Assessment of cHSA-PEO (2000)16-Gd in breast cancer xenografts on chicken chorioallantoic membrane @ 11.7T
Zhi Zuo1,2,3, Tatiana Syrovets4, Tao Wang5, Yu-zhou Wu5, Felicitas Genze4, Alireza Abaei3, Ina Vernikouskaya2, Wei-na Liu5, Gen-shan Ma1, Tanja Weil5, Thomas Simmet4, and Volker Rasche2,3

1Department of Cardiology, Zhongda Hospital, Medical School of Southeast University, Nanjing, China, People's Republic of, 2Department of Internal Medicine II, University Hospital Ulm, Ulm, Germany, 3Core Facility Small Animal MRI, Medical Faculty, Ulm University, Ulm, Germany, 4Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, Ulm, Germany, 5Institute of Organic Chemistry III, Ulm University, Ulm, Germany


High-resolution MRI and chicken chorioallantoic membrane (CAM) cancer xenografts model are employed for the assessment of bio-distribution of polypeptide hybrid biomaterials cHSA-PEO (2000)16-Gd. The breast carcinomas were planted on the CAM, which were intravascular injected with cHSA-PEO (2000)16-Gd, gadofosveset (Vasovist®) and gadobenate dimeglumine (Multihance®) before MR scanning. The detailed internal structures of tiny cancer were clearly highlighted, which was proven by the immune-histological analysis. Assessment of the bio-distribution of contrast agents renders feasible. The cHSA-PEO (2000)16-Gd has exhibited an improved specific tissue uptake by cancer cell xenografts and long lasting enhancement compared to traditional contrast agents.


As immune-deficient in vivo model, the CAM model is much cheaper, easier for tumor implantation compared to nude rodent model and has no ethical or legal concerns referring to European and USA experimental animal protection laws. It has gained great interest for quality testing of novel contrast agent (CA) materials designed for the early stage cancer diagnosis 1. With the recent developments in immobilization of the chicken embryo 2, non-invasive high-resolution imaging of the embryos and cancers planted on the CAM has rendered feasible. In this contribution it is investigated whether the CAM model could be used for the MRI assessment of a self-assembled Gd-DOTA conjugated protein based polypeptide copolymer cHSA-PEO (2000)16-Gd.

Methods and Materials

28 chicken embryos with planted mammalian carcinoma were investigated. The carcinomas were planted at day 7 after embryo incubation. Carcinomas grew for 9 days and imaging was performed at day 16. Prior to imaging, the embryos were cooled at 4℃ for 110 minutes for immobilization. All scanning was performed by a T1-weighted three-dimensional FLASH sequence with acquisition parameters as follows: TR/TE = 6/2 ms, matrix = 400×439×96, spatial resolution = 100×100×560 µm³ and NSA = 2. Eight chicken embryos were injected with different gadofosveset doses (5, 10, 20, 50, 60, 70, 80, and 90μl 0.5mmol/ml gadofosveset solution) for assessment of the optimal contrast agent dosage. Scanning was performed right after injection and the optimal dosage identified by analysis of the signal-to-noise ratio (SNR) in the umbilical vein. After optimal gadofosveset dosage was confirmed, 10 chick embryos were used for the assessment of the gadofosveset bio-distribution systemically injection. All embryos were scanned prior to, 30min, 3h, 20h and 40h after intravenous administration of CA. SNR data of different organs (vessels, allantoic fluid, liver, and brain) of chick embryos at different time points after contrast agent injection were measured and calculated. After all protocols were confirmed, novel MR contrast agent material was assessed and compared with gadofosveset and gadobenate dimeglumine.


MR images of post-injection embryos with different CA dose and resulting dose-SNR curves are provided in Figure 1. A dose of 80μl was chosen as optimal gadofosveset dosage for day 16 chicken embryos. Example MR images of gadofosveset bio-distribution and change of the mean SNR ± std. Dev in different embryo organs are provided in Figure 2. The bio-distribution of the injected gadofosveset can be clearly visualized with high spatial fidelity in ovo. From Figure 2 an early enhancement of the blood circulation can be appreciated followed by enhancement the liver parenchyma. Finally the allantoic fluid is enhanced. The enhancement variability in cancer cells xenograft over 40 hours after Vasovist intravenous injection and whole egg enhancement at 0.5 hour time point is shown in Figure 3. The cancer cells xenograft was enhanced almost from the same time with the blood pool. The correlation of immune-histological analysis of tumor xenografts and MR image is shown in Figure 4. A high correlation between the MR images and immune-histological analysis can be appreciated. The comparison of cancer xenograft enhancement by novel material and two traditional commercial CA is shown in Figure 5. Only in samples injected with cHSA-PEO (2000)16-Gd the enhancement is restricted to the cancer cells xenograft with persistent enhancement of up to 20 hours after injection.

Discussion and Conclusion

To our knowledge, this is the first report on the MR assessment of novel CA in CAM model with cancer cell xenograft with intravenous administration. The novel CA material cHSA-PEO (2000)16-Gd has exhibited an improved uptake by cancer cell xenografts and long lasting enhancement compared to traditional CA. With the protocol introduced, the CAM model presented great potential of initial testing of novel CA material thus maybe replacing some animal experiments.


No acknowledgement found.


1. Vargas A, Zeisser-Labouèbe M, Lange N, et al. The chick embryo and its chorioallantoic membrane (CAM) for the in vivo evaluation of drug delivery systems. Adv Drug Deliv Rev 2007 Sep 30;59(11):1162-76.

2. Zuo Z, Syrovets T, Genze F, et al. High-resolution MRI analysis of breast cancer xenograft on the chick chorioallantoic membrane. NMR Biomed 2015 Apr;28(4):440-7.


Figure 1: Images of chicken embryos post systemically-injection of Vasovist, at different doses and the resulting SNR of the umbilical veins.

Figure 2: Representative FLASH images and change of SNR ± std. Dev in different organs prior to, 0.5h, 3h, 20h and 40h after Vasovist injection (n=10). Vessels: red line, allantoic fluid: white line, liver parenchyma: yellow line and brain: blue line.

Figure 3: Enhancement variability in cancer cells xenograft over 40 hours after Vasovist intravenous injection and whole egg enhancement at 0.5 hour time point.

Figure 4: Correlation of immune-histological analysis of tumor xenografts and MR image. White arrow: vessel. Black arrow: loosen cells area.

Figure 5: Comparison between cHSA-PEO (2000)16-Gd (a), gadofosveset (b) and gadobenate dimeglumine (c). Arrows: cancer xenografts enhanced by CA.

Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)