IN VITRO CYTOTOXICITY BLADDER
STUDIES
IN
AND RENAL CELL CANCER*
MOSTAFA M. ELHILALI,
M.D.,
PH.D.
SHANKAR K. NAYAK, PH.D. From the Urology Department, Sherbrooke, Quebec, Canada
Sherbrooke University,
ABSTRACT - Attelnpts to obtain cell lines from bladder and renal cancer patients were done in 42 patients, with success to obtain primary cultures in 25 and long-term cultures in 7. Cell-mediated and humoral immunity was demonstrated in both tumors by microcytotoxicity in vitro. No cytotoxicity could be demonstrated against target cells obtainedfiom metastatic cancer tissue. Abrogation of cell-mediated immunity by serum factors was demonstrated. No recurrence was obtained in the group of patients showing positive lymphocytotoxicity during follow-up.
The possible role of the immune system in the life history of cancer is the subject of innumerable research projects at the present time. We will present in this article the results of in vitro cytotoxicity studies obtained in patients with bladder cancer and renal cell carcinoma. Material and Methods Target cells Human bladder cancer tissues were obtained from 31 patients. They were all histologically diagnosed as transitional cell cancers with 20 superficial and 11 infiltrating. Renal cell carcinoma tissues were obtained from 11patients: 9 were primary and 2 were metastatic. Control target cells were one cell line from normal bladder mucosa, one human fibroblast cell line from a prostatic cancer patient, and an established Hela cell line derived from human carcinoma of the cervix. In addition, for each tumor type cell lines from the other cancer were used as controls, namely, bladder cancer cells served as controls for renal cell cancer experiments and vice versa. Attempts were made to initiate cell cultures within two hours after tumor removal, primary *Supported by a grant from the National Cancer Institute of Canada.
488
cultures were prepared according to standard techniques. l Effector cells Blood samples (50 ml.) were obtained from 15 patients: 9 with bladder cancer and 6 with renal cell cancer. Blood samples were also obtained from 11 control subjects. Peripheral blood lymphocytes were separated and purified according to the method used by Hellstrom et ale2 Serum Sera were obtained from 18 patients: 9 with bladder cancer and 9 with renal cell cancer. Serum samples were obtained from 28 control subjects. All sera used in this study were inactivated at 56” C. for thirty minutes and sterilized by filtration through 0.22 Frn. millex filters. Sterilized sera were stored at - 20” C. Fresh and heat inactivated rabbit complement were used in all experiments. Donors for lymphocytes and sera were not receiving radiotherapy or chemotherapy at the time of testing. C ytotoxicity assays The microcytotoxicity technique described by Takasugi and Klein3 was used in all experiments.
UROLOGY
/ MAY1976
/ VOLUMEVII.
NUMBER5
TABLE I.
Target cells origin*
Bladder Transitional Cell Carcinoma Superficial Infiltrating Number of specimens Primary cultures Long-term cultures
20
11 9 (82) I (9)
9 (45) I (5)
Renal Cell Carcinoma Primary Metastatic
Totals
2 2 (loo) 2 (100)
L6) 3 (33)
42 25 7
*Numbers in parentheses indicate per cent
Target cell suspensions were prepared by trypsinization. The target cell to lymphocyte ratio used was 1: 100. Sera from each experimental or control group were diluted 1: 1 in medium. In serum cytotoxicity experiments undiluted fresh rabbit complement was added to half the wells, and inactivated rabbit complement was added to
cytes were added to respective wells. Calculations were done after fixation and staining as described. Results
the other halfand incubated for forty-five minutes before termination of the experiment. The mean count and the standard deviation of the target cells surviving incubation with different lymphocytes or sera were calculated. The results were then expressed as the percentage reduction in the cell count in the experimental group as compared with the controls. The statistical significance was established by Student’s t test and a P value less than 0.05 was taken as our confidence level.
Primary cultures were obtained in 9 of 20 cases when the tissues were obtained from superficial transitional cell carcinoma (Table I). When the tissues were obtained from infiltrating transitional cell carcinoma, primary cultures were obtained in 9 of 11 instances. Long-term cultures were obtained in 1 instance in each group. Renal cell carcinoma tissue culture attempts resulted in 5 of 9 primary cultures from primary tumors and 2 of 2 from metastatic cancer. Long-term cultures were obtained in 3 of 5 primary tumors and 2 of 2 metastatic.
