Summary                         

Mouse models are invaluable tools for radiotracer development and validation. They are, however, expensive, low throughput, and are constrained by animal welfare considerations. Here, the chicken chorioallantoic membrane (CAM) was assessed as an alternative to mice for preclinical cancer imaging studies. NCI-H460 FLuc cells grown in Matrigel on the CAM formed vascularized tumors of reproducible size without compromising embryo viability. By designing a simple method for vessel cannulation it was possible to perform dynamic PET imaging in ovo, producing high tumor-to-background signal for both ¹⁸F-2-fluoro-2-deoxy-D-glucose (¹⁸F-FDG) and (4S)-4-(3-¹⁸F-fluoropropyl)-L-glutamate (¹⁸F-FSPG). The pattern of ¹⁸F-FDG tumor uptake were similar in ovo and in vivo, although tumor-associated radioactivity was higher in the CAM-grown tumors over the 60 min imaging time course. Additionally, ¹⁸F-FSPG provided an early marker of both treatment response to external beam radiotherapy and target inhibition in ovo. Overall, the CAM provided a low-cost alternative to tumor xenograft mouse models which may broaden access to PET and SPECT imaging and have utility across multiple applications.

Results from nanoScan® PET/CT

To determine whether CAM-grown tumors were a viable alternative to mouse xenografts for molecular imaging applications, we performed dynamic ¹⁸F-FDG PET/CT imaging of NCIH460 FLuc tumors in ovo following i.v. injection (Fig. 2a). In ovo, ¹⁸F-FDG uptake was highest in the tumor, which was characterized by rapid initial delivery, reaching 7.0 ± 0.8% ID/g at 5 min, followed by a slower rate of uptake. We selected the yolk sac as the background ROI which was 0.7 ± 0.6% ID/g at 60 min, giving a tumor-to-background ratio of ~15. For comparison, dynamic in vivo PET/CT imaging was performed in immunocompromised mice implanted with subcutaneous NCI-H460 FLuc xenografts. Similarly to the chick CAM, rapid ¹⁸F-FDG tumor accumulation occurred over the initial 5 min, followed by a slower rate of retention over the proceeding 55 min. Tumor-associated radioactivity was lower in the mouse compared to the egg at 60 min p.i., reaching 6.0 ± 1.4% ID/g.

Fig. 2 Comparison of in ovo and in vivo ¹⁸F-FDG PET/CT imaging. a Representative in ovo ¹⁸F-FDG PET/CT images 40–60 min p.i.. White arrows indicate the tumor. b Representative in vivo sagittal, coronal and axial (insert) ¹⁸F-FDG PET/CT images 40–60 min p.i.. White arrows indicate the tumor. Br, brain; H, heart; K, kidney. c Comparison of in ovo and in vivo ¹⁸F-FDG tumor pharmacokinetics. d In ovo and in vivo healthy and tumor tissue ¹⁸F-FDG uptake, expressed as the area under the TAC. Data is expressed as the mean plus standard deviation.

Studies in chickens:

Prior to chick CAM tumor cell inoculation, fertilized Dekalb white eggs were stored at 12°C. To initiate embryo growth, freshly fertilized (E0) eggs were moved into an incubator, running at 37.6 °C and 50% humidity and set to tilt every 30 min. On E3, 3 days following the start of incubation, eggs were placed in a second incubator set to 37.6 °C and 50% humidity with no tilt setting. On E7, eggs were removed and inoculated with 3 × 10⁶ NCI-H460 FLuc cells. The eggs were then placed back in the incubator and kept until E14. On E14, a CAM vein was cannulated and 90 μL of a 1 mg/mL solution of the anesthetic medetomidine was pipetted on to the surface of the CAM. Eggs were left for 15 min at room temperature before receiving a bolus injection of ~3 MBq ¹⁸F-FDG or ¹⁸F-FSPG (<150 μL) on the imaging bed and washed through with 50 μL PBS. 60 min dynamic or 20 min static PET scans 40-60 min post injection (p.i.) were acquired using a Mediso NanoScan PET/CT system (1-5 coincidence mode, 3D reconstruction, attenuation-corrected, scatter-corrected). CT images were obtained for attenuation correction (180 projections, semi-circular acquisition, 50 kVp, 300 ms exposure time). The eggs were kept at 37 °C throughout the scan. Dynamic PET data were reconstructed into 19 bins of 4 × 15 s, 4 × 60 s, and 11 × 300 s (Tera-Tomo 3D; four iterations, six subjects, 400–600 keV, 0.3mm3 voxel size). VivoQuant software was used to analyze the reconstructed images. Regions of interest (ROIs) were drawn manually using 40-60 min summed PET images. Finally, time verses radioactivity curves (TACs) were generated, and area under time verses radioactivity curves (AUC) were calculated. For inhibition studies, eggs bearing NCI-H460 FLuc tumors received an intratumoral injection of IKE (2.5 mg/kg, in 5% DMSO, 95% PBS) 60 min prior to PET imaging, with control tumors left untreated.

Results show that:

• Tumors exhibited high but variable retention of ¹⁸F-FSPG (12.7 ± 5.8% ID/g at 60 min p.i) with a tumor to background ratio of 74 (Fig. 4b). ¹⁸F-FSPG retention was highest in the kidneys, with liver uptake comparable to the tumor (Fig. 4c), as confirmed by ex vivo biodistribution

Fig. 4 Dynamic ¹⁸F-FSPG PET imaging in ovo. a Representative in ovo ¹⁸F-FSPG PET/CT images 40–60 min p.i.. White arrows indicate the tumor. K, kidney. b TAC for tumor and yolk sac-associated 18F-FSPG retention in ovo. c AUC for major organs. Data is expressed as a mean plus standard deviation.

• To assess the utility of the chick CAM in mechanistic imaging studies, we treated eggs bearing NCI-H460 FLuc tumors with an intratumoral injection of the system xc - inhibitor IKE 60 min prior to imaging with ¹⁸F-FSPG PET/CT (Fig. 6a). As we showed previously, high ¹⁸F-FSPG retention was present in the control tumors (14.4 ± 3.9% ID/g at 60 min p.i) which was reduced by ~60% in IKE-treated tumors (5.5 ± 2.3% ID/g; n = 6; p = 0.004; Fig. 6b).

Fig. 6 Inhibition of system xc - reduces ¹⁸F-FSPG uptake. a Representative ¹⁸F-FSPG PET/CT image of an egg bearing both control and IKE-treated NCI-H460 FLuc in ovo tumors 40–60 min p.i. Orange circle shows the location of control tumor; blue circle shows the location of IKE-treated tumor. b ¹⁸F-FSPG TAC of control vs. IKEtreated tumors. Error bars represent one STD from the mean value.

In summary, results show that it is possible to reproducibly cultivate in ovo NSCLC tumors for imaging just 7 days after implantation. Dynamic PET imaging of these tumors was possible using a simple cannulation method without the requirement for microsurgery. Chick CAM tumors were avid for both ¹⁸F-FDG and ¹⁸F-FSPG, providing high signal-to-background ratios. This work supports the case for the use of the chick CAM as a more sustainable, low-cost substitute to tumor xenograft mouse models, and has the potential to both expedite novel radiotracer development and assess tumor response to treatment.

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