The FR-targeted gadolinium chelate P866, but not its non-FR targeted analog P1001, caused a significant increase in R1 relaxation rates of FR-positive ovarian tumors on delayed MR scan (1 hour post injection). Because R1 relaxation rates reflect the amount of contrast in the tumors, our results suggests a component of specific accumulation of FR-targeted P866 in FR-positive tumors. This is also supported by the in vitro study which showed decreased gadolinium concentration in cells incubated with P866 in the presence of competing free folate.
The FR-targeted iron-oxide agent P1048, when compared to its non-targeted analog P904, showed a trend of increase in R2 relaxation rates, corresponding to higher retained contrast, in FR-positive ovarian tumors on delayed MR scan (48 hours post injection), albeit not statistically significant (p = 0.09). The in vitro
study showed decreased iron-oxide concentration in cells incubated with P1048 in the presence of competing free folate, suggesting specificity of P1048 for the FR on the ovarian tumor cells in vitro
. When the ovarian tumors were imaged in vivo
, there may be more nonspecific uptake of the iron-oxide contrast agents due to non-specific leak of the iron-oxide particles into the tumor interstitium and the presence of other cell populations such as macrophages which are known to take up iron-oxide [31
]. This may partially explain why we were unable to find a statistically significant difference in contrast uptake between P1048 and P904 in vivo
. The detection of nonspecific uptake may be reduced by allowing the unbound iron-oxide particle to washout over time, which was the rationale behind imaging the tumors at 48 hours in our study. This process is dependent on the concentration gradient across the vasculature and the pharmacokinetics of the FR-targeted iron oxide particles. Thus, further modification of the contrast agents that allow higher interstitial accumulation and longer interstitial residence, along with optimization of the imaging efficiency, are needed to improve the FR-specific uptake into tumor cells and to improve sensitivity and specificity of the FR-targeted iron-oxide contrast agents.
Few other investigators have studied FR-targeted MR contrast agents for tumor imaging in vivo
. Wiener, Konda and colleagues described uptake of a gadolinium polyamidoamine (PAMAM) folate-dendrimer in FR-α positive ovarian cancers (OVCA 432) [24
]. More recently, Choi and colleagues reported iron oxide nanoparticles conjugated to folate as MR contrast agent for imaging of nasopharyngeal carcinomas (KB cells) [27
]. Our data are in agreement with those previous studies showing the feasibility of imaging FR-positive tumors in vivo
using targeted MR contrast agents. In addition, our data suggest active FR-targeting by P866 because we were able to compare the uptake of contrast in tumors between targeted- (P866) and non-targeted- (P1001) contrast agents. Those previous studies did not have non-targeted contrast agents available for comparison. Corot and colleagues have recently shown a specific uptake of P866 in a nasopharyngeal carcinoma tumor model (KB cells) in mice [33
]. The degree of specific uptake of P866 in the KB cells was much higher than what was observed in our ovarian tumor model in rats. The differences in P866 uptake may be attributed to a much higher level of FR-α expression in the KB cells than the IGROV-1 cells [34
As shown in , the specific uptake of FR-targeted contrast agents in vivo was better evaluated using a non-FR-targeted analog as a control rather than performing folate competition experiments. For in vivo folate competition studies, excessive amount of free folate would be required to saturate the metabolic pathways in the liver, therefore resulting in nonphysiological amount of folate in the blood. While our study suggested a specific retention of the FR-targeted contrast agent P866 in ovarian tumors, we did not investigate if the P866 was bound to FR on the cell surface or if some of the contrast agent was internalized into the cells. The mechanism is important with respect to the elimination pathway of this diagnostic agent as well as potential future designs of FR-targeted therapy.
The depiction of the FR-specific uptake in our study was close to the detection limit of MR imaging. The differences between the FR-targeted and non-targeted agents could only be detected by direct measurements of the R1 relaxation rates for the gadolinium agent rather than signal intensity measurements. It is known that the changes in the relaxation rates are proportional to the concentration of contrast agents in the tumors, and that the relaxation rates measurements are more sensitive than actual signal intensity measurements. Compared to the FR-targeted radionuclide and optical imaging techniques, MR imaging of cell surface receptors still faces many challenges mostly because of the relatively low signal yield of MR contrast agents. The gadolinium agent P866 used in our study is composed of folate coupled to a high relaxivity dimeric gadolinium chelate in order to reach a higher MR sensitivity. Similarly, P1048 is composed of folate moiety coupled to an iron oxide core in an attempt to improve the sensitivity of signal detection [35
]. Clearly our study represents work-in-progress, and further modification of the contrast agents, improvement in pulse sequences, and use of higher field MR scanners will be needed to improve the sensitivity of FR-specific uptake in tumors. Nonetheless, our current study serves as proof-of-concept and demonstrates the possibility of combining the specificity of receptor targeting with the improved anatomic resolution of MR imaging.
The impetus behind the FR-targeted MR contrast research is the potential clinical applications which include characterization and treatment monitoring of FR-positive tumors. FR-targeted contrast agents may allow more sensitive and specific diagnosis of FR-positive tumors by detecting additional sites of tumor that are missed on conventional imaging, and by better distinguishing between tumor and treatment-related fibrosis or scarring. Assessment of tumor FR level with targeted contrast agents may also lead to improved characterization of tumor aggressiveness, provide rational means of selecting patients who would most benefit from anti-folate therapy, and allow better treatment monitoring. We focused our studies on ovarian tumors because FR is over-expressed in 90% of ovarian tumors [6
], which serve as a good model to evaluate the feasibility of FR-targeted MR imaging. Analyses of human ovarian tumors to date have demonstrated considerable variability in the expression levels of FR among patients as well as heterogeneity within the same tumor [36
]. FR-specific MR contrast agents may estimate the level of over-expression of the receptors, which has been correlated directly with the biological aggressiveness such as the histological grade and S-phase fraction [6
]. The FR status has also been documented to reflect the tumor's response to chemotherapy [39
]. Although ovarian tumors are relatively rare in the pediatric population, our study is designed to test the feasibility and potential of FR-targeted MR imaging. The concept of FR-targeted MR contrast agents may be applied to various FR-positive tumors, including pediatric tumors such as choroid plexus tumors and ependymomas [8
], osteosarcoma [9
], and acute myelogenous leukemia [3
In conclusion, a specific accumulation of the FR-targeted gadolinium agent P866 was suggested in a FR-positive ovarian tumor model. Further development in the FR-targeted contrast agents and improvement in imaging efficiency are needed to improve the sensitivity and specificity of MR imaging of FR-positive tumors.