Journal of Surgical Oncology 7:479-483 (1975)
Migration Inhibition Test in Lung Cancer .......................................................................................... .......................................................................................... PATRICK C. MARABELLA, B . A . , LINDA L. ELIAS, M.S. , HIROSHI TAKITA, M.D., and JUN MINOWADA, M.D. Twenty patients with primary carcinoma of the lung were tested with migration inhibition test, using extract of cultured lung carcinoma of large cell type. Eight of 10 patients with localized disease had positive inhibition, while all 10 of the patients with disseminated disease had negative inhibition (x', p < 0.005). Two cases of large cell carcinoma (localized) showed extremely positive inhibition which may suggest the presence of cell type specific lung carcinoma antigen.
.......................................................................................... .......................................................................................... KEY WORDS: migration inhibition test, lung cancer, tumor extract
The primary purpose of this experiment was to study the migration inhibition test as an in vitro tool for evaluating the cell-mediated immunity in lung carcinoma. Furthermore, by using an extract of lung carcinoma, it was hoped that insight into tumor specific cellular immunity was to be gained.
MATERIALS AND METHODS Patients
A total of 20 previously untreated patients with lung carcinoma were evaluated. Ten of these, 8 male and 2 female, were found to have localized disease, while the remaining 10, all male, were diagnosed as disseminated disease. Of the 10 patients with localized disease, 5 were squamous cell carcinoma, 2 adenocarcinoma, 2 large cell carcinoma, and 1 alveolar cell carcinoma. In the group of 10 disseminated cases, 4 were oat cell carcinoma, 3 large cell carcinoma, 2 adenocarcinoma, and 1 squamous cell carcinoma. Also included were 5 normal healthy individuals as controls. Patrick Marabella and Hiroshi Takita, Department of Thoracic Surgery, New York State Department of Health, Roswell Park Memorial Institute, Buffalo, New York. Linda Elias, Department of'Genera1 Surgery, New York State Department of Health, Roswell Park Memorial Institute, Buffalo, New York. Jun Minowada, Department of Immunology and Immunochemistry, New York State Department of' Health, Roswell Park Memorial Institute, Buffalo, New Y ork. Address reprint requests t o Dr. Patrick Marabella, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, New York, 14203.
479
0 1975 Alan R. Liss,
Inc., 150 Fifth A v e n u e , N e w York, N . Y . 10011
480
Marabella et al.
Lung Tumor Extract A cultured cell line of large cell undifferentiated carcinoma of the lung, called Cha-Go Line (Rabson et al., 1973), was employed to obtain tumor extract. The cells are washed 3 times, resuspended in twice the volume of 0.9%NaCl solution, and are homogenized by sonification for 60 sec (20 sec for .3 times) at 4°C. After standing for 18 hr at 4"C, the homogenate is centrifuged at 1,300 g for 15 min. The protein concentration was estimated by measuring the optical density at a wave length of 280 nmoles. Migration Inhibition Test Sterile procedure was carried through as far as possible. Ten ml of peripheral blood were drawn into a plastic disposable syringe containing 400-600 units of preservative free sodium heparin (Abbott Laboratories, North Chicago, Illinois). The blood was mixed gently by inverting the syringe 10 times and was allowed to settle for 90 min at 37". After the erythrocytes had settled out, all but the last 0.5 ml of plasma was transferred into a 12 ml glass centrifuge tube and centrifuged at 250 g for 8 min at 20'. The plasma was then discarded, and the cell pellet was resuspended and washed with warmed RPMI 1640 3 times (this wash medium had neither calf serum nor the antibiotic mixture added to it). At the time of the third washing, a viability count using trypan blue was done. Over 90% viability was considered acceptable. After the last wash, the cell pellet was resuspended in RPMI 1640 containing 20% calf serum. This suspension was adjusted so that the heparinized (sodium) capillary tubes (1.2 X 75 mm) contained 1-3 million cells per capillary. The cell suspension was drawn into the capillary by mild suction and then sealed at one end with sterile Clay Adams seal-ease (Scientific Products, Edison, New Jersey). The capillaries were placed