International Journal of
Radiation Oncology biology
physics
www.redjournal.org
BRIEF REPORT AND OPINION
Is Indirect Cell Death Involved in Response of Tumors to Stereotactic Radiosurgery and Stereotactic Body Radiation Therapy? Chang W. Song, PhD,* Inhwan Park, BA,* L. Chinsoo Cho, MD,* Jianling Yuan, MD, PhD,* Kathryn E. Dusenbery, MD,* Robert J. Griffin, PhD,y and Seymour H. Levitt, MD* *Department of Therapeutic Radiology-Radiation Oncology, University of Minnesota Medical School, Minneapolis, Minnesota; and yDepartment of Radiation Oncology, University of Arkansas for Medical Sciences, Little Rock, Arkansas Received Mar 6, 2014, and in revised form Mar 14, 2014. Accepted for publication Mar 25, 2014.
Despite the increasing use of stereotactic radiosurgery (SRS) and stereotactic body radiation therapy (SBRT) in recent years, the biological principles underlying the impressive clinical effects of SRS and SBRT are still unclear. We previously hypothesized that hypofractionated irradiation of tumors with doses greater than 10 Gy per fraction cause severe vascular damage leading to indirect cell death, and suggested that such indirect cell death plays an important role in the response of tumors to SRS and SBRT (1-4). However, in a recent article, Brown et al (5) concluded that the standard radiobiological concepts are sufficient to explain the clinical results of SRS and SBRT, and that our previous data (3, 4) are only fragmentary to support our conclusion. Here, we report our new experimental data that strongly demonstrate that significant indirect cell death might occur in tumors treated with SRS or SBRT. We used HT-1080 human fibrosarcoma xenografts grown subcutaneously in the rear limb of nu/nu mice, and FSaII mouse fibrosarcoma grown subcutaneously in the rear limb of C3H mice. Tumors 6 to 7 mm in diameter were irradiated with 10 to 20 Gy of x-rays in a single dose, excised at different times after irradiation, and dispersed into single cells and the harvested cells were assayed for clonogenicity. Figure 1 shows the effect of 20 Gy on the surviving fraction of HT-1080 cells. The range of surviving fraction for
individual tumor was rather large, particularly 3 and 5 days after irradiation. Nevertheless, the mean or median values of cell survival on day 3 were markedly lower than those observed immediately after, irradiation indicating that significant secondary cell death occurred by 3 days. After a maximum nadir in cell survival on day 3, the mean and median values of cell survival slightly increased, suggesting that some repopulation or proliferation of tumor cells had occurred. However, in some tumors, the surviving fraction remained more than 2 logs lower than that immediately after irradiation for 5 days, indicating that secondary cell death continued for 5 days after irradiation. In Figure 2, the surviving fractions in FSaII tumors assayed immediately and 2 days after irradiation are compared. In the tumors exposed to 10 Gy, the surviving fraction immediately and 2 days after irradiation were similar. On the other hand, the surviving fractions 2 days after irradiation with 15 Gy or 20 Gy were markedly less than those immediately after irradiation. Further studies are in progress to reveal the cell survival on later days after irradiation in the FSaII tumor model. The results shown in Figures 1 and 2, together with our previous observations (3, 4), unequivocally indicate that irradiation with high dose per fraction induces indirect death in tumor cells. Such an indirect cell death after high-dose radiation is
Reprint requests to: Chang W. Song, PhD, Department of Therapeutic RadiologyeRadiation Oncology, University of Minnesota Medical School,
MMC 494, 424 Harvard St SE, Minneapolis, MN 55455. Tel.: (612) 6266852; E-mail: [email protected] Conflict of interest: none.
