Immunosurveillance of spontaneous lung adenomagenesis in mice

Euro.O.J. Canter Vol. 11, pp. 633-637. Pergamon Press 1975. Printed in Great Britain

Immunosurveillance of Spontaneous Lung Adenomagenesis in Mice* MARIA I. COLNAGHI Division of Experimental Oncology A, Istituto Nazionale per lo Studio e la Cura dei Tumori 20133 Milano, Italy. A b s t r a c t ~ B A L B /c mice repeatedly injected with syngeneic or allogeneic lymphoma cells blocked by mitomycin-C had a statistically significant decrease of lymphoma and increase of lung adenoma spontaneous incidence in comparison to untreated controls. When tested for the number of plaque-forming spleen cells, animals inoculated with mitomycin-Ctreated cells revealed a severe immunodepression suggesting a role of the immunosurveillance in adenomagenesis. Also, one group of mice injected with untreated aIlogeneic normal immunocompetent cells revealed an increased lung adenoma incidence most likely due to a graft versus host phenomenon; this result seems to exclude thefact that a direct carcinogenic effect of mitomycin-C on lung tissue could be responsiblefor the increased adenomagenesis.

centrifugation, for gross removal of the chemical, or after repeated washings. We found that mice inoculated with unwashed mitomycin-Ctreated cells or with untreated lymphoid cells of one of the allogeneic strains used, had a significantly higher lung adenoma incidence than untreated mice. It was subsequently found that, when injected without repeated washing, mitomycin-C caused a severe immunodepression.

INTRODUCTION

A NUMBER of experiments are presently on record on the role of immune surveillance in lung adenomagenesis in mice. Thymectomy or antilymphocytic serum have been shown to facilitate the spontaneous or chemicallyinduced onset of lung tumors [1-5], and it seems that lung adenoma development and impairment of the cellular immune response are closely related [6]. However, no antigenicity of lung adenomas was detected by in vivo tests [7, 8] whereas in vitro studies of cellmediated response produced evidence that lung adenomas possess tumor-associated antigens [9] whose strength was dependent on the immune status of the host in which the tumor arose [10]. In the course of a series of experiments on antigenicity of murine lymphomas, BALB/c mice were immunized, to obtain anti-lymphoma antisera, with untreated allogeneic normal lymphoid or lymphoma cells, or with syngeneic or allogeneic lymphoma cells blocked by mitomycin-C or by X-rays. Mitomycin-Ctreated cells were injected either after a single

MATERIAL AND M E T H O D S Mice BALB/c, C57BL/He and C3Hffemale inbred mice, maintained in our laboratory by brother x sister mating, were used. Tumors The thymic lymphosarcomas utilized for immunizations were induced by urethan in mice of the three strains and maintained by s.c. transplant in preirradiated syngeneic mice. Only tumors that arose in female mice were used. Inoculation with lymphoid cells Cell suspensions from pooled C3Hf, C57BL or BALB/c lymphoma or normal thymus and spleen cells were injected repeatedly in young

Accepted 30 May 1975. *This work was supported by a grant from Consiglio Nazionale delle Ricerche, Rome. 633

634

Maria I. Colnaghi

adult female BALB/c mice at 1- to 3-week intervals, each injection being done half s.c. and half i.p. The first inoculum was about 2 x 106 cells and the following doses increased twofold. Syngeneic and, in some experimental groups~ allogeneic lymphoma cells were blocked before inoculum by X-rays (10,000 R) or by mitomycin-C (Kyowa Hakko Kogyo Co., Ltd., Tokyo). In the latter case, 2 x 107 tumor cells suspended in 2 ml of Hank's balanced salt solution (HBSS) were added with 150pg mitomycin-C, incubated for 2 hr, centrifuged and resuspended in HBSS, without any additional washing, before being injected in the appropriate dose.

