Int. J. Cancer: 16, 413-426 (1975)
CELLULAR IMMUNITY TO TRANSITIONAL CELL CARCINOMA OF THE URINARY BLADDER. 111. EFFECTS OF HYDROSTATIC PRESSURE THERAPY
Carol O’TOOLEl , 4, K. HELMSTEIN z, P. PERLMANN and G. MOBERGER Department of Immunology, Wenner Gren Institute, Nortullsgatan 16, 113-45 Stockholm: Department of Surgery, Sodersjukhuset, Stockholm 38, and Department of Tumor Pathology, Karolinska Sjukhuset, Stockholm 60, Sweden
The efects ofhydrostatic pressure therapy on in vitro cellular cytotoxicity responses have been studied in 19 patients with transitional cell carcinoma of the bladder (TCC) . Cytotoxicity was quantitated against allogeneic targets in a microplate assay or by 5’chromium isotope release. Two types of reactivity were detected, the most common being a differential cytotoxicity for targets derived from TCC, in either short-term or long-term tissue culture. This reaction is operationally termed ‘‘ tumor-specific ”. Less frequently, a general cytotoxicity for targets of diverse histogenic origins was observed. For the present, this is termed ‘‘ non-specific ”. Nine patients were tested before pressure therapy and of these only two gave a specific reaction and one a non-specific reaction, while six were non-reactive. Eighteen patients were tested at varying intervals after treatment and of these 11 gave a specific reaction and one a non-specific effect, while six were non-reactive. Three individuals who were non-reactive prior to therapy had a specific reaction post therapy. Two who reacted specifically before therapy became non-reactive after therapy. Two cases remained non-reactive throughout and the single case which gave a non-specific efect before therapy became non-reactive post therapy. The results of serial in vitro testing for cytotoxicity are presented with individual case histories, tumor staging and grading and the clinical outcome of hydrostatic pressure therapy.
Prognosis in patients with transitional cell carcinoma is influenced by the clinical stage and histologic grade of malignancy of the tumor (degree of maturation). Thus, a correlation exists between the degree of cellular pleomorphism and the biological behavior of the neoplasms (Bergkvist et al., 1965; Esposti et al., 1970). Recently hydrostatic pressure therapy has been introduced as a therapeutic alternative to surgery and radiotherapy (Helmstein, 1972). This treatment has been shown to reduce blood supply to
the urinary bladder (Arvidsson and Helmstein, 1972) and a resulting selective necrosis of neoplastic tissue has been observed (Helmstein, 1972). Adjacent to necrosing neoplastic tissue, a dense infiltration of plasmo-lymphocytes was noted in a majority of the cases, (Helmstein and Moberger, unpublished). To elucidate whether the celiular infiltrates represented a therapyinduced immune response rather than mereIy a local inflammatory reaction, cytotoxicity tests were performed.
Received: April 29, 1975. Present address: Department of Microbiology and Immunology, UCLA Medical School, Los Angeles, Calif. 90024, USA.
413
O’TOOLE ET AL.
Changes in in vitro reactivity of peripheral lymphoid cells toward bladder tumor targets have previously been reported in some patients receiving pressure therapy (Bubenik et al., 19706). The post-therapy reactions were reported to subside within about 1 month; however, no further follow-up was presented on these cases.
repeatedly treated for recurrent disease by electrocoagulation. Several cases had undergone repeated pressure therapy prior to current testing (for details, Tables I-IV). Clinical data on cases KD, GF, ChM, P N and NK have been presented by Helmstein (1972).
