Cancer Immunol. lmmunother. 5, 145-147 (1978) a n e e r mmunolggyand mmunotherapy

© Springer-Verlag 1978

Cortisol Binding in Human Breast Cancer: Correlation with Antitumor Immunity

A. G. Fazekas, J. Marti, M. Flores, J. K. MacFarlane, and D. M. P. Thomson

Departments of Surgery and Immunology, McGill University, The Montreal General Hospital, Montreal, Canada

Summary. The intensity of cortisol binding was mea- sured in the cytosol fraction of the primary tumor ob- tained from 50 patients with stage I and H breast can- cer. The state of cellular antitumor immunity of the same patients was investigated by the tube leucocyte ad- herence inhibition (LAI) test, performed with peripheral blood leucocytes 1 -2 days preoperatively. It was found that the intensity of tumor cortisol binding correlates negatively with LAI values. Patients with high cortisol binding in their tumors have low LAI values, while low tumor cortisol binding is associated with higher antitu- mor immunity. The results suggest that high cortisol binding in the tumor might inhibit the tumor recognition process and~or the cellular immune defense mechanism and thus facilitate cancer development.

Studies performed in our laboratories revealed that the cytosols prepared from many human breast cancers dis- play high cortisol binding activity (Fazekas and Mac- Farlane, 1975). This binding is largely due to a macro- molecule that is remarkably similar to transcortin. It sediments at 4 S in the ultracentrifuge, has a dissociation constant (Ka) in the 10 -8 M range, and does not bind synthetic fluorinated corticosteroids (Fazekas ahd Mac- Farlane, 1977a). It is readily distinguished from a dexa- methasone-binding glucocorticoid receptor that is pres- ent in some tumors which has an 8 S sedimentation rate (Fazekas and MacFarlane, 1977a, b). Plasma transcor- tin as a source of cortisol binding activity could be ex- cluded (Fazekas and MacFarlane, 1977a) and the two macromoleeules were separated on Sephadex anion ex- change columns (Fazekas and MacFarlane, 1977c). This cortisol binder was detected in the culture media of human breast cancer cells grown in long term tissue cul- ture (Fazekas et al. 1978). It was also found that human

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breast cancer tissue contained elevated concentrations of endogenous cortisol as compared to normal breast (Fazekas and MacFarlane, 1977d).

Cortisol and its derivatives are effective suppres- sants of cellular immune reactions in both clinical and experimental situations (Fauci et al., 1976). Corticoste- roids may indirectly interfere with the antigen-process- ing functions of macrophages (DeSousa and Fachet, 1972). Moreover, recent evidence suggests that cortico- steroids suppress the recruitment of monocytes-macro- phages necessary for the expression of cellular immunity (Weston et al., 1973). Also, monocyte macrophage interaction with the soluble products of activated lymphocytes are antagonized by corticosteroids (Dimitriu, 1976).

The question arose, therefore, whether breast can- cers with high local levels of cortisol (or cortisol binding) might adversely affect the expression of antitumor im- munity in comparison to breast cancer with low cortisol levels. This paper reports a correlation between high cortisol binding in human mammary cancer tissue and depressed LAI reactivity, as expressed by the tube leu- kocyte adherence inhibition (LAI) assay (Grosser and Thomson, 1975). The cortisol binding and the tube LAI assays were performed independently in two separate laboratories by different persons in a double blind man- ner.

Methods

Tumor specimens were obtained from patients with histologically verified cancer of the breast (Stages I and II) undergoing breast biopsy or mastectomy. The presence of tumor tissue was verified in the specimens used by histological examination.

