Dose and Spatial Effects in Long-Distance Radiation Signaling In Vivo: Implications for Abscopal Tumorigenesis Mariateresa Mancuso, PhD, Paola Giardullo,

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Dose and Spatial Effects in Long-Distance Radiation Signaling In Vivo: Implications for Abscopal Tumorigenesis Mariateresa Mancuso, PhD, Paola Giardullo, PhD, Simona Leonardi, PhD, Emanuela Pasquali, MSc, Arianna Casciati, PhD, Ilaria De Stefano, PhD, Mirella Tanori, PhD, Simonetta Pazzaglia, PhD, Anna Saran, PhD International Journal of Radiation Oncology • Biology • Physics Volume 85, Issue 3, Pages 813-819 (March 2013) DOI: 10.1016/j.ijrobp.2012.07.2372 Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 1 Shielding geometries for detection of nontargeted central nervous system effects. (a) Ptch1+/− mice at postnatal day 2 (P2) were irradiated with 4-mm-thick individual lead cylinder shields designed to protect approximately one-third (SH) or two-thirds of the body (ie, SH1 or SH2). (b) Marked hair-growth delay at postnatal day 10 demarcates irradiated from shielded areas. (c) Schematic representation of P2 mouse brain. CB = cerebellum. (d) Histologic image of the anterodorsal cardinal lobe of P2 cerebellum. EGL = external granule layer; ML = molecular layer. International Journal of Radiation Oncology • Biology • Physics 2013 85, 813-819DOI: (10.1016/j.ijrobp.2012.07.2372) Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 2 Dose dependence of long-range bystander signaling in vivo. Dose response for apoptosis at 6 hours after 1, 2, or 3 Gy in the external granule layer of Ptch1+/− cerebellum (n=6) after whole-body (WB) irradiation (a) and after shielded (SH) irradiation (c). (b) Kaplan-Meier kinetic analysis of medulloblastoma in Ptch1+/− mice directly exposed to x-rays (1, 2, or 3 Gy) or (d) SH irradiated. (e) Western blot analysis of connexin43 (Cx43) expression in cerebella (n=3) at 3 hours after SH irradiation with 1, 2, or 3 Gy of x-rays or (f) after WB exposure to the scatter dose (0.036 Gy), compared with control mice. ***P<.0001; **P=.0021. International Journal of Radiation Oncology • Biology • Physics 2013 85, 813-819DOI: (10.1016/j.ijrobp.2012.07.2372) Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 3 Time and spatial effects of abscopal signals after 3 Gy. (a) Kinetics of appearance of apoptotic external granule layer cells in Ptch1+/− cerebella (n=6) at 3, 6, 9, and 18 hours postirradiation for all shielding geometries (ie, SH, SH1, and SH2). (b) Kaplan-Meier kinetic analysis of medulloblastoma after SH1 and SH2 irradiation compared with unirradiated mice. SH data are reported from Mancuso et al (8). International Journal of Radiation Oncology • Biology • Physics 2013 85, 813-819DOI: (10.1016/j.ijrobp.2012.07.2372) Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 4 Time and spatial effects of abscopal signals after 10 Gy. (a, left column) Representative images of 53BP1 immunohistochemical staining (30 min postirradiation) in the external granule layer (EGL) of SH-, SH1-, and SH2-10 Gy mice. Arrows: 53BP1 foci. (a, right column) Representative images of apoptotic response at 6 hours (ie, peak response). Inset: immunohistochemical analysis of caspase-3 activation. (b) Kinetics of appearance of apoptotic EGL cells of Ptch1+/− cerebella (n=6) at 3, 6, 9, and 18 hours postirradiation for all shielding geometries. (c) Histology of microtumor (MT) in Ptch1+/− cerebellum. (d) MT incidence in SH-, SH1-, and SH2-10 Gy asymptomatic mice of 8 weeks compared with age-matching mice WB exposed to the estimated scatter dose (0.1 Gy) or unirradiated. International Journal of Radiation Oncology • Biology • Physics 2013 85, 813-819DOI: (10.1016/j.ijrobp.2012.07.2372) Copyright © 2013 Elsevier Inc. Terms and Conditions