Characterizing candidate anticancer drugs through fluorescent imaging with the Azure c600

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In a recent publication from Mendel University in Zemedelska, Mitrevska et al used the Azure c600 as part of a study to characterize candidate anticancer drugs and compare the anticancer activity of platinum nanoparticles to cisplatin using a breast cancer model.

Cisplatin is a chemotherapeutic agent used to treat many types of solid tumors, including hematologic cancers.

Its anti-tumor effects result from platinum binding covalently to purine bases, leading to DNA damage and cell death. Because cisplatin and other platinum agents are associated with toxicity, researchers are searching for replacements to conventional platinum chemotherapy. 

Since the release of this publication, the cSeries Imaging Systems have been succeeded by the new Azure Imaging Systems. The upgraded systems are high performance instruments capable of NIR fluorescence, visible fluorescence, and chemiluminescence.

In ovo and ex ovo chick CAM assays

Chick chorioallantoic membrane (CAM) assays are used to study angiogenesis and tumor biology. CAM is used in research instances like these because they are faster and less expensive to use than mouse models. They provide an in vivo system to study tumor vascularization and metastasis.

To implant the tumor cells into the chorioallantoic membrane of the chick embryos, researchers must cut a hole in the shell (in ovo), or gently crack the egg into a dish (ex ovo). Tumor vascularization and growth can be studied in three dimensions over the course of several days. Mitrevska et al used both in ovo and ex ovo CAM assays in this study.

Comparing the effectiveness of platinum nanoparticles against cisplatin

This new work evaluates platinum nanoparticles (PtNPs) in a breast cancer xenograft model and compares their effectiveness to cisplatin. In addition to studying these effects on tumor growth, the authors looked at the effects of cisplatin and PtNPs on tumor metabolism. Previous work suggests that PtNPs exert effects through a different mechanism than cisplatin, forming complexes with DNA polymerase rather than DNA bases.

The activity of PtNP-10 and PtNP-40 (two types of PtNPs) against primary tumors from a human breast cancer cell line were compared to the activity of cisplatin. The Azure c600 was used in a study of ex ovo cultures inoculated with fluorescently labeled breast cancer cells. The developing tumors were treated with each of the anticancer agents, and the embryos were fluorescently imaged using the c600. The Azure c600 used was equipped with three RGB fluorescence channels to best detect fluorescent biomolecules in the visible range, Cy2/Cy3/Cy5.

Green fluorescent macrovisualization of MDA-MB-231 migration and colonization in the chick embryos by ex ovo CAM assay captured using fluorescent imaging on Azure c600 imager
Figure 2a from Mitrevska et al. Fluorescent visualization of MDA-MB-231 migration and colonization in the chick embryos by ex ovo CAM assay using Azure c600. Used to evaluate the antitumor and antimetastatic activity of platinum-based drugs in association with the impact onamino acid metabolism. Imaged on the Azure c600. Licensed under CC BY 4.0.

The fluorescent images captured (Figure 2A) show metastasis of the cancer cells throughout the chick embryos. Metastasis is reduced in the embryos treated with PtNPs compared to the untreated embryos and those treated with cisplatin.

The Ultimate Western Blot Imaging System

The Azure 600 offers laser technology with two IR detection channels enabling you to image more than one protein in an assay. It provides accurate and fast chemiluminescent detection, as well as the sensitivity, dynamic range, and linearity needed for model organism imaging and quantitative blot analysis.
Scientist choosing settings on Azure 600
The Azure 600 is the only system that offers two channel laser based IR detection, chemiluminescent detection with the speed and sensitivity of film, and the ability to image visible fluorescent dyes, standard EtBr and protein gels.

Additional experiments further characterized the effects of PtNPs and cisplatin in the CAM assays. These experiments described metabolic effects and changes in expression of TCA cycle enzymes. The Azure c600 was then used to visualize EtBr gels to confirm RNA integrity (Figure S1) and check for amplicon size and primer dimers after PCR as part of the gene expression studies.

Chemiluminescent Western blot showing RNA integrity verified using a bleach gel visualized using Azure c600
Figure S1 from Mitrevska et al. Gel visualized using the Azure c600 to show the isolation of RNA. Licensed under CC BY 4.0.
Validation of the amplicon size and control of primer-dimer formation, visualized by Azure c600 from Azure Biosystems
Figure S2 from Mitrevska et al. Amplicon size and control of primer-dimer formation were checked by gel electrophoresis, and visualized using Azure c600. Licensed under CC BY 4.0.

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Conclusion

The authors concluded that the CAM assay is a powerful model to characterize anticancer drugs. The amino acid metabolism in MDA-MB-231 cell culture is the most susceptible to PtNPs-10. Their experiments demonstrate PtNPs are more effective than cisplatin at inhibiting tumor growth and metastasis and suggest a different mechanism of action for PtNPs than cisplatin.

SOURCES

  1. Mitrevska K, Rodrigo MAM, Cernei N, et al. Chick chorioallantoic membrane (CAM) assay for the evaluation of the antitumor and antimetastatic activity of platinum-based drugs in association with the impact on the amino acid metabolism. Mater Today Bio. 2023;19:100570

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