Research ArticleCancer

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

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Science Translational Medicine  11 Mar 2015:
Vol. 7, Issue 278, pp. 278ra34
DOI: 10.1126/scitranslmed.aaa4214
  • 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 hours 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 analysis of variance (ANOVA) for genotype and time interaction followed by Bonferroni’s post hoc tests for pairwise comparisons between genotypes showed that the differences were not statistically significant. (C and D) Tumor growth curves (C) and time to volume tripling for sarcomas (D) in KPFRTVBaxFL/+ and KPFRTVBaxFL/FL mice after irradiation with 20 Gy (n = 8 mice per group). Two-tailed Student’s t test showed that the differences were 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 after irradiation with 50 Gy (n = 18 and 20 mice per group). Log-rank test showed that the differences were not statistically significant.

  • 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 hours after irradiation with 50 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 hours after irradiation with 50 Gy (n = 6 mice per group). Two-tailed Student’s t test, P < 0.05. (C and D) 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 50 Gy (n = 22 and 23 mice per group, respectively) (C) or four daily fractions of 20 Gy (n = 23 and 25 mice per group, respectively) (D). 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 four daily fractions of 20 Gy before 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 showed that the differences were not statistically significant. Asterisks represent statistically significant difference between the indicated groups.

  • 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 and C) Sarcoma growth curves (B) and Kaplan-Meier plot of local sarcoma control (C) defined as the absence of tumor volume tripling for primary sarcomas generated by intramuscular 4-hydroxytamoxifen injection into P7KPloxPAtmFL/+ and P7KPloxPAtmFL/FL mice after irradiation with 50 Gy (n = 13 mice per group). Several mice were euthanized before 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 statistically significant difference between the indicated groups.

  • Fig. 4. The PI3KK inhibitor BEZ235 preferentially radiosensitizes primary sarcomas compared to mouse heart tissue.

    (A and B) Immunofluorescence (A) and quantification of TUNEL-positive cells (B) in hearts and sarcomas from vehicle- or BEZ235-treated KPloxP mice 24 hours after irradiation with 20 Gy (n = 5 mice per group). Two-way ANOVA for tissue and treatment interaction followed by Bonferroni’s post hoc tests for pairwise comparison between treatments showed that the differences were statistically significant in sarcomas but not in hearts (P < 0.05). Scale bar, 100 μm. (C and D) Tumor growth curves (C) and time to volume tripling (D) of primary sarcomas in KPloxP mice treated on day 0 with vehicle or a single dose of BEZ235 (50 mg/kg) 2 hours 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 BEZ235 (50 mg/kg) 2 hours 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 for VPFL/+ Vehicle versus VPFL/FL Vehicle, VPFL/+ Vehicle versus VPFL/FL BEZ235, VPFL/+ BEZ235 versus VPFL/FL Vehicle, and VPFL/+ BEZ235 versus VPFL/FL BEZ235 (P < 0.05). Asterisks represent statistically significant difference between the indicated groups.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/278/278ra34/DC1

    Fig. S1. Deletion of Bax in primary tumor endothelial cells does not affect sarcoma initiation or growth.

    Fig. S2. Deletion of Bax in endothelial cells does not affect sarcoma growth delay after a curative dose of irradiation.

    Fig. S3. Loss of Bak and Bax in endothelial cells does not affect primary soft tissue sarcoma initiation or growth.

    Fig. S4. Deletion of Bak and Bax in endothelial cells does not affect primary sarcoma response to radiation therapy.

    Fig. S5. Deletion of Atm in endothelial cells does not affect sarcoma growth delay after curative doses of irradiation.

    Fig. S6. Pax7-CreER, but not adeno-Cre, efficiently recombines both AtmFL alleles in primary sarcomas in vivo.

    Fig. S7. Spectrum of histological responses for primary sarcomas in KPloxPAtmFL/FL mice after irradiation with 50 Gy.

    Fig. S8. BEZ235 inhibits Atm in primary sarcomas.

  • Supplementary Material for:

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

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

    *Corresponding author. E-mail: david.kirsch@duke.edu

    Published 11 March 2015, Sci. Transl. Med. 7, 278ra34 (2015)
    DOI: 10.1126/scitranslmed.aaa4214

    This PDF file includes:

    • Fig. S1. Deletion of Bax in primary tumor endothelial cells does not affect sarcoma initiation or growth.
    • Fig. S2. Deletion of Bax in endothelial cells does not affect sarcoma growth delay after a curative dose of irradiation.
    • Fig. S3. Loss of Bak and Bax in endothelial cells does not affect primary soft tissue sarcoma initiation or growth.
    • Fig. S4. Deletion of Bak and Bax in endothelial cells does not affect primary sarcoma response to radiation therapy.
    • Fig. S5. Deletion of Atm in endothelial cells does not affect sarcoma growth delay after curative doses of irradiation.
    • Fig. S6. Pax7-CreER, but not adeno-Cre, efficiently recombines both AtmFL alleles in primary sarcomas in vivo.
    • Fig. S7. Spectrum of histological responses for primary sarcomas in KPloxPAtmFL/FL mice after irradiation with 50 Gy.
    • Fig. S8. BEZ235 inhibits Atm in primary sarcomas.

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