Clin. exp. Immunol. (1975) 22, 126-132
FORMALINIZED TUMOUR CELLS IN THE LEUCOCYTE MIGRATION INHIBITION TEST C. E. ROSS, A. J. COCHRAN, D. E. HOYLE, R. M. GRANT AND R. M. MACKIE University Departments of Pathology and Dermatology, The Western Infirmary, Glasgow (Received 2 January 1975) SUMMARY
Immunization studies indicate that formalinized tumour cells retain at least part of their gross membrane structure and antigenicity; they are relatively easy to prepare and store well over a period of months. We have used formalinized tumour cells as antigen in the leucocyte migration inhibition test. Leucocyte migration inhibition occurred in leucocytes from thirtyeight out of sixty-nine malignant melanoma patients and from fifty-five out of eightyone breast cancer patients when in contact with formalinized tumour cells of a histologically similar type. Melanoma patients' and breast cancer patients' leucocytes were infrequently inhibited on contact with cells from histologically dissimilar human tumours and from xenogeneic mouse melanomas. Other advantages of formalinized cells over antigens prepared by homogenizing tumour tissue include a greater degree of inhibition and the ability to demonstrate a dose-response relationship between the ratio of leucocytes: tumour cells and the migration index. INTRODUCTION The leucocyte migration inhibition test (LMIT) (S0borg & Bendixen, 1967) was adapted from the macrophage migration technique (George & Vaughan, 1962) and there is increasing evidence that it correlates with cell-mediated immunity (CMI) in man. Most studies of tumour directed CMI in man by the LMIT have employed the supernatants of homogenized, fresh tumour tissue as 'antigens' (Andersen et al., 1970; Cochran et al., 1972; Segall et al., 1972; Mackie et al., 1972; Cochran et al., 1974). Such preparations inhibit the migration of leucocytes from a majority of patients with histogenetically similar tumours and frequently inhibit leucocytes from control donors (Mackie et al., 1972; Cochran et al., 1974). However, this type of 'antigen' may be non-specifically toxic at high concentrations (Wolberg, 1971) and the degree of inhibition at non-toxic levels is relatively low, the migration areas of inhibited cultures being only 20-30% less than those of non-inhibited control cultures. In addition dose-response curves are not consistently demonstrable and we have therefore sought more effective methods of antigen preparation. Correspondence: Dr C. E. Ross, Pathology Department, Western Infirmary, Glasgow, GIl 6NT.
126
Formalinized cells in the migration test
127
Various workers (Kronman et al., 1969; Churchill et al., 1972; Cerilli, Smith & Hattan, 1972; Anderson et al., 1973) have used intact cells in migration studies, but fresh cells have a limited availability, especially in studies of human tumours and may themselves be migratory (Cochran, 1971), complicating the assessment of tumour cell-leucocyte interaction. Cells stored deep-frozen in dimethyl sulphoxide have been employed (Anderson et al., 1973) but have low viability on thawing and require considerable time and moderately expensive apparatus for storage and recovery. Drake, Ungaro & Mardiney (1972) fixed EL-4 murine lymphoma cells with formalin and, in immunization studies, found them to be as antigenic and immunogenic as fresh tumour cells. This apparent preservation of membrane antigens suggested to us that formalin-fixed cells might be suitable for use in the LMIT.
MATERIALS AND METHODS Reagents. 10% buffered formalin: pH 7-2. 100 ml 40%. formaldehyde, 4 g NaH2 P04H20 and 6-5
g
Na2HPO4 were made up to 1 litre. Cellpreparation. Fresh tumour tissue, freed as far as possible from fat and stroma, was dispersed by finely chopping in physiological saline. The suspension was then filtered through 49-gauge stainless steel mesh. The resulting cells were washed with 0-850% NaCl solution and spun at 400 g for 15 min. Twenty-five volumes of 10% buffered formalin were added to the packed cells which were resuspended and left at room temperature (approx. 20'C) for at least 12 hr. The cells were washed twice with 0-85% NaCl solution and stored at 40C until required. LMIT. Human peripheral blood leucocytes, prepared as previously described (Cochran et al., 1972) were added to the formalinized cells at ratios of 100:1, 250:1, 500:1 and 1000:1 (leucocytes: tumour cells). Capillaries were filled with the cell mixture, sealed at one end with inert clay and spun at 200g for 5 min. The cell buttons were cut with a diamond at the cell-fluid interface and mounted horizontally in a spot of silicone grease on the base of disposable tissue culture plates. The wells were filled with Eagle's medium+ 100% FCS, closed with a coverslip and the completed plates incubated in air at 370C for 18-24 hr. Four capillaries were prepared for each leucocyte-tumour cell combination. The areas of cell migration were drawn using a drawing tube attached to a light microscope and measured by planimetry. The migration index was calculated by dividing the mean area of migration of test leucocytes + tumour cells by the mean area migration of leucocytes alone or of leucocytes mixed with tumour cells of a histogenetically different type to that of the leucocyte donor. Significance at the 5%Y level was assessed by the Mann-Whitney-Wilcoxon U test of ranking.
