Alkylphosphocholine analogs for broad-spectrum cancer imaging and therapy

Abstract

Many solid tumors contain an overabundance of phospholipid ethers relative to normal cells. Capitalizing on this difference, we created cancer-targeted alkylphosphocholine (APC) analogs through structure-activity analyses. Depending on the iodine isotope used, radioiodinated APC analog CLR1404 was used as either a positron emission tomography (PET) imaging ((124)I) or molecular radiotherapeutic ((131)I) agent. CLR1404 analogs displayed prolonged tumor-selective retention in 55 in vivo rodent and human cancer and cancer stem cell models. (131)I-CLR1404 also displayed efficacy (tumor growth suppression and survival extension) in a wide range of human tumor xenograft models. Human PET/CT (computed tomography) and SPECT (single-photon emission computed tomography)/CT imaging in advanced-cancer patients with (124)I-CLR1404 or (131)I-CLR1404, respectively, demonstrated selective uptake and prolonged retention in both primary and metastatic malignant tumors. Combined application of these chemically identical APC-based radioisosteres will enable personalized dual modality cancer therapy of using molecular (124)I-CLR1404 tumor imaging for planning (131)I-CLR1404 therapy.

Copyright © 2014, American Association for the Advancement of Science.

Figures

Fig. 1. APC analog chemical structures

CLR1404 could be synthesized as a radioactive compound (125I, 124I, or 131I), as a fluorescent analog (CLR1501), or as a near-infrared analog (CLR1502).

Fig. 2. Uptake of CLR1404 analogs in human cancer cell lines

(A) Fluorescence confocal microscopy shows CLR1501 (5 µM) uptake in multiple cancer types, and less uptake in normal human fibroblasts at 24 hours. Scale bar, 10 µm. (B) Coculture of human prostate cancer (PC-3) cells and normal human fibroblasts. Cells were treated with the fluorescent analog CLR1501 (5 µM) for 24 hours and counterstained with the nuclear stain, Hoechst 33342. Scale bar, 10 µm. (C) Quantitative uptake and retention of 125I-CLR1404 in three cancer cell lines derived from patients and the corresponding patient-matched normal cell lines. Data are means ± SEM (n = 3 independent experiments with three biological replicates each). *P < 0.001, analysis of variance (ANOVA) with post hoc Tukey test.

Fig. 3. CLR1501 fluorescent labeling of GSCs in vitro

(A) GSCs (line 99) were treated overnight with CLR1501. CLR1501 uptake was visualized with confocal microscopy and compared to matched serGBM, normal human astrocytes, and human NSCs. (B) Flow cytometry was performed to quantify CLR1501 uptake after 24 hours in GSC patient–specific lines 12.1, 22, 33, 44, and 99; serGBM lines 22, 33, 99, U251, and U87; NSCs; and astrocytes. Data are means ± SD of three independent experiments. *P < 0.05, Student’s t test compared to astrocyte. RFU, relative fluorescence unit. (C) CLR1501 labeling of the CD133+ subpopulation of GSCs was analyzed using flow cytometry. Data are means ± SD of three independent experiments. *P < 0.05 versus line-matched CD133+ or CD133− cells; #P < 0.05 versus NSCs, Student’s t test.

Fig. 4. 124I-CLR1404 uptake in a broad range of tumors

Images shown are micro-PET or PET/CT, or fused brain MRI (magnetic resonance imaging)/PET. Yellow arrows show xenografts; green arrow points to liver metastasis. (A) Xenografts of human cancer cell lines: brain (U87 in nude rat), colon (HCT-116, HT-29), triple-negative breast (MDA-MB-231), prostate (PC-3), pancreatic (BxPC3), and Ewing’s sarcoma. (B) Rodent tumor models: bladder (mouse SV40), breast (mouse 4T1), pancreatic (c-myc transgenic mouse), and brain (rat CNS-1).

Fig. 5. CLR1501 labeling of GSCs in vivo

CLR1501 was administered to mice harboring GSC-derived orthotopic gliomas 4 days before sacrifice. (A and B) Confocal microscopy verified CLR1501 uptake in histologically confirmed tumor [A: CLR1501 = green; B: hematoxylin and eosin (H&E), T = tumor]. (C to E) Immunohistochemistry for stem cell markers, including human-specific nestin (hNestin) (C), Olig2 (D), and CD15/SSEA-1 (E). DAPI (4′,6-diamidino-2-phenylindole) (blue) is shown in merged images. (F and G) Ex vivo flow cytometry analysis with CD133 (105 GSC-derived tumor-1) to quantify CD133+/CLR1501+ colocalization in GSC-derived and U251 xenografts. The percentage of marker-positive cells in total number of tested tumor cells is listed in (G).

Fig. 6. Tumor growth and animal survival after 131I-CLR1404 therapy

Nude mice harboring human tumor xenografts (n = 6 renal, 6 colorectal, 5 ovarian, and 9 breast) were administered a single 131I-CLR1404 dose (arrow). Control animals were administered a mass-equivalent CLR1404 dose (n = 6). Data are mean tumor volumes ± SEM. P values for tumor growth curves determined by one-way repeated-measurement ANOVA. P values for survival curves were constructed by Kaplan-Meier method and analyzed via log-rank test.

Fig. 7. Imaging of brain tumor patients and patient-derived xenografts

(A) Metastatic NSCLC patient. PET imaging 6 days after intravenous 124I-CLR1404 administration revealed three previously unknown brain lesions suspicious for metastases (3D reconstruction). Lesions were verified via contrast-enhanced MRI [T1–gadolinium (Gd), arrow], but not detected on surveillance 18F-FDG PET/CT, and present on 124I-CLR1404 PET (dotted line = axial PET section). (B to D) Grade IV glioma patient. A patient diagnosed with suspected left insular GBM on T1-gadolinium–enhanced (B) and T2-weighted (C) MRI was confirmed on surgical pathology. Preoperative PET imaging obtained 2 days after intravenous 124I-CLR1404 administration revealed heterogeneous tumor uptake in a different pattern than T1-contrasted MRI, with low background signal in venous sinus blood pool (arrows) and normal brain (tumor/brain ratio = 28) (D). (E to I) Fluorescent CLR1502 imaging 4 days after administration showed uptake and localization in line 107 GSC-derived xenografts (E). Histological confirmation of line 107 GSC-derived xenografts: H&E (F and G) and human-specific nestin staining (brown in H and I).