Mammary Carcinoma Arising in Mice Undergoing a Chronic Graft-Versus-Host Reaction 1,2,3

Transcription

1 Mammary Carcinoma Arising in Mice Undergoing a Chronic Graft-Versus-Host Reaction 1,2,3 John G. Gartner, 4,5 Thomas A. Seemayer,6 and Wayne S. Lapp 4,7 ABSTRACT -A graft-versus-host reaction (GVHR) was induced in 30 (CBA X A)F, mice by the iv injection of 50X10 6 parental strain A lymphoid cells. Solid tumors emerged in 5 of 10 experimental animals that survived beyond 14 months after the GVHR was initiated. The neoplasms were judged to be mammary carcinomas by light and electron microscopic examinations. C-type RNA viral structures were observed in some tumor cells. The neoplasms were successfully transplanted into syngeneic animals by either sc or ip injections of tumor cell suspensions. Tumor transfer to syngeneic mice was not possible if only spleen cells from tumorbearing animals were transferred. No tumors developed in an age-matched control group that received no treatment.-jnci 1984; 73: The G VHR induced across major or multiple minor histocompatibility barriers has served as a valuable means to study the immune system. The iv administration of a single dose of parental lymphoid cells into FJ hybrids is followed by marked immunosuppression that persists for an indefinite period (1). In addition to developing immune function abnormalities, mice maintained long term following the induction of chronic GVHR display a significant incidence of both donor and host B-cell lymphomas (2). The ease with which lymphomas can be induced makes this a valuable model for the study of lymphomagenesis, viral oncogenesis, and host responses to neoplasia. Previous reports suggested that an I-region incompatibility between the parent and F, was required to induce lymphoma development during the GVHR (2). We are currently investigating the development of neoplastic disease during the course of a GVHR. In the course of our studies we have utilized a parent-f, combination identical at the I-region. Using this combination, we did not observe the development of lymphoma, but we did detect an increased incidence of mammary carcinomas in female mice experiencing a chronic G VHR. invariably results in a chronic GVHR in which the animals live for a sufficient period of time to develop tumors. Donors and recipients were between 12 and 16 weeks old. All of the animals were obtained from a colony bred and maintained in our laboratory. All inbred strains in this colony are checked for syngeneity on a regular basis. Preparation of cell suspensions and induction of GVHR.-Single-cell suspensions were prepared from spleens and lymph nodes as described previously (1). Thirty female BAF 1 mice were given an iv injection of 50XI06 strain A female lymphoid cells. Tumor cell suspensions and spleens from tumor-bearing mice were prepared in the same manner as the spleen cells used to induce a G VHR. Control group.-twenty-five BAFJ female mice were set aside as a control group that received no treatment. These animals were periodically examined for tumor development for the duration of the experiment. In vivo and in vitro PFC responses to SRBC.-The direct splenic PFC response to SRBC was assayed by the technique of Cunningham and Szenberg (4) as modified in this laboratory (5-7). For the in vivo PFC response to SRBC, animals were immunized with 5X10 8 SRBC 4 days before being sacrificed. This immunization was performed 48 days after the GVHR was initiated. The in vitro PFC response to SRBC was analyzed by the Marbrook culture method as modi fed in this laboratory (8). With this method loxi06 spleen cells and 5X10 6 SRBC were cultured in 1 ml 1066 medium (Grand Island Biological Co., Grand Island, N.Y.) supplemented with 5% fetal bovine serum (Reheis Chemical Co., Kankakee, 111.). The culture vessels were incubated for 4 days at ABBREVIATIONS USED: Con A=concanavalin A; GVHR=graft-versushost reaction(s); LPS=lipopolysaccharide; PFC=plaque-forming cell(s); PHA = phytohemagglutinin; SRBC=sheep red blood cells. MATERIALS AND METHODS Animals.