Int. J. Cancer: 19, 474-481 (1977)
THE MACROPHAGE ELECTROPHORETIC MOBILITY TEST : RESULTS ON CARCINOMA OF THE COLON A N D RECTUM Dorothy GLAVES, James P. HARLOS and Leonard WEISS Dcpartment of Experimental Pathology, Roswell Park Memorial Institute, Buflalo, N Y 14263, USA
The macrophage electrophoretic mobility ( M E M ) test was performed on guinea-pig macrophages treated with the interaction products of encephalitogenic protein and peripheral lymphocytes f rom 44 patiefits with colorectal cancer and 33 healthy" controls. I n 54/60 tests involving patients, statistically significant reductions in electrophoretic mobilities were observed, compared with 12/33 in controls. O u r overall results on the reductions in macrophage mobilities by lymphocyte products are in accord with the work of some other workers, but not all. In contrast t o many other studies, the standard procedures used here t o express the results should permit an exchange of data on an international basis and allow a more rapid, general appraisal of the M E M test.
It has been reported that when lymphocytes from patients with cancer are incubated with tumor or brain extracts, the product of these interactions will reduce the net surface negativity of guinea-pig macrophages. This forms the basis of the macrophage electrophoretic mobility (MEM) test (Field and Caspary, 1971 ; Dickinson et al., 1973; Pritchard et al., 1973a, 6). In most previous publications on the MEM-test, results have been expressed in instrument-dependent " transit times ", with no clear indications of variance, and with the use of arbitrary data selection procedures. This approach has hindered both the comparison of results between different laboratories, and the general assessment of the potential clinical application of the test. In the present communication we have expressed mobilities in standard units, and compared them by means of standard statistical procedures without data selection. The main purpose of this communication is to invite comparison of our own results with those of others. We present data derived from a group of 44 patients with carcinoma of the colon or rectum, and discuss these results with particular emphasis on the variables in the electrokinetic characterization of macrophage populations. MATERIAL AND METHODS
Lymphocyte donors
Blood was collected from a group of 44 patients with colonic or rectal cancer, with and without overt metastases, some of whom were receiving chemotherapy. Control samples were collected from " healthy " volunteers with no history of malignancy.
Encephalitogenic factor (EF) This material was Prepared from fresh human brain, obtained at autopsy, by sequential chloroform/ methanol and acid extractions, according to the method of Kies (1965) which is similarto that used by CaSParY and Field (1971) for their preparation of EF. The final extract was stored at -80" C at a concentration of not less than 5 mg/ml in Hanks' balanced salt solution (HBSS). Lymphocytes Peripheral lymphocytes were isolated from at least 10 ml blood collected into heparinized " Vacutainer " tubes (Becton-Dickinson, Rutherford, New Jersey). The isolation procedure used by us was developed by Dr. M. Goldrosen (Goldrosen, 1976) as a modification of standard lymphocyte isolation techniques, and yielded preparations containing 95-99 % lymphocytes. To each 20ml of anticoagulated blood was added 0.5 ml of 0.04% ADP in 0.15 M NaCl and 0.5 ml of 0.5 % carbonyl iron in HBSS; samples were rotated slowly at 37" C for 45 minutes. The blood was then diluted 1:l with RPMI 1640 medium and lymphocytes were separated by centrifugation on discontinuous density gradients (English and Anderson, 1974). Lymphocytes were washed twice by centrifugation at 500 g for 15 min and resuspended in RPMI 1640. Finally cell suspensions were adjusted to contain 1 x lo6 viable lymphocytes per ml. Lymphocyte-EF interaction Two million lymphocytes were routinely incubated with 100,ug EF for 90min at room temperature, after which they were removed by centrifugation at 500 g for 15 min and the supernatants were stored at -20" C until assayed. Macrophage preparations Female Camm-Hartley guinea-pigs (Camm Research Institute, New Jersey) weighing 500-800 g, were used in these studies. Animals received 20 ml sterile light mineral oil by intraperitoneal injection, 6-10 days prior to harvesting. Anesthetized guineapigs were exsanguinated by cardiac puncture, and macrophages were aspirated from the abdominal cavity following intraperitoneal injection of 100 ml
Received: November 10, 1976 and in revised form January 19, 1977.