Plaque-forming cells (PFC) determination Groups of 3 BALB/c mice each were repeatedly inoculated with BALB/c or C3Hf mitomycin-C treated lymphoma cells or with C3Hf untreated lymphoid cells, and were injected with SRBC 7 days after the 1st, 3rd, 5th, and 9th immunizing inoculum or 40 days after the 9th inoculum, to test for the PFC capacity of their spleens. The number of PFC was determined following the technique of Jerne et al. [11]. Four days after immunization with SRBC, the spleen was removed and the nucleated cells of each spleen were counted and then processed in agar plates. Three plates were prepared from each spleen and the number of PFC was calculated as a mean of the 3 plates. Recording of tumor incidence Mice were controlled weekly, killed when in bad condition and autopsied. Lymphomas Table 1.

and lung adenomas were recorded and histology was made when necessary to confilm the gross diagnosis. RESULTS

Influence of hyperimmunization with untreated or blocked cells on lymphoma and lung adenoma onset In Table 1 the incidence of lymphomas and lung adenomas in the various groups of mice is reported. Most of the lymphomas were generalized lymphosarcomas of non-thymic origin. In comparison to untreated control mice, the lymphoma incidence was decreased in all 3 groups injected with mitomycin-Ctreated cells, the difference reaching a statistically significant level in 2 groups. It should be noted that the average age at death of the untreated controls was 107 weeks, whereas in none of the mitomycin-C treated groups was it over 90. Therefore, the observed decreased incidence of lymphoma could have been due to the shorter survival time since the average age at death for lymphoma was 109 weeks in the control group and varied from 72 to 86 weeks in the 3 mitomycin-C-treated groups. The lung adenoma incidence was much higher than in the controls in all the 3 groups injected with mitomycin-C-treated cells (P < 0.001) and in the group injected with untreated C3Hf thymus and spleen cells ( P < 0.01). The group injected with C57BL untreated lymphoid cells also showed an increased (though not significant) incidence of lung adenomas. The 3 groups injected with X-raytreated syngeneic or untreated allogeneic lymphoma cells were only slightly different

Effect of hyperimmunization of BALB/c female mice with normal or neoplastic cells of thymic origin on the spontaneous incidence of lymphoma and lung adenoma

i

Animals with

Injected cells -BALB/c lymphoma C3Hflymphoma C3Hfthymus+spleen C57BLlymphoma C57BL thymus+spleen BALB/c lymphoma C3Hflymphoma C57BL lymphoma

Cells treated with -X-rays ----Mitomycin-C Mitomycin-C Mitomycin-C

No. of Lymphoma Lung adenoma Both tumors mice No. % Mean age* No. % Mean age* No. % Mean age* 62 32 61 28 71 26 48 29 23

27 43 10 31 30 49 13 46 36 50 8 30 2 4§ 4 14"~ 6 26

109+29 20 32 96_+ 16 12 37 100_+12 26 42 114_+13 18 64+ 99+ 12 31 43 94-+ 12 13 50 78+ 8 34 70§ 86-+ 2 26 89§ 72_+36 18 78§

*Mean age at death _+S.D. t P < 0.05 (probability of statistical difference from the control). ~P < 0-01. §P < 0.001.

117+22 99_+ 12 101_+ 9 112_+ 9 99-+ 7 96-+ 9 79-+ 5 84-+ 5 85_+ 8

7 2 8 7 8 2 2 3 3

11 6 13 25 11 8 4 10 13

116_+16 112, 120 103_+12 107_+ 4 97_+ 3 93, 95 84, 85 86-+ 3 86_+ 11

635

Immunosurveillance of Lung Adenomagenesis in Mice from the controls. The increased lung adenoma incidence observed in the mitomycin-C-treated groups was strengthened by the shorter survival time. In all groups, some animals developed both lymphoma and lung adenoma as reported in Table 1, with no meaningful differences among the groups.

Effect of immunizations on plaque-forming spleen cells The number of PFC in the spleen of control or experimental mice is reported in Table 2. After the 1st and the 3rd inoculum no depression was seen and an even higher immune response to SRBC occurred in all treated groups than in the untreated controls. From the 5th to the 9th inoculum the 2 groups injected with mitomycin-C treated cells showed a striking decrease of the PFC number, whereas the group injected with untreated lymphoid cells revealed only a slight, not statistically significant reduction. Forty days after the last inoculum, the mice injected with syngeneic lymphoma cells appeared to be completely recovered, whereas the allogeneic lymphomainjected mice still showed a significantly reduced number of PFC. DISCUSSION