RESULTS (a) Malignant melanoma patients (Tables and 2) Sixty-nine patients with malignant melanoma were tested against at least one and up to five of six melanoma cell suspensions. The leucocytes of thirty-eight of the sixty-nine patients (550%) showed significant inhibition of migration on contact with melanoma cells. The leucocytes of nine of sixty-one members of the control group (15%) normal individuals and patients with other malignancies, were inhibited by melanoma cells. The above tests on melanoma patients employed homologous combinations of leucocytes and tumour cells: however, a similar proportion of autologous combinations showed migration inhibition, the leucocytes of six of ten melanoma patients being inhibited by their own tumour cells. When mouse melanoma cell suspensions (S91 and B16) were used as a specificity control only two of seven melanoma patients' leucocytes were inhibited. Two of ten melanoma patients' leucocytes and none of three normal donors' leucocytes showed migration inhibition when in contact with formalinized normal liver cells. 1
128
C. E. Ross et al.
TABLE 1. The effect of formalinized tumour cells on the migration of leucocytes from cancer patients and control donors Leucocyte donor
Melanoma patients Melanoma patients Control donors Breast cancer patients Breast cancer patients Control donors Melanoma patients Melanoma patients Normal donors
Formalinized cell
No. positive Percentage positive No. tested
Melanoma* Melanomat Melanoma*
38/69 6/10 4/31
55 60 13
Melanoma*
5/30
17
55/81 16/46 8/25 2/10 0/3
68 34 32 20
Breast cancer* Breast cancer* Breast cancer* Normal liver* Normal liver*
Homologous tumour cells. t Autologous tumour cells. *
TABLE 2. The effect of formalinized mouse melanoma cells on the migration of leucocytes from patients with malignant melanoma and breast carcinoma
Significant* migration inhibition at: Leucocyte donor
Melanoma patients Breast cancer patients
Melanoma patients Breast cancer patients *
Formalinized cells 100:1 S91 S91 B16 B16
2/5 0/1 0/2 0/4
200:1
500:1
1/4
0/5
1/4 0/7
0/3 0/7
Significance assessed by the Mann-Whitney-Wilcoxon U
test of ranking.
129
Formalinized cells in the migration test
When high numbers of inert cells are present (10-50 leucocytes: 1 tumour cell) non-specific inhibition occurs regardless of the source of leucocytes (Table 3). The ratio of motile leucocytes to inert cells is therefore important and the optimum ratio for each cell preparation should be assayed. Most of the specific inhibitions occurred at the ratios 100: 1 and 200: 1 and at these ratios the greatest difference between the test and control situations is seen (Fig. 1). A dose response curve was usually demonstrable and Fig. 2 shows the results obtained with one antigen when tested against forty-seven melanoma patients' and eighteen control donors' leucocytes. 60
I.0 40
0)
i
0
t
S
Fi20g.
B o sV+1 1
n~~~~~~~~~~~
C0 -0:
0:
0:
E
T. 2 X
1001
001
201
501
10
FIG. 1. The effect of exposing leucocytes from melanoma patients to formalinized melanoma cells at different ratios. Melanoma patients, hatched column; control donors, open column. FIG. 2. The effect of exposing leucocytes from forty-seven melanoma patients (continuous line) and eighteen control donors (dashed line) to formalinized melanoma cells from patient Bl. Dose-response curve.