Necrosis of tumor tissue can be expected to release varying amounts of material into the periphery and/or to the exterior per urethrum, the quantity involved being influenced by tumor mass and mode of necrosis, whether rapid or prolonged. Sequential studies in patients with bladder carcinoma treated by surgery have shown that critical amounts of tumor material are required to stimulate and maintain tumorspecific cytotoxicity (CMI). Successful removal of tumor leads to the disappearance of detectable CMI within a 4-week period after surgery. However, CMI was maintained after surgery in the presence of residual tumor (O’Toole et al., ‘972a3 1973b)‘ Radiotherapy to the bladder causes tumor necrosis in situ but concomitantly produces a generalized lymphopenia. Sequential studies in patients given preoperative radiotherapy followed by cystectomy have indicated that tumor-associated materials are disseminated as a result of radiation-induced necrosis (O’Toole et al., 1972a, 6, 19736). Hydrostatic pressure therapy may produce an analogous situation to radiationinduced necrosis but without the accompanying lymphopenia.
Hydrostatic pressure This was performed as described by Helmstein (1972), using epidural anesthesia. A rubber balloon was introduced into the bladder through a resectoscope and slowly filled with saline. Pressure inside the bladder was measured indirectly by the height of the infusion reservoir above the patient and was maintained at the level of the patient’s diastolic pressure for 5%-7 h.
MATERIAL AND METHODS
TCC patients
Nineteen individuals were studied; six females and 13 males. Their ages ranged from 29-81 with a mean of 67 years. Six patients were classified as having multiple papillomatosis. This was defined as a lesion with numerous superficially growing, recurrent papillary tumors involving extensive areas of the bladder mucosa. The remaining 13 had either solitary or multiple carcinomas; the stage distribution according to uIcc (1963) was as follows: T25 cases; T3-4 cases; and T 4 4 c a s e s . Malignancy grading was based on the system of Bergkvist et a/. (1965): M2-1 case; M3-7 cases; and M4-5 cases. The majority of these patients had a prolonged disease history and had been
414
Control blood donors
These included 23 normal, healthy persons agematched with the bladder tumor patients where possible. Seventeen clinical controls were tested before therapy; 2 with hypernephroma, 2 cases with carcinoma of the renal pelvis, 6 with carcinoma of the prostate, 1 with carcinoma of the breast, 1 with basal cell carcinoma and 5 with malignant melanoma of the skin. Target cells
Specimens of transitional cell carcinoma (TCC) and non-malignant bladder epithelium were obtained from transurethral resection material. The tissue was finely minced and placed in a thin film of medium 199 containing 20% heatinactivated fetal calf serum (FCS), antibiotics (100 IU penicillin and 100 pg streptomycin) and 0.3 mg glutamine/ml. When epithelial outgrowth occurred the cells were passaged by treatment with a solution of 0.02% EDTA-t0.05% trypsin. All cultures were passaged at least twice before being used as targets in the cytotoxicity assays, to ensure elimination of contaminating lymphoid elements. Cells derived from six primary cultures of TCC and from three non-malignant bladder epithelium specimens were used in this study. Primary cultures of lung fibroblasts were also tested. The tissue culture passage (TC) number is presented for each primary target in the results. Long-term cell lines were used as standard reference targets; these were T24 (Bubenik et a/., 1973) and RT4 (Rigby and Franks, 1970) derived from TCC; HCV-29 (J. Fogh) established from
IMMUNITY IN TCC OF THE BLADDER
nonmalignant bladder epithelium; MEL-1, a cell line from metastatic cutaneous melanoma (Unsgaard and O'Toole, 1975) and Chang liver cells (Chang, 1954). Preparation of effector cells
This was as described previously by O'Toole et al., (1972a, b, 1973a, b ; O'Toole, 1973). Thirty to 50ml of defibrinated blood were obtained from each donor and mixed with a 3 % gelatin solution at a ratio of 3:l v/v. The bulk of the erythrocytes were sedimented during incubation at 37" C for 60 min. The leukocyte-plasma supernatant was decanted off, added to a nylon-wool column and incubated at 37" C for 30 min in humidified air 5 % CO,. Non-adherent cells were then retrieved from the column by washing with Tris-buffered Hank's solution (TH) containing 2.5 % FCS. The cells were pelleted at 700 xg for 10 min and then washed twice with TH. After resuspension in 1-2ml TH+2.5% FCS, 0.83% Tris-NH,CI solution at 4" C was added in a 8:l volume ratio to the cells. The cell suspension was kept at 4" C for 5-10 min to lyse residual erythrocytes. The mixture was then diluted with an equal volume of TH+lO% FCS and the cells pelleted at 300xg for 10min. The cells were washed three times more with TH+2.5% FCS, then counted using Turk's stain; viability was estimated by trypan blue dye exclusion. Differential counts were performed on each effector-cell preparation stained with May-Grunwald Giemsa. The purity of the preparations from bladder cancer and control blood donors was similar to that reported by Unsgaard and O'Toole (1975).