Measurement of Cortisol Binding Activity. Cytosols were prepared from fresh tissue specimens within minutes after surgery. Tissues were homogenized in Tris-EDTA-dithiothreitol (TED) buffer (McGuire and DeLaGarza, 1973) and centrifuged at 100,000 g for

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1 h. Aliquots of the cytosols (0.2 mi/tube) were incubated at 4 ° C for 16 h with [1, 2, 6, 7] 3H-tort±sol (spec. act. 91 Ci/mmol; 0.5 pmol/tube). Nonspecific binding of tort±sol was accounted for by performing the same incubation in the presence of an excess of non- radioactive cortisol (blanks). Incubations were carried out in dupli- cate. After incubation, bound and free fractions were separated by dextran coated charcoal. Charcoal was sedimented by centrifuga- tion and supernatants were counted for radioactivity. The cortisol binding index (CBI) was calculated as follows: CBI (DPM Bound Cortisol-Blank DPM)/mg cytosol protein/100. The protein content of the cytosols was measured by the method of Lowry et al. (1951).

Endogenous tort±sol in the cytosol of tumors and adjacent nor- mal breast tissue was measured by radioimmunoassay, using the antiserum and procedure of New England Nuclear Corp., Boston, Massachusetts. Results were expressed as ng cortisol/g weight tissue. Regarding assay accuracy, the coefficient of variation of ten repli- cate cortisol determinations on a cytosol sample was 9.3%. The in- traassay variability was 7.2%. The starting amount of tissue was between 0.3 and 1.5 g, homogenized in 9-18 ml TED buffer.

For the tube LAI assay, washed buffy coat peripheral blood leukocytes (PBL) from patients with breast cancer were incubated in glass test tubes with a single breast tumor extract and control ex- tracts (Grosser and Thomson, 1975) in medium 199 at 37 ° C in a 5% CO2 humidified atmosphere. After 2 h of incubation the number of nonadherent cells was determined in a hemocytometer. Sensitized PBL of patients with breast cancer showed nonadherence in the pres- ence of breast cancer extract, but not when incubated with unrelated tumor extracts or normal breast tissue. Results were expressed as a nonadherence index (NAI).

Nonadherent cells with - Nonadherent cells with specific antigen nonspecific antigen

N A I - x 100. Nonadherent cells with nonspecific antigen

The LAI reactivity of the breast cancer patients was tested against a single allogeneic breast cancer extract, but not against primary tu- mors that had eortisol binding activity measured.

Results

The linear regression analysis of cortisol binding and

NAI values is illustrated in Fig. 1. The analysis was performed with data obtained from 50 unselected pa- tients with Stage I and Stage II carcinoma of the breast. The cortisol binding assays were carded out on the pri- mary tumors, while the tube LAI test was performed 1 - 2 days prior to the operation with buffy coat periph- eral blood leukocytes obtained from the patients. The results of the comparison of these two independent as- says suggest that breast tumors with high cortisol bind-

ing activity were associated with a depressed expression of LAI reactivity in these patients, as assayed by in vitro tube LAI. Conversely, low tort±sol binding in the tu- mors was associated with higher levels of LAI reactivity.

When levels of endogenous cortisol were assayed in the tumors and the adjacent normal breast tissue of 12 patients, the levels in the tumors were found to be gener- ally higher. Also, tumor cortisol binding values as ex- pressed by CBI and endogenous cortisol levels were positively correlated. Table 1 meaning that higher bind-

A. G. Fazekas et al.: Cortisol Binding in Human Breast Cancer

NA~ 150 "

130 -

110 i0 • 9O

• 00 • •

7O

50 ~ "

10 o . . . . . . . . . . . . . . . . . " _ - _ - : - , . . . .

1oo 2oo 3oo 4oo CBI

Fig. 1. Linear regression correlation of NAI and CBI values from 50 patients with Stage I-II breast cancer. (o~ = 60.78;/~ = -0.14; r = -0.48; N = 50; P < 0,001)

Table 1. The concentration of endogenous cortisol and cortisol bind- ing index (CBI) in human breast carcinoma and adjacent normal breast tissue (ANB)

Case Cortisol (ng/g tissue) CBI no.