in a 10 ml test tube containing a wad of cotton and centrifuged at 250 g for 8 min at 20". After centrifugation, the capillaries were cut 0.5 mm above the cell-liquid interface, and the cell portion was immediately placed in the migration chambers ("Lexy" Culture Chambers, Mini Lab, Divernay Laval, Quebec, Canada). One capillary was placed in each chamber and anchored at the closed tip by sterile silicone grease. Each chamber was closed by a coverslip and sealed with paraffin. Control chambers contained RPMI 1640 with 20% calf serum, while the test chambers contained the same medium with 10 p g of the Cha-Go cell extract added. Each test was done in triplicate or quadruplicate. All slides were incubated in a perfectly horizontal position at 37" for 16-20 hr. The cell migration pattern after this period of time was projected on an American Optical (AO) projection screen fitted over the A 0 microscope, and the outline of the migration area was traced on thin paper. The surface area of migration was calculated by a planimeter. The inhibition of mi-ration was evaluated by the following formula: Average area of Average area of control migration test migration Migration Inhibition = Average area of control migration
x
100
A migration index of greater than 15% indicated positive inhibition, while anything less was considered negative. This margin (1 5%) was allowed for biological and laboratory error. Also tested was lung tumor extract concentrations of 1,000 p g , but no significant
48 1
MIT in Lung Cancer
results were obtained. Toxicity at such a dose is a possible explanation for lack of significant results.
Skin Tests Delayed subcutaneous hypersensitivity skin tests were given to 8 patients with localized disease and 7 patients with disseminated. Nonspecific memory antigens injected intradermally were: Candida albicans (Hollister-Stier Labs, Spokane, Washington), purified protein derivative (PPD) intermediate strength containing 0.0001 mg PPD/O. 1 ml (Parke Davis Co., Detroit, Michigan), mumps (Eli Lilly Co., Indianapolis, Indiana), streptokinase-streptodornase (Varidase, Lederle Labs, Pearl River, New York). The diameter of the largest reaction was measured after 48 hr. Those reactions 2 1 cm were considered good, and those < lcm were considered poor reactions (Brugarolas and Takita, 1973).
RESULTS Eight of the 10 patients with localized disease were found to have positive inhibition that is, inhibition more than 15%. All of the patients with disseminated disease had negative inhibition less than 15% (x” p < 0.005). The 5 controls were all negative (Fig. 1). Of the 8 patients with localized disease who were skin tested 6 had good reactions 2 1 cm, while of the 7 with disseminated disease who were skin tested 6 had poor reaction < 1 cm, (Table I; x 2 , p < 0.05. In the two cases of localized large cell carcinoma, the degree of migration inhibition was the highest. However, in all 3 cases of disseminated large cell carcinoma, stimulation of migration was observed. -
DISCUSSION In previous studies the authors have extensively evaluated the status of nonspecific cellular immunity of lung cancer patients, utilizing delayed hypersensitivity skin testing, mixed lymphocyte culture, and T and B lymphocyte counts (Han and Takita, 1972; Brugarolas et al., 1973). We observed that those patients with relatively localized disease (without hematogenous metastasis) maintained competent nonspecific cellular immunity, but patients with disseminated disease (with heniatogenous metastasis) were uniformly found to have marked impairment of cellular immunity. Skin tests using nonspecific antigens employed in this study gave data consistent with previous findings. Our clinical trial with adjuvant immunotherapy to radical surgical resection in locally extensive lung carcinoma patients based on the above findings gave encouraging results (Takita and Brugarolas, 1973). A more important subject in immunological study of lung cancer patients is tumor specific immunity. For test of tumor specific immunity, skin tests may be applied using tumor antigens (Marabella and Takita, 1974), but their application to normal subjects or patients in remission may not be advisable. Mixed lymphocyte culture was found t o be unsuitable for study of tumor specific immunity (personal communication from Dr. T. Han with his permission.) To investigate such tumor specific immunity the migration inhibition test using
482
Marabella et al.