Assay for serum-blocking
Lymphocyte
effect
Microcytotoxicity assays were employed.3 The target cells were washed to remove unattached cells, and the serum to be studied for the blocking effect was added to respective wells. Serum was diluted 1:5 in these experiments. Plates were incubated for forty-five minutes in a carbon dioxide incubator. The control and test lympho-
TABLE
Target Cells Origin PATIENTS
WITH
BLADDER
Other cancers
other cancers Bladder cancer
Renal cancer Renal cancer Other cancers
Lymphocytes obtained from patients with cancer of the bladder were tested against bladder cancer target cells. This included both autochthonous and allogeneic combinations. Significant cytotoxicity was obtained in 86 per cent of assays (Table II). No significant cytotoxicity was obtained when lymphocytes obtained from other
cytotoxicity
No. of Experiments
No. of Assays
Significant Cytotoxicity (P cO.05) (W
CANCER
Bladder cancer Other cancers Bladder and
WITH
Lymphocyte
Effector Cells Origin
Bladder cancer Bladder cancer Normal bladder
PATIENTS
UROLOGY
II.
cytotoxicity
4 6 2
7 6 4
86 0 0
10
20
5
16 13
16 22
11
11
44 5 0
RENAL CELL CARCINOMA
Renal cancer Other cancers Renal cancer
/ MAY 1976 / VOLUME
VII, NUMBER 5
489
TABLE III. Target Cells Origin
Complement-dependent
Sera Origin
serum cytotoxicity
No. of Experiments
Significant No. of Cytotoxicity (P cO.05) Assays @)
PATIENTSWITHBLADDERCANCER Bladder cancer Bladder cancer Normalbladder Other cancers
Bladder cancer Other cancers Bladder and other cancers Bladder cancer
9 7 2
15 4
13
69
19
34
6
30
33 15 9
27 0
PATIENTSWITHRENALCELL CARCINOMA Renal cancer Renal cancer Other cancers
Renal cancer Other cancers Renal cancer
cancers were tested against bladder cancer target cells. Similarly no significant cytotoxicity was obtained when lymphocytes obtained from bladder cancer and other cancers were tested against normal bladder mucosa target cells. When lymphocytes obtained from bladder cancer patients were tested against other cancer cell lines, 5 per cent significant cytotoxicity was obtained. When lymphocytes obtained from patients with renal cell cancer were tested against renal cancer target cells (autochthonous and allogeneic), significant cytotoxicity was obtained in 44 per cent of assays (Table II). No significant cytotoxicity was obtained when the same lymphocytes were tested against other cancer target cells. When lymphocytes obtained from other cancer patients were tested against renal cancer target cells, significant cytotoxicity was obtained in 5 per cent of assays. No lymphocyte cytotoxicity could be demonstrated in the autochthonous group against cell lines obtained from metastatic tumor. Complement-dependent
serum cytotoxicity
Significant cytotoxicity was obtained in 69 per cent of assays when sera obtained from patients with bladder cancer were tested against bladder cancer target cells (Table III). No cytotoxicity was obtained when sera from bladder cancer or other cancer patients were tested against normal bladder mucosa target cells. Significant cytotoxicity was obtained in 27 per cent of assays when sera obtained from other cancers were tested against bladder cancer target cells. The opposite combination, namely, sera from bladder cancer patients, caused 6 per cent significant cytotoxicity when tested against other target cells from other cancers.
490
17
48
22 9
11
Significant cytotoxicity was obtained in 33 per cent of assays when sera obtained from renal cancer patients were tested against renal cancer target cells (Table III). This was obtained in 15 per cent of assays when sera from other cancer patients were tested against renal cancer target cells and 9 per cent of assays when sera from renal cancer patients were tested against target cells from other cancer patients. Serum
blocking
effect
The possible blocking effect of serum on lymphocyte cytotoxicity was evaluated in three experiments performed on one target cell of primary renal cell carcinoma origin. The lymphocyte cytotoxicity was abrogated by sera in both autochthonous and allogeneic combinations (Table IV). Comment In this study we have demonstrated the relative difficulty to obtain long-term tissue cultures from transitional cell carcinoma tissues. The relatively TABLE IV.
Blocking of lymphocyte cytotoxicity by sera fi-om renal cancer patients (target cell = HKT-13)
Lymphocytes Donor
Serum Donor
Autochthonous
Autochthonous
Allogeneic
Autochthonous
Allogeneic
Allogeneic
Significant Blocking Activity (P=%0l) Yes (P=
STUDIES
IN
AND RENAL CELL CANCER*
MOSTAFA M. ELHILALI,
M.D.,
PH.D.
