Microbeam radiation therapy alters vascular architecture and tumor oxygenation and is enhanced by a galectin-1 targeted anti-angiogenic peptide

Abstract

In this study, we sought to determine the therapeutic potential of variably sized (50 μm or 500 μm wide, 14 mm tall) parallel microbeam radiation therapy (MRT) alone and in combination with a novel anti-angiogenic peptide, anginex, in mouse mammary carcinomas (4T1)--a moderately hypoxic and radioresistant tumor with propensity to metastasize. The fraction of total tumor volume that was directly irradiated was approximately 25% in each case, but the distance between segments irradiated by the planar microbeams (width of valley dose region) varied by an order of magnitude from 150-1500 μm corresponding to 200 μm and 2000 μm center-to-center inter-microbeam distances, respectively. We found that MRT administered in 50 μm beams at 150 Gy was most effective in delaying tumor growth. Furthermore, tumor growth delay induced by 50 μm beams at 150 Gy was virtually indistinguishable from the 500 μm beams at 150 Gy. Fifty-micrometer beams at the lower peak dose of 75 Gy induced growth delay intermediate between 150 Gy and untreated tumors, while 500 μm beams at 75 Gy were unable to alter tumor growth compared to untreated tumors. However, the addition of anginex treatment increased the relative tumor growth delay after 500 μm beams at 75 Gy most substantially out of the conditions tested. Anginex treatment of animals whose tumors received the 50 μm beams at 150 Gy also led to an improvement in growth delay from that induced by the comparable MRT alone. Immunohistochemical staining for CD31 (endothelial cells) and αSMA (smooth muscle pericyte-associated blood vessels as a measure of vessel normalization) indicated that vessel density was significantly decreased in all irradiated groups and pericyte staining was significantly increased in the irradiated groups on day 14 after irradiation. The addition of anginex treatment further decreased the mean vascular density in all combination treatment groups and further increased the amount of pericyte staining in these tumors. Finally, evidence of tumor hypoxia was found to decrease in tumors analyzed at 1-14 days after MRT in the groups receiving 150 Gy peak dose, but not 75 Gy peak dose. Our results suggest that tumor vascular damage induced by MRT at these potentially clinically acceptable peak entrance doses may provoke vascular normalization and may be exploited to improve tumor control using agents targeting angiogenesis.

FIG. 1

Panel A: A multi-slit collimator fractionates high-energy synchrotron X rays into parallel planar microbeams of variable beam sizes and spacing. In this experiment, 500 μm and 50 μm beams with 2000 μm and 200 μm center-to-center spacing, respectively, were generated (schematic, left side, not to scale). Gafchromic film captured the exit dose pattern for dosimetry and alignment (right side). Panel B: Magnification of the exit dose patterns for 500 μm and 50 μm beams with 1.5 mm and 0.15 mm spacing, respectively. Panel C: Relative dose plots from the Monte Carlo estimation for the MRT irradiation set-up. Left panel, 8 beams from the 500 μm beam pattern, middle panel, full spectrum of the 50 μm pattern, and, right panel, a section of the center portion of the 50 μm beam pattern.

FIG. 2

4T1 tumor growth after 75 Gy (panels A and B) or 150 Gy (panels C and D) alone or when combined with 20 mg/kg/day Anginex treatment starting 2 h before 500 μm or 50 μm MRT exposure and continuing until the end of observation. Each group contained 3–4 mice and the bars indicate 1 SEM. Tumors receiving MRT were all significantly delayed in tumor growth (P < 0.01) except for the 500 μm beam, 75 Gy group (P=0.5). When anginex treatment was added to MRT, there was a substantial change in tumor growth delay in the 500 μm, 75 Gy group (panel B), (P=0.07) and the 50 μm, 150 Gy group (panel D) (P=0.06). Tumor growth after 500 μm, 150 Gy or 50 μm, 75 Gy MRT was not altered with the addition of anginex treatment.

FIG. 3

Examples of immunohistochemical staining of 4T1 tumor tissue for vessel density (anti-CD31, red) or pericytes (anti-αSMA, green). Mice were sacrificed at day 14 after MRT exposure and tumors were excised for staining and analysis.

FIG. 4

Pimonidazole positive staining in tumors over 2 weeks after MRT at 150 Gy peak dose with varying beam width. The average staining intensity from three untreated tumors is indicated at time 0, and the other time points are the average staining intensity of 1–2 tumors treated with each beam size.