Radiation Sensitivity of Cultured Human Glioblastoma Cells 1
Radiation Biology
Leo E. Gerweck, Ph.D., Paul L. Kornbllth, M.D., 2 Peg Burlett, B.S., Janice Wang, A.B., and Susan Sweigert, B.S. Glial cells from 2 normal brains and 9 astrocytomas were cultured in vitro. The most rapidly proliferating cell lines were obtained from Grades III-IV astrocytomas. Normal glia proliferated slowly and growth ceased after 20 to 40 generations. The response of 3 Grade III-IV lines to graded doses of radiation was determined by colony formation and end point dilution technique. Two of the 3 lines were relatively radioresistant compared to other human cell lines; this may partially account for the radiation resistance of high grade astrocytoma tumors. INDEX TERMS:
Brain neoplasms. Glioma. Neoplasms, experimental • Radiosensitivity
Radiology 125:231-234, October 1977
IGH GRADE ASTROCYTOMAS are among the least re-
H
TABLE
sponsive tumors commonly treated by ionizing radiation. The lethal prognosis for patients with these tumors is not substantially altered by wide variation in total dose, fraction size, field size, and treatment time. Combinations of radiation with chemotherapy and surgery can now extend patient survival to almost a year, but survival for 5 years is virtually nonexistent (1). Interestingly, treatment failure is due to local recurrence rather than metastasis. Possible explanations for recurrence include: a relatively large fraction of clonogenic tumor cells, a significant fraction of hypoxic cells, absent or negligible tumor host immunogenicity, and radioresistance of the tumor cells. Little information has been published on the radiation sensitivity of cultured glial cells. The slow growth rate and low plating efficiency of these cells have hampered previous attempts at quantitative in vitro culture (2). Weischelbaum et al. (3) obtained survival curve parameters of Do ~ 135 and n ~ 1.5 for long term cultured human glioblastoma cells. Leith et al. (4) reported a Do dose of 160 and extrapolation number of 8 for cultured rat glial sarcoma cells. While the rat glial cells were relatively radioresistant, the survival curve data for human cells lies in the mid-range of previously reported survival curve parameters (5). We present data on the radiation sensitivity of these human tumor cells.
I:
GROWTH RATES OF CLN..TURED GLIAL CELL LINES
HISTOLOGICAL GRADE (KERHONAN)
POPULATION DOUBLING TIME' -HOURS
40-50 50-60 60-70 70-80 80-90 90-100 >100
Normal Glia
1
Grades I-II Grades III-IV
2
*
2
1
Onlymaximum growth rates are listed.
to 2 splitting ratios for approximately 5 passages. During the first few passages the tissue fragments were removed by settling. Splitting ratios were gradually increased to maintain cells in the exponential growth phase. Single cell suspensions were obtained with 0.25 % trypsin in Ca"" and Mg++ free Hanks solution containing EDTA. Population doubling times were determined by counting with an electronic cell counter the number of trypsin-suspended and supernatant cells during the exponential growth phase. The following procedure was used to determine if lethally irradiated feeder cells increased the fraction of single cells which formed colonies: for each cell line 6 groups of flasks were prepared with a constant number (0 to 100,000) of lethally irradiated feeder cells (3000 rads) of the same cell line in each group. Four replicates in each group were plated with 250 nonirradiated cells. In 2 groups an additional 8 flasks were plated. The flasks were incubated up to 4 weeks for colony development. The medium was changed at days 7 and 14 in half the flasks, and 4 flasks were removed to determine the number of colonies and number of cells/colony every 7 days. The remaining flasks were removed from the incubator and the colonies were scored when the number of colonies/flask became constant. The following procedure was used to evaluate the x-ray
MATERIALS AND METHODS
Primary cultures were obtained from biopsies of patients at Massachusetts General Hospital. The explant was immediately rinsed in Hanks balanced salt solution, dissected free of necrotic tissues, and minced into 1-2mm pieces. The fragments were cultured in a T-75 plastic flask containing Ham's F-10 plus 15% fetal calf serum, in an atmosphere of 5% CO2 , The cells were subcultured with 1
1 From The Edwin L. Steele Laboratory of Radiation Biology, Department of Radiation Medicine, Massachusetts General Hospital, Harvard Medical School, Boston. Accepted for publication in March 1977. Supported by NIH Grants CA07368, CA05163, and CA13311. 2 Neurosurgical Service, Massachusetts General Hospital, Harvard Medical School. emt
231
232
LEO E. GERWECK AND OTHERS
October 1977
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FEEDER CELLS XfO+~ Fig. 1. The effect of added feeder cells on the fraction of single cells which form colonies (plating efficiency). Replicate flasks were plated with 250 cells plus a variable number of lethally irradiated (3000 rad) feeder cells, and incubated for colony growth at 37°C. The medium was exchanged at 7 and 14 days (dashed line) in half the flasks. lines A3 and A7 were fixed and stained for colony enumeration after 20 days incubation, and A2 was evaluated after 25 days incubation. The mean and standard error are plotted.