25 -
10 -
5X
0,
-5 -10-15-
8
8
-
0
0 0
rn
I ,
-
0
8
0
P'
-20 -25
-
-30 -35 4 0
I
a 0
Ez z-
D
w
0 W
0
0
Pig. 1. Migration inhibition test in lung cancer patients.
lung tumor extract was employed. This test has been shown to be a well established in vitro tool in evaluating cell-mediated immunity (Soborg and Bendixen, 1967; Andersen et al., 1969; Segall et al., 1972). The results obtained were in correlation with the previous studies of nonspecific cellular immunity. Negative inhibition in disseminated cases can be explained by marked impairment of cellular immunity, whereas positive inhibition in localized cases suggests competent cellular immunity. Considering the cell type of the lung tumor extract (large cell), it is of interest that the localized cases of this same cell type had more positive inhibition than the localized cases of different cell types. Furthermore, the disseminated large cell cases were more often negative than other disseminated cases.
MIT i n L u n g Cancer
48 3
TABLE I. Results of Migration Inhibition Test and Skin Test in 20 Lung Carcinoma Patients and 5 Controls
Normal
Localized
Disseminated
Patient
% Inhibition
H.T. L.E. T.F. P.M. A. B.
13.2 4.8 - 8.6 - 1.2 - 9.0 -
S.M. A.R. M.Z. C. E. B.M. A. S. E.G. c.0. E.W. E.S.
2.8 15.2 + 25.3 + 0.5 19.2 + 19.2 + 24.7 + 17.2 + 18.1 + 19.7 +
Squamous Adenocarcinoma Large cell Squamous Squamous Alveolar Large cell Squamous Squamous Adenocarcinoma
1.3 8.7 1.7 5.4 8.4 - 2.7 -38.1 -38.9 4.4 - 1.5 -
Squamous Adenocarcinoma Oat cell Oat cell Oat cell Large cell Large cell Large cell Adenocarcinoma Oat cell
L.H. M.G. D. H. P.S. A.T. J.M. C.O. G.B. G. B. E.C.
-
Cell Type
Skin Test Reaction (diameter in mm)
-
-
-
-
-
-
-
-
-
-
18 24 20 15 5 -
6 10 15 -
9 10 7 0 -
9 -
9 0
It may be too premature to state, but the above findings may suggest the presence of a lung tumor specific antigen and that there may also be a histologically specific difference.
REFERENCES Andersen, V., Bendixen, U., and Schmidt, T. (1969). An in vitro demonstration o f cellular immunity against autologous mammary carcinoma in man. Acta. Med. Scand. 186: 101-103. Brugarolas, A., Han, T., Takita, H., and Minowada, J. (1973). Immunological assays in lung cancer. N.Y. State J. Med. 73:747-750. Brugarolas, A., and Takita, H. (1973). Immunological status in lung cancer. Chest 64:427-430. Han, l., and Takita, H. (1972). Immunological impairment in bronchogenic carcinoma: A study of lymphocyte response to phytohemagglutinin. Cancer 30:616-619. Marabella, P., and Takita, H. (1975). Skin test with tumor extract in bronchogenic carcinoma. J. Surg. Oncol. 7:205-212. Rabson, A.S., Rosen, S.W., Tashjian, A.H., and Page, W. (1973). Production of human chorionic gonadotropin in vitro by a cell line derived from ;1 carcinoma o f the lung. J. Nat. Cancer Inst. 50:669-672. Segall, A., Weiler, P., Genin, J., Lacour, J., and Lacour, F. (1972). In vitro study o f cellular immunity against autochthonous human cancer. Int. J. Cancer 9:417-425. Soborg, M., and Bendixen, U. (1967). Human lymphocyte migration as a parameter of hypersensitivity. Acta. Med. Scand. 181:247. Takita, H., and Brugarolas, A. (1973). Adjuvant immunotherapy for bronchogenic carcinoma: Preliminary results. Cancer Chemother. Rep. 4: 293-298.
