Immunologic heterogeneity of tumor cell subpopulations from a single mouse mammary tumor.

Five subpopulations (66, 67, 68H, 168, and 4.10LM) obtained from a single BALB/cfC3H mammary adenocarcinoma were used to assess intratumor immunologic heterogeneity. BALB/c and BALB/cfC3H mice were immunized with each of the subpopulations, and lymph node cells (LNC) from immunized animals were tested for cell-mediated immunity (CMI) to each subpopulation in vitro by chromium release and microcytotoxicity tests and in vivo by Winn assays. The immunogenic character of the subpopulations differed markedly. The pattern of cross-reactivity indicated that at least two determinants were involved, one of which was probably a viral antigen. The viral antigen was expressed on 4 subpopulations (66, 68H, 168, and 4.10LM). The other determinant was immunogenic in both BALB/c and BALB/cfC3H mice and was expressed on 4 subpopulations (66, 67, 168, and 4.10lm). Thus 1 subpopulation (68H) expressed only the viral antigen, 1 (67) expressed only the other antigen, and 3 (66, 168, and 4.10LM) expressed both. Expression of the determinants showed qualitative and quantitative variations. Quantitative differences were noted by the relative effectiveness of the subpopulations to induce CMI and by the relative sensitivities to LNC-mediated killing. Qualitative differences were indicated by the occurrence of unidirectional cross-reactivities between some pairs of subpopulations; a determinant could be expressed so that the subpopulation could induce cytotoxic cells but not be sensitive to them or vice versa.

Differential sensitivity of metastatic versus nonmetastatic mammary tumor cells to macrophage-mediated cytostasis.

Sensitivity to macrophage-mediated cytostasis was determined with 4 tumor cell lines derived from a single, spontaneously arising mouse mammary tumor. Cytostasis was measured in a 48-hour [3H]thymidine-incorporation assay with the use of maleic vinyl ether (pyran) fraction 2 (MVE-2)-elicited peritoneal macrophages as effector cells. Metastatic tumor lines 66 and 410.4 were less sensitive than nonmetastatic lines 67 and 168. Pretreatment of tumor cells with indomethacin for 24 hours before assay increased the cytostatic sensitivity of the metastatic tumor lines but did not affect that of the nonmetastatic tumor lines. Addition of 100 ng lipopolysaccharide (LPS)/ml to the assay mixture of MVE-2-primed macrophages and tumor cells or pretreatment of macrophages with LPS markedly lessened the differences in cytostatic sensitivity among the metastatic and nonmetastatic lines. Pretreatment of tumor cells with indomethacin plus addition of LPS during the effector phase of the assay completely abrogated differences in sensitivity. These results suggest that differences in sensitivity of metastatic versus nonmetastatic tumor cells to macrophage cytostasis are due to both tumor cell (prostaglandin) and effector cell (activation state) factors.

Effect of chemical carcinogen treatment on the immunogenicity of mouse mammary tumors arising from hyperplastic alveolar nodule outgrowth lines.

The experiments reported herein examined the effects of chemical carcinogen treatment of precursor hyperplastic alveolar nodules on the immunogenicity of subsequent mammary tumors in mice. The results of in vitro immunogenicity assays were compared with those of in vivo transplantation resistance tests. This comparison demonstrated that the in vitro assays predicted qualitatively and quantitatively the results from the in vivo test. In addition, the data suggested that the immunogenicity of a tumor depends on events occurring during the development of precursor hyperplastic alveolar nodules rather than during the development of subsequent mammary tumors.

Activity of lymphoid cells separated from mammary tumors in blastogenesis and Winn assays.

Lymphoid cells isolated from mouse mammary tumors by isokinetic gradients were not stimulated in vitro by either phytohemagglutinin or a soluble, tumor-associated antigen extract even though splenocytes from the tumor-bearing mice were responsive to both. In in vivo Winn assays, lymphoid cells isolated from tumors markedly stimulated tumor growth rate. The effect on growth rate was abrogated by exposure of the isolated lymphoid cells to antilymphocyte serum and complement.

Induction of antitumor immunity by interleukin-2 gene-transduced mouse mammary tumor cells versus transduced mammary stromal fibroblasts.