sensitivity of the cells: monolayer cultures on plastic were irradiated with 250 kVp x-rays with HVL of 1.5 mm Cu, at a dose rate of approximately 100 rad/min. The cells were cooled to 22°C Y2 hour before irradiation, and irradiated on a rotating Masonite block. Within 3 minutes following irradiation, the cells were warmed to 37°C by submersion in a water bath. The cells were usuallytrypsinized, counted, and plated in 25 cm 2 plastic flasks for colony development 2 hours after, or 6 hours before irradiation. Lethally irradiated feeder cells were added at the time of plating. Four to 8 replicate flasks were plated for each dose level. Following 15-26 days incubation, the cells were rinsed with saline, fixed with methyl alcohol, and stained with crystal violet. The number of colonies was determined by counting circular cell aggregates with a cell density of 10X background, containing greater than 50 cells. Multiplicity which never exceeded 1.2 was used to adjust survival to the single cell level. The mean and standard error of survival was obtained from duplicate experiments and survival curves were fit to the data by eye.
Fig. 2. The lethal response of line A7 determined by 3 assay methods. Cells were trypsinized after irradiation and an appropriate number of cells was plated in replicate flasks for colony development (.). Cells were plated prior to irradiation (0). A serially diluted number of cells in flasks was plated after irradiation (X). See Methods for additional temperature and growth conditions.
Two procedures were followed to evaluate the possibility that the 15-25 days incubation would deplete the medium and therefore invalidate the results: (a) the medium was replaced during colony development, and the number of colonies obtained was compared to cultures without nutrient replenishment. (b) A serial dilution technique was used in which the maximum number of control and treated cells which yielded no colonies was compared; the possibility of subcolony formation was eliminated because survival was determined from cell numbers which yielded no colonies. The glial properties of the irradiated cell lines have been confirmed by several methods: (a) the presence of glial fibrils (6); (b) 8-100 protein (7); (c) membrane potential time constant (8); and (d) by immunological techniques (9). RESULTS
The maximum population doubling time of 9 astrocytomas and 2 normal glial cell lines, determined after 5-10 in vitro passages, is shown in TABLE I. Cultures derived from higher grade (III and IV) astrocytomas proliferated rapidly while low grade astrocytomas proliferated slowly in culture, and growth usually ceased after 10-12 passages. The use of McCoy's 5a medium, and various concentrations of calf and fetal calf serum, did not affect the eventual cessation of proliferation.
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Radiation Biology
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233
RADIATION SENSITIVITY OF CULTURED HUMAN GLIOBLASTOMA CELLS
Vol. 125
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600
800
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Fig. 3. Radiation cell survival curve for line A3, for cells trypsinized and re-plated after irradiation.
The 3 most rapidly proliferating cell lines (TABLE I) were evaluated for their capacity to form colonies from single cells. The results are presented in Figure 1. The addition of lethally irradiated feeder cells of up to 50 X 103 cells/ flask (2 X 103/cm2 and 10 X 103/ml medium) increased plating efficiency in all cases. Nutritional depletion did not inhibit colony formation except at the higher feeder cell concentration. Flasks containing 100,000 feeder cells had smaller and fewer colonies. In subsequent experiments a total of 50,000 live and lethally irradiated cells was plated. The lethal response of these cells to single doses of radiation is shown in Figures 2,3, and 4. Figure 2 shows the survival curve for line A7, a Grade'" astrocytoma, after 13 in vitro passages. The lethal response was determined by 3 methods: cells were trypsinized after irradiation, plated before irradiation, or plated after irradiation followed by serial dilution. All 3 methods yielded similar results. The lethal response of line A3, a Grade IV astrocytoma, after 12 in vitro passages is shown in Figure 3. The responses of lines A3 and A7 are similar; follOWing a dose of 600 rads the surviving fractions of lines A7 and A3 were O. 17 and 0.13 respectively. In contrast to the short-term cultured lines, cells of line A2, another Grade IV astrocytoma culture, are radiosensitive after 180 in vitro passages (Fig. 4). Cell lines with population doubling times greater than 60-70 hours did not yield reproducible results with the techniques used in this study.
400
Fig. 4. Survival for line A2, determined by the method described in the text and in Figure 3.