Int J Radiation Oncol Biol Phys, Vol. 89, No. 4, pp. 924e925, 2014 0360-3016/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ijrobp.2014.03.043
Volume 89 Number 4 2014
Indirect tumor cell death by SRS and SBRT
100
HT-1080
FSall Tumor
1.0
20 GY
925
Day 0
Surviving Cell Fraction
Day 2
Surviving Cell Fraction
10-1
10-2
0.1
0.01
0.001
0.0001
10-3
10 Gy Mean
0
20 Gy
Dose
Median
10-4
15 Gy
1
2
3
4
5
Days
Fig. 1. Effect of 20 Gy in a single dose on the surviving fraction in HT-1080 human sarcoma xenografts in nu/nu mice. The surviving fraction in each tumor was determined from the number of cells harvested from each tumor and their clonogenicity. The mean and 1 standard error of the mean (closed circle) and median value (triangle) for each group are shown. likely due to radiation-induced vascular damage and ensuing deterioration of the intratumor microenvironment (2, 5). Enhancement of antitumor immunity has been suggested to be involved in the response of tumors to ablative radiation therapy (6). However, given that indirect cell death occured rather soon (within 2-3 days) after irradiation, it is unlikely that immune reactions played an important role in the secondary cell death that we observed. Furthermore, indirect cell death also occurred in HT-1080 xenografts grown in immunologically compromised nu/nu mice, which further demonstrates that immune activation was not a major player in the induction of secondary cell death in the present study. It has been known that there are as many as 108 to 109 clonogenic cells in a 1-g tumor (7). A calculation based on conventional radiobiological principles indicates that the radiation doses currently used in SRS and SBRT, that is, 20-60 Gy in 1 to 5 fractions, are apparently insufficient to directly sterilize all of the clonogenic cells in a tumors as small as 1 g, assuming that 10% to 20% of tumor cells are hypoxic (8, 9). The fact that SRS and SBRT are highly effective for achieving complete control of many tumors strongly indicates that SRS and SBRT sterilize significant fractions of tumor cells by mechanisms other than directly killing all tumor cells. The
Fig. 2. Surviving fractions of FSaII tumor cells immediately (day 0) and 2 days after irradiation with 10 Gy, 15 Gy, and 20 Gy in a single dose. Surviving cell fractions were determined as shown in Fig. 1. The mean of 9 to 12 tumors and 1 standard error of the mean for each group are shown. evidence shown in the present study, together with previous reports (3, 4) clearly demonstrate that secondary cell death that is most likely caused by deterioration of the tumor physiology is involved in the response of tumors to high doseper-fraction SRS and SBRT.
References 1. Song CW, Park HJ, Griffin RJ, et al. Radiobiology of stereotactic radiosurgery and stereotactic body radiation therapy. In: Levitt SH, et al., editors. Technical Basis of Radiation Therapy, Medical Radiology, Radiation Oncology. Berlin: Springer-Verlag; 2012. p. 51-61. 2. Park HJ, Griffin RJ, Hui S, et al. Radiation-induced vascular damage in tumors: Implications of vascular damage in ablative hypofractionated radiotherapy (SBRT and SRS). Radiat Res 2012;177:311-327. 3. Song CW, Cho LC, Yuan J, et al. Radiobiology of stereotactic body radiation therapy/stereotactic radiosurgery and the linear-quadratic mode. Int J Radiat Oncol Biol Phys 2013;87:18-19. 4. Clement JJ, Takanka N, Song CW. Tumor reoxygenation and postirradiation vascular changes. Radiology 1978;127:799-803. 5. Brown JM, Carlson DJ, Brenner DJ. The tumor radiobiology of SRS and SBRT: Are more than the 5 Rs involved? Int J Radiat Oncol Biol Phys 2014;88:254-262. 6. Lee Y, Auh SL, Wang Y, et al. Therapeutic effects of ablation on local tumor require CR8þ T cells: Changing strategies for cancer treatment. Blood 2009;114:589-595. 7. Monte UD. Does the cell number 109 still really fit one gram of tumor tissue? Cell Cycle 2009;8:505-506. 8. Fowler JF, Wolfgan AT, Fenwik JD, et al. A challenge to traditional radiation oncology. Int J Radiat Oncol Biol Phys 2004;60:1241-1256. 9. Brown JM, Diehn M, Loo BW. Stereotactic ablative radiotherapy should be combined with a hypoxic cell radiosensitizer. Int J Radiat Oncol Biol Phys 2010;78:323-327.