Our results demonstrate that some manipulation of the immune apparatus resulting in inadequate immunocompetence may interfere with the process of spontaneous tumorigenesis. Mitomycin-C, one of the antibiotics discovered among the fermentation products of one species of Streptomyces [12], has been demonstrated to have chemotherapeutic effectiveness against experimental as well as human tumors and to cause in different mammalian species analogous

pathologic lesions in hematopoietic tissue [13]. These effects are probably mediated by the ability of the chemical to inhibit DNA synthesis and cell division and for this property it is commonly used to block neoplastic cells for preventing their in vivo growth in immunizing procedures. The reduced lymphoma incidence we have observed, if not due to shorter survival, might have been caused by the mitomycin-C acting as a prophylactic chemotherapeutic agent by killing cells involved in the spontaneous neoplastic transformation. The reduction of the splenic PFC after repeated immunizing inoculum confirmed that even the low quantity of mitomycin-C remaining in the injected cell suspensions could exert damage on the cells of the immunocompetent compartment. The consequent severe immunodepression could be responsible for the increased lung adenoma incidence, indicating that immunological surveillance operates during the spontaneous onset of this type of tumor. Cellmediated, but not humoral, response has been found to control chemical induction of lung adenomas [3, 6]. We are unable to say what type of immune response controlled the spontaneous adenomagenesis. The direct correlation between the reduction of PFC, that is of antibody producing cells, and the increase of adenoma incidence, does not necessarily indicate that adenomagenesis is under humoral control since cells directly involved in antibody production (B cells) as cells with helper activity (T cells), which are responsible also for the cell-mediated response, both are essential for anti-SRBC response. Another explanation for the increased number of lung adenomas, could be a specific carcinogenic activity of the chemical for lung tissue, although only induction of sarcomas has

Table 2. Plaqueforming cells in spleen of BALB/c mice immunized against mitomycin-C-treatedlymphoma cells or untreated thymus cells Injected cells -BALB/c lymphoma, Mitomycin-C-treated C3Hf lymphoma, Mitomycin-C-treated C3Hf thymus and spleen untreated

1

No. of PFC _+S E x I x 10 6 nucleated spleen cells after 3 5 7 9

1103+290

1450_+257

1750 + 200

1604 _+215

32 _+2~.

2023_+490

1310_+224 2355_+335

2140_+ 190 t

1450_+246

1885_+185

1387_+145

190 _+56++

I 14 _+83 §

1226 _+203

370_+61~

29_+8§

17_+5§

216_+92++

1011_+104

1059_+330

1448_+134

i

*40 days after the 9th inoculum. ~'P < 0.05 (probability of statistical difference from the control). ++P < 0.01. §P < 0.001.

1457_+201

9* inocula

1362_+82

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Maria L Colnaghi

been reported until now [14]. This seems to be excluded by the fact that an increased adenomagenesis in comparison to controls was also found in groups injected with untreated normal allogeneic immunocompetent cells, although the difference reached significance level only in mice injected with C 3 H f cells. It has already been reported [15] that outbred mice repeatedly injected with lymphoid cells from mice of the same colony had an increased urethaninduced lung adenoma incidence, attributed to a graft-host reaction. It seems from our experiment that when normal immunocompetent cells were injected, a graft-host phenomenon decreasing the ability of the immune apparatus to exert a control over neoplastic transformation, may also interfere with spontaneous adenomagenesis. It is true that

mice injected with C 3 H f lymphoid cells had a statistically non-significant decrease of PFC. However, the cell-mediated control of carcinogenesis might be such a delicate phenomenon that it can be affected even by a weak grafthost reaction, unable to interfere with antibody production. It is interesting to note that the lung adenoma incidence was higher in BALB/c mice injected with C 3 H f lymphoid cells, which can recognize 9 alien determinants of the host H-2 system, than in mice injected with C57BL ceils, recognizing 6 alien determinants only. In conclusion, our results indicate that an immunosuppression caused by mitomycin-Ctreated cells, or a prolonged disturbance of the immune apparatus exerted by untreated allogeneic immunocompetent cells markedly influence lung adenomagenesis.

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