer.

Spontaneous

release of 51Cr was determined

by

suspensions were prepared by trypsin digestion of nonnecrotic tumor tissue (19). Viability of cells was more than 95%, as assessed by phase-

incubating the target cells in medium alone, and it ranged from 5 to 9%. Maximum release of 51Cr was determined after addition of 0.1 ml Triton

contrast microscopy and trypan blue exclusion. TOÂ»Assay for s.c. Tumor Take. This assay is described in detail in a previous paper (25). Five days after receiving 650 rads WBI, mice were given s.c. injections in 4 sites overlying the abdomen of 10' to 105 viable FSA cells diluted in 0.1 ml Hsu's medium (Grand Island Biological Co.,

X-100 to 0.1 ml target cells, and it ranged from 85 to 95%. The percentage of 51Cr was determined according to the formula:

Grand Island, NY). Thereafter mice were examined 2 to 3 times a week for tumor development up to 60 days after tumor cell transplantation. Tumor take was expressed in TDso values.

% of experimental

release - % of spontaneous

% of maximal release - % of spontaneous

release release

WBI, CY, and WR-2721

Colonization of Lung by i.v. Injected Cells (Experimental Metastasis)

For WBI, mice were exposed to -y-rays in groups of 5 in a Lucite container. The single-source 137Csunit delivered a dose rate of 223 rads/

To produce tumor mÃ©tastasesin the lung, mice that had received 400 to 800 rads WBI or CY 5 days earlier were given i.v. injections of 2.5 x 10* or 5 x 10* FSA cells suspended in 0.5 ml Hsu's medium. Mice were

min. CY (Mead Johnson & Co., Evans ville, IN) was dissolved in distilled water at concentrations ranging from 5 to 25 mg/ml, and was adminis tered i.p. at doses of 50 to 250 mg/kg body weight. WR-2721 was

killed 14 days after tumor cell injection, their lungs were removed, and lung lobes were separated and fixed in Bouin's solution. Colonies of tumor cells (mÃ©tastases) appeared as white, round nodules on the surface of the yellowish lung, and were counted with the naked eye. Tumor Response to Irradiation

dissolved in 0.9% sodium chloride solution and injected i.p. in the dose of 400 mg/kg 30 min before WBI or CY treatment. The pH of the sodium chloride solution was 6.0, but immediately upon addition of WR-2721, it became 7.0, and remained at that value for 4 hr. WR-2721 was obtained from the Drug Synthesis and Chemistry Branch, Developmental Thera peutics Program, Division of Cancer Treatment, National Cancer Insti tute, Bethesda, MD. WR-2721 and CY were injected into animals within

The TCOÂ» assay was used to assess tumor response to ionizing radiation. Normal mice or mice that had been exposed to 600 rads WBI 1 day earlier received injections in the right hind thighs of 5 x 10s FSA

1 hr after having been dissolved.

cells. When tumors had grown to 8 mm in diameter, the tumor-bearing leg was exposed to single doses of -y-radiation, which was delivered from a dual-source 137Cs unit at a dose rate of 879 rads/min. During

RESULTS

irradiation the mice were not anesthetized, but they were immobilized in a jig. The tumor was centered in the circular radiation field, 3 cm in diameter. Mice were checked for the presence of tumor at the irradiated site at 9- to 12-day intervals for up to 100 days. TCDso values were

The following experiment was performed to determine whether WR-2721 can protect against WBI-induced enhancement of s.c. tumor take. Mice were given i.p. injections of 400 mg WR-2721/ kg 30 min before they were exposed to 650 rads WBI. Five days later, they were given injections s.c. of FSA cells in the range of 101 to 105 cells/mouse. Tumor take is shown in Table 1. WBI

computed by the logit method of analysis (10). Determination of Tumor Macrophages The assay was described in detail in a previous paper (24). Macro phages in tumor cell suspensions were identified through their ability to form rosettes following incubation with sensitized calf erythrocytes. The erythrocytes were sensitized with rabbit anti-calf RBC antibody; 0.1 ml tumor cells at 106/ml were added to 0.1 ml 1.7% sensitized RBC, spun, and incubated for 30 min at room temperature. The percentage of macrophages in the suspension was determined after staining nucleated cells with crystal violet. NK Cell Activity Splenic NK cell activity was determined using allogenic T-cell lympnoma YAC-1 cells as targets in the 51Cr release assay (16). Spleen cells were mixed with tumor cells in ratios of 100:1 and incubated in 0.2-ml volumes of Hsu's medium with 20% fetal calf serum in the wells of round-bottomed microtiter plates in a 5% CO2 atmosphere for 4 hr. After incubation, the plates were centrifuged at 250 x g for 10 min, and 0.1 ml was removed from each well and counted in an Auto-Gamma scintil-