Tumor ANB Tumor ANB

32 51.0 43.0 145 90 36 27.5 23.2 258 166 39 28.3 8.6 260 49 44 14.8 5.1 175 20 53 34.8 16.3 210 121 55 31.9 20.9 146 181 67 18.8 6.7 216 59 76 56.6 18.0 139 88 80 36.1 18.8 105 37 90 14.7 12.2 165 125

114 135.9 22.9 89 23 117 44.0 19.2 254 41

Means±SEM 41.2±9.4 17.9±2.8 180±17 83±15

Cortisol was measured in tumor cytosols by radioimmunoassay. Values are expressed as ng/g tissue weight. Specific binding of tort±- sol (CBI) expressed as described at Fig. 1 Differences between tumor and ANB values are statistically signifi- cant Pcortisol < 0.02; P CBI <( 0.001 Tumor cortisol binding and endogenous cortisol content are posi- tively correlated (r = 0.40; P < 0.05)

ing activity is associated with higher cortisol levels in tumor tissue. Cortisol binding, measured in three benign lesions (fibroadenoma) of the breast, was found to be

low (CBI: 19, 23, and 79).

Discussion

Leukocyte adherence inhibition (LAI) is an assay based on the finding that nonsensitized leukocytes from both

A. G. Fazekas et al.: Cortisol Binding in Human Breast Cancer 147

cancer patients and control subjects adhere to glass sur- faces, whereas leukocytes from cancer patients but not from control subjects, when mixed in vitro with antigen- ic extracts of tumors of the same histologic type, under- go a diminution in their normal adherence to glass sur- faces (Halliday and Miller, 1972), By the tube LAI assay adapted and modified in this laboratory, we showed that LAI assays measure one aspect of the ex- pression of antitumor immunity (Grosser and Thomson, 1975). The phenomenon was shown to be mediated by circulating monocytes armed with cytophilic antitumor antibody (Grosser et al., 1976). Monocytes of patients with early cancer, in general, showed L AI reactivity, whereas monocytes of patients with advanced cancer were unreactive (Grosser and Thomson, 1976).

The mechanism by which tumors with high cortisol binding activity depress L AI reactivity is unclear. Possi- bly, a milieu rich in cortisol binding protein(s) within the cancer cell leads to high free cortisol levels in the micro- environment of the tumor which is then available to in- teract with the cells of the host essential for the initiation of an immune response.

Stress associated with surgery produces high sys- temic levels of corticosteroids, and this was observed to depress LAI reactivity (Flores et al., 1976). Similarly, cortisol, added in vitro to the L AI test, had an inhibitory effect on the reaction at levels of cortisol (18 and 36 p,g/100 ml) associated with stress (Flores et al., 1976). These levels were higher, but comparable to endogenous levels of cortisol found in human breast cancer tissue (Fazekas and MacFarlane, 1977d) (0 .2-13.6 p,g/100 g). Also, Riley (1975) observed that stress increased the risk o f mammary cancer in mice. Again, this stress may result in increased eorticosteroid output, high systemic plasma concentrations that could adversely impair host immune defenses. Recently, Amaral and Werthamer (1976) described the presence of transcortin-like mate- rial in human breast cancer, and have shown that it inhibited the phytohemagglutinin-induced stimulation of D N A synthesis of cultured T-lymphocytes in vitro.

The results of the present study suggest that local elevation o f cortisol (coupled to its binding proteins) within tumors may be associated with the depression of the development and expression of antitumor immunity. Ectopic production of hormones and fetal macromole- cules by a large variety of tumors is well known (Neville and Symington, 1975). Detection of specific cortisol- binding macromolecules in human breast cancer, and their effect on systemic antitumor immunity, may intro- duce a new aspect into the endocrine and immunological relationship of human neoplasms.

Acknowledgements: This research was supported by the Medical Re- search Council of Canada, the National Cancer Institute of Canada and the Cancer Research Society, Inc. of Montreal.

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Received April 13, 1977/In amended form May 30, 1978/Accepted July 13, 1978