+
Microcytotoxicity assay for cell-mediated immunity
Target cells were prepared from monolayer cultures by EDTA-trypsin treatment, followed by three washings in medium 199 containing 10% FCS. The cells were seeded into Falcon 3034 microplates (Falcon Plastics, Oxnard, Calif., USA) to give optimally 50 cells/we1l in a 1Opl volume. Plating efficiency varied from 2040% for primary cultures and 90% or greater for established cell lines. The plates were incubated at 37" C in humidified air with 5 % CO, for 6-12 h to allow cell adhesion. The medium was then removed and replaced with either fresh medium 199 containing 10% FCS, antibiotics and glutamine alone, or with effector-cell preparations.
Each donor's lymphoid cells were tested at two or more ratios to the target cells. The tests were incubated 36-40 h at 37" C in humidified air with 5 % CO,. At termination of the experiments, the plates were washed gently with phosphate-buffered saline PH 7.2 and then stained with May-Griinwald Giemsa. Residual targets were counted visually. Calculation of cytotoxicity
The arithmetic mean of residual targets in wells which had contained patients' lymphoid cells was compared with that in wells having contained control donors' cells at equivalent ratios. The significance of differences was estimated by Student's t-test with pG0.05. Cytotoxicity is expressed as percent reduction (1 -P/C) x 100; where P = mean residual targets after incubation with bladder patients' lymphoid cells; and C = mean residual targets after incubation with control donors' lymphoid cells; at equivalent concentrations. Tumor-specific cytotoxicity refers to a significant effect produced by a given donor's effector cells only on targets of a common histogenic origin. Non-specific cytotoxicity refers to a significant effect produced on targets of more than one histogenic origin. Isotope release assay
Target-cell suspensions were prepared from monolayers as described above, 0.5 x lo6 cells in 3 ml of medium 199 with 10% FCS antibiotics and glutamine were labelled with 50 pCi sodium chromate (specific activity 100-300 mCi/mg Cr) for 3-4 h at 37" C in humidified air+5% CO,. The cells were then washed three times with medium 199+10 % FCS. Viability was estimated by trypan blue dye exclusion. Targets were added to 15 ml conical centrifuge tubes; using 1,000 cells/tube. Effector cells were added at a ratio of 1OO:l to the targets; the total incubation volume was 1 ml/tube and the incubation time was 15-20 h. Duplicate tubes were prepared for each effector-target combination and appropriate effector-cell-free controls. The total isotope released into the supernatant was compared to the total incorporated into the targets; variation between duplicates was +4%.
41 5
O’TOOLE ET AL.