(b) Breast carcinoma patients (Tables 1 and 2) Leucocytes from eighty-one patients with breast carcinoma were tested against formalinized breast carcinoma cells. Fifty-five of these patients' leucocytes (67%) showed significant migration inhibition on contact with at least one such cell suspension. However, twenty-four of seventy-one control donors' leucocytes (34%/) also showed migration inhibition when tested with the same breast cancer cell preparations. Breast carcinoma patients' leucocytes were infrequently inhibited by human melanoma-derived cells (5/30-l17%) and were not inhibited by mouse melanoma cells (0/5). (c) Comparison offormalinized cells and supernatant antigens (Table 4) Formalinized cells consistently produced greater inhibition of leucocyte migration than did supernatant antigens prepared from the same tumour. Table 4 gives two examples typical of the results obtained.
130
C. E. Ross et al.
TABLE 3. The effect of a high ratio of non-migrating: migrating cells on the leucocyte migration test
Leucocyte donor
Formalinized cells
J.L. (breast cancer)
M.I. (breast cancer)
B. (normal control)
M.I. (breast cancer)
*
Ratio leucocytes: tumour Migration cells index 100:1 50:1 20:1 10:1
0 95 0-71* 0-45* 0-28*
100:1 50:1 20:1 10:1
0-83 0-86 0.55* 0-48*
P< 005 using Mann-Whitney-Wilcoxon U test of ranking.
TABLE 4. A comparison of the effect of formalinized cells and supernatant antigen, from the same tumour specimen, on the migration of leucocytes from patients with malignant melanoma and breast carcinoma
Leucocyte: tumour cell Leucocyte ratio donor McA. D.
100:1 500:1 1000:1 100:1 500:1 1000:1
Migration index (MI) 0-76* 0-76* 0 90
0.35* 0.65* 0.70*
Antigen protein
(pug) 100 50 25 100 50 25
Migration index 0.91
0.79* 0-83 0-83 0-80 10
* P< 005 using Mann-Whitney-Wilcoxon U test of ranking.
Formalinized cells in the migration test
131
DISCUSSION These results show a distinct difference in the frequency of migration inhibition of leucocytes from patients with malignant melanoma and control donors' leucocytes when exposed to formalinized melanoma cells. Control leucocytes were infrequently inhibited by melanoma cells and the majority of such inhibitions occurred when the number of tumour cells present was high. HL-A antigens are unlikely to be the cause of the inhibitions seen in this study as inhibition occurred in a similar proportion of autologous and homologous tests. Furthermore, fixation studies have shown that when Moloney leukaemia virus (MLV) induced murine lymphoma cells are fixed in formalin for over 2 hr their H-2a antigenicity is markedly reduced while MLV related antigens are relatively well preserved (Gatti, Ostborn & Fagraeus, 1974). Advantages of using fixed cells as antigen in the LMIT are the greater inhibition obtained as compared to that induced by supernatant antigen, and the dose-related response observed in the majority of tests. A further advantage is that the membrane remains physically intact during formalinization and it is therefore the plasma membrane surface antigens which are presented to the leucocytes. These antigens seem likely to include biologically active tumour associated antigens (TAA). In contrast when tumour cells are disrupted for antigen preparation the content of the supernatant is largely unknown unless differential centrifugation is carried out. Different formalinized cell suspensions varied considerably in their ability to cause migration inhibition. This may reflect low antigenicity of the tumour, or the variable presence of substances masking the antigens of the cell surface. Substances such as adsorbed Ig or endogenous sialic acid present on the cell surface may prevent sensitized lymphocytes from recognizing and reacting with tumour-associated antigens. Problems have been encountered using cell suspensions derived from breast carcinoma. Although a high number of breast cancer patients showed migration inhibition the frequency of inhibition of control leucocytes was also fairly high. Several possibilities were considered to explain these findings. First, formalin released by the tumour cells might be toxic to the leucocytes and thus abolish their motility, although this would seem unlikely in view of the results obtained in the melanoma group. However, after repeated washing ( x 4) of the formalinized tumour cells, some inhibition of control leucocytes was still seen. Clumping of cells was also considered, but even after several months of storage very few clumps were present and no clumps contained more than eight to ten cells. The frequency of clumps did not correlate with the occurrence of 'non-specific' inhibition. The most probable explanation is that it is more difficult to obtain a clean, homogeneous cell suspension from breast cancer tissue than from melanoma tissue. The fibrous nature of the stroma of most breast cancers results in a suspension which contained a mixture of cell types and this will therefore constitute a less specific antigen. Dispersal of cells by mechanical means may also result in cell surface damage, affecting the antigenicity of the cells. However, fixed tumour cells appear to retain specific antigenicity and with improved methods of preparation they may be useful in studies of human tumour immunology. Such cells could be used as sources of specific antigen in lymphocyte transformation techniques. In studies of humoral immunity formalinized cells could perhaps be used as target cells in membrane immunofluorescence, and immune adherence tests.