BACKGROUND: Tumor cell-targeted cytokine gene transfer has been used to generate tumor cell vaccines, but this approach is limited by the need to establish and implant live tumor cells. PURPOSE: The purpose of this study was to determine if stromal fibroblasts could be used as an alternative vehicle for delivery of the cytokine interleukin-2 (IL-2) into the tumor microenvironment. We attempted to establish the feasibility of (a) genetic immunotherapy in a mammary tumor system and (b) engineering stromal fibroblasts as well as tumor cells. We compared the effects of tumor cell-mediated and stromal fibroblast-mediated local IL-2 expression on the generation of antitumor immune responses. METHODS: Retroviral vectors containing a human IL-2 gene were used to transduce a mouse mammary tumor line, 4TO7, and an immortalized but nontumorigenic fibroblast line established from syngeneic mammary fatpads. Expression of the IL-2 gene in transduced cells was determined by measuring IL-2 secretion, by RNA-polymerase chain reaction, and by immunochemistry. Groups of 5-12 BALB/c mice were injected with either 4TO7 cells or various doses of IL-2-secreting 4TO7 cells (4TO7-IL-2); tumor growth was monitored. To test whether local IL-2 expression by transduced cells could influence the growth of unmodified tumor cells, we determined tumor development in groups of mice treated with 4TO7 cells co-injected with either 4TO7-IL-2 cells or IL-2-secreting fibroblasts. RESULTS: 4TO7-IL-2 cells induced active immunity able to reject the immunizing tumor and to resist challenge with parental 4TO7 cells on the contralateral side. Mice pretreated with 4TO7-IL-2 were significantly protected compared with untreated control animals or mice pretreated with irradiated 4TO7 cells. The immunity induced by 4TO7-IL-2 cells did not protect against challenge with another subline, 4T1, which was derived from the same spontaneously arising mammary tumor as 4TO7. Co-injection of 4TO7 cells with 4TO7-IL-2 cells reduced tumorigenicity, whereas co-injection of 4TO7 cells with IL-2 secreting fibroblasts did not. CONCLUSION: Our results suggest that induction of anti-tumor immune response by local IL-2 production is most effective when the helper cytokine is secreted by the tumor cell. IMPLICATION: Our studies caution against the use of IL-2 gene-transduced syngeneic stromal cells as an alternative strategy of gene therapy for cancer. However, they may allow study of the mechanisms of tumor antigen recognition and the possible involvement of co-stimulatory signals for effective tumor vaccination by gene-modified cells.

Racial differences in breast cancer patients.

One thousand seventy-eight patients diagnosed with primary breast cancer were examined for racial differences in histopathologic and clinical parameters. There were no observed differences in tumor histopathologic type or tumor endocrine status between races. There were no differences with respect to time to breast tumor recurrence observed between black and white patients. However, differences were observed in factors that contributed to tumor stage at diagnosis and to tumor grade. Survival differences observed in univariant analysis of blacks vs. whites were explainable by the presence of more severe skin involvement, tumor grade, and tumor size at diagnosis in the black patients.

Presurgical serum immunoglobulin concentrations and the prognosis of operable breast cancer in women.

Serum concentrations of IgG, IgA, IgM, IgE, and allergen-specific IgE were measured in presurgical serum samples from 400 women admitted to a multidisciplinary study of primary breast cancer. The relationships between the serum immunoglobulins and patient survival were analyzed with the use of a Cox proportional hazards linear model. After adjustment for TNM stage, tumor histopathologic grade, and estrogen receptor (E2R) status, lower IgM concentrations were associated with longer survival. Lower IgE concentrations were also associated with longer survival, but only in patients whose tumors were E2R positive. IgG and IgA were not related to survival. Serum IgM and IgE concentrations, allergen-specific IgE scores, and the tumor E2R status were combined to construct a three-level risk classification that was more prognostic than any of the individual components. Cox model analysis demonstrated that this combination of immunologic and hormonal variables provided significant new information beyond that obtained from TNM staging and histopathologic grading of the tumors (P = .01). This new information may be useful to physicians in advising patients with primary, operable breast cancer about the relative risks and benefits of adjuvant therapy and in designing clinical trials of adjuvant therapy.

Xenograft model of progressive human proliferative breast disease.

