[Gann, 66, 167-174; April, 1975] IN VITRO CELLULAR RESPONSES TO AUTOLOGOUS TUMOR EXTRACT DETECTED BY INHIBITION OF MACROPHAGE MIGRATION*1 Tsuyoshi AKIYOSHI, Akira HATA, and Hideo TSUJI Department of Surgery, Institute of Balneotherapeutics, Kyushu University*2 Indirect macrophage migration technique was employed to study in vitro cellular immune response to autologous tumor extract in man. Cellular hypersensitivity to autologous tumor extract was detected in 21 (26%) of 80 patients with various carcinomas preoperatively by means of the in vitro production of macrophage migration inhibitory factor (MIF). The relative frequency of MIF production in these patients, varied inversely with progress of the diseases. The follow-up studies revealed postoperative decline of the cellular hypersensitivity after curative operation. These findings indicate that this assay can be applied for detecting cellular hypersensitivity to autologous tumor extracts in clinical situation. Cellular immune responses are considered to be playing a major role in immunologic resistance to tumor. Several in vitro methods have been utilized to monitor this cellular immunity. The macrophage migration technique is now well established as a specific and sensitive indicator of cellular hypersensitivity status in animals and man. Several investigations on experimental tumors in animals4,16) demonstrated that this technique is applicable to the study of cellular immunity to tumor. In clinical situation, it has been reported that autologous tumor extracts inhibit migration of leucocytes from some patients with malignant tumor.1,2,6) However, other work17) has shown that some preparations of human tumor extracts are cytotoxic to migrating leucocytes. In the present study, we have used a clinically applicable assay for detecting cellular immunity in man based on the method of Thor et al.15) and Rocklin et al.,11,12) i.e., indirect migration inhibition test, to study the cellular immune response of patients to their own tumor extract. MATERIALS AND METHODS Patient Selection Eighty patients with various carcinomas were studied. As shown in Tables I and III, curative operation was performed on 50 patients with localized tumor. The remaining 30 patients were in advanced stage of the disease at the time of surgery and non-curative operation was undertaken. In all these patients, blood samples were drawn before surgery and cultures for the production of macrophage migration inhibitory factor (MIF) were set up on the day of operation. As indicated in Table I, subsequent blood samples were obtained at 2- to 4-week intervals thereafter in 43 patients. In this study, patients who had been treated with antitumor chemotherapy or radiation therapy before or during the study were excluded. In 33 patients, normal tissue from the tumor-containing organ was obtained for use as a control. Extract of autologous leucocytes was also used as a control in another 34 patients (Table II). Preparation of Normal Tissue, Tumor, and Leucocyte Extracts Pieces of tumor, 2-3cm3, and normal tissue of the tumor-containing organ were obtained from surgical specimens under a sterile condition. These were treated with penicillin and streptomycin, and washed thoroughly with sterile phosphate-buffered saline (PBS). These tissues were then minced with forceps, suspended in cold PBS, and homoeenized in Virtis 4.5 hmmn- *1 This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education.
centrifuged for 10min at 600g. Sterility of the supernatants was checked by incubating 0.5-ml aliquots in thioglycollate broth and contaminated preparations were excluded from the evaluation in this study. The leucocytes were obtained from patients by sedimentation of heparinized peripheral blood. These leucocyte preparations were washed with PBS and sonified for 15sec in PBS. The protein concentration of these extracts was T. AKIYOSHI, ET AL. determined by Lowry's method.10) These extracts Migration index= testing. Production of MIF The method describcd by Rocklin and David11) was used for the production of MIF, with modification in several ways. Peripheral blood (25-50ml) was drawn into a heparinized syringe from patients with carcinoma. The 60min while the syringe was in an upright position. The leucocyte-rich plasma was drawn and centrifuged for 5min at 200g. The leucocyte pellet was suspended in TC-199 medium and passed through a column of Tetoron (polyester fiber, Toray Co., Tokyo) to separate the lymphocytes. The cells were washed three times with TC-199 medium and resuspended in the medium containing penicillin (100U/ml) and streptomycin (100 for culture. The cell preparation contained 80-95% of lymphocytes. These cells were essentially 100%. viable as assessed by the Trypan Blue dye exclusion. Normal tissue, tumor, or leucocyte extracts were added to the lymphocyte suspension at a concentration indicated in Tables I and II. After CO2 in air, the cells were sedimented by centrifugation. The collected