on the T24 cell line (Figs. 1 and 2). This phenomenon has been documented previously, (O’Toole et al., 1972a, b, 1973a, b, 1974; O’Toole, 1973; Bubenik et al., 1973). A cross-reactivity between neoplastic cells derived from transitional epithelium was first described by Bubenik et a[., (1970a, b). Conversely, cells derived from nonmalignant bladder epithelium are not preferentially affected by lymphoid cells from patients with bladder carcinoma, as shown in Tables VI and XI. This confirms previous data (Bubenik et al., 1970a, b ; O’Toole et a/., 1973a, b, 1974; O’Toole, 1973). All tests were performed on allogeneic target cells, with a single exception. Patient Ch.M. was tested in addition on his autologous tumor cells in primary culture (Tu 15) (Fig. 1 ) . When tested 3 and 8 days after pressure therapy, this patient showed no reaction against either autologous (Tu 15) or allogeneic (T 24) targets. By 16 days post therapy, the patient did react against allogeneic bladder tumor cells from either a primary
RESULTS
Details of the cytotoxicity reactions obtained are presented in Tables V-XI. Quantitation of the response was achieved in the microplate assay by titrating the effector:target-cell ratio for each donor/target combination. Tumor-specific reactions were observed in both the microplate and 51chromium isotope release assays. Both methods gave qualitatively comparable results. However, the values obtained in the isotope release assay were quantitatively lower than in the micro plate test (Table XI). This may be explained by the lower effector :target-cell ratio and shorter incubation time used. When a 24 h incubation is used for the microplate assay, the two methods give a closer approximation (O’Toole, unpublished data). Target-cell specificity
Target cells from primary cultures derived from TCC gave results comparable with those obtainde
g100 m
Tu18
Y
a O‘O
50
n
3
8 Time post Therapy
16
h
23 days
4 months
RECURRENCE AT 6 MONTHS
FIGURE1
Ch.M. tested after being pressure-treated for the sixth time (17.11.70). For clinical details see Table I. Tul5 autologous tumor targets primary culture TC.3 Tu18 allogeneic primary culture TCC TC5. T24 allogeneic cell line derived from TCC. Columns represent percentage reduction obtained with effector/ target-cell ratios of 500:l and 250:l.
416
IMMUNITY IN TCC OF THE BLADDER
Tu 21
9 days Pre
5days Post Therapy
7weeks
8 weeks TUMOUR FREE 1.5 Years
FIGURE 2
K.St. tested before and after first pressure therapy (3.12.70),and after the second given on 25.5.71. For clinical details see Table 11, Tu18, TC6; Tu21, TC4 and Tu22, TC3; allogeneic primary cultures derived from TCC. Other details as in Figure 1.
culture Tu 18 or from the cell line T 24. Unfortunately, his autologous cells were no longer available for testing at this time. The results were similar 23 days after therapy. When retested 4 months after therapy, the patient still reacted against T 24. This case (previously reported as K.M.) was extensively studied by Bubenik et al. (1970b). In sequential tests, his leukocytes were found to react selectiveiy against bladder tumor targets. At that time, the post-therapy response was found to subside by 50 days after pressure therapy (Bubenik et al., 19706). When studied by us 1 year later, the patient was developing more frequent recurrences and eventually succumbed to metastatic disease. Incidence of cytotoxicity reactions before pressure therapy Nine patients were tested before receiving pressure therapy. Three had previously been treated by the same method. These patients had a prior history of TCC cancer ranging from 1 to
15 years; no case was freshly diagnosed or without previous treatment. The clinical details on each patient are presented in Tables I-IV. Among these patients, only two cases gave a tumor-specific reaction. Details on one such case, GF, are presented in Table VIII. Effector cells from patient S.J. had an equally potent reductive effect on targets from bladder tumor and of non-bladder origin (Table X). This patient was suspected of having metastatic disease prior to therapy and did develop widespread disease within 3 months after therapy. It has been previously observed that effector-cell preparations from the majority of patients with metastatic tumors show a non-specific reaction on target cells of diverse histogenic origins (Unsgaard and O'Toole, 1975). The low incidence of tumorspecific reactivity in this group of patients prior to therapy contrasts with that reported for patients with the same diagnosis and a shorter disease history, who had received no previous treatment (O'Toole et al., 1972a,b, 19736).
417
O'TOOLE ET AL. TABLE I PATIENTS WITH MULTIPLE PAPILLOMATOSlS CMI Patient
Duration Of disease
Clinical observations post therapy Before treatment
Time post pressure
K.G.L.