BACKGROUND: Progression of proliferative breast disease has been associated with increased risk for development of invasive carcinoma. Cell lines have been developed to facilitate the study of this process. Human cell line MCF10A originated from spontaneous immortalization of breast epithelial cells obtained from a patient with fibrocystic disease, and cell lines MCF10AneoN and MCF10AneoT were created by stable transfection of these cells with the neomycin-resistance gene and either the HRAS gene or the mutated T-24 HRAS gene, respectively. PURPOSE: Our goal was to develop an experimental model of progressive human proliferative breast disease. METHODS: MCF10A, MCF10AneoN, and MCF10AneoT cells were injected subcutaneously into the dorsal flank of male nude/beige (C57/BALB/c nu/nu bg/bg) mice (12 mice for each cell type). These mice were examined periodically for formation and persistence or growth of palpable nodules. One mouse per group was killed 1 week after cell injection; thereafter, mice were observed as long as possible. Cells were recovered from palpable lesions by enzymatic dissociation of the excised lesions. Cells re-established in tissue culture from a week-14 tumor (MCF10AneoT.TG1) were injected into 12 male nude/beige mice. Southern blot hybridization analysis of the HRAS gene locus and cytogenetic analyses were performed. RESULTS: Transplanted MCF10A and MCF10AneoN cells formed transient, small palpable nodules that regressed and disappeared during the 4th and 5th weeks. In 10 of the 12 mice, T-24 HRAS gene-transfected MCF10A cells (MCF10AneoT) formed small, flat nodules that persisted for at least 1 year. Three of these xenografts became carcinomas. One (removed 7 weeks after transplantation) was an undifferentiated carcinoma composed of polygonal cells with large, vesicular nuclei and numerous mitoses. The second (removed after 14 weeks) was an invasive squamous cell carcinoma. The third (removed after 56 weeks) was a moderately differentiated adenocarcinoma. Initially, xenografts of MCF10AneoT.TG1 cells showed intraductal proliferative changes; after 23 weeks, the lesions showed histologic features resembling those seen in atypical hyperplasia of the human breast, and later lesions showed characteristics of carcinoma in situ. The MCF10 lineage of cells of three MCF10AneoT.TG1 xenografts was confirmed by DNA fingerprinting and karyotype analysis. CONCLUSIONS: MCF10AneoT and MCF10AneoT.TG1 comprise a transplantable xenograft model that produces a broad spectrum of human proliferative breast disease. IMPLICATIONS: The reproducible establishment of representative stages in early breast cancer progression from the MCF10 model offers a new opportunity to analyze critical events of carcinogenesis and progression in breast cancer.

Motility and tumoricidal activity of interleukin-2-stimulated lymphocytes.

The motility of murine splenic lymphocytes stimulated nonspecifically by recombinant interleukin 2 (RIL-2) was studied in a three-dimensional collagen-gel system. Nonadherent BALB/c splenic lymphocytes were cultured in medium containing Cetus RIL-2 (700 to 1000 units/ml) or excipient control. They were then allowed to locomote randomly for 16 to 18 h into slabs of type I rat tail collagen gel. The gels were digested with collagenase, and total lymphocyte populations and motile subpopulations were collected and compared with respect to their lymphokine-activated killer activity (measured as 4-h cytotoxicity against the natural killer-resistant mammary adenocarcinoma line 410.4), their natural killer activity (measured as 4-h cytotoxicity versus lymphoma YAC-1), and their subset distribution (defined by immunofluorescence). Some of the slabs were not digested but fixed for measurement of leading-front distance. RIL-2-stimulated lymphocyte populations displayed greater motility than unstimulated populations; the mean leading front distance was 2.4 times greater, and the percentage of cells exhibiting motility was approximately doubled. The most motile RIL-2-stimulated cells, however, were not the most tumoricidal. Motile subpopulations displayed approximately 25 to 60% lower lymphokine-activated killer activity than did the total populations from which they were derived. Natural killer activity followed a similar pattern. Motile subpopulations contained a lower proportion of asialo-GM1+ and T-null cells than did total populations and a higher proportion of L3T4+ cells. Chemokinetic stimulation with alpha-interferon increased overall motility, but the lymphokine-activated killer activity of the motile subpopulation was still lower than that of the total population. Lymphocyte motility is important in the infiltration of tumors and other inflammatory lesions. The results indicate that the most tumoricidal lymphocytes in RIL-2-stimulated populations may not be the best tumor infiltrators, and that the tumoricidal activity of circulating lymphocytes may be a misleading indicator of the effectiveness of immunotherapy.

Relationships of prostaglandin E and natural killer sensitivity to metastatic potential in murine mammary adenocarcinomas.

