Efficacy of Cold Atmospheric Plasma vs. Chemotherapy in Triple-Negative Breast Cancer: A Systematic Review

Breast cancer is a growing disease, with a high worldwide incidence and mortality rate among women. Among the various types, the treatment of triple-negative breast cancer (TNBC) remains a challenge. Considering the recent advances in cold atmospheric plasma (CAP) cancer research, our goal was to evaluate efficacy data from studies based on chemotherapy and CAP in TNBC cell lines and animal models. A search of the literature was carried out in the PubMed, Web of Science, Cochrane Library, and Embase databases. Of the 10,999 studies, there were fifty-four in vitro studies, three in vivo studies, and two in vitro and in vivo studies included. MDA-MB-231 cells were the most used. MTT, MTS, SRB, annexin-V/propidium iodide, trypan blue, and clonogenic assay were performed to assess efficacy in vitro, increasing the reliability and comprehensiveness of the data. There was found to be a decrease in cell proliferation after both chemotherapy and CAP; however, different protocol settings, including an extensive range of drug doses and CAP exposure times, were reported. For both therapies, a considerable reduction in tumor volume was observed in vivo compared with that of the untreated group. The treatment of TNBC cell lines with CAP proved successful, with apoptosis emerging as the predominant type of cellular death. This systematic review presents a comprehensive overview of the treatment landscape in chemotherapy and CAP regarding their efficacy in TNBC cell lines.


Introduction
Cancer is a growing disease worldwide.In 2020, breast cancer (BC) had highest incidence among cancers in women and was responsible for the largest number of deaths across all age groups [1].The expressions of estrogen receptor-, progesterone receptor-, and human epidermal growth factor receptor-related protein are primary determinants of BC biology.This profile, which is associated with various high-throughput techniques, is used for BC stratification, prognosis, and treatment [2].Triple-negative breast cancer (TNBC) represents approximately 15-20% of all breast cancer molecular subtypes [3].It is characterized by the absence of these three types of receptors [4] and tends to have a worse prognosis [5].Accurate molecular classification of TNBC is crucial for risk stratification [6,7].Six TNBC molecular subtypes have been proposed, each one with its own features and responses to standard treatment: two different basal-like types (basal-like 1 and basal-like 2), immunomodulatory, mesenchymal, mesenchymal stem-like, and luminal androgen receptors [8,9].These updates have allowed for personalized treatment with enhanced specificity.Currently, TNBC therapy responds to conventional chemotherapy and monoclonal antibodies, for example, pembrolizumab and avelumab, in the presence of specific biological markers such as programmed death-ligand 1 [10,11].According to the European Society for Medical Oncology (ESMO) and American Society for Clinical Oncology (ASCO) guidelines for the treatment of TNBC, (neo)adjuvant chemotherapy drugs are used in almost all cases [11,12].There are different regimens in use, including doxorubicin or epirubicin in combination with cyclophosphamide and paclitaxel or docetaxel in combination with carboplatin.If residual disease or the presence of the BRCA gene mutations is positive, capecitabine or olaparib is also included as an option [11,12].Unfortunately, TNBC treatment continues to be a challenge, and new approaches are still needed.Some patients present insufficient response, others develop resistance, and treatments are frequently associated with adverse effects [13].
The plasma state, also known as the fourth state of matter, has enough energy to ionize a significant amount of positive and negative particles [14].There are two types of plasma, namely thermal and non-thermal; the latter is frequently referred to as "cold atmospheric plasma" (CAP).CAP is characterized as non-thermal because the heavy particles are at room temperature.Several methods for producing it have been described [15].CAP has recently been studied as a potential cancer treatment, with evidence obtained in several malign neoplasms both in vitro and in vivo [16][17][18][19][20].Some authors have demonstrated CAP selectivity to tumoral cells compared to non-malignant counterparts, highlighting its potential in cancer treatment [21][22][23].Some mechanisms have been proposed to explain the effects of CAP, namely properties of ultraviolet radiation [24], electric fields that may affect cellular permeabilization by increasing calcium permeability [25], and reactive species that may alter the intracellular redox state, triggering critical cellular responses [20].Thus, it is crucial to understand the potential of CAP when compared to chemotherapy drugs.Based on the population, intervention, comparison, outcome, and study design (PICOS) criteria, this study aimed to systematically review the literature to determine whether CAP can be as effective as chemotherapy in the treatment of TNBC.Since CAP therapy is not yet a clinically approved treatment, TNBC cell lines and animal models were chosen to compare the cytotoxic effects of CAP with chemotherapeutic agents selected according to ESMO and ASCO guidelines [11,12].Specifically, doxorubicin, epirubicin, cyclophosphamide, paclitaxel, docetaxel, carboplatin, capecitabine, and olaparib were considered for this study.These chemotherapeutic drugs are the most commonly used in clinical practice.Therefore, the main aim of this systematic review was to answer the PICO question: Is cold atmospheric plasma as effective as chemotherapy in the treatment of triple-negative breast cancer?