Effect on WBI-induced Enhancement of s.c. Tumor Take.

reduced TD50 values by a factor of approximately 2 logs, 2.0 x 102 cells as compared to 5.1 x 104 cells in control animals. Treatment of mice with WR-2721 prior to WBI almost abolished the enhancing effect of irradiation on tumor take, increasing the TOÂ»value to 1.4 x 104 cells. Mice treated with only WR-2721 exhibited no change in tumor take. Protection against Radiation- and CY-induced

Enhance

ment of Metastasis Formation in the Lung. Pretreatment of mice with WBI (18, 19) and CY (reviewed in Ref. 22) increases the formation of metastatic nodules in the lung generated by tumor cells injected i.v. The results of the 2 following experiments showed that WR-2721 can greatly protect mice against this effect of WBI and CY (Chart 1). Mice were exposed to graded doses of WBI or CY 5 days before they were given i.v. injections of FSA cells. In addition, groups of mice were given 400 mg of WR-2721/kg i.p. 30 min before WBI or CY. In the WBI experi ment, mice were given 5 x 104 FSA cells i.v.; in the experiment

Table 1 Effect of WR-2721 on s.c. take of FSA in WBI mice Mice were WBI with 650 rads 5 days before they were given s.c. injections of FSA cells. WR-2721 (400 mg/kg Â¡.p.)was given 30 min prior to WBI.

^ Tumor take after injection of following no. of tumor cells

x 101

recipientsNone

101

2a WR-2721 2/122/123X1033/12 4/11 9/123x10*2/12 3/1210/1210*4/12 11/12 650 rads WBI WR-2721 + 650 rads WBI10Â»9/1 2/1210a11/12 2/123x10*1/11 0/121028/12 0/123

0/12

3/12

TOÂ» limits)5.1 value (95% confidence x104(2.3x103-1.1 X10") 5.2x10* (1-9* 10M.4X 10") 2.0 x 10* (6.5 x 10' -6.4 x 102) 1.4 x 10Â« (5.1 x 10M.O x 10*)

' Number of tumor takes/total number of injected s.c. sites.

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L. Mitaset al. the time of analysis for macrophage content. Tumors were generated by 5 x 105 FSA cells injected into the right thighs of mice exposed to WBI 1 day earlier. WR-2721 (400 mg/kg) was given 30 min before irradiation. Thus, tumors in WBI mice, at the time of local tumor irradiation, had more tumor cells than did tumors in mice not exposed to WBI, which could be a contributing factor to the increased TCDso value in WBI mice (see "Discus sion").

200

IRRADIATIONDOSE (rad)

DOSE OF CYCLOPHOSPHAMIOF(mg/kg)

Chart 1. Protection by WR-2721 against WBI- and CY-induced enhancement of FSA colonies in the lung. Mice were exposed to graded doses of WBI or CY 5 days before i.v. injection of FSA cells. Mice treated with WBI received 5 x 104and those treated with CY received 2.5 x 10* FSA cells. O, controls; â€¢,WR-2721treated mice. WR-2721 at the dose of 400 mg/kg was given i.p. 30 min before WBI or CY. Number of lung mÃ©tastases was determined 14 days after tumor cell injection. Protection factors comparing the slopes of the curves were 2.5 for WBI treatment and 1.8 for CY treatment. Points, means; oars, S.E.

Protection of NK Cell Activity. Since NK cells play an impor tant role in antitumor resistance (13), particularly in that directed against circulating tumor cells (11 ), it was important to determine whether WR-2721 can protect these cells against damage by irradiation and CY. Mice were treated with WR-2721 (400 mg/ kg i.p.) 30 min before they were exposed to graded doses of WBI or CY. One day later, NK cell activity of these mice, as well as mice exposed to WBI or CY, was assayed; the results were expressed as the ratio of lytic activity for 106 spleen cells to that

with CY, they were given 2.5 x 104 FSA cells. The number of

of the same number of spleen cells from untreated mice. Both WBI (Chart 2) and CY (Chart 3) suppressed NK cell activity, the suppression increasing as doses of the agents were increased. Treatment of mice with WR-2721 reduced this effect of WBI and CY. For example, at the lytic activity level of 0.5, the protection factor was 1.6 against WBI and 1.4 against CY.

mÃ©tastaseswas determined 14 days after tumor cell injection. There was a radiation- and CY dose-dependent increase in