The levels of two prostaglandins (prostaglandins E and F) have been determined in a series of murine mammary lesions ranging from preneoplastic, hyperplastic alveolar nodules to highly metastatic adenocarcinomas. A highly positive correlation was seen between high levels of prostaglandin E and high tumorigenicity and metastatic potential. In addition, spontaneous metastasis of two highly metastatic tumors was partially inhibited by p.o. administration of indomethacin from the time of s.c. tumor transplantation until removal of the primary tumor at a limited size. Further, mammary tumor cells of differing metastatic potential were susceptible to polyinosinic-polycytidylic acid activated spleen lymphocytes in vitro. Cells of metastatic tumor lines (410.4 and 66) were more resistant to killing than were cells of two non-metastatic tumor lines (168 and 410). The sensitivity of all target cells was increased when endogenous prostaglandin synthesis was prevented by the addition of indomethacin (1 microM) but was not affected by the lipoxygenase inhibitor nordihydroguaiaretic acid.

Correlation of frequency of induced mutation and metastatic potential in tumor cell lines from a single mouse mammary tumor.

Spontaneous mutation rates were determined in mouse mammary tumor subpopulation lines that differ in metastatic phenotype. Although there was almost a 9-fold difference in spontaneous rates to ouabain resistance among the three lines tested, the difference did not correlate with ability to metastasize. Similarly a 10-fold difference in spontaneous rates to 6-thioguanine resistance did not correlate with metastatic ability. In contrast, the frequency of ethyl methanesulfonate-induced mutations was associated with metastatic potential. Thus, ethyl methanesulfonate only induced significant numbers of 6-thioguanine resistant colonies in 66 and 410.4 cells, the only 2 of 5 lines tested that spontaneously metastasize at high frequency, and of ouabain resistant colonies in 66, 410.4, and 168 cells, the only lines tested that produce experimental lung metastases after i.v. injection. Differential sensitivity to induced mutation was not correlated with differences in plating efficiency, wild type sensitivity to ethyl methanesulfonate, 6-thioguanine, or ouabain toxicity, ploidy, cell shape, cell size, or ability to engage in metabolic cooperation.

Heterogeneity of expression and induction of mouse mammary tumor virus antigens in mouse mammary tumors.

Seven spontaneous BALB/cfC3H mouse mammary tumors were heterogeneous in expression of murine mammary tumor virus-associated cell surface antigens. To determine the basis of this heterogeneity, cells from spontaneous tumors and from five subpopulations isolated from a single spontaneous tumor were examined for expression of viral antigens under both conventional conditions and conditions known to induce synthesis of murine mammary tumor virus antigens (5-iodo-2'-deoxyuridine and dexamethasone). Induction with 5-iodo-2'-deoxyuridine resulted in further manifestation of the antigenic heterogeneity of spontaneous tumors. The five subpopulations from a single tumor differed in the amount of viral antigens present in untreated and in induced cultures. Coculturing showed that viral antigen expression was independent in each subpopulation within a heterogeneous mixture and was not influenced by the presence of other subpopulations with different potentials for viral antigen synthesis. The expression of murine mammary tumor virus structural antigens, a protein with a molecular weight of 28,000 and a glycoprotein with a molecular weight of 52,000, differed within the heterogeneous subpopulations, and was noncoordinate. The data suggest that the antigenic heterogeneity in spontaneous tumors reflects the existence of cells within them that differ in both expression of viral antigens and in their response to inducers of viral antigen synthesis.

Effect of sequence of administration of methotrexate, leucovorin, and 5-fluorouracil on mammary tumor growth and survival in syngeneic C3H mice.

The administration of methotrexate (1mg/kg), leucovorin (1 mg/kg), and (after a 1-hr interval) 5-fluorouracil (50 mg/kg) selectively suppresses antibody production in C3H mice without inhibiting, but even stimulating, cell-mediated immunity. The effect of this regimen, given at weekly intervals, was tested on the growth of recently arising syngeneic C3H/HeJ mammary tumors. Inhibition of growth was found in three types of experiments: (a) when treatment was begun 2 days after tumor implantation, (b) when it was begun 3 weeks after implantation, and (c) when it was begun after surgical enucleation of the tumor. The extent of the effect varied from tumor to tumor, but in all cases tumor incidence or growth was significantly inhibited. Comparison was made between the above regimen and two other sequences of administration of the same drugs, which are only weakly immunosuppressive of antibody production and which do not stimulate cell-mediated immunity. Survival of tumor-bearing mice was greater with the administration of methotrexate, then leucovorin, followed by 5-fluorouracil, than it was with the other two administration schedules.