Methods
This systematic review was developed and reported following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [26], and its protocol was registered in the International Prospective Register of Systematic Reviews-PROSPERO-with the number CRD42023414394.The research question was built according to the PICO methodology, as described in Table 1.

Search Strategy
The literature search was performed in four databases, namely Medline (through PubMed), Web of Science (all databases), Embase, and Cochrane Library.The search formulas used for each database are presented in the Supplemental Materials.No restriction on publication date was applied, and the English, Portuguese, Spanish, and French language filters were used.The search was completed on 13 February 2023.A manual search of the reference lists of relevant studies was performed to find additional potentially relevant studies.The search results were imported to the reference management program Mendeley Reference Manager© v2.80.1 (Mendeley Ltd., London, UK), and duplicate results were removed.

Inclusion and Exclusion Criteria
Two independent reviewers critically assessed the eligibility of studies for inclusion, first by title and abstract and later by evaluating the full text.In case of uncertainty or discrepancies regarding eligibility, a third reviewer was consulted, and a decision was made by consensus.In the eligibility phase, only in vitro and in vivo studies were considered, according to the following inclusion criteria: (1) cell lines and animal models of TNBC; (2) CAP treatment; (3) treatment with chemotherapy drugs selected according to the most recent European and American Society for Medical Oncology Clinical Practice Guidelines (ESMO and ASCO 2021), specifically doxorubicin or epirubicin or cyclophosphamide or paclitaxel or docetaxel or carboplatin or capecitabine or olaparib, and (4) papers whose main goal was to study the cell viability/proliferation of both treatments alone or in combination or in vivo tumor regression, measured as volume or histopathological changes.The exclusion criteria were as follows: (1) other study types, (2) new drugs, (3) resistant cell lines, (4) delivery methods or other formulations, (5) patient samples, (6) non-approved pharmacological combinations, (7) no report of cell viability/proliferation or tumor regression, (8) other experimental models, and (9) other types of studies whose main aim was not to evaluate the efficacy of treatments.

Data Extraction
For studies that met the inclusion criteria, the following information was collected: (1) authors and year of publication; (2) study model (i.e., triple-negative cell line(s) or animal model(s)), (3) chemotherapy treatment (i.e., chemotherapy agent, concentration drug, periodicity of drug administration and/or how it was carried out), (4) CAP treatment (i.e., plasma source, features of machine as voltage, and exposure times), ( 5) combination treatments with chemotherapy and CAP, (6) methods for cytotoxicity assay and/or histopathological assessment, and (7) cell viability/proliferation and in vivo tumor regression measurements.

Quality Assessment
The risk of bias of the in vitro studies was evaluated with Toxicological Data Reliability Assessment Tool (ToxRTool), which provides guidance on assessing the consistency and quality of toxicologic data [27].The methodological quality of in vivo studies was checked by assessing the risk of bias with the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) risk-of-bias tool [28].Two independent authors evaluated the quality assessment methodology of eligible studies included in this systematic review.

