In Vivo Radiobiological Investigations with the TOP-IMPLART Proton Beam on a Medulloblastoma Mouse Model

Abstract

Protons are now increasingly used to treat pediatric medulloblastoma (MB) patients. We designed and characterized a setup to deliver proton beams for in vivo radiobiology experiments at a TOP-IMPLART facility, a prototype of a proton-therapy linear accelerator developed at the ENEA Frascati Research Center, with the goal of assessing the feasibility of TOP-IMPLART for small animal proton therapy research. Mice bearing Sonic-Hedgehog (Shh)-dependent MB in the flank were irradiated with protons to test whether irradiation could be restricted to a specific depth in the tumor tissue and to compare apoptosis induced by the same dose of protons or photons. In addition, the brains of neonatal mice at postnatal day 5 (P5), representing a very small target, were irradiated with 6 Gy of protons with two different collimated Spread-Out Bragg Peaks (SOBPs). Apoptosis was visualized by immunohistochemistry for the apoptotic marker caspase-3-activated, and quantified by Western blot. Our findings proved that protons could be delivered to the upper part while sparing the deepest part of MB. In addition, a comparison of the effectiveness of protons and photons revealed a very similar increase in the expression of cleaved caspase-3. Finally, by using a very small target, the brain of P5-neonatal mice, we demonstrated that the proton irradiation field reached the desired depth in brain tissue. Using the TOP-IMPLART accelerator we established setup and procedures for proton irradiation, suitable for translational preclinical studies. This is the first example of in vivo experiments performed with a "full-linac" proton-therapy accelerator.

Keywords: apoptosis; cerebellum; dosimetry; linac; medulloblastoma; preclinical mouse models; proton therapy; small-field irradiation.

Figure 1

Bragg curve profiles of the PBP (A) and SOBP (B) obtained from the PL images stored in LiF crystals and their best-fitting curves. (C) Energy spectra corresponding to the best-fitting curves shown in (A,B). In (A,B), the peaks at zero depth are ascribed to light scattering at the crystal edge and were therefore excluded from the fitting process.

Figure 2

X-axis normalized dose profiles of the irradiated EBT3 films (solid blue line) and the relevant smoothed data (dashed red line) of (A) mouse tumor with 20 mm collimator diameter and SOBP = 19 mm, and (B) neonatal mouse brain with 8 mm collimator diameter and SOBP = 3 mm. The insets show the selected ROIs used to extract the curves.

Figure 3

Schematic representation of the employed MB tumor model. Fragments of Ptch1^+/−-derived tumor were subcutaneously propagated in the right flank of C57Bl6/J mice (A). Once the tumors reached 1400–2400 mm³, mice were irradiated or sham-exposed. Immunostaining for cleaved-caspase-3 in MBs 4 h after proton irradiation with 8 Gy ((B–D); magnification 4×). Caspase-3 staining shows that with a SOBP of 7 mm dose delivery can be successfully restricted to the upper half of the tumor (C) or administered to the whole tumor mass when a 19 mm SOBP was used (D); unirradiated control (B); black arrows indicate mouse skin. High power views of caspase-3 staining ((E–G); magnification 40×). * Black asterisk in (F) represents the exposed upper tumor part. * Red asterisk in (F) shows a lack of caspase-3 immunoreactivity, indicating tissue sparing from irradiation. (H) Representative immunoblotting for caspase-3 in tumors irradiated entirely or for half of the volume and relative representation of densitometric immunoblot analysis (I). The number of mice used for Western blot analyses is indicated in the graphs (n), Student’s t-test was used for the statistical analysis.

Figure 4

Representative immunostaining for cleaved-caspase-3 in MBs at 4 h after irradiation with 8 Gy of protons with a SOBP of 19 mm (46.4 MeV maximum energy) (B) or photons of 250 kVp (C). Unirradiated control (A). (A–C) are 20× magnification. Representative immunoblotting for caspase-3 in MBs at 4 h after irradiation in the same conditions reported above (D). (E) Graphic representation of densitometric immunoblot analysis in (D). The number of mice used for Western blot analyses is indicated in the graphs (n), Student’s t-test was used for the statistical analysis.

Figure 5

Immunostaining for cleaved-caspase-3 in brain sections from P5 mouse 4 h post-irradiation with 6 Gy of protons with a SOBP of 8 mm (29.5 MeV maximum energy) (A). Caspase-3 staining was especially detected in the EGL of the cerebellum (Cb), in the hippocampus (H), and in the rostral migratory stream (RMS) (B), compared to unexposed control (C). Serial cutting of sagittal brain sections showing the presence of caspase-3 labeling up to the distal end of the Cb (D). B and C are 10× and D is 4×.

Figure 6

Immunostaining for cleaved-caspase-3 of brain sections at P5 after irradiation with 6 Gy of protons with SOBP of 3 mm (16.2 MeV maximum energy) (A) or 8 mm (29.5 MeV maximum energy) (B). Dashed vertical lines in A and B indicate the area of cut used to separate the posterior and anterior brain parts during the freezing collection. Dashed areas in A indicate H and RMS regions. Representative immunoblotting for caspase-3 in the posterior (C) or anterior brain part (D) and relative representation of densitometric immunoblot analyses (E,F). The number of mice used for Western blot analyses is indicated in the graphs (n), Student’s t-test was used for the statistical analysis.

Figure 7

(A) Schematic layout of the TOP-IMPLART accelerator with maximum output energy of 55.5 MeV; Details of the irradiation end station set-up illustrating the schematic layout (B) and photograph (C); in picture (C) the target region hosts the mD dosimeter and its support for a dose assessment irradiation run.

Figure 8

SRIM (Stopping and Range of Ions in Matter) output for the RS of 1.5 mm setup on target; Energy of 46.4 MeV, Energy spread of 373.7 keV: (A) Beam spot; (B) energy distribution; (C) horizontal distribution; (D) vertical distribution.

Figure 9

Calibration of dose measurement for the 2D-IC readings (in arbitrary unit, AU) against the microDiamond dosimeter (in Gy) for delivered doses in the range 2.7 to 8 Gy; this calibration corresponds to the 8 mm collimator configuration (neonatal brain irradiation).