4 years
Not tested
Observed duration
CELLULAR IMMUNITY TO TRANSITIONAL CELL CARCINOMA OF THE URINARY BLADDER. 111. EFFECTS OF HYDROSTATIC PRESSURE THERAPY
Carol O’TOOLEl , 4, K. HELMSTEIN z, P. PERLMANN and G. MOBERGER Department of Immunology, Wenner Gren Institute, Nortullsgatan 16, 113-45 Stockholm: Department of Surgery, Sodersjukhuset, Stockholm 38, and Department of Tumor Pathology, Karolinska Sjukhuset, Stockholm 60, Sweden
The efects ofhydrostatic pressure therapy on in vitro cellular cytotoxicity responses have been studied in 19 patients with transitional cell carcinoma of the bladder (TCC) . Cytotoxicity was quantitated against allogeneic targets in a microplate assay or by 5’chromium isotope release. Two types of reactivity were detected, the most common being a differential cytotoxicity for targets derived from TCC, in either short-term or long-term tissue culture. This reaction is operationally termed ‘‘ tumor-specific ”. Less frequently, a general cytotoxicity for targets of diverse histogenic origins was observed. For the present, this is termed ‘‘ non-specific ”. Nine patients were tested before pressure therapy and of these only two gave a specific reaction and one a non-specific reaction, while six were non-reactive. Eighteen patients were tested at varying intervals after treatment and of these 11 gave a specific reaction and one a non-specific effect, while six were non-reactive. Three individuals who were non-reactive prior to therapy had a specific reaction post therapy. Two who reacted specifically before therapy became non-reactive after therapy. Two cases remained non-reactive throughout and the single case which gave a non-specific efect before therapy became non-reactive post therapy. The results of serial in vitro testing for cytotoxicity are presented with individual case histories, tumor staging and grading and the clinical outcome of hydrostatic pressure therapy.
Prognosis in patients with transitional cell carcinoma is influenced by the clinical stage and histologic grade of malignancy of the tumor (degree of maturation). Thus, a correlation exists between the degree of cellular pleomorphism and the biological behavior of the neoplasms (Bergkvist et al., 1965; Esposti et al., 1970). Recently hydrostatic pressure therapy has been introduced as a therapeutic alternative to surgery and radiotherapy (Helmstein, 1972). This treatment has been shown to reduce blood supply to
the urinary bladder (Arvidsson and Helmstein, 1972) and a resulting selective necrosis of neoplastic tissue has been observed (Helmstein, 1972). Adjacent to necrosing neoplastic tissue, a dense infiltration of plasmo-lymphocytes was noted in a majority of the cases, (Helmstein and Moberger, unpublished). To elucidate whether the celiular infiltrates represented a therapyinduced immune response rather than mereIy a local inflammatory reaction, cytotoxicity tests were performed.
Received: April 29, 1975. Present address: Department of Microbiology and Immunology, UCLA Medical School, Los Angeles, Calif. 90024, USA.
413
O’TOOLE ET AL.
Changes in in vitro reactivity of peripheral lymphoid cells toward bladder tumor targets have previously been reported in some patients receiving pressure therapy (Bubenik et al., 19706). The post-therapy reactions were reported to subside within about 1 month; however, no further follow-up was presented on these cases.
repeatedly treated for recurrent disease by electrocoagulation. Several cases had undergone repeated pressure therapy prior to current testing (for details, Tables I-IV). Clinical data on cases KD, GF, ChM, P N and NK have been presented by Helmstein (1972).