Study Selection
A total of 19,364 studies were obtained for analysis, with 4614 articles from PubMed (4507 regarding chemotherapy drugs plus 107 on CAP treatment), 6308 articles from Web of Science (6016 regarding chemotherapy drugs plus 292 on CAP treatment), 7308 articles from Embase (7219 regarding chemotherapy drugs plus 89 on CAP treatment), and 1134 from Cochrane Library (1107 regarding chemotherapy drugs plus 27 on CAP treatment).Before the screening, duplicate articles (8365) were removed using Web Manager (Clarivate™), leading to 10,999 records.Of these, 10,927 were excluded based on title and abstract analysis, resulting in 72 studies for full-text reading.Despite attempts to obtain all full-text records, only 66 were available to assess eligibility.Of these, seven articles were excluded for not meeting the inclusion criteria (two did not examine any drugs included in our review, one performed a mathematical analysis not encompassed in the inclusion criteria, and four did not evaluate cell viability/proliferation as defined in the PICO strategy).Thus, 59 articles were included in this systematic review for full-text reading and analysis, with publication dates from 1986 to 2023.
The PRISMA flow diagram, which summarizes the study selection performed in this systematic review, is shown in Figure 1.

Studies' Characteristics
Table 2 summarizes the main results observed in the studies included.In this systematic review, 59 studies were considered.Of these, 54 were in vitro studies, 3 were in vivo, and 2 articles included in vitro and in vivo experiments.The outcomes regarding the different types of studies are reported separately.We found articles on all chemotherapy drugs considered, as well as CAP treatment.Concerning CAP therapy, we identified two distinct methods: directly administered CAP and plasma-activated media (PAM), an indirect approach consisting of previously exposed solutions.Moreover, a table quantifying the percentage of inhibition induced by the therapies administered was compiled and included in the Supplemental Materials (Table S1).

Studies' Characteristics
Table 2 summarizes the main results observed in the studies included.In this systematic review, 59 studies were considered.Of these, 54 were in vitro studies, 3 were in vivo, and 2 articles included in vitro and in vivo experiments.The outcomes regarding the different types of studies are reported separately.We found articles on all chemotherapy drugs considered, as well as CAP treatment.Concerning CAP therapy, we identified two distinct methods: directly administered CAP and plasma-activated media (PAM), an indirect approach consisting of previously exposed solutions.Moreover, a table quantifying the percentage of inhibition induced by the therapies administered was compiled and included in the Supplemental Materials (Table S1).CAP: Effects were seen after 20 s of treatment and 72 h of incubation.More pronounced effects were seen after 60 s.After 60 s, a 4.5-fold growth reduction occurred from 4 to 120 h of incubation.PAM: The results showed a slightly lower anti-proliferative potential for PAM than for CAP.From 4 to 120 h, cell growth was reduced threefold.At 48 h of incubation, the cell growth reduced significantly from the control at two conditions.

MTS assay
All CAP-treated groups showed significantly inhibited cell proliferation after 3 and 5 days (p < 0.01).(1) The anti-tumor capacity increased as the treatment time exposure increased and decreased as the cell seeding confluence decreased.
(2) One minute after PAM, proliferation decreased as the size of the wells decreased.The effect of treatment was reduced 2/3 in the 48-well plate compared to the 6-well plate.
(3) Relative viability significantly increased as the volume of media increased from 1 to 4 mL.

Tumor monitoring
All mice in the control group died within 27 days, and all mice from the CAP direct group survived to the last day.The 30-day survival of mice in the CAP group was significantly higher than that of the PAM group (p = 4.9 × 10 −4 ).Both treatments significantly inhibited tumor growth (CAP: p = 0.044 for CAP; PAM: p = 0.017).However, the growth of the tumors in the PAM group was more suppressed than that in the CAP group.

CAP Treatment
This section describes the analysis of 19 studies.Regarding PAM treatment, the solutions used were millipore water, cell culture medium, and Ringer's solution.A decrease of cell proliferation to 20% (p = 0.001) was observed in a volume of 150 µL and 200 µL of PAM (millipore water-based) [72].The cell viability was reduced to 0.41 and 0.46 in MDA-MB-231 and MDA-MB-468 cells, respectively, compared to control cells after 5 minutes of PAM treatment (medium-based) [79].The viable cells significantly decreased from 80.50 ± 1.59% to 65.00 ± 3.39% after 120 seconds of CAP treatment in HCC1806 cells [20], while another study demonstrated a reduction of more than 50% in the MDA-MB-231 cell line [52].Chen et al. reported a reduction of cell viability of approximately 27.4% and 14.7%, respectively, with argon or helium gas flow.CAP decreased cell viability in a dose-dependent manner with statistical significance, as described in Ma et al. [55].The viability of the MDA-MB-231 cell line decreased to under 40% or 50% after 5 minutes of CAP or PAM exposure, respectively [44].Furthermore, apoptosis seemed to be the most prevalent type of cell death [20,55,65].In general, CAP and PAM demonstrated an effective anti-tumoral effect in short exposure times, such as 60 and 120 seconds; however, some authors tested longer exposure times, mainly in PAM treatment.