132
C. E. Ross et al.
The mechanism of the leucocyte migration test is not fully elucidated, but if properly controlled enough evidence exists to indicate that it is a useful in vitro method of detecting CMI (Cochran et al., 1973). The study described here does not attempt to define the mechanism of leucocyte migration inhibition mediated by formalin-fixed intact tumour cells. Studies designed to elucidate this further are in progress. This study was conducted with the assistance of grants from the Secretary of State for Scotland and the McMillan Research Funds of the University of Glasgow.
REFERENCES ANDERSEN, V., BJERRUM, O., BENDIXEN, G., SCHI0DT, RUSSELL, J.M. (1974) Sensitisation to tumourT. & DISSING, I. (1970) Effect of autologous associated antigens in human breast carcinoma. mammary tumour extracts on human leucocytes Int. J. Cancer, 14, 19. migration in vitro. Int. J. Cancer, 5, 357. DRAKE, W.P., UNGARO, P.C. & MARDINEY, M.R. ANDERSON, J.M., KELLY, F., WOOD, S.E., RODGER, (1972) Preservation of cellular antigenicity of K.D. & FRESNEY, R.I. (1973) Evaluation of tumour cells by the use of formalin fixation. leucocyte functions six years after tumour autoCancer Res. 32, 1042. graft in human mammary cancer. Brit. J. Cancer, GATrI, R.A., OSTBORN, A., FAGRAEUS, A. (1974) 28, supplement 1, 83. Selective impairment of cell antigenicity by fixaCERIHLI, J., SMITH, M.C. & HATTAN, D. (1972) The tion. J. Immunol. 113, 1361. detection of cellular immunity to tumour cells by GEORGE, M. & VAUGHAN, J.H. (1962) In vitro cell the macrophage migration technique. Transmigration as a model for delayed hypersensitivity. plantation, 14, 125. Proc. Soc. exp. Biol. (N. Y.), 111, 514. CHURCHILL. W.H., ZBAR, B., BELLI, J.A. & DAVID, KRONMAN, B.S., WEPSIC, H.T., CHURCHILL, W.H., J.R. (1972) Detection of cellular immunity to ZBAR, B., BORSOS, T. & RAPP, H.J. (1969) Tumour tumour antigens of a guinea-pig hepatoma by specific antigens detected by inhibition of macroinhibition of macrophage migration. J. nat. Cancer phage migration. Science, 165, 296. Inst. 48, 541. MACKIE, R.M., SPILG, W.G.S., THOMAS, C.E. & COCHRAN, A.J. (1971) Tumour cell migration. Europ. COCHRAN, A.J. (1972) Cell mediated immunity in J. clin. biol. Res. 16, 44. patients with malignant melanoma. Brit. J. Derm. COCHRAN, A.J., SPILG, W.G.S., MACKIE, R.M. & 87, 523. THOMAS, C.E. (1972) Postoperative depression of SEGALL, A., WEILER, O., GENIN, J., LACOUR, J. & cell mediated immunity in patients with malignant LACOUR, F. (1972) In vitro study of cellular disease. Brit. med. J. iv, 67. immunity against autochthonous human cancer. COCHRAN, A.J., MACKIE, R.M., THOMAS, C.E., Int. J. Cancer, 9, 417. GRANT, R.M., CAMERON-MOWAT, D.E. & SPILG, S0BORG, M. & BENDIXEN, G. (1967) Human lymphoW.G.S. (1973) Cellular immunity to breast carcyte migration as a parameter of hypersensitivity. cinoma and malignant melanoma. Brit. J. Cancer, Acta med. scand. 181, 247. 28, supplement 1, 77. WOLBERG, W.H. (1971) Inhibition of migration of human autogenous and allogeneic leukocytes by COCHRAN, A.J., GRANT, R.M., SPILG, W.G., MACKIE, R.M., Ross, C.E., HOYLE, D.E. & extracts of patients' cancers. Cancer Res. 31, 798.