Necrosis of tumor tissue can be expected to release varying amounts of material into the periphery and/or to the exterior per urethrum, the quantity involved being influenced by tumor mass and mode of necrosis, whether rapid or prolonged. Sequential studies in patients with bladder carcinoma treated by surgery have shown that critical amounts of tumor material are required to stimulate and maintain tumorspecific cytotoxicity (CMI). Successful removal of tumor leads to the disappearance of detectable CMI within a 4-week period after surgery. However, CMI was maintained after surgery in the presence of residual tumor (O’Toole et al., ‘972a3 1973b)‘ Radiotherapy to the bladder causes tumor necrosis in situ but concomitantly produces a generalized lymphopenia. Sequential studies in patients given preoperative radiotherapy followed by cystectomy have indicated that tumor-associated materials are disseminated as a result of radiation-induced necrosis (O’Toole et al., 1972a, 6, 19736). Hydrostatic pressure therapy may produce an analogous situation to radiationinduced necrosis but without the accompanying lymphopenia.
Hydrostatic pressure This was performed as described by Helmstein (1972), using epidural anesthesia. A rubber balloon was introduced into the bladder through a resectoscope and slowly filled with saline. Pressure inside the bladder was measured indirectly by the height of the infusion reservoir above the patient and was maintained at the level of the patient’s diastolic pressure for 5%-7 h.
MATERIAL AND METHODS
TCC patients
Nineteen individuals were studied; six females and 13 males. Their ages ranged from 29-81 with a mean of 67 years. Six patients were classified as having multiple papillomatosis. This was defined as a lesion with numerous superficially growing, recurrent papillary tumors involving extensive areas of the bladder mucosa. The remaining 13 had either solitary or multiple carcinomas; the stage distribution according to uIcc (1963) was as follows: T25 cases; T3-4 cases; and T 4 4 c a s e s . Malignancy grading was based on the system of Bergkvist et a/. (1965): M2-1 case; M3-7 cases; and M4-5 cases. The majority of these patients had a prolonged disease history and had been
414
Control blood donors
These included 23 normal, healthy persons agematched with the bladder tumor patients where possible. Seventeen clinical controls were tested before therapy; 2 with hypernephroma, 2 cases with carcinoma of the renal pelvis, 6 with carcinoma of the prostate, 1 with carcinoma of the breast, 1 with basal cell carcinoma and 5 with malignant melanoma of the skin. Target cells
Specimens of transitional cell carcinoma (TCC) and non-malignant bladder epithelium were obtained from transurethral resection material. The tissue was finely minced and placed in a thin film of medium 199 containing 20% heatinactivated fetal calf serum (FCS), antibiotics (100 IU penicillin and 100 pg streptomycin) and 0.3 mg glutamine/ml. When epithelial outgrowth occurred the cells were passaged by treatment with a solution of 0.02% EDTA-t0.05% trypsin. All cultures were passaged at least twice before being used as targets in the cytotoxicity assays, to ensure elimination of contaminating lymphoid elements. Cells derived from six primary cultures of TCC and from three non-malignant bladder epithelium specimens were used in this study. Primary cultures of lung fibroblasts were also tested. The tissue culture passage (TC) number is presented for each primary target in the results. Long-term cell lines were used as standard reference targets; these were T24 (Bubenik et a/., 1973) and RT4 (Rigby and Franks, 1970) derived from TCC; HCV-29 (J. Fogh) established from
IMMUNITY IN TCC OF THE BLADDER
nonmalignant bladder epithelium; MEL-1, a cell line from metastatic cutaneous melanoma (Unsgaard and O'Toole, 1975) and Chang liver cells (Chang, 1954). Preparation of effector cells
This was as described previously by O'Toole et al., (1972a, b, 1973a, b ; O'Toole, 1973). Thirty to 50ml of defibrinated blood were obtained from each donor and mixed with a 3 % gelatin solution at a ratio of 3:l v/v. The bulk of the erythrocytes were sedimented during incubation at 37" C for 60 min. The leukocyte-plasma supernatant was decanted off, added to a nylon-wool column and incubated at 37" C for 30 min in humidified air 5 % CO,. Non-adherent cells were then retrieved from the column by washing with Tris-buffered Hank's solution (TH) containing 2.5 % FCS. The cells were pelleted at 700 xg for 10 min and then washed twice with TH. After resuspension in 1-2ml TH+2.5% FCS, 0.83% Tris-NH,CI solution at 4" C was added in a 8:l volume ratio to the cells. The cell suspension was kept at 4" C for 5-10 min to lyse residual erythrocytes. The mixture was then diluted with an equal volume of TH+lO% FCS and the cells pelleted at 300xg for 10min. The cells were washed three times more with TH+2.5% FCS, then counted using Turk's stain; viability was estimated by trypan blue dye exclusion. Differential counts were performed on each effector-cell preparation stained with May-Grunwald Giemsa. The purity of the preparations from bladder cancer and control blood donors was similar to that reported by Unsgaard and O'Toole (1975).