Migration Inhibition Test in Lung Cancer .......................................................................................... .......................................................................................... PATRICK C. MARABELLA, B . A . , LINDA L. ELIAS, M.S. , HIROSHI TAKITA, M.D., and JUN MINOWADA, M.D. Twenty patients with primary carcinoma of the lung were tested with migration inhibition test, using extract of cultured lung carcinoma of large cell type. Eight of 10 patients with localized disease had positive inhibition, while all 10 of the patients with disseminated disease had negative inhibition (x', p < 0.005). Two cases of large cell carcinoma (localized) showed extremely positive inhibition which may suggest the presence of cell type specific lung carcinoma antigen.
.......................................................................................... .......................................................................................... KEY WORDS: migration inhibition test, lung cancer, tumor extract
The primary purpose of this experiment was to study the migration inhibition test as an in vitro tool for evaluating the cell-mediated immunity in lung carcinoma. Furthermore, by using an extract of lung carcinoma, it was hoped that insight into tumor specific cellular immunity was to be gained.
MATERIALS AND METHODS Patients
A total of 20 previously untreated patients with lung carcinoma were evaluated. Ten of these, 8 male and 2 female, were found to have localized disease, while the remaining 10, all male, were diagnosed as disseminated disease. Of the 10 patients with localized disease, 5 were squamous cell carcinoma, 2 adenocarcinoma, 2 large cell carcinoma, and 1 alveolar cell carcinoma. In the group of 10 disseminated cases, 4 were oat cell carcinoma, 3 large cell carcinoma, 2 adenocarcinoma, and 1 squamous cell carcinoma. Also included were 5 normal healthy individuals as controls. Patrick Marabella and Hiroshi Takita, Department of Thoracic Surgery, New York State Department of Health, Roswell Park Memorial Institute, Buffalo, New York. Linda Elias, Department of'Genera1 Surgery, New York State Department of Health, Roswell Park Memorial Institute, Buffalo, New York. Jun Minowada, Department of Immunology and Immunochemistry, New York State Department of' Health, Roswell Park Memorial Institute, Buffalo, New Y ork. Address reprint requests t o Dr. Patrick Marabella, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, New York, 14203.
479
0 1975 Alan R. Liss,
Inc., 150 Fifth A v e n u e , N e w York, N . Y . 10011
480
Marabella et al.
Lung Tumor Extract A cultured cell line of large cell undifferentiated carcinoma of the lung, called Cha-Go Line (Rabson et al., 1973), was employed to obtain tumor extract. The cells are washed 3 times, resuspended in twice the volume of 0.9%NaCl solution, and are homogenized by sonification for 60 sec (20 sec for .3 times) at 4°C. After standing for 18 hr at 4"C, the homogenate is centrifuged at 1,300 g for 15 min. The protein concentration was estimated by measuring the optical density at a wave length of 280 nmoles. Migration Inhibition Test Sterile procedure was carried through as far as possible. Ten ml of peripheral blood were drawn into a plastic disposable syringe containing 400-600 units of preservative free sodium heparin (Abbott Laboratories, North Chicago, Illinois). The blood was mixed gently by inverting the syringe 10 times and was allowed to settle for 90 min at 37". After the erythrocytes had settled out, all but the last 0.5 ml of plasma was transferred into a 12 ml glass centrifuge tube and centrifuged at 250 g for 8 min at 20'. The plasma was then discarded, and the cell pellet was resuspended and washed with warmed RPMI 1640 3 times (this wash medium had neither calf serum nor the antibiotic mixture added to it). At the time of the third washing, a viability count using trypan blue was done. Over 90% viability was considered acceptable. After the last wash, the cell pellet was resuspended in RPMI 1640 containing 20% calf serum. This suspension was adjusted so that the heparinized (sodium) capillary tubes (1.2 X 75 mm) contained 1-3 million cells per capillary. The cell suspension was drawn into the capillary by mild suction and then sealed at one end with sterile Clay Adams seal-ease (Scientific Products, Edison, New Jersey). The capillaries were placed in a 10 ml test tube