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Radiation Biology
Leo E. Gerweck, Ph.D., Paul L. Kornbllth, M.D., 2 Peg Burlett, B.S., Janice Wang, A.B., and Susan Sweigert, B.S. Glial cells from 2 normal brains and 9 astrocytomas were cultured in vitro. The most rapidly proliferating cell lines were obtained from Grades III-IV astrocytomas. Normal glia proliferated slowly and growth ceased after 20 to 40 generations. The response of 3 Grade III-IV lines to graded doses of radiation was determined by colony formation and end point dilution technique. Two of the 3 lines were relatively radioresistant compared to other human cell lines; this may partially account for the radiation resistance of high grade astrocytoma tumors. INDEX TERMS:
Brain neoplasms. Glioma. Neoplasms, experimental • Radiosensitivity
Radiology 125:231-234, October 1977
IGH GRADE ASTROCYTOMAS are among the least re-
H
TABLE
sponsive tumors commonly treated by ionizing radiation. The lethal prognosis for patients with these tumors is not substantially altered by wide variation in total dose, fraction size, field size, and treatment time. Combinations of radiation with chemotherapy and surgery can now extend patient survival to almost a year, but survival for 5 years is virtually nonexistent (1). Interestingly, treatment failure is due to local recurrence rather than metastasis. Possible explanations for recurrence include: a relatively large fraction of clonogenic tumor cells, a significant fraction of hypoxic cells, absent or negligible tumor host immunogenicity, and radioresistance of the tumor cells. Little information has been published on the radiation sensitivity of cultured glial cells. The slow growth rate and low plating efficiency of these cells have hampered previous attempts at quantitative in vitro culture (2). Weischelbaum et al. (3) obtained survival curve parameters of Do ~ 135 and n ~ 1.5 for long term cultured human glioblastoma cells. Leith et al. (4) reported a Do dose of 160 and extrapolation number of 8 for cultured rat glial sarcoma cells. While the rat glial cells were relatively radioresistant, the survival curve data for human cells lies in the mid-range of previously reported survival curve parameters (5). We present data on the radiation sensitivity of these human tumor cells.
I:
GROWTH RATES OF CLN..TURED GLIAL CELL LINES
HISTOLOGICAL GRADE (KERHONAN)
POPULATION DOUBLING TIME' -HOURS
40-50 50-60 60-70 70-80 80-90 90-100 >100
Normal Glia
1
Grades I-II Grades III-IV
2
*
2
1
Onlymaximum growth rates are listed.
to 2 splitting ratios for approximately 5 passages. During the first few passages the tissue fragments were removed by settling. Splitting ratios were gradually increased to maintain cells in the exponential growth phase. Single cell suspensions were obtained with 0.25 % trypsin in Ca"" and Mg++ free Hanks solution containing EDTA. Population doubling times were determined by counting with an electronic cell counter the number of trypsin-suspended and supernatant cells during the exponential growth phase. The following procedure was used to determine if lethally irradiated feeder cells increased the fraction of single cells which formed colonies: for each cell line 6 groups of flasks were prepared with a constant number (0 to 100,000) of lethally irradiated feeder cells (3000 rads) of the same cell line in each group. Four replicates in each group were plated with 250 nonirradiated cells. In 2 groups an additional 8 flasks were plated. The flasks were incubated up to 4 weeks for colony development. The medium was changed at days 7 and 14 in half the flasks, and 4 flasks were removed to determine the number of colonies and number of cells/colony every 7 days. The remaining flasks were removed from the incubator and the colonies were scored when the number of colonies/flask became constant. The following procedure was used to evaluate the x-ray
MATERIALS AND METHODS
Primary cultures were obtained from biopsies of patients at Massachusetts General Hospital. The explant was immediately rinsed in Hanks balanced salt solution, dissected free of necrotic tissues, and minced into 1-2mm pieces. The fragments were cultured in a T-75 plastic flask containing Ham's F-10 plus 15% fetal calf serum, in an atmosphere of 5% CO2 , The cells were subcultured with 1
1 From The Edwin L. Steele Laboratory of Radiation Biology, Department of Radiation Medicine, Massachusetts General Hospital, Harvard Medical School, Boston. Accepted for publication in March 1977. Supported by NIH Grants CA07368, CA05163, and CA13311. 2 Neurosurgical Service, Massachusetts General Hospital, Harvard Medical School. emt
231
232
LEO E. GERWECK AND OTHERS
October 1977
30
20
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20
RAO
10
o
o
10
25
50
80
100
FEEDER CELLS XfO+~ Fig. 1. The effect of added feeder cells on the fraction of single cells which form colonies (plating efficiency). Replicate flasks were plated with 250 cells plus a variable number of lethally irradiated (3000 rad) feeder cells, and incubated for colony growth at 37°C. The medium was exchanged at 7 and 14 days (dashed line) in half the flasks. lines A3 and A7 were fixed and stained for colony enumeration after 20 days incubation, and A2 was evaluated after 25 days incubation. The mean and standard error are plotted.