DISCUSSION

metastasis formation, so that higher doses produced a larger enhancement. WR-2721 significantly reduced this effect of WBI and CY; protection factors comparing the slopes of the experi mental and control curves (Chart 1) were 2.5 for WBI and 1.8 for CY. Effect on Tumor Radioresponse in WBI Mice. The radiation response of immunogenic tumors, including FSA, is dependent on the immune competence of the tumor host, and is therefore reduced for tumors growing in WBI mice (31, 32). The next experiment was designed to test whether WR-2721 can protect against the WBI-induced decrease in tumor radiocurability. WR2721 in the dose of 400 mg/kg was given to mice 30 min before they were exposed to 600 rads WBI. One day later, the right hind thighs of these mice, as well as those of mice that received WBI or WR-2721 only, and untreated mice, were given injections of 5 x 105 FSA cells. When tumors grew to 8 mm in diameter, which occurred at about 9 days after transplantation for both normal and WBI mice, regardless of whether the mice were given WR-2721 or not, they were exposed to local tumor irradiation with single doses of 3400 to 7000 rads. Tumor regression and regrowth was followed for up to 100 days after irradiation, and the curative effect was expressed in TCDSOvalues (Table 2). WBI increased the TCDSOvalue from 4160 rads in normal mice to 5950 rads. WR-2721 treatment did not change the TCD.Â»value of normal mice (TCDso 4380), but it reduced the TCDso value in WBI mice to 5210 rads. Therefore, there was some abolition of the WBI effect by WR-2721 treatment. Effect of WR-2721 on WBI-induced Depletion of Tumor Macrophages. One of us observed earlier that FSA growing in WBI mice contain fewer normal cells than the same-size tumors growing in normal mice (15). Results presented in Table 3 show that WBI reduces the percentage of macrophages in FSA from the control value of about 17 to 3%. WR-2721 prevented this effect of WBI entirely; tumors in this group had 19.4% macro phages. Leg tumors were between 8 and 9 mm in diameter at

Results presented in this paper show that WR-2721 can, to a considerable degree, protect antitumor resistance mechanisms against damage inflicted by ionizing radiation and the alkylating agent CY. WBI (1) and CY (4) are strongly immunosuppressive, and as such they can compromise immune mechanisms that mediate different antitumor responses. In the present study, WBI promoted "take" of tumor cells when these were injected either s.c. or i.v., and CY promoted colonization of the lungs by tumor TableZ Effect of WR-2721on the radioresponse of FSAin WBImice Mice were exposed to WBI with 600 rads 1 day before they were given injections of 5 x 10s FSA cells into the right hind thighs. WR-2721, at a dose of 400 mg/kg, was given i.p. 30 min before tumor cell injection. When tumors grew to 8 mm in diameter, they were exposed to single doses of >-rays, ranging from 3400 to 7000 rads. TCDsovalues were determined at 100 days after tumor irradiation. Treatment of tumor cell recipients Exposure to WBI

Treatment with WR-2721

TCDÂ«, values (rads) 4160(3730-4630)"

Yes

4380(4040-4750)

5950(5510-6410) WBI 5210(4860-5580) WBI Yes 8 Numbers in parentheses, 95% confidence limits.

Table3 Effect of WR-2721on WBI-induceddepletion of macrophages in FSA Mice were given WBI, 600 rads, 1 day before they were given injections of 5 x 10s FSA cells into the right hind thighs. WR-2721, at a dose of 400 mg/kg, was given i.p. 30 min before WBI. When tumors grew to 8 to 9 mm in diameter, they were assayed for the percentage of Fc receptor-positive cells (macrophages). Three tumors were analyzed in each group. Treatment of tumor ceHrecipients

% of Fc receptor-positive cells in FSA

None WR-2721 WBI

17.4 Â±0.7

WR-2721 + WBI

19.4 Â±1.4

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17.1Â±1.6 3.0 Â±0.6

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8OC U O

extent, to protection of NK cells. It should be noted, however, that enhancement of lung colonization by i.v. injected tumor cells in mice treated with CY or ionizing radiation is only partially due to immunosuppression, and that other factors, including the tissue damage in particular, are important (22). Thus, protection by WR-2721 against CY- or WBI-induced enhancement of artifi

1.0

0.5

LU

o:

cial mÃ©tastasescould also be mediated by means other than the prevention of damage of the immune system. Furthermore, WR2721 is very effective in protection of hematolymphopoietic stem cells against ionizing radiation (36) and alkylating agents (33). Consequently, replacement of mature lymphoid cells involved in any type of immune reaction, which are damaged by the above cytotoxic agents, would be more rapid and more complete with the addition of WR-2721 .