475
MEM TEST
cold HBSS containing 5 IU heparin per ml. The cell suspension was centrifuged at 220g for 5 min and resuspended in 5 ml Tris-buffered ammonium chloride (Boyle, 1968) for 2-5 min to lyse contaminating erythrocytes. This procedure reduces clumping of the macrophage suspension and does not affect cell electrophoretic mobility measurements. The cells were then washed three times by repeated centrifugation at 220g for 5 min, resuspended in cold RPMI 1640, and adjusted to contain 1 x 10' trypan-blueexcluding macrophages per ml RPMI 1640. Macrophage-lymphocyte product interaction Two ml of macrophage suspension containing 1 x lo7 celIs/ml were incubated for 90 min at 37" C with an equal volume of supernatant from patients' or control lymphocytes previously incubated with EF. Electrophoretic mobility measurements were made on coded samples. Electrophoresis apparatus Apparatus of the cylindrical tube type was used throughout, as basically described by Seaman (1965). The tube was immersed in a water bath maintained at 25" C, with grey, sintered platinum electrodes at each end. A constant potential was maintained between the electrodes by means of an electronic power pack. The viewing microscope was focused on the so-called " stationary layer " of the electrophoresis tube, and only cells in sharp focus were measured. Cells were timed in transit between vertical lines in an eyepiece grid, the distance between these lines having been previously determined for the apparatus, using a micrometer slide. Each cell was made to move in both directions by current reversal. For a knowl-
edge of transit time and distance covered, the velocity of cells is calculable. In converting these data to electrophoretic mobilities, i.e. pm per second per volt per cm of electrical potential gradient, the voltage gradient between the ends of the capillary tube must be accurately determined for each tube. This is done once for each tube, by filling it with a standard potassium chloride solution, and measuring the specific resistance with a Wayne-Kerr bridge. From a knowledge of the cross-section of the capillary tube, its electrical length may be calculated from the specific resistance. Thus a constant, K, for a particular tube in a specific piece of apparatus is determined and electrophoretic mobility, U, is related to mean transit time, t, by: U = K/t In all experiments, macrophages were suspended in synthetic medium RPMI 1640 (Moore et al., 1967) at 25" C, when its viscosity is 0.915 centipoises. Before each run, the correct functioning of the apparatus was checked by means of standard reference particles, in the present case human erythrocytes, which have a mean electrophoretic mobility of - 1.08 ,um volt -%ec -l cm at 25" C, in RPMI 1640. Measurements on macrophages were confined to those containing one or more oil droplets, and which were >15 p n in diameter-a procedure followed by Shenton et al. (1973) and others. In each experiment, the following samples were assayed: (1) macrophages in RPMI 1640 alone (control macrophages), (2) macrophages exposed to patients' lymphocyte-EF interaction products (test macrophages) and (3) macrophages exposed to normal volunteer lymphocyte-EF interaction products (normal macrophages).
TABLE I MACROPHAGE ELECTROPHORETIC MOBILITY ASSAYS ON PATIENTS WITH GASTROINTESTINAL CANCER
Patient
1 2
Age
56 26
Sex
M F
Chemotherapy
+ + +
3
70
M
-
-
4 5 6 7
50 68 67 48
F M M
M
++ + + + -
Disease history
Metastases present Metastases 8 weeks post op. Metastases 16 weeks post op. No recurrence No recurrence No recurrence Residual disease Metastases present Metastases present Metastases present Metastases present Metastases 4 weeks post op.
