Article Figures & Data
Figures
- FIGURE 1.
Gastrointestinal transit of [223Ra]RaCl2 and radiobiologic effects. (A) [223Ra]RaCl2 autoradiography of mouse gastrointestinal tract. (B) Signal intensity profiles from stomach to cecum displaying isotope migration (approximately 200 mm/animal) at indicated time points. Shown are upper (stomach) and lower (cecum) compartments and complete organ signal quantification, over time. (C) Immunofluorescence of duodenum and colon sections after treatment (left) and colonic section of saline control mouse (right). For each micrograph, stain and color are indicated: 4,6-diamidino-2-phenylindole (DAPI) for nuclei, phalloidin for cytoskeleton, terminal deoxynucleotidyl transferase 2′-deoxyuridine, 5′-triphosphate nick end labeling (TUNEL) marker for apoptosis, and γ-H2A histone family member X (γ-H2AX) for DNA damage. DLU = dynamic light units; GI = gastrointestinal.
- FIGURE 2.
Active 223Ra transport through human gastrointestinal organoids. (A) Schematic representation of enteroid monolayers grown on permeable Transwell. (B) 223Ra passage to apical compartment normalized to initial dose in basolateral media, revealing temperature-dependent mechanism, with undifferentiated cryptlike enteroids. (C) Increased 223Ra transport measured using differentiated over undifferentiated enteroids as function of time (P < 0.05). (D) 223Ra flux normalized to untreated well as measured through caco-2 monolayers coincubated with library of 52 ion-channel inhibitors or activators. Caco-2 monolayer integrity was confirmed by pre- and postradioactive incubation measuring transepithelial/transendothelial electrical resistance and Lucifer yellow (Lucifer Yellow Permeability < 1.5%) readings. Average differential of radioactive counts (n = 3) has been normalized to 223Ra flux baseline exempt from treatment (no. 12 control well). Amiloride (red) and NS-1619 (green) proceeded for in vivo validation.
- FIGURE 3.
Evaluation of selected ion channel modulators with [223Ra]RaCl2. (A) Radioactive organ distribution of healthy male C57BL/6 mice randomized in 3 cohorts (n = 6) given amiloride before [223Ra]RaCl2, NS-1619 before [223Ra]RaCl2, and saline before [223Ra]RaCl2. Several organs displayed significant differences in 223Ra uptake, including 1.5-fold higher bone localization with amiloride. Differences in scale can be seen for 15-min data (values reported in supplemental materials). (B) 223Ra bone activity uptake (%IA/g) at 15 min for NS-1619 combination is half that of control and amiloride-treated groups. In contrast, amiloride-treated group shows significantly higher bone uptake than does control group (*P < 0.05). Upper gastrointestinal radioactive uptake reflects higher content for NS-1619–treated animals at 15 min (*P < 0.05). Kidney uptake across cohorts at 15-min time point shows decrease for amiloride group. (C and D) Whole-organ distribution (C) and bone focus over 10-d time course (D) comparing amiloride combination to [223Ra]RaCl2; activity uptake at 24 h significantly differs between the 2 groups (P = 0.035).
- FIGURE 4.
Tumor growth inhibition and monitoring of C4-2B bone-inoculated animals treated with amiloride, combination of amiloride plus [223Ra]RaCl2, and [223Ra]RaCl2 alone. Average bioluminescent radiance measured using luciferase-expressing C4-2B cells inoculated in tibia shaft shows superior tumor growth inhibition for combination cohort. *P < 0.05 at days 19, 21, and 29 comparing [223Ra]RaCl2 with combination. Animals on combination lost less weight and regained that mass faster than with either agent alone (Supplemental Fig. 6). Representative radiograph of tibia 35 d after injection demonstrating degradation for control amiloride as compared with radiotherapy and combination cohorts.
- FIGURE 5.
Toxicologic effects of single or combination treatment: blood chemistry markers for control saline, [223Ra]RaCl2 or amiloride alone, and combination at 1, 7, and 19 d after administration. For [223Ra]RaCl2 and combined therapy, amiloride, ALP, and creatinine present significant drop at 7 d as compared with control saline cohort, with no other noticeable differences found. Kidney pathology by hematoxylin and eosin (H & E), Masson trichrome, and periodic acid–Schiff (PAS) staining at 20 d after treatment indicates no morphologic differences. Weight monitoring demonstrated indistinguishable gain for all groups. ALP units are U/L. ALb = albumin (g/L); ALT = alanine amino transferase (U/L); AMY = amylase (U/L); TBu = total bilirubin (μmol/L); BUN = blood urea nitrogen (mmol/L); Ca = calcium (mmol/L); PHOS = phosphate (mmol/L); CRT = total protein (g/L); Glob = globulin (g/L).
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Supplemental Data
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