-Mice of inbred strains A (H-2a) and CBA (H-2k) and the (CBA X A)FJ hybrid (BAFJ) were used. H-2a is a recombinant between H-2k and H-2d. Animals bearing the H-2a genotype express the k-allele in the K and I-regions of the H-2 genetic region and express the d-allele in the S- and D-regions (3). Since strain A and strain CBA are identical at the K- and I-regions of the H-2 complex, the injection of parental strain A cells into the BAF, hybrid induces a GVHR against the H-2Dk antigen and against multiple minor histocompatibility antigens. This graft-versus-host combination I Received July 26, 1983; revised June 4, 1984; accepted June 18, Supported by grants from the National Cancer Institute of Canada, Medical Research Council of Canada, and from the McGill University-Montreal Children's Hospital Research Institute. 3This is publication No of the McGill University-Montreal Children's Hospital Research Institute. 4 Department of Physiology, McGill University, 3655 Drummond St., Montreal, PQ, Canada H3G I Y6. 5 Medical Research Council of Canada Fellow. 6 Department of Pathology, Montreal Children's Hospital, McGill University, 2300 Tupper St., Montreal, Canada H3H IP3. 7 Address reprint requests to Dr. Lapp JNCI. VOL. 73. NO.5. NOVEMBER 1984

2 1120 Gartner, Seemayer, and Lapp 37 C in a 5% CO 2 humidified atmosphere after which an aliquot of 0.1 ml cell suspension was removed and assayed for the direct PFC response to SRBC. This procedure was done 115 days after GVHR induction. Mitogen responses.-mitogen responses to Con A [Pharmacia (Canada) Ltd., Dorval, Quebec, Canada], PHA (Wellcome purified PHA-HA16; Well come Reagents Ltd., Wellcome Research Laboratories, Beckenham, England), and Escherichia coli LPS (Difco Laboratories, Detroit, Mich.) were ascertained by the method described by Kirchner et al. (9). This method consisted of the culture of 3XI06 spleen cells/ml in RPMI-1640 medium with optimum concentrations of mitogens (PHA, Con A, and LPS) in triplicate cultures in microtiter plates. After 48 hours in culture each well was pulsed with 1 /-lci [3H]thymidine and incubated for another 16 hours. The cultures were then harvested and counted in an LKB Rackbeta liquid scintillation counter. The mean counts per minute (cpm) for the triplicate samples and the stimulation index were calculated. The stimulation index was calculated from cpm mitogen stimulation/cpm no mitogen. Pathologic study.-experimental animals were examined periodically for tumor development and signs of morbidity. When tumors were observed, the animals were sacrificed by ether inhalation and autopsied. Portions of fresh tumor were minced in sterile dishes, placed in Hanks' solution, and transplanted intraperitoneally and subcutaneously into normal BAF] recipients. Spleen cells from tumor-bearing mice were similarly transferred into syngeneic animals. Tissue samples from all tumors as well as representative tissues from complete autopsies were fixed in 10% buffered Formalin and processed for light microscopy. Fragments of tumor were also diced in chilled 3% glutaraldehyde in cacodylate buffer and processed for transmission electron microscopy. Tissue from transplanted tumors in normal F] mice as well as from complete autopsies of both transplanted F] hosts and non-tumor-bearing, GVHR-experiencing mice was similarly processed. At the conclusion of the study (17 mol all control animals (25 mice) were sacrificed, autopsied, and examined thoroughly for the presence of tumors. A complete histologic study was performed in 8 animals. In addition to these studies, all grossly visible mammary tissue was excised and examined histologically for either malignant or premalignant microscopic changes. All remaining GVHR-experiencing mice were also sacrificed at this time and examined for the presence of tumors. RESULTS Of the 30 experimental animals in which a GVHR was initiated, 18 survived to 9 months post GVHR induction. By 14 months, only 10 animals remained. No grossly apparent tumors were present in animals dying before 14 months. Tumors emerged in 5 experimental animals 14, 15, and 17 months after the GVHR was