Therapeutic perturbation of the tumor ecosystem in reconstructed heterogeneous mouse mammary tumors.

We have measured the response to methotrexate in vivo of paired mixtures of sister subpopulation lines from a mouse mammary tumor, as a model of drug response of a heterogeneous tumor. The subpopulation lines differed in intrinsic sensitivity to methotrexate. Response was measured both as growth delay and as a shift in tumor cell population distribution toward the more resistant cell line. We found differences between two pairs of cell lines in growth delay: line 66 plus 4T07 mixtures tended to be as responsive as was line 4T07 (the more sensitive line) alone, whereas line 168 plus 4T07 mixtures tended to be less responsive than line 4T07 alone. With both paired mixtures, the tumors arising after treatment tended to contain more line 66 or line 168 than did untreated tumors, but this shift was extremely variable among individual tumors. Within most treatment groups, there was no correlation between the growth rate of individual mixed tumors and the final tumor cell distribution. Likewise, between experiments, there was no correlation between the amount of growth delay in mixed tumors and the final tumor cell distribution. Thus, the cellular composition of treated tumors did not directly reflect the response to therapy.

Mutagenic activity of tumor-associated macrophages in Salmonella typhimurium strains TA98 and TA 100.

Suspensions of cells from a series of strain BALB/cfC3H mouse mammary tumors, and adherent and nonadherent cells from the tumors, were tested for their ability to increase the mutation rate of Salmonella typhimurium tester strains TA98 and TA100. Significant increases were seen with cells from three of four tumor lines tested on the TA98 strain and with one of four tested on the TA100 strain. The mutagenic activity was due primarily to cells in the adherent fractions which were greatly enriched for macrophages.

Associated effects of bromocriptine on neoplastic progression of mouse mammary preneoplastic hyperplastic alveolar nodule line C4 and on hyperplastic alveolar nodule-infiltrating and splenic lymphocyte function.

Progression of the mouse mammary preneoplastic hyperplastic alveolar nodule (HAN) line C4 to carcinoma can be enhanced by stimulators and depressed by inhibitors of host lymphocyte function (W.Z. Wei et al., Cancer Res., 49: 2709-2715, 1989). The purpose of the present study was to ask whether prolactin (PRL), a regulator of both mammary epithelial and lymphoid cells, might be a factor in the association between lymphocytic function and HAN progression. Daily administration of bromocriptine, a suppressor of pituitary PRL secretion, increased the latency period and decreased the incidence of tumor development in HAN bearing mice. Bromocriptine treatment suppressed in vitro responsiveness of HAN-infiltrating lymphocytes and, to some extent, spleen cells, to T- and B-cell mitogens, without altering the relative proportion of lymphocytic subsets. Suppression could be partially reversed by PRL treatment. Natural killer cell activity of HAN-infiltrating lymphocytes was also reduced by bromocriptine. In vitro incubation with anti-PRL antisera inhibited both lymphocyte mitogen responsiveness and natural killer activity in a concentration-dependent manner. PRL reversed this inhibition also. Altogether, these results demonstrate a correlation among tumor development, PRL levels, and lymphocyte function and suggest that an immune-endocrine network involving PRL may play a role in C4 HAN progression.

Preferential growth of mammary tumors in intact mammary fatpads.

Four transplantable tumors, three (66, 410, and 168cl) isolated from a spontaneously occurring strain BALB/cfC3H mammary tumor and one (D2) arising from a BALB/c hyperplastic alveolar nodule were found to grow better in mammary fatpads than at s.c. sites. Furthermore, tumor growth was better (p less than 0.05) in intact mammary glands than in cleared mammary fatpads for the D2, 410, and 66 tumors (168cl was not tested). The role of immunity in these differences was investigated using the highly immunogenic 410 tumors. Tumor 410 induced equally effective immunity to subsequent challenge whether it was implanted s.c. or in intact fatpads. Furthermore, in immunized animals, Tumor 410 was rejected equally well when the challenge site was intact fatpad as when s.c. Similarly, Tumor 410 induced immunity after implantation into cleared fatpads and, in immunized animals, was rejected when the challenge site was the cleared fatpad. We thus found no evidence that the mammary fatpad is immunologically privileged, as compared to the s.c. site, with respect to tumor transplantation antigens.

Factors affecting growth and drug sensitivity of mouse mammary tumor lines in collagen gel cultures.