FORMALINIZED TUMOUR CELLS IN THE LEUCOCYTE MIGRATION INHIBITION TEST C. E. ROSS, A. J. COCHRAN, D. E. HOYLE, R. M. GRANT AND R. M. MACKIE University Departments of Pathology and Dermatology, The Western Infirmary, Glasgow (Received 2 January 1975) SUMMARY
Immunization studies indicate that formalinized tumour cells retain at least part of their gross membrane structure and antigenicity; they are relatively easy to prepare and store well over a period of months. We have used formalinized tumour cells as antigen in the leucocyte migration inhibition test. Leucocyte migration inhibition occurred in leucocytes from thirtyeight out of sixty-nine malignant melanoma patients and from fifty-five out of eightyone breast cancer patients when in contact with formalinized tumour cells of a histologically similar type. Melanoma patients' and breast cancer patients' leucocytes were infrequently inhibited on contact with cells from histologically dissimilar human tumours and from xenogeneic mouse melanomas. Other advantages of formalinized cells over antigens prepared by homogenizing tumour tissue include a greater degree of inhibition and the ability to demonstrate a dose-response relationship between the ratio of leucocytes: tumour cells and the migration index. INTRODUCTION The leucocyte migration inhibition test (LMIT) (S0borg & Bendixen, 1967) was adapted from the macrophage migration technique (George & Vaughan, 1962) and there is increasing evidence that it correlates with cell-mediated immunity (CMI) in man. Most studies of tumour directed CMI in man by the LMIT have employed the supernatants of homogenized, fresh tumour tissue as 'antigens' (Andersen et al., 1970; Cochran et al., 1972; Segall et al., 1972; Mackie et al., 1972; Cochran et al., 1974). Such preparations inhibit the migration of leucocytes from a majority of patients with histogenetically similar tumours and frequently inhibit leucocytes from control donors (Mackie et al., 1972; Cochran et al., 1974). However, this type of 'antigen' may be non-specifically toxic at high concentrations (Wolberg, 1971) and the degree of inhibition at non-toxic levels is relatively low, the migration areas of inhibited cultures being only 20-30% less than those of non-inhibited control cultures. In addition dose-response curves are not consistently demonstrable and we have therefore sought more effective methods of antigen preparation. Correspondence: Dr C. E. Ross, Pathology Department, Western Infirmary, Glasgow, GIl 6NT.
126
Formalinized cells in the migration test
127
Various workers (Kronman et al., 1969; Churchill et al., 1972; Cerilli, Smith & Hattan, 1972; Anderson et al., 1973) have used intact cells in migration studies, but fresh cells have a limited availability, especially in studies of human tumours and may themselves be migratory (Cochran, 1971), complicating the assessment of tumour cell-leucocyte interaction. Cells stored deep-frozen in dimethyl sulphoxide have been employed (Anderson et al., 1973) but have low viability on thawing and require considerable time and moderately expensive apparatus for storage and recovery. Drake, Ungaro & Mardiney (1972) fixed EL-4 murine lymphoma cells with formalin and, in immunization studies, found them to be as antigenic and immunogenic as fresh tumour cells. This apparent preservation of membrane antigens suggested to us that formalin-fixed cells might be suitable for use in the LMIT.
MATERIALS AND METHODS Reagents. 10% buffered formalin: pH 7-2. 100 ml 40%. formaldehyde, 4 g NaH2 P04H20 and 6-5
g
Na2HPO4 were made up to 1 litre. Cellpreparation. Fresh tumour tissue, freed as far as possible from fat and stroma, was dispersed by finely chopping in physiological saline. The suspension was then filtered through 49-gauge stainless steel mesh. The resulting cells were washed with 0-850% NaCl solution and spun at 400 g for 15 min. Twenty-five volumes of 10% buffered formalin were added to the packed cells which were resuspended and left at room temperature (approx. 20'C) for at least 12 hr. The cells were washed twice with 0-85% NaCl solution and stored at 40C until required. LMIT. Human peripheral blood leucocytes, prepared as previously described (Cochran et al., 1972) were added to the formalinized cells at ratios of 100:1, 250:1, 500:1 and 1000:1 (leucocytes: tumour cells). Capillaries were filled with the cell mixture, sealed at one end with inert clay and spun at 200g for 5 min. The cell buttons were cut with a diamond at the cell-fluid interface and mounted horizontally in a spot of silicone grease on the base of disposable tissue culture plates. The wells were filled with Eagle's medium+ 100% FCS, closed with a coverslip and the completed plates incubated in air at 370C for 18-24 hr. Four capillaries were prepared for each leucocyte-tumour cell combination. The areas of cell migration were drawn using a drawing tube attached to a light microscope and measured by planimetry. The migration index was calculated by dividing the mean area of migration of test leucocytes + tumour cells by the mean area migration of leucocytes alone or of leucocytes mixed with tumour cells of a histogenetically different type to that of the leucocyte donor. Significance at the 5%Y level was assessed by the Mann-Whitney-Wilcoxon U test of ranking.