Macrophage electrophoretic mobility (pm sec-'volt-' cm) Test
Control
Percentage difference
-0.79 (f .016) -0.85 (i.016)
-0.91 (k .022) -1.05 (i.014)
-13 -18
0.001 0.001
-0.86 (+ .014)
-1.09 (+ .018)
-21
0.001
-0.96 -0.95 -0.90 -0.96 -0.94 -0.92 -0.94 -1.00 -0.97
-19 -12 -10 -20 -10 -11 -12 -12 -14
0.001 0.001 0.001 0.001 0.001 0.01 0.001 0.001
-0.78 -0.83 -0.81 -0.77 -0.85 -0.82 -0.83 -0.88 -0.84
(k .014) (+ .014)
(k .014) (+ .015) (+ ,015) (+ .018)
( 5 .017) (+ .015) (& .013)
(* .018) (f .022) ( f .018) (+. 018) (& .014) (k .019) (& .015) (i.012) (+ .017)
p
THE MACROPHAGE ELECTROPHORETIC MOBILITY TEST : RESULTS ON CARCINOMA OF THE COLON A N D RECTUM Dorothy GLAVES, James P. HARLOS and Leonard WEISS Dcpartment of Experimental Pathology, Roswell Park Memorial Institute, Buflalo, N Y 14263, USA
The macrophage electrophoretic mobility ( M E M ) test was performed on guinea-pig macrophages treated with the interaction products of encephalitogenic protein and peripheral lymphocytes f rom 44 patiefits with colorectal cancer and 33 healthy" controls. I n 54/60 tests involving patients, statistically significant reductions in electrophoretic mobilities were observed, compared with 12/33 in controls. O u r overall results on the reductions in macrophage mobilities by lymphocyte products are in accord with the work of some other workers, but not all. In contrast t o many other studies, the standard procedures used here t o express the results should permit an exchange of data on an international basis and allow a more rapid, general appraisal of the M E M test.
It has been reported that when lymphocytes from patients with cancer are incubated with tumor or brain extracts, the product of these interactions will reduce the net surface negativity of guinea-pig macrophages. This forms the basis of the macrophage electrophoretic mobility (MEM) test (Field and Caspary, 1971 ; Dickinson et al., 1973; Pritchard et al., 1973a, 6). In most previous publications on the MEM-test, results have been expressed in instrument-dependent " transit times ", with no clear indications of variance, and with the use of arbitrary data selection procedures. This approach has hindered both the comparison of results between different laboratories, and the general assessment of the potential clinical application of the test. In the present communication we have expressed mobilities in standard units, and compared them by means of standard statistical procedures without data selection. The main purpose of this communication is to invite comparison of our own results with those of others. We present data derived from a group of 44 patients with carcinoma of the colon or rectum, and discuss these results with particular emphasis on the variables in the electrokinetic characterization of macrophage populations. MATERIAL AND METHODS
Lymphocyte donors
Blood was collected from a group of 44 patients with colonic or rectal cancer, with and without overt metastases, some of whom were receiving chemotherapy. Control samples were collected from " healthy " volunteers with no history of malignancy.
Encephalitogenic factor (EF) This material was Prepared from fresh human brain, obtained at autopsy, by sequential chloroform/ methanol and acid extractions, according to the method of Kies (1965) which is similarto that used by CaSParY and Field (1971) for their preparation of EF. The final extract was stored at -80" C at a concentration of not less than 5 mg/ml in Hanks' balanced salt solution (HBSS). Lymphocytes Peripheral lymphocytes were isolated from at least 10 ml blood collected into heparinized " Vacutainer " tubes (Becton-Dickinson, Rutherford, New Jersey). The isolation procedure used by us was developed by Dr. M. Goldrosen (Goldrosen, 1976) as a modification of standard lymphocyte isolation techniques, and yielded preparations containing 95-99 % lymphocytes. To each 20ml of anticoagulated blood was added 0.5 ml of 0.04% ADP in 0.15 M NaCl and 0.5 ml of 0.5 % carbonyl iron in HBSS; samples were rotated slowly at 37" C for 45 minutes. The blood was then diluted 1:l with RPMI 1640 medium and lymphocytes were separated by centrifugation on discontinuous density gradients (English and Anderson, 1974). Lymphocytes were washed twice by centrifugation at 500 g for 15 min and resuspended in RPMI 1640. Finally cell suspensions were adjusted to contain 1 x lo6 viable lymphocytes per ml. Lymphocyte-EF interaction Two million lymphocytes were routinely incubated with 100,ug EF for 90min at room temperature, after which they were removed by centrifugation at 500 g for 15 min and the supernatants were stored at -20" C until assayed. Macrophage preparations Female Camm-Hartley guinea-pigs (Camm Research Institute, New Jersey) weighing 500-800 g, were used in these studies. Animals received 20 ml sterile light mineral oil by intraperitoneal injection, 6-10 days prior to harvesting. Anesthetized guineapigs were exsanguinated by cardiac puncture, and macrophages were aspirated from the abdominal cavity following intraperitoneal injection of 100 ml
Received: November 10, 1976 and in revised form January 19, 1977.