SHANKAR K. NAYAK, PH.D. From the Urology Department, Sherbrooke, Quebec, Canada
Sherbrooke University,
ABSTRACT - Attelnpts to obtain cell lines from bladder and renal cancer patients were done in 42 patients, with success to obtain primary cultures in 25 and long-term cultures in 7. Cell-mediated and humoral immunity was demonstrated in both tumors by microcytotoxicity in vitro. No cytotoxicity could be demonstrated against target cells obtainedfiom metastatic cancer tissue. Abrogation of cell-mediated immunity by serum factors was demonstrated. No recurrence was obtained in the group of patients showing positive lymphocytotoxicity during follow-up.
The possible role of the immune system in the life history of cancer is the subject of innumerable research projects at the present time. We will present in this article the results of in vitro cytotoxicity studies obtained in patients with bladder cancer and renal cell carcinoma. Material and Methods Target cells Human bladder cancer tissues were obtained from 31 patients. They were all histologically diagnosed as transitional cell cancers with 20 superficial and 11 infiltrating. Renal cell carcinoma tissues were obtained from 11patients: 9 were primary and 2 were metastatic. Control target cells were one cell line from normal bladder mucosa, one human fibroblast cell line from a prostatic cancer patient, and an established Hela cell line derived from human carcinoma of the cervix. In addition, for each tumor type cell lines from the other cancer were used as controls, namely, bladder cancer cells served as controls for renal cell cancer experiments and vice versa. Attempts were made to initiate cell cultures within two hours after tumor removal, primary *Supported by a grant from the National Cancer Institute of Canada.
488
cultures were prepared according to standard techniques. l Effector cells Blood samples (50 ml.) were obtained from 15 patients: 9 with bladder cancer and 6 with renal cell cancer. Blood samples were also obtained from 11 control subjects. Peripheral blood lymphocytes were separated and purified according to the method used by Hellstrom et ale2 Serum Sera were obtained from 18 patients: 9 with bladder cancer and 9 with renal cell cancer. Serum samples were obtained from 28 control subjects. All sera used in this study were inactivated at 56” C. for thirty minutes and sterilized by filtration through 0.22 Frn. millex filters. Sterilized sera were stored at - 20” C. Fresh and heat inactivated rabbit complement were used in all experiments. Donors for lymphocytes and sera were not receiving radiotherapy or chemotherapy at the time of testing. C ytotoxicity assays The microcytotoxicity technique described by Takasugi and Klein3 was used in all experiments.
UROLOGY
/ MAY1976
/ VOLUMEVII.
NUMBER5
TABLE I.
Target cells origin*
Bladder Transitional Cell Carcinoma Superficial Infiltrating Number of specimens Primary cultures Long-term cultures
20
11 9 (82) I (9)
9 (45) I (5)
Renal Cell Carcinoma Primary Metastatic
Totals
2 2 (loo) 2 (100)
L6) 3 (33)
42 25 7
*Numbers in parentheses indicate per cent
Target cell suspensions were prepared by trypsinization. The target cell to lymphocyte ratio used was 1: 100. Sera from each experimental or control group were diluted 1: 1 in medium. In serum cytotoxicity experiments undiluted fresh rabbit complement was added to half the wells, and inactivated rabbit complement was added to
cytes were added to respective wells. Calculations were done after fixation and staining as described. Results
the other halfand incubated for forty-five minutes before termination of the experiment. The mean count and the standard deviation of the target cells surviving incubation with different lymphocytes or sera were calculated. The results were then expressed as the percentage reduction in the cell count in the experimental group as compared with the controls. The statistical significance was established by Student’s t test and a P value less than 0.05 was taken as our confidence level.
Primary cultures were obtained in 9 of 20 cases when the tissues were obtained from superficial transitional cell carcinoma (Table I). When the tissues were obtained from infiltrating transitional cell carcinoma, primary cultures were obtained in 9 of 11 instances. Long-term cultures were obtained in 1 instance in each group. Renal cell carcinoma tissue culture attempts resulted in 5 of 9 primary cultures from primary tumors and 2 of 2 from metastatic cancer. Long-term cultures were obtained in 3 of 5 primary tumors and 2 of 2 metastatic.
