The results of our study suggest that the 4kc
model, derived using the ITS parameter-estimation method, is most appropriate for describing 64
Cu-DOTA-RGD PET data. summarizes the model discrimination process that was applied to the 2k, 3k, 4k, and 4kc
structures. We were able to rule out the 2k and 4k structures by AIC analysis () of each model fitted to the initial dynamic PET scans (). The 2k model structure yielded the lowest AICs for tumor time–activity curves in which concentration of free αv
was low(blocked and A431 studies), suggesting that only a single compartment is required to describe 64
Cu-DOTA-RGD kinetics in αv
-negative tissues. Likewise, the 3k and 4kc
models yielded the lowest AICs for tumor time–activity curves in which density of free αv
was high (U373 and U87 studies), suggesting that 2 compartments are needed to describe 64
Cu-DOTA-RGD kinetics in αv
-positive tissues. Implementation of a fixed value of k4
is supported by the ITS parameter-estimation process, in which k4
converges to 0.00938 min−1
for all studies under consideration (), and by graphical analysis, in which Patlak uptake (Ki
) and Logan volume of distribution (Vd
) are highly correlated (RS
≈ 0.90, not shown) across all studies, suggesting little variability in tracer dissociation rate (k4
), because Ki
) and Vd
) when calculated from fitted model parameters. The 3k model structure is ruled out by fitting 3k and 4kc
models to 60-min dynamic scans plus the 20-h postinjection scan, where the 4kc
structure yields lower AICs (not shown); the 3k model also provides a less accurate prediction of the 20-h postinjection data, compared with 2k, 4k and 4kc
structures (). Although the PET-derived input function, u(t), is corrected for partial-volume effects, errors due to spillover, delay, and dispersion are assumed to be negligible and are not accounted for in the present study. The impact of spillover, in particular, is expected to be small, because uptake of αv
-binding RGD peptides by the myocardium is minimal (4
Model discrimination process by which 4kc model, compared with 2k, 3k, and 4k models, was determined to be most appropriate for describing in vivo 64Cu-DOTA-RGD kinetics in mouse models that carry αvβ3-positive tumors.
By using the 20-h postinjection scans, we were able to estimate kint, the rate of irreversible tracer internalization. The black curve in depicts a model fit to selected 20-h data, with the inset showing the first 60 min of data and fit; the gray curve illustrates the effect of setting kint to zero, in which a negligible shift from the original fitted black curve is observed (, inset). The effect of setting kint to zero suggests that although the internalization rate is high enough to produce significant retention of the tracer over a 20-h period, the rate does not confound apparent tracer retention because of specific binding to αvβ3 (k3) during the first 60–90 min after tracer injection. Even when fitting the model to data collected over a 20-h period, only a slight decrease in estimated value of k3 is observed with kint included in the model structure, compared with k3 estimated via a model fit to the 60-min dynamic scan with kint set to zero (). Because tracer metabolite analysis has not yet been performed at 20 h after injection, it is possible that we overestimated 64Cu-DOTA-RGD plasma concentration at the late static scan. This overestimation would affect the accuracy of the input function past 60 min and thus the confidence level of the estimated value of kint. To assess the effect a lower actual tracer plasma concentration 20 h after injection might have on conclusions drawn from the fitted model, we lowered 20-h plasma values calculated from the reconstructed PET image by 2 orders of magnitude and then reestimated the input function as described in the “Parameter Estimation” section. As expected, the resulting kint values are higher than those listed in ; however, the fitted model suggests that tracer internalization does not confound the apparent specific binding capacity of the tumor during the first 60 min after tracer injection, even when plasma concentration of tracer at 20 h after injection is assumed to be close to zero. That is, the result is similar to that shown in .
and show that the average estimated value of kint is much higher in the blocked studies than in the nonblocked studies, suggesting that a greater fraction of αvβ3-bound peptide may be internalized per unit of time when the 10 mg/kg dose of cold peptide is coinjected with 64Cu-DOTA-RGD. The αvβ3 integrin is activated by the binding of substrate, which stimulates recycling of the integrin from the cell surface to the intracellular compartment. This process occurs when the integrin binds RGD sequences on molecules such as fibronectin or fibrinogen—analogous to the interaction between the integrin and 64Cu-DOTA-RGD described by our model—and may explain why internalization rates are higher in tumors exposed to a large bolus of integrin substrate in the form of coinjected cold peptide, compared with tumors exposed to tracer alone. The effect of kint on apparent tracer kinetics in blocked studies is similar to the effect seen in nonblocked studies ().
Estimated values of specific volume of distribution () appear to be strongly correlated with concentration of available αvβ3 binding sites within the tumor (); with the exception of a single A431 study, VS increases in parallel with αvβ3 expression from baseline (blocked studies) to low (A431), intermediate (U373), and high (U87) expression. Estimated values of nondisplaceable (nonspecific) tumor uptake are approximately constant, regardless of αvβ3 status (; ). Along with the AIC analysis presented in , which suggests a 1-compartment tissue model is most optimal for describing tracer kinetics in low αvβ3-expressing tissue, these data support the putative physiologic significance of the 2-compartment tissue model and associated parameters for 64Cu-DOTA-RGD, in which compartment q1(t) represents accumulation of tracer in the tumor resulting from nonspecific transport mechanisms such as extravasation (K1), tissue efflux (k2), and nonspecific binding (K1/k2). Compartment q2(t) represents accumulation in tumor due to specific binding (k3) and dissociation (k4) of tracer from αvβ3. Additionally, the internalization term kint was introduced and is required to fit the model to tracer kinetics measured over a 20-h period (not shown).
calculated from a previous patient study involving 18
) closely match our results. Although variability across patients is not represented here, mean patient VND
is virtually identical to VND
calculated from our mouse studies and patient VS
is similar to values calculated for the U373 tumor, which expresses αv
at an intermediate level. This similarity suggests that the methods and model developed here for kinetic analysis of 64
Cu-DOTA-RGD may be readily applied to patient data.
A high correlation (RS = 0.92) between the ratio of tracer concentration in the tumor at 60 min after injection to tracer concentration in plasma at 10 min [CT(60)]/[CP(10)] and specific volume of distribution (VS) () was gleaned from an extensive analysis of correlations between model microparameters (VB, K1, k2, k3, k4), macroparameters (VS, VND, Ki, Vd), and tracer uptake at discrete time points (10, 30, and 60 min). This correlation, along with the aforementioned discussion of patient VS and VND values (), suggests that magnitude of αvβ3 expression could be estimated in a clinical setting on the basis of a blood sample taken at 10 min after injection and a single static PET scan at 60 min.