475
MEM TEST
cold HBSS containing 5 IU heparin per ml. The cell suspension was centrifuged at 220g for 5 min and resuspended in 5 ml Tris-buffered ammonium chloride (Boyle, 1968) for 2-5 min to lyse contaminating erythrocytes. This procedure reduces clumping of the macrophage suspension and does not affect cell electrophoretic mobility measurements. The cells were then washed three times by repeated centrifugation at 220g for 5 min, resuspended in cold RPMI 1640, and adjusted to contain 1 x 10' trypan-blueexcluding macrophages per ml RPMI 1640. Macrophage-lymphocyte product interaction Two ml of macrophage suspension containing 1 x lo7 celIs/ml were incubated for 90 min at 37" C with an equal volume of supernatant from patients' or control lymphocytes previously incubated with EF. Electrophoretic mobility measurements were made on coded samples. Electrophoresis apparatus Apparatus of the cylindrical tube type was used throughout, as basically described by Seaman (1965). The tube was immersed in a water bath maintained at 25" C, with grey, sintered platinum electrodes at each end. A constant potential was maintained between the electrodes by means of an electronic power pack. The viewing microscope was focused on the so-called " stationary layer " of the electrophoresis tube, and only cells in sharp focus were measured. Cells were timed in transit between vertical lines in an eyepiece grid, the distance between these lines having been previously determined for the apparatus, using a micrometer slide. Each cell was made to move in both directions by current reversal. For a knowl-
edge of transit time and distance covered, the velocity of cells is calculable. In converting these data to electrophoretic mobilities, i.e. pm per second per volt per cm of electrical potential gradient, the voltage gradient between the ends of the capillary tube must be accurately determined for each tube. This is done once for each tube, by filling it with a standard potassium chloride solution, and measuring the specific resistance with a Wayne-Kerr bridge. From a knowledge of the cross-section of the capillary tube, its electrical length may be calculated from the specific resistance. Thus a constant, K, for a particular tube in a specific piece of apparatus is determined and electrophoretic mobility, U, is related to mean transit time, t, by: U = K/t In all experiments, macrophages were suspended in synthetic medium RPMI 1640 (Moore et al., 1967) at 25" C, when its viscosity is 0.915 centipoises. Before each run, the correct functioning of the apparatus was checked by means of standard reference particles, in the present case human erythrocytes, which have a mean electrophoretic mobility of - 1.08 ,um volt -%ec -l cm at 25" C, in RPMI 1640. Measurements on macrophages were confined to those containing one or more oil droplets, and which were >15 p n in diameter-a procedure followed by Shenton et al. (1973) and others. In each experiment, the following samples were assayed: (1) macrophages in RPMI 1640 alone (control macrophages), (2) macrophages exposed to patients' lymphocyte-EF interaction products (test macrophages) and (3) macrophages exposed to normal volunteer lymphocyte-EF interaction products (normal macrophages).
TABLE I MACROPHAGE ELECTROPHORETIC MOBILITY ASSAYS ON PATIENTS WITH GASTROINTESTINAL CANCER
Patient
1 2
Age
56 26
Sex
M F
Chemotherapy
+ + +
3
70
M
-
-
4 5 6 7
50 68 67 48
F M M
M
++ + + + -
Disease history
Metastases present Metastases 8 weeks post op. Metastases 16 weeks post op. No recurrence No recurrence No recurrence Residual disease Metastases present Metastases present Metastases present Metastases present Metastases 4 weeks post op.
Macrophage electrophoretic mobility (pm sec-'volt-' cm) Test
Control
Percentage difference
-0.79 (f .016) -0.85 (i.016)
-0.91 (k .022) -1.05 (i.014)
-13 -18
0.001 0.001
-0.86 (+ .014)
-1.09 (+ .018)
-21
0.001
-0.96 -0.95 -0.90 -0.96 -0.94 -0.92 -0.94 -1.00 -0.97
-19 -12 -10 -20 -10 -11 -12 -12 -14
0.001 0.001 0.001 0.001 0.001 0.01 0.001 0.001
-0.78 -0.83 -0.81 -0.77 -0.85 -0.82 -0.83 -0.88 -0.84
(k .014) (+ .014)
(k .014) (+ .015) (+ ,015) (+ .018)
( 5 .017) (+ .015) (& .013)
(* .018) (f .022) ( f .018) (+. 018) (& .014) (k .019) (& .015) (i.012) (+ .017)
p
