Abstract
1357
Objectives The enhanced permeability and retention (EPR) effect is a tumor phenomenon, which is proposed to be the result of fast-growing leaky tumor vessels and decreased lymphatic drainage. While the EPR effect was first described 30 years ago, most work has been performed with either invasive or ex vivo methods. We developed radiolabeled liposomes with long circulating half lives that allow for in vivo investigations as well as quantification of the EPR effect in pre-clinical and clinical tumors. Concurrently, we also developed PET radiotracers for other key markers of the tumor microenvironment including a 68Ga-RGD radiotracer, which targets the αVβ3 integrin on immature blood vessels. In the present study we aimed to characterize the EPR effect by PET. To do so, we performed and compared the EPR effect (64Cu liposomes) with immature tumor vessels (68Ga-RGD) to see whether the latter was deterministic for the level of EPR effect.
Methods Human colorectal adenocarcinoma (COLO 205) cells were inoculated bilaterally in the subcutaneous thigh area of 8 NMRI nude mice and similarly, mouse colon carcinoma (CT26) cells were inoculated in 12 BALB/c mice. Tumors were allowed to grow for two weeks prior to the experimental procedures. 68Ga-RGD microPET/CT was performed on day one at 1 hour post injection (pi.). On day two and three liposome-encapsulated-64Cu microPET/CT was performed at 1 hour pi. and 24 hours pi. To validate PET results, the mice were re-injected with 68Ga-RGD and euthanized after the 24 hour liposome-encapsulated-64Cu PET/CT session, and all tumors were cut into multiple small 5-15mg pieces. These were then ex vivo gamma counted twice with approximately 21 hours apart to exploit the different half lives of 68Ga and 64Cu. All PET scans were reconstructed using a MAP reconstruction algorithm and the PET-CT images were rigidly co-registered. PET uptake was quantified using standardized uptake values (SUV) and expressed as mean and maximum ± SD. Gamma counts were converted to % of injected dose/g tissue (%ID/g). Uptake was linearly correlated using Spearmann’s nonparametric correlation analysis.
Results The 24 hour PET SUVmax uptake of 64Cu-liposome was positively correlated with SUVmax uptake of 68Ga-RGD for both CT26 (r=0.46, p=0.008) and COLO 205 (r=0.57, p=0.029) tumors. The 64Cu-liposome and 68Ga-RGD SUVmean uptake did not correlate for neither CT26 (p=0.68) nor COLO 205 (p=0.19) tumors. The gamma counted 68Ga-RGD and 64Cu-liposome %ID/g was positively correlated for both CT26 (r=0.44, p<0.0001) and COLO 205 (r=0.40, p=0.002) tumors. The pieces from each tumor showed very diverse %ID/g levels for both 64Cu and 68Ga demonstrating a high degree of micro regional heterogeneity.
Conclusions The neo-angiogenic activity was found to be positively correlated with the EPR-effect based on maximum SUV and gamma counting levels. This indicates that the EPR-effect is in fact dependent on the level of immature blood vessel density. The correlation was moderate to strong, which indicates that other factors than immature vessels also may contribute to the degree of EPR effect. With this study, we have shown a non-invasive method for characterizing how the tumor microenvironment may affect the EPR-effect, which will be relevant for performing interventional studies over time. Future studies should investigate how treatments targeting tumor vessels affect PET uptake of both 68Ga-RGD and 64Cu-liposomes.