A series of mouse mammary tumor subpopulation lines were compared for growth properties and sensitivity to chemotherapeutic drugs when grown as boluses in a collagen gel matrix versus in monolayer culture. Although the cell lines exhibited characteristic rates of bolus expansion in collagen, this growth was not paralleled by an exponential increase in cell number with time. Cell boluses contained a higher proportion of cells in G0-G1 phases of the cell cycle than did the same cell lines in monolayer cultures. Histological examination revealed areas of necrosis in boluses. Thus cells growing in collagen cultures resembled cells growing as solid tumors and cells from other three-dimensional culture systems. The growth of cell boluses in collagen gel cultures was reduced nonexponentially by melphalan, methotrexate, and 5-fluorouracil in contrast to the exponential decrease in growth measured in cloning assays. The lowest concentration to which cells first responded to drug was in general similar for collagen gel assays and for cloning assays. The rank order of sensitivity of different cell lines in the two assays was identical for methotrexate (four cell lines), similar for melphalan (four of five lines), but quite different for 5-fluorouracil. In contrast to cloning assays cell boluses continued to grow, albeit at a reduced rate, in the presence of high drug concentrations. This was not due to either diminished drug availability in collagen gel or drug penetration into the bolus.

Macrophage-mediated induction of drug-resistant variants in a mouse mammary tumor cell line.

The ability of macrophages to induce drug-resistant variants was studied in an in vitro macrophage-tumor cell coculture system utilizing the hypoxanthine-guanine phosphoribosyl transferase locus as measured by resistance to 6-thioguanine. Tumor cells of mouse mammary tumor line 66 were sensitive to macrophage induction of thioguanine resistance as shown by an increase in the frequency of thioguanine-resistant variants which arose following macrophage coculture to levels at least 5- to 10-fold above the spontaneous frequency. Detection of increased numbers of variants depended upon the macrophage:tumor cell ratio, with 50:1 or greater being necessary. The activity of the macrophages was dependent upon their activation stage. The induction of drug-resistant variants could be inhibited by oxygen radical scavengers. The basis for the emergence of thioguanine-resistant cells appeared to be induction of new variants rather than selection of preexisting resistant cells from the parental population, since thioguanine-sensitive and -resistant cells were equally sensitive to macrophage-mediated toxicity. In six of the six macrophage-induced variants tested, resistance was associated with loss of hypoxanthine-guanine phosphoribosyl transferase activity. The reverse variation frequency at the hypoxanthine-guanine phosphoribosyl transferase locus in five macrophage-induced variants was low and similar to that of a stable ethyl methanesulfonate-induced, thioguanine-resistant line. Macrophages isolated directly from growing mammary tumors, as well as activated peritoneal macrophages, were capable of inducing thioguanine resistance in line 66 cells.

Characterization of lymphocytic infiltrates in normal, preneoplastic, and neoplastic mouse mammary tissues.

Lymphocytic infiltrates were isolated from normal, preneoplastic, and neoplastic mouse mammary tissues. The surface markers on the infiltrating lymphocytes were characterized by immunofluorescent staining and flow cytometry. Preneoplastic and neoplastic tissues contained 10- to 20-fold more in situ lymphocytes than did the normal pregnant gland. Most of these lympocytes were T-cells. Relative to the T-cells in normal gland, the T-cells in C4 preneoplastic hyperplastic alveolar nodules and their spontaneous tumors have shifted in favor of the killer-suppressor subpopulation. This shift of T-cell subpopulations was a localized phenomenon and was not seen in the lymph nodes of hyperplastic alveolar nodules and tumor bearing mice. C4 lesion infiltrating cells also contained a subpopulation of lymphocytes that expressed 5- to 6-fold more LFA-1 antigen (lymphocyte function associated antigen-1) than did normal lymph node cells. The infiltrating lymphocytes of mammary tumors from cloned cell lines, on the contrary, had the same staining profile as did the lymphocytes from normal gland. Since most studies with human breast cancer infiltrates have demonstrated increased killer/suppressor T-cells and the presence of activated lymphocytes (J. Hurlimann and P. Saraga, Int. J. Cancer, 35: 753-762, 1985; H.L. Whitwell, H.P.A. Hughes, M. Moore, and A. Ahmed, Br. J. Cancer, 49: 161-172, 1984; and J.A. Ledbetter, R.V. Rouse, H. Spedding Micklem, and L. Herzenberg, J. Exp. Med., 152: 280-295, 1980) the C4 hyperplastic alveolar nodules and spontaneous tumor system may be a more relevant model for studying breast cancer infiltrates.