containing a wad of cotton and centrifuged at 250 g for 8 min at 20". After centrifugation, the capillaries were cut 0.5 mm above the cell-liquid interface, and the cell portion was immediately placed in the migration chambers ("Lexy" Culture Chambers, Mini Lab, Divernay Laval, Quebec, Canada). One capillary was placed in each chamber and anchored at the closed tip by sterile silicone grease. Each chamber was closed by a coverslip and sealed with paraffin. Control chambers contained RPMI 1640 with 20% calf serum, while the test chambers contained the same medium with 10 p g of the Cha-Go cell extract added. Each test was done in triplicate or quadruplicate. All slides were incubated in a perfectly horizontal position at 37" for 16-20 hr. The cell migration pattern after this period of time was projected on an American Optical (AO) projection screen fitted over the A 0 microscope, and the outline of the migration area was traced on thin paper. The surface area of migration was calculated by a planimeter. The inhibition of mi-ration was evaluated by the following formula: Average area of Average area of control migration test migration Migration Inhibition = Average area of control migration
x
100
A migration index of greater than 15% indicated positive inhibition, while anything less was considered negative. This margin (1 5%) was allowed for biological and laboratory error. Also tested was lung tumor extract concentrations of 1,000 p g , but no significant
48 1
MIT in Lung Cancer
results were obtained. Toxicity at such a dose is a possible explanation for lack of significant results.
Skin Tests Delayed subcutaneous hypersensitivity skin tests were given to 8 patients with localized disease and 7 patients with disseminated. Nonspecific memory antigens injected intradermally were: Candida albicans (Hollister-Stier Labs, Spokane, Washington), purified protein derivative (PPD) intermediate strength containing 0.0001 mg PPD/O. 1 ml (Parke Davis Co., Detroit, Michigan), mumps (Eli Lilly Co., Indianapolis, Indiana), streptokinase-streptodornase (Varidase, Lederle Labs, Pearl River, New York). The diameter of the largest reaction was measured after 48 hr. Those reactions 2 1 cm were considered good, and those < lcm were considered poor reactions (Brugarolas and Takita, 1973).
RESULTS Eight of the 10 patients with localized disease were found to have positive inhibition that is, inhibition more than 15%. All of the patients with disseminated disease had negative inhibition less than 15% (x” p < 0.005). The 5 controls were all negative (Fig. 1). Of the 8 patients with localized disease who were skin tested 6 had good reactions 2 1 cm, while of the 7 with disseminated disease who were skin tested 6 had poor reaction < 1 cm, (Table I; x 2 , p < 0.05. In the two cases of localized large cell carcinoma, the degree of migration inhibition was the highest. However, in all 3 cases of disseminated large cell carcinoma, stimulation of migration was observed. -
DISCUSSION In previous studies the authors have extensively evaluated the status of nonspecific cellular immunity of lung cancer patients, utilizing delayed hypersensitivity skin testing, mixed lymphocyte culture, and T and B lymphocyte counts (Han and Takita, 1972; Brugarolas et al., 1973). We observed that those patients with relatively localized disease (without hematogenous metastasis) maintained competent nonspecific cellular immunity, but patients with disseminated disease (with heniatogenous metastasis) were uniformly found to have marked impairment of cellular immunity. Skin tests using nonspecific antigens employed in this study gave data consistent with previous findings. Our clinical trial with adjuvant immunotherapy to radical surgical resection in locally extensive lung carcinoma patients based on the above findings gave encouraging results (Takita and Brugarolas, 1973). A more important subject in immunological study of lung cancer patients is tumor specific immunity. For test of tumor specific immunity, skin tests may be applied using tumor antigens (Marabella and Takita, 1974), but their application to normal subjects or patients in remission may not be advisable. Mixed lymphocyte culture was found t o be unsuitable for study of tumor specific immunity (personal communication from Dr. T. Han with his permission.) To investigate such tumor specific immunity the migration inhibition test using
482
Marabella et al.