supernatants were dialysed against 0.9% NaCl, redialysed in cold against distilled water, and concentrated by lyophilization. The lyophilized supernatants were resuspended in TC-199 medium, containing 20% calf serum and the antibiotics, at two-fold concentration of the original volume, sterilized by Millipore filtration, and adjusted to ph 7.4 with CO2 in air. In this experiment, controls to each test supernatant were prepared. These consisted of the supernatant from the lymphocyte cultures incubated without the extracts to which the extracts were added after removal of lymphocytes and all the steps for test supernatants were carried through, including dialysis and concentration. These control media were assayed on the same set of peritoneal exudate cells as the test supernatants. Migration Inhibition Assay Peritoneal exudate cells from normal guinea pigs were induced by sterilized liquid paraffin, collected, and packed into the capillary tubes as described by Rocklin and David.11) Two capillary tubes of packed peritoneal exudate cells were placed each in a Mackaness-type chamber which was filled with reconstituted test or control supernatant to be assayed. The area of migration was projected through a microscope prism onto a paper and measured by a planimeter. Percentage of macrophage migration (migration index) was calculated as follows: average area of test supernatant average area of control supernatant Production of MIF from lymphocytes contacted with autologous tumor extracts was studied in 80 patients on 152 occasions, as shown in Table I. As controls, autologous normal tissue extracts were used for the study in 33 of these patients simultaneously and the lymphocytes from another 34 patients were incubated with autologous leucocyte extracts. The results of migration inhibition assay in all these patients are shown in Tables I and II. Average migration indices (MI) of the supernatants from control cultures were 14.0 from cultures with normal tissue extracts in 33 patients, whereas supernatants from lymphocytes of 80 patients cultured with autologous tumor extracts on the day of This average value was significantly different. (by Student's t-test) from the value obtained from autologous leucocyte extracts (P<0.005) or the value from autologous normal tissue extracts (P<0.001). The lines which represent two standard deviations below the mean value obtained from control cultures were drawn at 75 from autologous leucocyte extracts and at 73 from autologous normal tissue extracts. As shown in Fig. 1 and Table IV, MI values below 75 were found in 21 of 80 supernatants from cultures with autologous tumor extracts. MI values below 73 were found in 18 out of 80 patients. These results indicate that lymphocytes from these patients were considered to
CELLULAR IMMUNITY TO TUMOR Table I. Summary of Migration Inhibition Assay Cultured with Autologous Tumor Extract and Normal Tissue Extract
T. AKIYOSHI, ET AL. Table I. (Continued)
CELLULAR IMMUNITY TO TUMOR Table 1. (Continued) a) Period between obtaining tumor specimen and setting up cultures. b) Protein concentration of tumor extract in lymphocyte culture. c) Protein concentration of normal tissue extract in lymphocyte culture. 66(2) 1975 171
T. AKIYOSHI, ET AL. Table II. Summary of Migration Inhibition Assay Cultured with Autologous Leucocyte Extract Table III. MIF Production in Response to Autologous Tumor Extract by Extent of the Disease ** The difference is significant at P<0.05. Table IV. Summary of MIF Production in Response to Autologous Tumor Extract a) Protein concentration of leucocyte extract in lymphocyte culture have produced MIF in response to autologous tumor extracts. In these patients, reactivity of their lymphocytes to autologous tumor extract detected by MIF production was examined in relation to the progress of the diseases. As shown in Table III, lymphocytes from only three of 30 patients who had non-curative operation Fig. 1. Distribution of MIF production 172 Gann
CELLULAR IMMUNITY TO TUMOR at this time, despite preoperative non-reactivity. All other patients who underwent curative operation were without signs of recurrence during the study. Fig. 2. Results of follow-up studies after curative operation in patients whose lymphocytes reacted with autologous tumor extract preoperatively because of the extent of their diseases produced MIF in response to tumor extract preoperatively, while 18 of 50 patients who underwent curative operation had circulating lymphocytes that proved to be sensitive to the tumor extract. Successive assays of migration inhibition were performed postoperatively in 43 of these patients 2-8 weeks after surgery. Lymphocytes from 12 of 43 patients produced MIF in response to autologous tumor extract preoperatively. As shown in Table I and Fig. 2, sensitivity to the extract disappeared within 2-4 weeks after curative operation in all these cases. Of the remaining 31 patients, whose lymphocytes did not react to autologous tumor extract before