induced. All control animals survived the experiment. No tumors were seen in these animals, and histologic examination of the rudimentary mammary tissue in the milk lines revealed no pathologic abnormalities (table 1). In vivo and in vitro PFC data are shown in table 2 and demonstrate that mice experiencing a GVHR were unable to mount a PFC response to SRBC when tested on days 48 and 115 after the G VHR was initiated. Mitogen responses are also shown in table 2, which illustrates that on day 48 both Con A and LPS responses were severely suppressed. The PHA response was also diminished but not to the extent of the Con A and LPS responses. On day 115 after GVHR induction both PHA and Con A responses were severely suppressed (near background levels), whereas the LPS response was only moderately diminished. Six neoplasms developed in 5 experimental animals. The tumors were situated in subcutaneous tissue along either mammary line; in 1 animal, 2 tumors were present. Histologic examination revealed cystic, papillary, trabecular, cribriform, and solid epithelial growth patterns (figs. la, IB). In the tumor tissues mitoses were numerous, imparting an anaplastic appearance to the neoplasms. Electron microscopy confirmed the epithelial nature of the neoplasms, since cellular junctions were present between neoplastic cells and because basal lamina formation was often seen at the tumorstromal interface. In each neoplasm numerous C-type RNA viral structures were observed within some tumor cells and in some extracellular spaces (figs. 2A, 2B). Transplantation of the primary tumors either intraperitoneally or subcutaneously into normal BAF] recipients resulted in the development of secondary tumors at the site of injection (table 3). Upon histologic examination (light and electron microscopic), these transplanted tumors were similar to the primary neoplasms except that a greater degree of anaplasia was noted in the transplanted tumors. Complete autopsies in these animals revealed no other pathologic abnor- TABLE 1.-Incidence of mammary carcinoma appearing in mice e,rperiencing a GVHR and in normal m1:ce between 425 and 515 days after GVHR induction Experimental group No. of animals surviving/total No. of animals No. of animals with tumors/total No. of animals Total No. of tumors GVHR 10/30 5/10 6 a P<'05 h P<.005 h Normal 25/25 0/25' 0 a One animal developed 2 tumors, one in the anterior right mammary gland and the other in the posterior left mammary gland. When transferred into normal BAFI female mice, these 2 tumors displayed different growth rates. b P-value comparing the frequency of tumor-bearing animals and the total number of tumors between animals experiencing a GVHR and normal mice. The P-values were determined by the chi-square test for independence of attributes. 'All animals were autopsied and examined histologically at the end of the experiment for the presence of mammary tumors. JNC], VOL. 73, NO.5, NOVEMBER ]981

3 Experimental group TABLE 2.-Immune funct1:on of experimental and control animals Mammary Carcinoma in GVH Reactive Mice 1121 Days Mitogen response a post Mean PFC ± SE GVHR None PHA Con A LPS Normal ±12.1X10,b 18.9± ± ± ±1.2 (13.7) (7.1) (2.4) GVHR 48 Ob 7.3± ± ± ±0.0 (6.0) (1.0) (0.7) Normal ±22.5 ' 20.0± ± ± ±3.4 (6.9) (9.8) (3,9) GVHR 115 0' 6.9± ± ± ±1.9 (1.2) (2.4) (2.9) a Mean cpm ± SE x 10~'. Stimulation indices are shown in parentheses. b Mean in vivo PFC ± SE response per spleen. Mice were immunized 4 days earlier with SRBC. A minimum of 2 animals was included in each group. e Mean in vitro PFC response per culture vessel. Spleen cells (10X10 6 ) were cultured with SRBC for 4 days. A minimum of 2 animals was included in each group, and 4 culture vessels were used for each animal. mali ties. Transplantation of spleen cells from mice bearing the primary tumor either subcutaneously or intraperitoneally into normal BAF I female mice failed to induce either mammary or lymphoid tumors (table 3), although lymphoid infiltrates were observed in the lung, salivary gland, and kidney. Autopsies in all 5 GVHR-experiencing, tumorbearing animals documented multifocal lymphoid infiltrates of variable intensity in the mesentery, salivary glands, pancreas, liver, lungs, kidneys, and adrenal glands. The thymus of each animal was dysplastic, which has been described in previous studies from this laboratory (l0, 11). Lymph nodes and spleen were characterized by hyperplasia of B-cell-dependent zones. No pathologic abnormalities were recorded in the TABLE 3.