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. 2015 Mar 11;7(278):278ra34.
doi: 10.1126/scitranslmed.aaa4214.

Tumor cells, but not endothelial cells, mediate eradication of primary sarcomas by stereotactic body radiation therapy

Everett J Moding  1 , Katherine D Castle  1 , Bradford A Perez  2 , Patrick Oh  2 , Hooney D Min  2 , Hannah Norris  2 , Yan Ma  2 , Diana M Cardona  3 , Chang-Lung Lee  2 , David G Kirsch  4
Affiliations

Tumor cells, but not endothelial cells, mediate eradication of primary sarcomas by stereotactic body radiation therapy

Everett J Moding et al. Sci Transl Med. .

Abstract

Cancer clinics currently use high-dose stereotactic body radiation therapy as a curative treatment for several kinds of cancers. However, the contribution of vascular endothelial cells to tumor response to radiation remains controversial. Using dual recombinase technology, we generated primary sarcomas in mice with _targeted genetic mutations specifically in tumor cells or endothelial cells. We selectively mutated the proapoptotic gene Bax or the DNA damage response gene Atm to genetically manipulate the radiosensitivity of endothelial cells in primary soft tissue sarcomas. Bax deletion from endothelial cells did not affect radiation-induced cell death in tumor endothelial cells or sarcoma response to radiation therapy. Although Atm deletion increased endothelial cell death after radiation therapy, deletion of Atm from endothelial cells failed to enhance sarcoma eradication. In contrast, deletion of Atm from tumor cells increased sarcoma eradication by radiation therapy. These results demonstrate that tumor cells, rather than endothelial cells, are critical _targets that regulate sarcoma eradication by radiation therapy. Treatment with BEZ235, a small-molecule protein kinase inhibitor, radiosensitized primary sarcomas more than the heart. These results suggest that inhibiting ATM kinase during radiation therapy is a viable strategy for radiosensitization of some tumors.

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Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Deletion of Bax from mouse endothelial cells does not affect primary sarcoma response to radiation therapy. (A) Representative immunofluorescence for CD31 and TUNEL in sarcomas from KPFRTVBaxFL/+ and KPFRTVBaxFL/FL mice 4 and 48 h after irradiation with 20 Gy. Areas enclosed by dashed lines are shown at higher magnification in the insets. Scale bar, 100 μm. (B) Quantification of CD31 and TUNEL double-positive cells in sarcomas from KPFRTVBaxFL/+ and KPFRTVBaxFL/FL mice at the indicated time points after irradiation with 20 Gy (n=4 mice per group). Two-way ANOVA for genotype and time interaction followed by Bonferroni’s post hoc tests for pairwise comparisons between genotypes not statistically significant. (C) Tumor growth curves and (D) time to volume tripling for sarcomas in KPFRTVBaxFL/+ and KPFRTVBaxFL/FL mice after irradiation with 20 Gy (n=8 mice per group). Two-tailed student’s t test not statistically significant. (E) Kaplan-Meier plot of local sarcoma control defined as the absence of tumor volume tripling for sarcomas in KPFRTVBaxFL/+ and KPFRTVBaxFL/FL mice following irradiation with 50 Gy (n=18 and 20 mice per group). Log rank test not statistically significant.
Fig. 2
Fig. 2
Sensitizing sarcoma endothelial cells to radiation does not affect local control of primary sarcomas. (A) Representative immunofluorescence for CD31 and TUNEL in sarcomas from KPFRTVAtmFL/+ and KPFRTVAtmFL/FL mice 24 h after irradiation with 20 Gy. Areas enclosed by dashed lines are shown at higher magnification in the insets. Scale bar, 100 μm. (B) Quantification of CD31 and TUNEL double-positive cells in sarcomas from KPFRTVAtmFL/+ and KPFRTVAtmFL/FL mice 24 h after irradiation with 20 Gy (n=6 mice per group). Two-tailed student’s t test, P<0.05. Kaplan-Meier plot of local sarcoma control (defined as the absence of tumor volume tripling) for sarcomas in KPFRTVAtmFL/+ and KPFRTVAtmFL/FL mice after irradiation with (C) 50 Gy (n=22 and 23 mice per group) or (D) four daily fractions of 20 Gy (n=23 and 25 mice per group). One KPFRTVAtmFL/FL mouse developed an abdominal metastasis 8 weeks after irradiation with 50 Gy, and two KPFRTVAtmFL/+ mice died of unknown causes at 7 and 10 weeks after irradiation with 4 daily fractions of 20 Gy prior to sarcoma tripling. Thus, these mice were scored as locally controlled until the time points at which they either developed metastasis or died. Log rank tests not statistically significant. Asterisk represents a statistically significant difference between the indicated groups.
Fig. 3
Fig. 3
Sensitizing tumor cells to radiation increases local control of primary sarcomas after radiation therapy. (A) Clonogenic survival of primary sarcoma cell lines from P7KPloxPAtmFL/+ and P7KPloxPAtmFL/FL mice (n=3 independent cells lines per genotype). Two-way ANOVA for genotype and dose interaction, P<0.05, followed by Bonferroni’s post hoc tests for pairwise comparisons between genotypes after 2 and 4 Gy, P<0.05. (B) Sarcoma growth curves and (C) Kaplan-Meier plot of local sarcoma control defined as the absence of tumor volume tripling for primary sarcomas generated by intramuscular 4-hydroxy-tamoxifen injection into P7KPloxPAtmFL/+ and P7KPloxPAtmFL/FL mice after irradiation with 50 Gy (n=13 mice per group). Several mice were euthanized prior to sarcoma tripling because of the development of second sarcomas at other locations in the body, presumably from systemic tamoxifen-mediated Cre recombination. These mice were scored as locally controlled until the time point of second tumor formation. Log rank test, P<0.05. Asterisks represent a statistically significant difference between the indicated groups.
Fig. 4
Fig. 4
The PI3KK inhibitor BEZ235 preferentially radiosensitizes primary sarcomas compared to mouse heart tissue. (A) Immunofluorescence and (B) quantification of TUNEL-positive cells in hearts and sarcomas from vehicle- or BEZ235-treated KPloxP mice 24 h after irradiation with 20 Gy (n=5 mice per group). Two-way ANOVA for tissue and treatment interaction, P<0.05, followed by Bonferroni’s post hoc tests for pairwise comparison between treatments in hearts not statistically significant, but in sarcomas, P<0.05. Scale bar, 100 μm. (C) Tumor growth curves and (D) time to volume tripling of primary sarcomas in KPloxP mice treated on day 0 with vehicle or a single dose of 50 mg/kg BEZ235 2 h before irradiation with 20 Gy (n=8 mice per group). Two-tailed student’s t test, P<0.05. (E) Kaplan-Meier plots of myocardial necrosis–free survival for mice that lacked p53 in endothelial cells (VPFL/FL) or mice that retained p53 in endothelial cells (VPFL/+), both of which were treated with vehicle or 50 mg/kg BEZ235 2 h before whole-heart irradiation with 12 Gy (n=7 to 9 mice per group). Two VPFL/FL mice treated with vehicle were censored because they developed tumors before developing myocardial necrosis. Log rank test VPFL/+ Vehicle vs. VPFL/FL Vehicle, VPFL/+ Vehicle vs. VPFL/FL BEZ235, VPFL/+ BEZ235 vs. VPFL/FL Vehicle, and VPFL/+ BEZ235 vs. VPFL/FL BEZ235, P<0.05. Asterisks represent a statistically significant difference between the indicated groups.
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