Assay for serum-blocking
Lymphocyte
effect
Microcytotoxicity assays were employed.3 The target cells were washed to remove unattached cells, and the serum to be studied for the blocking effect was added to respective wells. Serum was diluted 1:5 in these experiments. Plates were incubated for forty-five minutes in a carbon dioxide incubator. The control and test lympho-
TABLE
Target Cells Origin PATIENTS
WITH
BLADDER
Other cancers
other cancers Bladder cancer
Renal cancer Renal cancer Other cancers
Lymphocytes obtained from patients with cancer of the bladder were tested against bladder cancer target cells. This included both autochthonous and allogeneic combinations. Significant cytotoxicity was obtained in 86 per cent of assays (Table II). No significant cytotoxicity was obtained when lymphocytes obtained from other
cytotoxicity
No. of Experiments
No. of Assays
Significant Cytotoxicity (P cO.05) (W
CANCER
Bladder cancer Other cancers Bladder and
WITH
Lymphocyte
Effector Cells Origin
Bladder cancer Bladder cancer Normal bladder
PATIENTS
UROLOGY
II.
cytotoxicity
4 6 2
7 6 4
86 0 0
10
20
5
16 13
16 22
11
11
44 5 0
RENAL CELL CARCINOMA
Renal cancer Other cancers Renal cancer
/ MAY 1976 / VOLUME
VII, NUMBER 5
489
TABLE III. Target Cells Origin
Complement-dependent
Sera Origin
serum cytotoxicity
No. of Experiments
Significant No. of Cytotoxicity (P cO.05) Assays @)
PATIENTSWITHBLADDERCANCER Bladder cancer Bladder cancer Normalbladder Other cancers
Bladder cancer Other cancers Bladder and other cancers Bladder cancer
9 7 2
15 4
13
69
19
34
6
30
33 15 9
27 0
PATIENTSWITHRENALCELL CARCINOMA Renal cancer Renal cancer Other cancers
Renal cancer Other cancers Renal cancer
cancers were tested against bladder cancer target cells. Similarly no significant cytotoxicity was obtained when lymphocytes obtained from bladder cancer and other cancers were tested against normal bladder mucosa target cells. When lymphocytes obtained from bladder cancer patients were tested against other cancer cell lines, 5 per cent significant cytotoxicity was obtained. When lymphocytes obtained from patients with renal cell cancer were tested against renal cancer target cells (autochthonous and allogeneic), significant cytotoxicity was obtained in 44 per cent of assays (Table II). No significant cytotoxicity was obtained when the same lymphocytes were tested against other cancer target cells. When lymphocytes obtained from other cancer patients were tested against renal cancer target cells, significant cytotoxicity was obtained in 5 per cent of assays. No lymphocyte cytotoxicity could be demonstrated in the autochthonous group against cell lines obtained from metastatic tumor. Complement-dependent
serum cytotoxicity
Significant cytotoxicity was obtained in 69 per cent of assays when sera obtained from patients with bladder cancer were tested against bladder cancer target cells (Table III). No cytotoxicity was obtained when sera from bladder cancer or other cancer patients were tested against normal bladder mucosa target cells. Significant cytotoxicity was obtained in 27 per cent of assays when sera obtained from other cancers were tested against bladder cancer target cells. The opposite combination, namely, sera from bladder cancer patients, caused 6 per cent significant cytotoxicity when tested against other target cells from other cancers.
490
17
48
22 9
11
Significant cytotoxicity was obtained in 33 per cent of assays when sera obtained from renal cancer patients were tested against renal cancer target cells (Table III). This was obtained in 15 per cent of assays when sera from other cancer patients were tested against renal cancer target cells and 9 per cent of assays when sera from renal cancer patients were tested against target cells from other cancer patients. Serum
blocking
effect
The possible blocking effect of serum on lymphocyte cytotoxicity was evaluated in three experiments performed on one target cell of primary renal cell carcinoma origin. The lymphocyte cytotoxicity was abrogated by sera in both autochthonous and allogeneic combinations (Table IV). Comment In this study we have demonstrated the relative difficulty to obtain long-term tissue cultures from transitional cell carcinoma tissues. The relatively TABLE IV.
Blocking of lymphocyte cytotoxicity by sera fi-om renal cancer patients (target cell = HKT-13)
Lymphocytes Donor
Serum Donor
Autochthonous
Autochthonous
Allogeneic
Autochthonous
Allogeneic
Allogeneic
Significant Blocking Activity (P=%0l) Yes (P=