RESULTS (a) Malignant melanoma patients (Tables and 2) Sixty-nine patients with malignant melanoma were tested against at least one and up to five of six melanoma cell suspensions. The leucocytes of thirty-eight of the sixty-nine patients (550%) showed significant inhibition of migration on contact with melanoma cells. The leucocytes of nine of sixty-one members of the control group (15%) normal individuals and patients with other malignancies, were inhibited by melanoma cells. The above tests on melanoma patients employed homologous combinations of leucocytes and tumour cells: however, a similar proportion of autologous combinations showed migration inhibition, the leucocytes of six of ten melanoma patients being inhibited by their own tumour cells. When mouse melanoma cell suspensions (S91 and B16) were used as a specificity control only two of seven melanoma patients' leucocytes were inhibited. Two of ten melanoma patients' leucocytes and none of three normal donors' leucocytes showed migration inhibition when in contact with formalinized normal liver cells. 1
128
C. E. Ross et al.
TABLE 1. The effect of formalinized tumour cells on the migration of leucocytes from cancer patients and control donors Leucocyte donor
Melanoma patients Melanoma patients Control donors Breast cancer patients Breast cancer patients Control donors Melanoma patients Melanoma patients Normal donors
Formalinized cell
No. positive Percentage positive No. tested
Melanoma* Melanomat Melanoma*
38/69 6/10 4/31
55 60 13
Melanoma*
5/30
17
55/81 16/46 8/25 2/10 0/3
68 34 32 20
Breast cancer* Breast cancer* Breast cancer* Normal liver* Normal liver*
Homologous tumour cells. t Autologous tumour cells. *
TABLE 2. The effect of formalinized mouse melanoma cells on the migration of leucocytes from patients with malignant melanoma and breast carcinoma
Significant* migration inhibition at: Leucocyte donor
Melanoma patients Breast cancer patients
Melanoma patients Breast cancer patients *
Formalinized cells 100:1 S91 S91 B16 B16
2/5 0/1 0/2 0/4
200:1
500:1
1/4
0/5
1/4 0/7
0/3 0/7
Significance assessed by the Mann-Whitney-Wilcoxon U
test of ranking.
129
Formalinized cells in the migration test
When high numbers of inert cells are present (10-50 leucocytes: 1 tumour cell) non-specific inhibition occurs regardless of the source of leucocytes (Table 3). The ratio of motile leucocytes to inert cells is therefore important and the optimum ratio for each cell preparation should be assayed. Most of the specific inhibitions occurred at the ratios 100: 1 and 200: 1 and at these ratios the greatest difference between the test and control situations is seen (Fig. 1). A dose response curve was usually demonstrable and Fig. 2 shows the results obtained with one antigen when tested against forty-seven melanoma patients' and eighteen control donors' leucocytes. 60
I.0 40
0)
i
0
t
S
Fi20g.
B o sV+1 1
n~~~~~~~~~~~
C0 -0:
0:
0:
E
T. 2 X
1001
001
201
501
10
FIG. 1. The effect of exposing leucocytes from melanoma patients to formalinized melanoma cells at different ratios. Melanoma patients, hatched column; control donors, open column. FIG. 2. The effect of exposing leucocytes from forty-seven melanoma patients (continuous line) and eighteen control donors (dashed line) to formalinized melanoma cells from patient Bl. Dose-response curve.