sensitivity of the cells: monolayer cultures on plastic were irradiated with 250 kVp x-rays with HVL of 1.5 mm Cu, at a dose rate of approximately 100 rad/min. The cells were cooled to 22°C Y2 hour before irradiation, and irradiated on a rotating Masonite block. Within 3 minutes following irradiation, the cells were warmed to 37°C by submersion in a water bath. The cells were usuallytrypsinized, counted, and plated in 25 cm 2 plastic flasks for colony development 2 hours after, or 6 hours before irradiation. Lethally irradiated feeder cells were added at the time of plating. Four to 8 replicate flasks were plated for each dose level. Following 15-26 days incubation, the cells were rinsed with saline, fixed with methyl alcohol, and stained with crystal violet. The number of colonies was determined by counting circular cell aggregates with a cell density of 10X background, containing greater than 50 cells. Multiplicity which never exceeded 1.2 was used to adjust survival to the single cell level. The mean and standard error of survival was obtained from duplicate experiments and survival curves were fit to the data by eye.
Fig. 2. The lethal response of line A7 determined by 3 assay methods. Cells were trypsinized after irradiation and an appropriate number of cells was plated in replicate flasks for colony development (.). Cells were plated prior to irradiation (0). A serially diluted number of cells in flasks was plated after irradiation (X). See Methods for additional temperature and growth conditions.
Two procedures were followed to evaluate the possibility that the 15-25 days incubation would deplete the medium and therefore invalidate the results: (a) the medium was replaced during colony development, and the number of colonies obtained was compared to cultures without nutrient replenishment. (b) A serial dilution technique was used in which the maximum number of control and treated cells which yielded no colonies was compared; the possibility of subcolony formation was eliminated because survival was determined from cell numbers which yielded no colonies. The glial properties of the irradiated cell lines have been confirmed by several methods: (a) the presence of glial fibrils (6); (b) 8-100 protein (7); (c) membrane potential time constant (8); and (d) by immunological techniques (9). RESULTS
The maximum population doubling time of 9 astrocytomas and 2 normal glial cell lines, determined after 5-10 in vitro passages, is shown in TABLE I. Cultures derived from higher grade (III and IV) astrocytomas proliferated rapidly while low grade astrocytomas proliferated slowly in culture, and growth usually ceased after 10-12 passages. The use of McCoy's 5a medium, and various concentrations of calf and fetal calf serum, did not affect the eventual cessation of proliferation.
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Radiation Biology
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RADIATION SENSITIVITY OF CULTURED HUMAN GLIOBLASTOMA CELLS
Vol. 125
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600
800
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Fig. 3. Radiation cell survival curve for line A3, for cells trypsinized and re-plated after irradiation.
The 3 most rapidly proliferating cell lines (TABLE I) were evaluated for their capacity to form colonies from single cells. The results are presented in Figure 1. The addition of lethally irradiated feeder cells of up to 50 X 103 cells/ flask (2 X 103/cm2 and 10 X 103/ml medium) increased plating efficiency in all cases. Nutritional depletion did not inhibit colony formation except at the higher feeder cell concentration. Flasks containing 100,000 feeder cells had smaller and fewer colonies. In subsequent experiments a total of 50,000 live and lethally irradiated cells was plated. The lethal response of these cells to single doses of radiation is shown in Figures 2,3, and 4. Figure 2 shows the survival curve for line A7, a Grade'" astrocytoma, after 13 in vitro passages. The lethal response was determined by 3 methods: cells were trypsinized after irradiation, plated before irradiation, or plated after irradiation followed by serial dilution. All 3 methods yielded similar results. The lethal response of line A3, a Grade IV astrocytoma, after 12 in vitro passages is shown in Figure 3. The responses of lines A3 and A7 are similar; follOWing a dose of 600 rads the surviving fractions of lines A7 and A3 were O. 17 and 0.13 respectively. In contrast to the short-term cultured lines, cells of line A2, another Grade IV astrocytoma culture, are radiosensitive after 180 in vitro passages (Fig. 4). Cell lines with population doubling times greater than 60-70 hours did not yield reproducible results with the techniques used in this study.
400
Fig. 4. Survival for line A2, determined by the method described in the text and in Figure 3.
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