Radiation Oncology biology
physics
www.redjournal.org
BRIEF REPORT AND OPINION
Is Indirect Cell Death Involved in Response of Tumors to Stereotactic Radiosurgery and Stereotactic Body Radiation Therapy? Chang W. Song, PhD,* Inhwan Park, BA,* L. Chinsoo Cho, MD,* Jianling Yuan, MD, PhD,* Kathryn E. Dusenbery, MD,* Robert J. Griffin, PhD,y and Seymour H. Levitt, MD* *Department of Therapeutic Radiology-Radiation Oncology, University of Minnesota Medical School, Minneapolis, Minnesota; and yDepartment of Radiation Oncology, University of Arkansas for Medical Sciences, Little Rock, Arkansas Received Mar 6, 2014, and in revised form Mar 14, 2014. Accepted for publication Mar 25, 2014.
Despite the increasing use of stereotactic radiosurgery (SRS) and stereotactic body radiation therapy (SBRT) in recent years, the biological principles underlying the impressive clinical effects of SRS and SBRT are still unclear. We previously hypothesized that hypofractionated irradiation of tumors with doses greater than 10 Gy per fraction cause severe vascular damage leading to indirect cell death, and suggested that such indirect cell death plays an important role in the response of tumors to SRS and SBRT (1-4). However, in a recent article, Brown et al (5) concluded that the standard radiobiological concepts are sufficient to explain the clinical results of SRS and SBRT, and that our previous data (3, 4) are only fragmentary to support our conclusion. Here, we report our new experimental data that strongly demonstrate that significant indirect cell death might occur in tumors treated with SRS or SBRT. We used HT-1080 human fibrosarcoma xenografts grown subcutaneously in the rear limb of nu/nu mice, and FSaII mouse fibrosarcoma grown subcutaneously in the rear limb of C3H mice. Tumors 6 to 7 mm in diameter were irradiated with 10 to 20 Gy of x-rays in a single dose, excised at different times after irradiation, and dispersed into single cells and the harvested cells were assayed for clonogenicity. Figure 1 shows the effect of 20 Gy on the surviving fraction of HT-1080 cells. The range of surviving fraction for
individual tumor was rather large, particularly 3 and 5 days after irradiation. Nevertheless, the mean or median values of cell survival on day 3 were markedly lower than those observed immediately after, irradiation indicating that significant secondary cell death occurred by 3 days. After a maximum nadir in cell survival on day 3, the mean and median values of cell survival slightly increased, suggesting that some repopulation or proliferation of tumor cells had occurred. However, in some tumors, the surviving fraction remained more than 2 logs lower than that immediately after irradiation for 5 days, indicating that secondary cell death continued for 5 days after irradiation. In Figure 2, the surviving fractions in FSaII tumors assayed immediately and 2 days after irradiation are compared. In the tumors exposed to 10 Gy, the surviving fraction immediately and 2 days after irradiation were similar. On the other hand, the surviving fractions 2 days after irradiation with 15 Gy or 20 Gy were markedly less than those immediately after irradiation. Further studies are in progress to reveal the cell survival on later days after irradiation in the FSaII tumor model. The results shown in Figures 1 and 2, together with our previous observations (3, 4), unequivocally indicate that irradiation with high dose per fraction induces indirect death in tumor cells. Such an indirect cell death after high-dose radiation is
Reprint requests to: Chang W. Song, PhD, Department of Therapeutic RadiologyeRadiation Oncology, University of Minnesota Medical School,
MMC 494, 424 Harvard St SE, Minneapolis, MN 55455. Tel.: (612) 6266852; E-mail: [email protected] Conflict of interest: none.