(b) Breast carcinoma patients (Tables 1 and 2) Leucocytes from eighty-one patients with breast carcinoma were tested against formalinized breast carcinoma cells. Fifty-five of these patients' leucocytes (67%) showed significant migration inhibition on contact with at least one such cell suspension. However, twenty-four of seventy-one control donors' leucocytes (34%/) also showed migration inhibition when tested with the same breast cancer cell preparations. Breast carcinoma patients' leucocytes were infrequently inhibited by human melanoma-derived cells (5/30-l17%) and were not inhibited by mouse melanoma cells (0/5). (c) Comparison offormalinized cells and supernatant antigens (Table 4) Formalinized cells consistently produced greater inhibition of leucocyte migration than did supernatant antigens prepared from the same tumour. Table 4 gives two examples typical of the results obtained.
130
C. E. Ross et al.
TABLE 3. The effect of a high ratio of non-migrating: migrating cells on the leucocyte migration test
Leucocyte donor
Formalinized cells
J.L. (breast cancer)
M.I. (breast cancer)
B. (normal control)
M.I. (breast cancer)
*
Ratio leucocytes: tumour Migration cells index 100:1 50:1 20:1 10:1
0 95 0-71* 0-45* 0-28*
100:1 50:1 20:1 10:1
0-83 0-86 0.55* 0-48*
P< 005 using Mann-Whitney-Wilcoxon U test of ranking.
TABLE 4. A comparison of the effect of formalinized cells and supernatant antigen, from the same tumour specimen, on the migration of leucocytes from patients with malignant melanoma and breast carcinoma
Leucocyte: tumour cell Leucocyte ratio donor McA. D.
100:1 500:1 1000:1 100:1 500:1 1000:1
Migration index (MI) 0-76* 0-76* 0 90
0.35* 0.65* 0.70*
Antigen protein
(pug) 100 50 25 100 50 25
Migration index 0.91
0.79* 0-83 0-83 0-80 10
* P< 005 using Mann-Whitney-Wilcoxon U test of ranking.
Formalinized cells in the migration test
131
DISCUSSION These results show a distinct difference in the frequency of migration inhibition of leucocytes from patients with malignant melanoma and control donors' leucocytes when exposed to formalinized melanoma cells. Control leucocytes were infrequently inhibited by melanoma cells and the majority of such inhibitions occurred when the number of tumour cells present was high. HL-A antigens are unlikely to be the cause of the inhibitions seen in this study as inhibition occurred in a similar proportion of autologous and homologous tests. Furthermore, fixation studies have shown that when Moloney leukaemia virus (MLV) induced murine lymphoma cells are fixed in formalin for over 2 hr their H-2a antigenicity is markedly reduced while MLV related antigens are relatively well preserved (Gatti, Ostborn & Fagraeus, 1974). Advantages of using fixed cells as antigen in the LMIT are the greater inhibition obtained as compared to that induced by supernatant antigen, and the dose-related response observed in the majority of tests. A further advantage is that the membrane remains physically intact during formalinization and it is therefore the plasma membrane surface antigens which are presented to the leucocytes. These antigens seem likely to include biologically active tumour associated antigens (TAA). In contrast when tumour cells are disrupted for antigen preparation the content of the supernatant is largely unknown unless differential centrifugation is carried out. Different formalinized cell suspensions varied considerably in their ability to cause migration inhibition. This may reflect low antigenicity of the tumour, or the variable presence of substances masking the antigens of the cell surface. Substances such as adsorbed Ig or endogenous sialic acid present on the cell surface may prevent sensitized lymphocytes from recognizing and reacting with tumour-associated antigens. Problems have been encountered using cell suspensions derived from breast carcinoma. Although a high number of breast cancer patients showed migration inhibition the frequency of inhibition of control leucocytes was also fairly high. Several possibilities were considered to explain these findings. First, formalin released by the tumour cells might be toxic to the leucocytes and thus abolish their motility, although this would seem unlikely in view of the results obtained in the melanoma group. However, after repeated washing ( x 4) of the formalinized tumour cells, some inhibition of control leucocytes was still seen. Clumping of cells was also considered, but even after several months of storage very few clumps were present and no clumps contained more than eight to ten cells. The frequency of clumps did not correlate with the occurrence of 'non-specific' inhibition. The most probable explanation is that it is more difficult to obtain a clean, homogeneous cell suspension from breast cancer tissue than from melanoma tissue. The fibrous nature of the stroma of most breast cancers results in a suspension which contained a mixture of cell types and this will therefore constitute a less specific antigen. Dispersal of cells by mechanical means may also result in cell surface damage, affecting the antigenicity of the cells. However, fixed tumour cells appear to retain specific antigenicity and with improved methods of preparation they may be useful in studies of human tumour immunology. Such cells could be used as sources of specific antigen in lymphocyte transformation techniques. In studies of humoral immunity formalinized cells could perhaps be used as target cells in membrane immunofluorescence, and immune adherence tests.
