There is a significant amount of research into pharmaceutical compounds for chemotherapy. Cryo-imaging techniques enable researchers to qualitatively assess the effectiveness of new compounds.
Doctors and researchers are investigating ways to reduce the side effects of chemotherapy medications without impacting their effectiveness. Cisplatin is a common and widely used medication that has successfully increased the cure rate of a range of cancers, including breast, lung, brain and testicular cancers, but it is not without an array of side effects from nausea to kidney failure. The aim of this research, therefore, is to increase the effectiveness of the drug enabling a reduction in the dose and thus toxicity. In this article, a collective of researchers from universities and research hospitals in Barcelona reveal the results of their trials of the effectiveness of two novel methods to reduce the toxicity of Cisplatin, and the cutting edge imaging techniques they used to make these observations.
Cisplatin (cisPt) is a platinum-based complex that has been widely used as a therapeutic cancer treatment agent since the late 1970s. Its method of action is to bind to the DNA double helix within the cell, where it can distort the DNA structure and inhibit its replication and repair. This has proven to be extremely effective against carcinoma-type tumours, but is also highly toxic and any dose large enough to be effective has significant side effects. There have been various studies investigating the ability to decrease the toxicity of cisPt while maintaining maximum effectiveness, from adjusting the dosage and treatment time to the use of additives and inhibitors alongside cisPt.
In this work the authors combine these two paths by using two different bonding agents with cisPt at very low doses. They used gold nanoparticles (AuNP) and tricine, a compound used in buffer solutions with high ionic strength, and quantified their effectiveness through a series of cryo-microscopy techniques.
TEM (left) and cryo-SXT of carcinoma cells. The coloured regions indicate the mitochondria (yellow), the nucleus (blue), endosomes and/or lysosomes (purple) and vacuoles (green). Note in particular the excess mitochondria and condensation on the cells treated with CisPt- Tricine, indicating effective cell necrosis. (Figure compiled from reference 1 used under CC BY 4.0)
In order to observe the treated cells, the researchers used fluorescence microscopy, TEM and cryo-soft X-ray tomography (cryo-SXT) to image cryogenically frozen cell samples. The cells were first vitrified in a
cryo-plunger system. They were then imaged on a fluorescence microscope while being kept vitrified at liquid nitrogen temperatures using a Linkam CMS196 stage. The frozen grids were then transported to the beamline at ALBA synchrotron for cryo-SXT. They also used flow cytometry to analyse cellular damage.
In brief, they took A431NS human cutaneous squamous-cell carcinoma (cSCC) cells and used flow cytometry and cryo-imaging to identify the effect of the modified cisPt on the cells in comparison to control samples of pure cisPt. They found that while the combination of AuNP and cisPt were as effective as pure cisPt in damaging cancerous cells, they did not offer significant improvement. On the other hand, the addition of tricine enhanced the effectiveness of cisPt and increased cell death, which was highlighted in the cryo-SXT images by the prominent presence of mitochondria and chromatin condensation on the cell nucleus, both of which are indications of cell damage.
These results indicate that the addition of tricine facilitates effective therapeutic use of cisPt at lower doses than before, which bodes well for lower-impact chemotherapy cancer treatment. This contributes to the improvement of chemotherapy medications, made possible in part by the ability to accurately image vitrified cellular samples through cryogenic techniques such as cryo-fluorescence microscopy using the Linkam CMS196 stage and cryo-SXT.
References
S. Gil, et al. "Multiparametric analysis of the effectiveness of cisplatin on cutaneous squamous carcinoma cells using two different types of adjuvants" (2020) PloS one 15.3 (2020): e0230022. DOI:10.1371/journal.pone.0230022
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