PF:1.6

0.1 0.05

O

WBI

â€¢ WR2721 + WBI

0.01 O 100

300

600

IRRADIATION

900

DOSE

1200

(rad)

Chart 2. Effect of WR-2721 on WBI-induced suppression of NK cell activity of spleen cells. The activity was measured by the ability of spleen cells from mice treated with WBI to lyse in vitro cultured YAC-1 cells. Spleerrtarget cell ratio was 100:1. WBI was delivered 1 day before testing the NK cell activity. WR-2721 (400 mg/kg) was given i.p. 30 min before irradiation. PF, protection factor.

1.0

0.5

PF: 1.4

o

o.

CY alone

â€¢ WR2721 + CY

0

50

100

150

DOSE OF CYCLOPHOSPHAMIDE

200

250

(mg/kg)

Chart 3. Effect of WR-2721 on CY-induced suppression of NK-cell activity of spleen cells. The activity was measured by the ability of spleen cells from mice treated with CY 1 day earlier to lyse in vitro cultured YAC-1 cells. Spleen:target cell ratio was 100:1. WR-2721 (400 mg/kg) was given i.p. 30 min before CY. PF, protection factor.

cells injected i.v. WR-2721

protected

strongly against these

effects of radiation and CY, a protection most likely mediated via protection of the immune system. The evidence, already reported (12,34), shows that WR-2721 afforded a high degree of protec tion against radiation to spleen cells capable of forming hemolytic plaques, and to lymphocytes that mediate cell cytotoxicity. The data that we present here show that WR-2721 can significantly protect the activity of NK lymphocytes against both ionizing radiation (Chart 2) and CY (Chart 3). NK cells can influence various manifestations of tumor growth (13), but they are partic ularly effective in elimination of tumor cells from the circulation (11). This suggests that the presently observed efficient reduc tion of WBI- and CY-induced enhancement of tumor nodule formation in the lung by WR-2721 might be ascribed, to a certain

The immune system can also influence the curability of tumors exposed to irradiation in such a way that its suppression de creases and its stimulation increases tumor radiocurability (re viewed in Ref. 17). In accordance with this are data presented in Table 2, which show that 8-mm FSA responded to local tumor irradiation less well if grown in WBI mice than in normal mice. WBI delivered before tumor cell transplantation is likely to com promise both afferent and efferent components of the immune response. That the efferent response is compromised is shown by the reduced influx of normal host cells, including macro phages, after WBI (8, 15). In the present study, WBI reduced macrophage infiltration of tumors from 17 to only 3% (Table 3). Thus, the same size tumors have a higher absolute number of tumor cells if they grow in WBI mice. The greater number of tumor cells that have to be killed could be a factor, in addition to the suppression of the antitumor immune responses responsible for reduced radiocurability of tumors in WBI animals. Treatment of mice with WR-2721 fully protected them against the WBIinduced drop in macrophage infiltration and made tumors more responsive to localized irradiation. Since WR-2721 was incapable of complete restoration of tumor radiocurability, it is possible that in our experimental settings WR-2721 could not fully protect the immune system, at least the components that were involved in this manifestation of antitumor resistance. Because of its preferential protection of normal tissues, WR2721 can increase therapeutic gain under many experimental settings when combined with radio- or chemotherapy. Factors responsible for this differential protection are many, including delayed and reduced entry of WR-2721 into solid tumors, due to either compromised tumor vasculature (20, 28), to intrinsic inability of tumor cells to actively absorb this compound (35), or to reduced ability of WR-2721 to exert protection under hypoxic conditions (6, 7). Protection of the immune system is an additional factor by which WR-2721 can increase therapeutic gain. This mechanism will be operative in settings in which tumor growth and its response to irradiation or chemotherapy is influenced by the immune system, and in which these treatments are immunosuppressive. Immunosuppression by cytotoxic agents can also make tumor hosts more sensitive to infections. Therefore, WR-2721 should, by virtue of its protection of the immune system, prevent infection, and in this manner also be of thera peutic benefit. In conclusion, WR-2721 can protect the immune system against damage by irradiation and alkylating drugs, and thus prevent many undesirable manifestations of immunosuppression caused by these agents. Although the extent of protection we

JUNE 1984

2385


L.Milaset al. reported herein could be different under settings other than those of the present study, our results show that this compound can be strongly protective of the immune system, and as such has potential to be of benefit when combined with tumor radio- and chemotherapy. ACKNOWLEDGMENTS We wish to thank RozarmeGoddard for her assistance in the preparation of this manuscript. We are grateful to Lane Watkins and his staff for the supply and care of the mice used in these studies.

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