25 -
10 -
5X
0,
-5 -10-15-
8
8
-
0
0 0
rn
I ,
-
0
8
0
P'
-20 -25
-
-30 -35 4 0
I
a 0
Ez z-
D
w
0 W
0
0
Pig. 1. Migration inhibition test in lung cancer patients.
lung tumor extract was employed. This test has been shown to be a well established in vitro tool in evaluating cell-mediated immunity (Soborg and Bendixen, 1967; Andersen et al., 1969; Segall et al., 1972). The results obtained were in correlation with the previous studies of nonspecific cellular immunity. Negative inhibition in disseminated cases can be explained by marked impairment of cellular immunity, whereas positive inhibition in localized cases suggests competent cellular immunity. Considering the cell type of the lung tumor extract (large cell), it is of interest that the localized cases of this same cell type had more positive inhibition than the localized cases of different cell types. Furthermore, the disseminated large cell cases were more often negative than other disseminated cases.
MIT i n L u n g Cancer
48 3
TABLE I. Results of Migration Inhibition Test and Skin Test in 20 Lung Carcinoma Patients and 5 Controls
Normal
Localized
Disseminated
Patient
% Inhibition
H.T. L.E. T.F. P.M. A. B.
13.2 4.8 - 8.6 - 1.2 - 9.0 -
S.M. A.R. M.Z. C. E. B.M. A. S. E.G. c.0. E.W. E.S.
2.8 15.2 + 25.3 + 0.5 19.2 + 19.2 + 24.7 + 17.2 + 18.1 + 19.7 +
Squamous Adenocarcinoma Large cell Squamous Squamous Alveolar Large cell Squamous Squamous Adenocarcinoma
1.3 8.7 1.7 5.4 8.4 - 2.7 -38.1 -38.9 4.4 - 1.5 -
Squamous Adenocarcinoma Oat cell Oat cell Oat cell Large cell Large cell Large cell Adenocarcinoma Oat cell
L.H. M.G. D. H. P.S. A.T. J.M. C.O. G.B. G. B. E.C.
-
Cell Type
Skin Test Reaction (diameter in mm)
-
-
-
-
-
-
-
-
-
-
18 24 20 15 5 -
6 10 15 -
9 10 7 0 -
9 -
9 0
It may be too premature to state, but the above findings may suggest the presence of a lung tumor specific antigen and that there may also be a histologically specific difference.
REFERENCES Andersen, V., Bendixen, U., and Schmidt, T. (1969). An in vitro demonstration o f cellular immunity against autologous mammary carcinoma in man. Acta. Med. Scand. 186: 101-103. Brugarolas, A., Han, T., Takita, H., and Minowada, J. (1973). Immunological assays in lung cancer. N.Y. State J. Med. 73:747-750. Brugarolas, A., and Takita, H. (1973). Immunological status in lung cancer. Chest 64:427-430. Han, l., and Takita, H. (1972). Immunological impairment in bronchogenic carcinoma: A study of lymphocyte response to phytohemagglutinin. Cancer 30:616-619. Marabella, P., and Takita, H. (1975). Skin test with tumor extract in bronchogenic carcinoma. J. Surg. Oncol. 7:205-212. Rabson, A.S., Rosen, S.W., Tashjian, A.H., and Page, W. (1973). Production of human chorionic gonadotropin in vitro by a cell line derived from ;1 carcinoma o f the lung. J. Nat. Cancer Inst. 50:669-672. Segall, A., Weiler, P., Genin, J., Lacour, J., and Lacour, F. (1972). In vitro study o f cellular immunity against autochthonous human cancer. Int. J. Cancer 9:417-425. Soborg, M., and Bendixen, U. (1967). Human lymphocyte migration as a parameter of hypersensitivity. Acta. Med. Scand. 181:247. Takita, H., and Brugarolas, A. (1973). Adjuvant immunotherapy for bronchogenic carcinoma: Preliminary results. Cancer Chemother. Rep. 4: 293-298.