-Incidence of tumor growth in normal BAFJ mice following transfer of tumor cells or spleen cells from spontaneous tumor-bearing, GVHR-experiencing mice into normal F J mice No. of mice Type of Obsertissue vation with tumors/ total No. of trans- period, ferred a days mice given injections 1 Tumor 163 4/4 0/4 b Spleen 163 0/4 3/4 2 Tumor 152 3/3 0/3 b Spleen 152 0/3 2/3 b 3 Tumor 260 3/3 2/3 Spleen 260 0/3 2/3 4i' Tumor 150 2/2 0/2 4ii' Tumor 150 2/2 0/2 5 Tumor 165 2/3 1/3 Spleen 165 0/3 3/3 Total Tumor 16/17 3/17 Spleen 0/13 10/13 Tumor- bearing donor No. of mice surviving 150 days/ total No. of mice a A minimum of 10XIO' tumor cells and 15x10 6 spleen cells was transferred from tumor-bearing mice into normal BAF, mice at wk of age. bone animal from each of these groups was sacrificed between 85 and 96 days after transfer, and a complete autopsy was performed., See footnote a, table 1. heart, thyroid gland, parathyroid gland, or bone marrow. Metastases from the mammary tumor were not identified. Autopsies of 5 non-tumor-bearing, GVHR-experiencing animals disclosed lymphoid infiltrates in sites similar to those in the tumor-bearing animals. In addition, atypical lymphoid hyperplasia was identified in parotid and mesenteric lymphoid tissues of I animal. Control animals that were autopsied at the conclusion of the experiment revealed no histologic abnormalities. DISCUSSION These results show that when a chronic GVHR is induced in BAFJ mice by the injection of parental strain A lymphoid cells, the animals become immunosuppressed and later display a relatively high incidence of mammary carcinoma. In this model G VHR is directed against both the D-region of the H-2 complex and multiple minor histocompatibility antigens. The K- and the I-regions in this combination are identical. Despite the relatively low-grade nature of the GVHR, mortality was high inasmuch as only 10 of 30 mice survived beyond 14 months after the GVHR was induced. In 5 of these long-term survivors, mammary carcinoma was observed between 14 and 17 months after the GVHR was initiated. Although this study represents a relatively small group of mice, the incidence of mammary tumor appearance is highly significant when compared with the incidence in the control group (P<.005). Confirmation of this observation must now be done with the use of congenic inbred H-2 recombinant or mutant strains of mice in which the alleles at the H-2 subregions are well defined. Electron microscopy, in addition to confirming the epithelial origin of these polymorphic, and at times anaplastic, neoplasms, disclosed the presence of C-type RNA virus particles in the cytoplasm of tumor cells. No control animals developed neoplasms. That these JNC], VOL. 73, NO. '" NO\'EMBER 1984

4 1122 Gartner, Seemayer, and Lapp tumors were not lymphomas was further underscored by the fact that none of these tumors could be transplanted into BAF, syngeneic recipients by transfer of spleen cells from tumor-bearing animals. In previous studies of GVHR-associated lymphomagenesis, most of the lymphomas that emerged either arose in or metastasized to spleen, and at least 50% of these lymphomas could be transplanted by transferring spleen cells (2). The development of mammary carcinomas in the BAF, mice following GVHR induction with parental strain A cells is of some interest, since nulliparous strain A and CBA mice do not have a high incidence of mammary carcinomas (12). Only the older multiparous mice of this strain are at increased risk for these tumors. That the animals used in this experiment did not develop lymphomas is noteworthy, and a possible explanation for this finding may lie in the observation