In vitro and in vivo evaluation of the safety and efficacy of a novel liquid fiducial marker for image-guided radiotherapy

Abstract

The true extent of a tumor is difficult to visualize, during radiotherapy, using current modalities. In the present study, the safety and feasibility of a mixture of N-butyl cyanoacrylate and lipiodol (NBCA/Lip) was evaluated in order to investigate the optimal combination for application as a fiducial marker for radiotherapy. Four combinations of NBCA/Lip injection (1:1-0.1, 1:1-0.15, 1:3-0.1 and 1:3-0.15 ml) were injected into the subcutaneous tissue of BALB/c mice. The changes in gross histopathology, body weight, skin score, marker volume, neutrophil and macrophage counts were observed to analyze the effects of the different mixing ratios and injection volumes, in order to identify the best combination. Evaluation according to the International Organization for Standardization criteria was further conducted in order to test the biocompatibility of the mixture, including an acute systematic assay with mice, cytotoxicity with L929 fibroblasts and delayed-type hypersensitivity tests with guinea pigs and an intradermal test with rabbits. The results revealed that at the seventh week, 42 markers (42/48; 87.5%) were still visible using computed tomography (CT) imaging. No serious adverse effects were observed throughout the study period; however, the combination of 1:1-0.1 ml had the lowest body weight and worst skin score. A review of the histopathological reaction to NBCA/Lip revealed a combination of acute inflammation, chronic inflammation, granulation tissue, foreign-body reaction and fibrous capsule formation. The 1:1 NBCA combination ratio resulted in the most intense tissue repair reaction and a slower degradation rate of markers. In general, the combination of 1:3-0.15 ml had a better fusion with local tissue, maintained a stable imaging nodule on CT images for 7 weeks and the final biocompatibility test demonstrated its safety. Overall, the findings of the present study demonstrated NBCA/Lip as a safe and feasible fiducial marker, when using the 1:3-0.15 ml combination.

Keywords: biocompatibility; esophageal cancer; image-guided radiotherapy; liquid fiducial marker; n-butyl cyanoacrylate.

Figure 1.

Experimental design chart. Part 1 shows the macroscopic evaluation; part 2 shows the microscopic evaluation; part 3 shows the biocompatibility evaluation.

Figure 2.

Measurements of each treatment group during the 7-week observation period. (A) Weight; (B) skin score; (C) remaining size of nodules; (D) neutrophil count; and (E) macrophage count; (F) CT HU. CT, computed tomography; HU, Hounsfield Unit.

Figure 3.

Nodules on CT images in mouse of 1:3–0.15 ml at different time points. (A) 1, (B) 3, (C) 5 and (D) 7 weeks post implantation. Arrows indicate the nodules of markers in subcutaneous tissue of mice in part 1.

Figure 4.

H&E and immunohistochemical images (CD68 and LY6G) of N-butyl cyanoacrylate/lipiodol implantation into subcutaneous tissue during the acute inflammatory phase at 2 weeks post implantation from mice in part 2 in four treatment groups. Massive granulation tissue formation with inflammatory cell infiltration surrounding the material treated with 1:1 (A and B). Scattered granulation tissue around the embedded material treated with 1:3 (C and D). H&E, the first row. CD68-positive cells indicate macrophages (brown), the second row. LY6G-positive cells indicate neutrophils (brown), the third row. H&E, hematoxylin and eosin staining; I, inflammatory cells; C, capillary; G, granulation tissue; MC, material cavity.

Figure 5.

H&E and immunohistochemical (CD68 and LY6G) images of N-butyl cyanoacrylate and lipiodol implantation in subcutaneous tissue during the chronic inflammatory phase at 7 weeks from mice in part 2 in four treatment groups. Fibrous capsules from granulation tissue at the tissue repair phase. Thick layered fibrous capsule with massive macrophages surrounding the materials treated with 1:1–0.1 ml (A) and 1:1–0.15 ml (B). Thin layered fibrous capsule tissue around the material in low ratio of 1:3–0.1 ml (C) and 1:3–0.15 ml (D). Seldom neutrophils were observed at the tissue repair phase. CD68-positive cells indicate macrophages (brown). LY6G-positive cells indicate neutrophils (brown). H&E, hematoxylin and eosin staining; G, granulation tissue; MC, material cavity; C, capillary; I, inflammatory cells; FC, fibrous capsule; F, fibroblasts; FBGC, foreign body giant cell. Magnification, ×200.