Int J Radiation Oncol Biol Phys, Vol. 89, No. 4, pp. 924e925, 2014 0360-3016/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ijrobp.2014.03.043
Volume 89 Number 4 2014
Indirect tumor cell death by SRS and SBRT
100
HT-1080
FSall Tumor
1.0
20 GY
925
Day 0
Surviving Cell Fraction
Day 2
Surviving Cell Fraction
10-1
10-2
0.1
0.01
0.001
0.0001
10-3
10 Gy Mean
0
20 Gy
Dose
Median
10-4
15 Gy
1
2
3
4
5
Days
Fig. 1. Effect of 20 Gy in a single dose on the surviving fraction in HT-1080 human sarcoma xenografts in nu/nu mice. The surviving fraction in each tumor was determined from the number of cells harvested from each tumor and their clonogenicity. The mean and 1 standard error of the mean (closed circle) and median value (triangle) for each group are shown. likely due to radiation-induced vascular damage and ensuing deterioration of the intratumor microenvironment (2, 5). Enhancement of antitumor immunity has been suggested to be involved in the response of tumors to ablative radiation therapy (6). However, given that indirect cell death occured rather soon (within 2-3 days) after irradiation, it is unlikely that immune reactions played an important role in the secondary cell death that we observed. Furthermore, indirect cell death also occurred in HT-1080 xenografts grown in immunologically compromised nu/nu mice, which further demonstrates that immune activation was not a major player in the induction of secondary cell death in the present study. It has been known that there are as many as 108 to 109 clonogenic cells in a 1-g tumor (7). A calculation based on conventional radiobiological principles indicates that the radiation doses currently used in SRS and SBRT, that is, 20-60 Gy in 1 to 5 fractions, are apparently insufficient to directly sterilize all of the clonogenic cells in a tumors as small as 1 g, assuming that 10% to 20% of tumor cells are hypoxic (8, 9). The fact that SRS and SBRT are highly effective for achieving complete control of many tumors strongly indicates that SRS and SBRT sterilize significant fractions of tumor cells by mechanisms other than directly killing all tumor cells. The
Fig. 2. Surviving fractions of FSaII tumor cells immediately (day 0) and 2 days after irradiation with 10 Gy, 15 Gy, and 20 Gy in a single dose. Surviving cell fractions were determined as shown in Fig. 1. The mean of 9 to 12 tumors and 1 standard error of the mean for each group are shown. evidence shown in the present study, together with previous reports (3, 4) clearly demonstrate that secondary cell death that is most likely caused by deterioration of the tumor physiology is involved in the response of tumors to high doseper-fraction SRS and SBRT.
References 1. Song CW, Park HJ, Griffin RJ, et al. Radiobiology of stereotactic radiosurgery and stereotactic body radiation therapy. In: Levitt SH, et al., editors. Technical Basis of Radiation Therapy, Medical Radiology, Radiation Oncology. Berlin: Springer-Verlag; 2012. p. 51-61. 2. Park HJ, Griffin RJ, Hui S, et al. Radiation-induced vascular damage in tumors: Implications of vascular damage in ablative hypofractionated radiotherapy (SBRT and SRS). Radiat Res 2012;177:311-327. 3. Song CW, Cho LC, Yuan J, et al. Radiobiology of stereotactic body radiation therapy/stereotactic radiosurgery and the linear-quadratic mode. Int J Radiat Oncol Biol Phys 2013;87:18-19. 4. Clement JJ, Takanka N, Song CW. Tumor reoxygenation and postirradiation vascular changes. Radiology 1978;127:799-803. 5. Brown JM, Carlson DJ, Brenner DJ. The tumor radiobiology of SRS and SBRT: Are more than the 5 Rs involved? Int J Radiat Oncol Biol Phys 2014;88:254-262. 6. Lee Y, Auh SL, Wang Y, et al. Therapeutic effects of ablation on local tumor require CR8þ T cells: Changing strategies for cancer treatment. Blood 2009;114:589-595. 7. Monte UD. Does the cell number 109 still really fit one gram of tumor tissue? Cell Cycle 2009;8:505-506. 8. Fowler JF, Wolfgan AT, Fenwik JD, et al. A challenge to traditional radiation oncology. Int J Radiat Oncol Biol Phys 2004;60:1241-1256. 9. Brown JM, Diehn M, Loo BW. Stereotactic ablative radiotherapy should be combined with a hypoxic cell radiosensitizer. Int J Radiat Oncol Biol Phys 2010;78:323-327.
