Cold atmospheric plasma for breast cancer treatment: what next?

Abstract

Breast cancer (BC) has the highest incidence rate among all cancers worldwide, responsible for almost 2.3 million new cases reported in 2022.1 Moreover, it stands as one of the most fatal cancer types, emerging as the leading cause of death among women.1 In recent years, the global burden of BC has experienced a notable increase, driven by factors such as population growth and aging, as well as changes in risk factor profiles and advancements in cancer diagnosis. In fact, future projections indicate that BC burden will increase in women.2 BC, a complex and heterogeneous disease, can be categorized based on the expression of specific biomarkers, including the estrogen receptor, progesterone receptor, and human epidermal growth factor 2 (HER2)-enriched receptor. There are four distinct molecular types: luminal A, luminal B, HER2, and triple-negative BC.2 The treatment of BC focuses on the development of targeted strategies that are stratified according to the molecular subtypes, as well as tumor progression and grade, to provide the most effective and efficient treatment for patients. Currently, targeted therapies are specifically used in distinct subtypes.2 Luminal subtypes benefit from endocrine therapies that either directly target estrogen receptor, such as tamoxifen, or reduce estrogen levels, such as aromatase inhibitors.2 For the HER2 subtype, anti-HER2 therapy is used and, for the triple-negative subtypes, chemotherapy with taxanes and anthracyclines is the predominant treatment.2 Although effective, BC treatment remains incredibly challenging. Some patients have an inadequate therapeutic response with disease progression, others develop drug resistance, and treatments are often associated with side effects.2 Consequently, new approaches are still needed, and it is imperative to prioritize the development of novel selective therapies aiming to enhance personalized treatment for each patient’s tumor characteristics. Among the developed new therapies, the use of plasma has emerged as one of the most promising. Plasma potential is being explored in various fields of biomedicine, including dentistry, dermatology, oncology, infection control, and sterilization. Regarding oncology, it is being investigated for cancer treatment, as a potentially selective option that is likely to complement the existing therapies and increase their efficacy.3,4 Plasma, the fourth state of matter, emerges through the ionization of a gas, which acquires enough energy to displace the electrons.5 This dynamic mixture of particles comprising free electrons, positively and negatively charged ions, and neutral charge particles is an efficient electrical conductor that is influenced by electromagnetic fields.3 Plasma can be divided into atmospheric plasma and low-pressure plasma based on the exposure pressure. In atmospheric plasma, there is a higher frequency of collisions among particles since the distance between the particles is shorter than in low-pressure plasmas.3,5 Notably, low-pressure plasma, characterized by a more robust charge, is unsuitable for applications in human cells or tissues.3 Atmospheric plasmas can be classified into thermal and non-thermal, depending on the thermal motions of the electrons and heavy particles. In thermal plasmas, the particles are in thermal equilibrium and reach high temperatures. In contrast, in non-thermal plasmas, also known as cold atmospheric plasma (CAP), the particles are not in equilibrium, and the electrons have significantly higher temperatures than the heavy particles.3,5 CAP can be generated through three different types of devices, based on the method of plasma production: dielectric barrier discharge, which produce direct discharges of plasma; plasma jets, which produce indirect discharges; or hybrid devices, which combine both methods. In a plasma jet device, plasma is transported by a carrier gas, such as helium, argon, or oxygen, and the sample is only treated by the plasma. Dielectric barrier discharge devices can produce plasma directly in the air, with the sample becoming part of the plasma discharge. However, when comparing both devices, the plasma produced by a dielectric barrier discharge device is more uniform, while the discharge from a plasma jet is more directional. Dielectric barrier discharge devices are also limited to producing only low-pressure plasma, which has a higher risk of damaging the sample during treatment due to direct contact with the plasma. Hybrid plasma devices generate a discharge directly without affecting the sample, minimizing the risk of damage. All devices share the same physical principles and components promoting plasma generation between two electrodes.3 Nevertheless, all these devices are limited to surface structures being necessary to develop new ones capable of being directly applied in internal and subcutaneous structures.3,4,6 As previously refereed, several investigations have demonstrated the potential of CAP in oncology.3 The observed effects seem to be triggered by reactive species production, ultraviolet light generation, and the electromagnetic fields generated by CAP.3 Reactive oxygen and nitrogen species enter tumor cells through aquaporins. Since these cells have a lower amount of cholesterol in their membranes and higher levels of aquaporins, they become more susceptible to oxidative stress induced by reactive oxygen and nitrogen species, leading to an increase in lipid peroxidation. Consequently, intracellular oxidative stress surpasses the threshold of the oxidative defense system, leading to cell death.4 In addition, ultraviolet radiation and electric fields are capable of inducing DNA damage and causing cell cycle arrest.6 Nevertheless, the cellular pathways underlying the anti-cancer effects are still largely unclear.3 Currently, no significant secondary effects are associated with CAP’s action. However, the efficacy of CAP varies based on factors such as the distance between the plasma source and the cells, the voltage and the duration of the exposure treatment and the specific tumour type.3,4,6 In recent studies, it was demonstrated that direct application of CAP in BC cell lines exhibited a time- and dose-dependent effect on cell viability.6,7 It was also revealed that CAP and plasma-activated medium (PAM) exposure led to the disorganization of the actin cytoskeleton, resulting in an alteration in cell morphology. Significant downregulation of protein expression associated with cancer metastasis was also observed, suggesting a potential inhibitory effect on cancer dissemination.7 Moreover, CAP treatment induced