made by Gleichmann et al. (2) that I-region differences are a requisite for GVHR-associated lymphomagenesis. GVHR directed only at K and D end H-2 products rarely result in lymphomas. Mammary carcinomas following G VHR were described on two occasions (13, 14). In these studies the GVHR was induced in H-2-identical animals across multiple minor histocompatibility differences alone. In one of these reports the increased incidence of mammary tumors was attributable not to the GVHR but rather to the transfer of mammary tumor virus with the donor cells obtained from a strain that had a high incidence of mammary carcinoma (14). Although both strains used in our experiments had a low incidence of mammary tumor virus, we cannot exclude the possibility that virus may have been transferred with the parental lymphoid inoculation. An extensive study would be required to resolve this issue. These results document yet another serious consequence of chronic graft-versus-host disease. Female mice experiencing a GVHR, in which an I-region incompatibility does not exist, may not develop lymphomas; however, they appear to be at a significantly higher risk for developing mammary carcinoma than are normal mice. Further studies are in progress on this experimental model. REFERENCES (1) LAPP WS, WECHSLER A, KONGSHAVN PA. Immune restoration of mice immunosuppressed by a graft-versus-host reaction. Cell Immunol 1974; 11: , (2) GLEICHMANN E, GLEICHMANN H, WOLKE W. Autoimmunization and Iymphomagenesis in parent F 1 combinations differing at the major histocompatibility complex: Model for spontaneous disease caused by altered self antigens? Transplant Rev 1976; 31: (3) SHREFFLER DC. Composition of recombinant haplotypes of the H-2 gene complex: Mouse. In: Altman PL, Katz DD, eds, Inbred and genetically defined strains of laboratory animals. Part I: Mouse and rats. Bethesda, Md.: FASEB, 1979: (4) CUNNINGHAM AJ, SZENBERG A, Further improvements in the plaque technique for detecting single antibody forming cells. Immunology 1968; 14: (5) EUE R, LAPP WS. Graft-versus-host induced immunosuppression: Mechanism of depressed T-cell helper function in vitro, Cell Immunol 1977; 34: (6) PARTHENAIS E, LAPP WS, Evidence for two types of non-specific suppressor cells activated by the graft-versus-host reaction in mice, Scand J Immunol 1978; 7: , (7) TREIBER W, LAPP WS. Stimulation of humoral immune responses to a thymic-independent antigen in mice immunosuppressed by a graft-versus-host reaction. Cell Immunol 1978; 37: , (8) EUE R, LAPP WS. Apparent T-cell function of bone marrow cell from mice experiencing a graft-versus-host reaction. Cell Immunol 1976; 21: (9) KIRCHNER H, MUCHMORE AV, CHUSED TM, HOLDEN HT, HERB ERMAN RB. Inhibition of proliferation of lymphoma cells and T lymphocytes by suppressor cells from spleens of tumorbearing mice. J Immunol1975; 114: (10) SEE MAYER TA, LAPP WS, BOLANDE RP. Thymic involution in murine graft-versus-host reaction: Epithelial injury mimicking human thymic dysplasia. Am J Pathol 1977; 88: (11) --. Thymic epithelial injury in graft-versus-host reactions following adrenalectomy. Am J Pathol 1978; 93: (12) SQUARTINI F. Tumors of the mammary gland. In: Turusov VS, ed. Pathology of tumors in laboratory animals. Vol II. Tumors of the mouse, Lyon: IARC, 1979: (13) HAYS EF. Graft-versus-host reactions and the viral induction of mouse lymphoma. Cancer Res 1972; 32: (14) ROSSI GB, FRIEND C. Lymphomas in mice: Failure of induction after a graft-versus-host reaction. Science 1970; 167: JNCI, VOL 73, NO..0, NOVEMBER 1984

5 Mammary Carcinoma in GVH Reactive Mice 1123 FIGURE I.-Light photomicrographs of representative tumor sections. They show the cribriform (IA) and poorly differentiated (IB) growth patterns of mammary carcinoma. Hematoxylin and eosin. X 200 JNCI. VOL. 73. NO.5. NOVEMBER 1984

6 1124 Gartner, Seemayer, and Lapp FIGURE 2.-Electron photomicrograph of mammary carcinoma. Epithelial features (2A) and intracytoplasmic C-type RNA viral particles (2B) are illustrated. X 4,500 (fig. 2A); X 10,000 (fig. 2B) JNC[, VOL. 7.~, NO.5, NOVEMBER 1984