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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - CAP-CANCER (Cold atmospheric plasma treatment for effective cancer cell apoptosis)

Teaser

Summary

Some of the most useful tools at the hands of a surgeon operating on a cancer patient are energy sources such as Lasers, monopolar and bipolar electrocautery. Those tools deliver heat (they increase the temperature) to the targeted (usually diseased tissue such as cancerous etc.) tissue, cutting it, coagulating (burning and stopping the blood flow) and ablating it (evaporating it). Despite their wide use, there are two major challenges with thermal energy tools, namely non-selectivity and collateral damage. Collateral damage translates into longer recovery time and pain, and into the possibility of serious injury to critical tissue and in some cases abandonment of treatment. It will therefore be highly desirable to have a surgical energy source that does not cause collateral thermal damage and can selectively treat the cancerous tissue without affecting the healthy one. Such a source would have significant implications in the surgical treatment of cancer. It would result in less pain and faster recovery times for the patient and in the ability to cure forms of inoperable cancer. This project deals with a new type of energy known as cold atmospheric plasma (CAP) which has shown promising results exhibiting almost no thermal collateral damage and also selectivity in treatment. CAP is inexpensive, easy to work with and opens up the possibilities of biomedical applications. Because CAP is non-thermal, the absence of collateral thermal damage is well understood and intuitive. The selectivity aspect of CAP though, is not well understood and this is the focus of this proposed research. The overall objective of the project is to aid in the understanding of the phenomenon of the selectivity of CAP. It achieves that by developing numerical models that help understand the complex mechanisms of CAP with tissue and by conducting biological experiments where cancerous and non-cancerous cells are treated with CAP. This work showed that CAP could be understood as interacting with tissue in two ways that are interconnected. The first being an electric field and the second being a slew of reactive oxygen and nitrogen species (RS) such as NO, OH, O, H2O2 etc. The electric field causes the cells to open up (through a phenomenon known as electroporation) and that allows the RS to enter into the cells. Once a sufficient number of RS enter, the cell undergoes apoptosis (controlled and slow death). The work of CAP-CANCER showed that it is possible to adjust the plasma parameters to increase the effects electroporation and therefore apoptosis for cancerous cells. Furthermore, it also showed that cancerous cells are more susceptible to electroporation than healthy cells and this could account for the selectivity of the treatment.

Work performed

The work included both theoretical and experimental work with helium based CAP. It started with the development of the numerical model of CAP interacting with a dielectric surface and with subsequent experiments to validate that model. It was found that when a small amount of oxygen (0.1%) is added to the helium two major things happen. First, the shape of the plasma changes from ring (or donut) to solid and second, the induced electric field on the dielectric surface almost doubles in strength. This is a significant result, since higher electric fields mean higher chance for electroporation of the cells and consequently higher amounts of reactive species or even therapeutic drugs successfully introduced into the cells. Then the numerical model was extended to include interacting with cells. The intracellular structure (of normal tissue and a tissue affected by cancer) and their electrical properties was incorporated in the numerical model and the interaction with CAP was investigated. The theoretical work was followed with experimental work where cancerous breast line cell lines MDA-MB-231, and MCF7 the cancerous and the healthy breast line MCF-12F, MCF10A where exposed to plasma conditions. The effects of the CAP treatment were quantified using flow cytometry.
Biological Sample Treatment: This work and training was discussed from the beginning of the project but the majority of the training was carried over in the second year of CAP-CANCER. We started with cultivating the cells in the proper media. The cell cultures were seeded in 12 well plates, at a density of 100.000 cells/well and allowed to grow overnight, being maintained in a CO2 incubator (5% CO2, 37Â°C) prior to the experiments. In the experiments the cells were treated with pure Helium and Helium with 1000 ppm of O2 (He-O2) plasma jet for comparison. The electrical parameters where 6 kV square pulse with 50% duty cycle at 15 kHz repetition rate and the helium flow through the 4 mm (diameter) glass tube where the plasma is generated was 2.5 litres/min. For the He-O2 plasma, an additional 2.5 ml/min was added to the gas. The plasma treatment lasted between 60s to 240s of continual exposure. After the cells were treated, they were stored and the effects of the plasma were quantified at 8 and 24 hours using an annexin V-FITC/propidium iodide assay. Our experimental results showed that the plasma treatment is effective for the cancer lines and in the case of the MCF7 the experiments showed that there is an increase in the efficiency of the treatment with the addition of 1000 ppm (or 0.1%) of oxygen. This is consistent with the numerical results that showed the generation of higher electric fields and therefore an increase of electroporation, and therefore of apoptosis and destruction, for the cells.
Exploitation and dissemination: The results of the project were communicated through the webpage of the project. In addition to that, the fellow participated at events for both the scientific community and for the public. The fellow participated in the European Researchers Night in 2016 (at the beginning of the project) and 2018 (the end of the project). The results were also disseminated and the fellow had the chance to discuss them with other researchers at a total of six (6) international conferences/workshops including the 6th and 7th International Conference of Plasma Medicine and the 5th International Workshop on Plasma for Cancer Treatment. The results were also published in peer review journals (two of them are under review).

Final results

The numerical simulations and the accurate models developed will allow UCY scientist and others to use that model for determining the optimal conditions for the electrical parameters, for adjusting for the shape of the plasma bullet (that controls the interaction surface) and determining the effect of oxygen and water impurities to the helium plasma gas. Future work will include exploration of a novel treatment of cancer combining CAP and therapeutic drugs. The impact for Europe for any effective cancer treatment will be significant as it is estimated there are over 2 million cancer related deaths per year in the European Union (EU).