surgery, MIF production was not detected, either after curative or non-curative operation within 4-8 weeks in all these patients, except in one. In the latter (Case No. 29), a recurrent tumor appeared within 8 weeks after curative gastrectomy and lymphocytes of the patient responded well to autologous tumor extract DISCUSSION The detection of MIF produced by human lymphocytes in response to antigens by assay on guinea pig macrophages has been proved to be a useful tool in the study of cellular hypersensitivity. Since its introduction, this technique has been successfully applied to several clinical problems. The results of the present investigation indicate that this assay system is applicable to the study for detecting cellular hypersensitivity to tumor extract in man. By using the leucocyte migration assay, Andersen et al.2) found an inhibited migration of leucocytes from breast cancer patients in the presence of autologous tumor extract in 8 of 22 patients. However, other work17) has shown that some preparations from human tumors are cytotoxic to migrating leucocytes. In our present assay system, non-specific effect of tumor extracts on macrophage migration, as well as the spontaneous release of materials from lymphocytes which might affect migration, was taken into account by using the optimal control as described by others.8,11) Moreover, at the concentration of tumor extract we have used, cytotoxic effect of the extracts was not detected when viability of the cells in culture with tumor extract was compared with those in culture without the extract after 48hr of incubation period. The present study demonstrated that lymphocytes from 21 of 80 patients with carcinoma produced MIF in response to autologous tumor extract and, therefore, these patients exhibited cellular hypersensitivity to autologous tumor extract. These results are almost similar to those which have been obtained by several investigators9,14) using the assay of delayed cutaneous hypersensitivity to autologous tumor extracts. 66(2) 1975 173
T. AKIYOSHI, ET AL. In some studies,5,13) immunological responsiveness was frequently found to be decreased in patients with malignant tumor, particularly in those with advenced tumor. The results of our study have shown that lymphocytes from 18 of 50 patients with localized tumor but only 3 of 30 patients with disseminated tumor produced MIF in response to autologous tumor extract. It is of particular interest that the level of cellular hypersensitivity to autologous tumor extract varied inversely with progress of the disease. The results of the present follow-up studies appear to indicate that cellular hypersensitivity to tumor extract measured by migration inhibition assay disappeared within 2 to 4 weeks after curative operation. These rapid fall of cellular hypersensitivity after removal of tumor may be due to the same mechanism as that of abrupt decline of cellular immunity following initial peak which was observed after tumor or skin allografting.3,7) Presumably these are attributable, at least in part, to the decrease of antigenic stimulus by a tumor or allograft. These overall results indicate that this assay can be used for detecting cellular hypersensitivity to tumor extract in man and to the study of cellular immune response of patients to their own tumor. These results also indicate that it might be one of the most practical methods of assessing cellular immunity to tumor in clinical situation. It seems reasonable at this time to utilize such an index of cellular hypersensitivity as an aid in follow-up work and as a guide for individualization of therapy. (Received November 14, 1974) REFERENCES 1) Akiyama, T., Tamaura, N., Kenjo, T., Ohmura, T., Matsuda, R., Keio J. Med., 20, 15 (1971). 2) Andersen, V., Bjerrum, O., Bendixon, G., Schiodt, T., Dissing, I., Int. J. Cancer, 5, 357 (1970). 3) Biesecker, J. L., Transplantation, 15, 289 (1973). 4) Cerilli, J., Smith, M. C., Surg. Gynecol. Obstet., 134, 739 (1972). 5) Eilber, F. R., Morton, D. J., Cancer, 25, 362 (1970). 6) Falk, R. E., Mann, P., Langer, B., Arch. Surg., 107, 261 (1973). 7) Friedman, H., Transplantation, 11, 288 (1971). 8) Hilberg, R. W., Balcerzak, S. P., LoBuglio, A. F., Cell. Immunol., 7, 152 (1973). 9) Hughes, L. E., Lytton, B., Br. Med. J., 1, 209 (1964). 10) Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J., J. Biol. Chem., 193, 265 (1951). 11) Rocklin, R. E., David, J. R., "In Vitro Methods in Cell-mediated Immunity," ed. B. R. Bloom and P. R. Glade, p. 281 (1971). Academic Press, Inc., New York and London. 12) Rocklin, R. E., Meyers, O. L., David, J. R., J. Immunol., 104, 95 (1970). 13) Solowey, A. G., Rapaport, F. T., Surg. Gynecol. Obstet., 121, 756 (1965). 14) Stewart, T. H. M., Cancer, 23, 1368 (1969). 15) Thor, D. E., Jureziz, R. E., Veach, S. R., Miller, E., Dray, S., Nature, 219, 755 (1968). 16) Vaage, J., Jones, R. D., Brown, B. W., Cancer Res., 32, 680 (1972). 17) Wolberg, W. H., Cancer Res., 31, 798 (1971). 174 Gann