Introduction: Radioiodinated NM404 (12-(4-iodophenyl)-octadecylphosphocholine), a second-generation phospholipid ether analog, has displayed remarkable tumor selectivity in rodent tumor models. This novel agent is retained within tumor cells for a prolonged time, thus allowing for clinical imaging applications that may enhance preoperative staging. Phospholipid ether analogs appear to be a substrate for phospholipase D (PLD) but not phospholipase A or C based on preliminary data. We hypothesized that the mechanism of prolonged retention of NM404 is likely due to a decrease in its breakdown secondary to either decreased PLD1 or PLD2 expression.
Methods: Three human colorectal cancer cell lines, DLD-1, HT-29 and LS-180, were simultaneously implanted subcutaneously (1 x 106 cells) into SCID mice. When tumor size reached 0.5 cm, animals were injected i.v. with 124I-NM404 (80-100 μCi in 0.1 ml). MicroPET and microCT images were obtained at 24 and 72 hrs. post injection, while quantification of NM404 retention was determined by ROI analysis of the PET scans. Cell lines were analyzed for PLD1 and PLD2 mRNA using quantitative PCR. qPCR for the PLD gene transcripts involved 50 cycles of amplification by real-time PCR to give PCR products of 560 bp for PLD1 and 557 bp for PLD2.
Results: On microPET imaging, tumor conspicuity was excellent in all 3 tumor types at both 24 and 72 hours post injection. For NM404 retention, tumor-to-muscle ratios of 11.1, 6.0, and 4.0 were obtained for LS180, DLD-1, and HT-29, respectively. qPCR results from three separate experiments for PLD1 and PLD2 in the colorectal cancer cell lines were determined and expressed as a ratio to the level of gene expression in the normal CCD-18co colorectal cells after normalizing to the S26 house-keeping gene. There was no difference in PLD1 expression in the colorectal cell lines compared to CCD-18co. However, expression of PLD2 was markedly decreased in all three cell lines, with PLD2 values of 0.05 ± 0.02 (p = 0.006): 0.47 ± 0.19 (p = 0.083); and 0.14 ± 0.06 (p = 0.021) for LS-180, DLD-1, and HT-29, respectively. Thus a decreased PLD2 expression level correlated with increased retention levels of NM404.
Conclusion: Human colorectal tumors showed excellent tumor conspicuity when utilizing 124I-NM404 microPET. This appears to be associated with a decrease in expression of PLD2. Further evaluation of the mechanism of selective retention of NM404 is needed in order to define a molecular profile that may classify tumors as more sensitive to NM404, enabling the selection of a subset of patients that would benefit from preoperative staging with NM404.