+
Microcytotoxicity assay for cell-mediated immunity
Target cells were prepared from monolayer cultures by EDTA-trypsin treatment, followed by three washings in medium 199 containing 10% FCS. The cells were seeded into Falcon 3034 microplates (Falcon Plastics, Oxnard, Calif., USA) to give optimally 50 cells/we1l in a 1Opl volume. Plating efficiency varied from 2040% for primary cultures and 90% or greater for established cell lines. The plates were incubated at 37" C in humidified air with 5 % CO, for 6-12 h to allow cell adhesion. The medium was then removed and replaced with either fresh medium 199 containing 10% FCS, antibiotics and glutamine alone, or with effector-cell preparations.
Each donor's lymphoid cells were tested at two or more ratios to the target cells. The tests were incubated 36-40 h at 37" C in humidified air with 5 % CO,. At termination of the experiments, the plates were washed gently with phosphate-buffered saline PH 7.2 and then stained with May-Griinwald Giemsa. Residual targets were counted visually. Calculation of cytotoxicity
The arithmetic mean of residual targets in wells which had contained patients' lymphoid cells was compared with that in wells having contained control donors' cells at equivalent ratios. The significance of differences was estimated by Student's t-test with pG0.05. Cytotoxicity is expressed as percent reduction (1 -P/C) x 100; where P = mean residual targets after incubation with bladder patients' lymphoid cells; and C = mean residual targets after incubation with control donors' lymphoid cells; at equivalent concentrations. Tumor-specific cytotoxicity refers to a significant effect produced by a given donor's effector cells only on targets of a common histogenic origin. Non-specific cytotoxicity refers to a significant effect produced on targets of more than one histogenic origin. Isotope release assay
Target-cell suspensions were prepared from monolayers as described above, 0.5 x lo6 cells in 3 ml of medium 199 with 10% FCS antibiotics and glutamine were labelled with 50 pCi sodium chromate (specific activity 100-300 mCi/mg Cr) for 3-4 h at 37" C in humidified air+5% CO,. The cells were then washed three times with medium 199+10 % FCS. Viability was estimated by trypan blue dye exclusion. Targets were added to 15 ml conical centrifuge tubes; using 1,000 cells/tube. Effector cells were added at a ratio of 1OO:l to the targets; the total incubation volume was 1 ml/tube and the incubation time was 15-20 h. Duplicate tubes were prepared for each effector-target combination and appropriate effector-cell-free controls. The total isotope released into the supernatant was compared to the total incorporated into the targets; variation between duplicates was +4%.
41 5
O’TOOLE ET AL.