cell death primarily by apoptosis.6,7 Additionally, there was an accumulation of cells in the G2/M phase of the cell cycle, indicating reduced replication ability and lower survival rates. Furthermore, the researchers, observed a new balance in the redox state of the MCF7 cell line following CAP exposure, as well as a lack of proportional reduction in metabolic activity when compared to cell viability.5 CAP treatment has also been studied in other types of cancer including glioblastoma and lung cancer cell lines. In these studies, CAP treatment altered the mitochondrial membrane potential leading to increased expression of pro-apoptotic gene expression as well as a decrease in cell viability.3 Another study demonstrated an increase in poly ADP-ribose polymerase-1 activity in lung cancer cell line A549 derived from higher oxidative stress after CAP treatment.6 Another study has also mentioned the anti-proliferative potential of CAP treatment in leukemia, cervical, gastric, head and neck and pancreatic cancers.8 CAP can be used as a monotherapy or in combination with established therapeutic regimens, enhancing their efficacy and potentially allowing for dose reduction. The effectiveness of PAM in combination with tamoxifen was investigated in MCF7 and MDA-MB-231 cell lines. The results revealed a significant decrease in cell viability and proliferative capacity with the combined treatment compared to monotherapy, indicating that PAM has the potential to enhance the sensitivity of the existing therapies, acting as a neoadjuvant therapy.9 CAP also demonstrated ability to sensitize cells that had become resistant to tamoxifen therapy by reversing the dysregulated expression of genes induced by tamoxifen.10 Another relevant finding was CAP’s ability to promote anti-tumoral activity while presenting minimal effects on non-malignant cells in vitro. Moreover, mice receiving CAP treatment exhibited prolonged survival times and improved outcomes regarding the promotion of antitumor immune response and inhibition of tumor recurrence.11 The promising results of CAP and PAM cancer treatment encourage deepening the understanding of the mechanisms of action, including the modulation of biochemical pathways, such as those associated with the hallmarks of cancer, cellular and physiological effects, potential resistance induction, and interaction with other therapies. Also, few authors explored CAP on BC stem cells.6 If CAP and PAM are proven effective in targeting this critical subpopulation, their therapeutic value would be significantly enhanced. It is also imperative to explore strategies to maximize CAP effects. The evaluation of the synergistic effects of combining CAP treatment with existing cancer therapies, such as chemotherapy, radiation therapy, and immunotherapy, is essential. Combining CAP with conventional treatments may enhance their efficacy, reduce treatment resistance, and minimize side effects.8 Also, CAP integrated with physical fields, such as ultrasound or electroporation, can further enhance its therapeutic potential. For example, ultrasound can be used to enhance the penetration of CAP-generated reactive species into tissues, while electroporation can facilitate the uptake of therapeutic agents following CAP treatment. These combination approaches could improve treatment outcomes by targeting tumors through complementary mechanisms. The use of breast human tumor samples will be a complementary tool that provides valuable insights into CAP mechanisms of action, given the heterogeneity of BC disease. In patient-derived xenograft models, human breast cancer tissue samples are transplanted into immunodeficient mice to recapitulate the tumor microenvironment and study tumor growth and treatment response. Treating patient-derived xenograft models with CAP will allow researchers to evaluate its efficacy in vivo and assess its potential for clinical translation.8 These preclinical studies enlighten CAP’s therapeutic effects and will help guide the design of future clinical trials. Despite CAP’s introduction in the cancer field around 2010 and its continuous advancement, clinical trials involving CAP are growing. In 2019, U.S. Food and Drug Administration (FDA) agency approved the first clinical trial (NCT04267575) using CAP for oncotherapy in combination with surgical resection in patients with stage IV or recurrent solid tumors, including BC. The results showed safety and selectivity, reducing the risk of recurrence.12 Recently, the number of ongoing clinical trials using CAP has growing considering its different biomedical applications.12 Ultimately, with the promising results already demonstrated, we hypothesize that, in the future, the specific properties of CAP and PAM can be used and translated, considering different delivery approaches.3-10 Hydrogels and different formulations of PAM solutions targeting specifically BC might be a low-cost, effective, and safe option for BC treatment. Furthermore, biocompatible nanoparticles-encapsulated PAM might emerge as a specific delivery system to tumor cells. We speculated that a nanoparticle loaded with PAM would target the tumor cell using specific receptors, ensuring the efficacy and selectivity of the treatment. Such advances also allow us to think about less invasive delivery methods, such as an injectable solution or even a tablet for oral administration, harnessing their full potential for BC patients and society. In our view, the collaboration between academia and the pharmaceutical industry is an asset for the scale-up of CAP treatment. CAP research has been increasing in recent years, with promising results in BC treatment and encouraging more investigation. Translating findings into clinical trials will be a crucial step toward validating the efficacy and safety of CAP in cancer treatment. Collaborative efforts between researchers, clinicians, biomedical engineers and industry partners are needed to design and conduct rigorous clinical studies evaluating the effectiveness of CAP alone or in combination with other therapies in different cancer types. Foundation for Science and Technology (FCT) supports the Center for Innovative Biomedicine and Biotechnology (CIBB) through the Strategic Projects UIDB/04539/2020 (https://doi.org/10.54499/UIDB/04539/2020) and UIDP/04539/2020 (https://doi.org/10.54499/UIDP/04539/2020) and the Associated Laboratory funding LA/P/0058/2020 (https://doi.org/10.54499/LA/P/0058/2020). This work was supported by the Project CARBONCT, 2022.03596.PTDC (https://doi.org/10.54499/2022.03596.PTDC). A PhD fellowship granted by FCT supports CAF (2022.12228.BD).

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