132
C. E. Ross et al.
The mechanism of the leucocyte migration test is not fully elucidated, but if properly controlled enough evidence exists to indicate that it is a useful in vitro method of detecting CMI (Cochran et al., 1973). The study described here does not attempt to define the mechanism of leucocyte migration inhibition mediated by formalin-fixed intact tumour cells. Studies designed to elucidate this further are in progress. This study was conducted with the assistance of grants from the Secretary of State for Scotland and the McMillan Research Funds of the University of Glasgow.
REFERENCES ANDERSEN, V., BJERRUM, O., BENDIXEN, G., SCHI0DT, RUSSELL, J.M. (1974) Sensitisation to tumourT. & DISSING, I. (1970) Effect of autologous associated antigens in human breast carcinoma. mammary tumour extracts on human leucocytes Int. J. Cancer, 14, 19. migration in vitro. Int. J. Cancer, 5, 357. DRAKE, W.P., UNGARO, P.C. & MARDINEY, M.R. ANDERSON, J.M., KELLY, F., WOOD, S.E., RODGER, (1972) Preservation of cellular antigenicity of K.D. & FRESNEY, R.I. (1973) Evaluation of tumour cells by the use of formalin fixation. leucocyte functions six years after tumour autoCancer Res. 32, 1042. graft in human mammary cancer. Brit. J. Cancer, GATrI, R.A., OSTBORN, A., FAGRAEUS, A. (1974) 28, supplement 1, 83. Selective impairment of cell antigenicity by fixaCERIHLI, J., SMITH, M.C. & HATTAN, D. (1972) The tion. J. Immunol. 113, 1361. detection of cellular immunity to tumour cells by GEORGE, M. & VAUGHAN, J.H. (1962) In vitro cell the macrophage migration technique. Transmigration as a model for delayed hypersensitivity. plantation, 14, 125. Proc. Soc. exp. Biol. (N. Y.), 111, 514. CHURCHILL. W.H., ZBAR, B., BELLI, J.A. & DAVID, KRONMAN, B.S., WEPSIC, H.T., CHURCHILL, W.H., J.R. (1972) Detection of cellular immunity to ZBAR, B., BORSOS, T. & RAPP, H.J. (1969) Tumour tumour antigens of a guinea-pig hepatoma by specific antigens detected by inhibition of macroinhibition of macrophage migration. J. nat. Cancer phage migration. Science, 165, 296. Inst. 48, 541. MACKIE, R.M., SPILG, W.G.S., THOMAS, C.E. & COCHRAN, A.J. (1971) Tumour cell migration. Europ. COCHRAN, A.J. (1972) Cell mediated immunity in J. clin. biol. Res. 16, 44. patients with malignant melanoma. Brit. J. Derm. COCHRAN, A.J., SPILG, W.G.S., MACKIE, R.M. & 87, 523. THOMAS, C.E. (1972) Postoperative depression of SEGALL, A., WEILER, O., GENIN, J., LACOUR, J. & cell mediated immunity in patients with malignant LACOUR, F. (1972) In vitro study of cellular disease. Brit. med. J. iv, 67. immunity against autochthonous human cancer. COCHRAN, A.J., MACKIE, R.M., THOMAS, C.E., Int. J. Cancer, 9, 417. GRANT, R.M., CAMERON-MOWAT, D.E. & SPILG, S0BORG, M. & BENDIXEN, G. (1967) Human lymphoW.G.S. (1973) Cellular immunity to breast carcyte migration as a parameter of hypersensitivity. cinoma and malignant melanoma. Brit. J. Cancer, Acta med. scand. 181, 247. 28, supplement 1, 77. WOLBERG, W.H. (1971) Inhibition of migration of human autogenous and allogeneic leukocytes by COCHRAN, A.J., GRANT, R.M., SPILG, W.G., MACKIE, R.M., Ross, C.E., HOYLE, D.E. & extracts of patients' cancers. Cancer Res. 31, 798.