on the T24 cell line (Figs. 1 and 2). This phenomenon has been documented previously, (O’Toole et al., 1972a, b, 1973a, b, 1974; O’Toole, 1973; Bubenik et al., 1973). A cross-reactivity between neoplastic cells derived from transitional epithelium was first described by Bubenik et a[., (1970a, b). Conversely, cells derived from nonmalignant bladder epithelium are not preferentially affected by lymphoid cells from patients with bladder carcinoma, as shown in Tables VI and XI. This confirms previous data (Bubenik et al., 1970a, b ; O’Toole et a/., 1973a, b, 1974; O’Toole, 1973). All tests were performed on allogeneic target cells, with a single exception. Patient Ch.M. was tested in addition on his autologous tumor cells in primary culture (Tu 15) (Fig. 1 ) . When tested 3 and 8 days after pressure therapy, this patient showed no reaction against either autologous (Tu 15) or allogeneic (T 24) targets. By 16 days post therapy, the patient did react against allogeneic bladder tumor cells from either a primary
RESULTS
Details of the cytotoxicity reactions obtained are presented in Tables V-XI. Quantitation of the response was achieved in the microplate assay by titrating the effector:target-cell ratio for each donor/target combination. Tumor-specific reactions were observed in both the microplate and 51chromium isotope release assays. Both methods gave qualitatively comparable results. However, the values obtained in the isotope release assay were quantitatively lower than in the micro plate test (Table XI). This may be explained by the lower effector :target-cell ratio and shorter incubation time used. When a 24 h incubation is used for the microplate assay, the two methods give a closer approximation (O’Toole, unpublished data). Target-cell specificity
Target cells from primary cultures derived from TCC gave results comparable with those obtainde
g100 m
Tu18
Y
a O‘O
50
n
3
8 Time post Therapy
16
h
23 days
4 months
RECURRENCE AT 6 MONTHS
FIGURE1
Ch.M. tested after being pressure-treated for the sixth time (17.11.70). For clinical details see Table I. Tul5 autologous tumor targets primary culture TC.3 Tu18 allogeneic primary culture TCC TC5. T24 allogeneic cell line derived from TCC. Columns represent percentage reduction obtained with effector/ target-cell ratios of 500:l and 250:l.
416
IMMUNITY IN TCC OF THE BLADDER
Tu 21
9 days Pre
5days Post Therapy
7weeks
8 weeks TUMOUR FREE 1.5 Years
FIGURE 2
K.St. tested before and after first pressure therapy (3.12.70),and after the second given on 25.5.71. For clinical details see Table 11, Tu18, TC6; Tu21, TC4 and Tu22, TC3; allogeneic primary cultures derived from TCC. Other details as in Figure 1.
culture Tu 18 or from the cell line T 24. Unfortunately, his autologous cells were no longer available for testing at this time. The results were similar 23 days after therapy. When retested 4 months after therapy, the patient still reacted against T 24. This case (previously reported as K.M.) was extensively studied by Bubenik et al. (1970b). In sequential tests, his leukocytes were found to react selectiveiy against bladder tumor targets. At that time, the post-therapy response was found to subside by 50 days after pressure therapy (Bubenik et al., 19706). When studied by us 1 year later, the patient was developing more frequent recurrences and eventually succumbed to metastatic disease. Incidence of cytotoxicity reactions before pressure therapy Nine patients were tested before receiving pressure therapy. Three had previously been treated by the same method. These patients had a prior history of TCC cancer ranging from 1 to
15 years; no case was freshly diagnosed or without previous treatment. The clinical details on each patient are presented in Tables I-IV. Among these patients, only two cases gave a tumor-specific reaction. Details on one such case, GF, are presented in Table VIII. Effector cells from patient S.J. had an equally potent reductive effect on targets from bladder tumor and of non-bladder origin (Table X). This patient was suspected of having metastatic disease prior to therapy and did develop widespread disease within 3 months after therapy. It has been previously observed that effector-cell preparations from the majority of patients with metastatic tumors show a non-specific reaction on target cells of diverse histogenic origins (Unsgaard and O'Toole, 1975). The low incidence of tumorspecific reactivity in this group of patients prior to therapy contrasts with that reported for patients with the same diagnosis and a shorter disease history, who had received no previous treatment (O'Toole et al., 1972a,b, 19736).
417
O'TOOLE ET AL. TABLE I PATIENTS WITH MULTIPLE PAPILLOMATOSlS CMI Patient
Duration Of disease
Clinical observations post therapy Before treatment
Time post pressure
K.G.L.
4 years
Not tested
Observed duration