CAP Treatment and Chemotherapy
Interestingly, CAP treatment and chemotherapy drugs, specifically paclitaxel and olaparib, were combined in two studies [19,51].Olaparib showed a tendency to improve the efficacy of CAP in all cell lines [51].Moreover, the chemosensitivity to paclitaxel (0.01 µM) was improved after 15 seconds of CAP exposure.According to clonogenic assay, the combined treatment decreased the number of colonies to a number similar to or even smaller than that specific to PAM or paclitaxel [19].

In Vivo Studies
To obtain animal models, the strains CB-17 of severe combined immunodeficiency mice [56,61,70] and Balb/c were used [79,84] and were inoculated with the TNBC cell line MDA-MB-231.

Chemotherapy
As described by Man et al., cyclophosphamide was administered following two regimens.The group continuously administered 25 mg/kg of cyclophosphamide (low concentration) via drinking water showed an initial reduction of tumor size with no weight loss for 50 days.However, the group of mice treated in cycles of 6 days with 450 mg/kg/cycle (150 mg/kg/injection every other day) demonstrated severe weight loss and death one week after starting the therapy [56].Munõz et al., demonstrated that 20 mg/kg/day via drinking water starting on the 14th day reduced the volume of tumor compared to the control group.The percentage of necrosis increased from the control group (78%) to the treated group (85%), and no weight loss or other signs of toxicity were observed [61].Another study demonstrated that cyclophosphamide (20 mg/kg/day) added to capecitabine (100 mg/kg) significantly increased survival compared to the control [70].

CAP Treatment
CAP treatment was administered directly into animal tumors, and PAM injection of PBS showed significant inhibition of tumor growth (p = 0.044 and p = 0.017, respectively).In the comparison of both approaches, the survival of mice in the CAP treatment group was significantly higher than that in the PAM group (p = 4.9 × 10 −4 ).In addition, all control mice died within 27 days, while all mice from the CAP group survived until the end of the experiment (30 days) [84].Another study showed that the tumor volume was inhibited by PAM injection, and tumor weight dropped considerably after treatment (from 4.053 g to 0.787 g, p = 4.69 × 10 −4 ).No visible adverse effects were observed [79].

Quality Assessment
In terms of quality assessment, most studies presented an unclear bias, determined by the SYRCLE tool regarding in vivo studies, as described in Figure 2 and detailed in Supplemental Table S2.According to these bias tool guidelines, only three parameters were correctly reported for all studies, specifically, incomplete outcome data, selective outcome reporting, and other sources of bias.Moreover, 20% of articles presented no data on random outcome assessment, as shown in Figure 2.For other studies, the ToxRTool protocol was used, and articles were categorized based on this score.We observed scores between 11 and 14 as the most prevalent, where the studies were considered reliable with restrictions.Although five articles were classified as not reliable, eleven studies were classified under the criterion of reliability without restrictions.The risk assessment for individual study bias is represented in Figure 3 and detailed in Supplemental Table S3.
classified under the criterion of reliability without restrictions.The risk assessment for individual study bias is represented in Figure 3 and detailed in Supplemental Table S3.classified under the criterion of reliability without restrictions.The risk assessment for individual study bias is represented in Figure 3 and detailed in Supplemental Table S3.

Discussion
The clinical complexity of treating TNBC often requires a tailored approach due to its aggressive nature and poor prognosis compared to other molecular subtypes [85][86][87].Chemotherapy is a well-established treatment indicated in clinical practice regarding TNBC [10,11]; however, it has associated adverse effects.CAP has been investigated across a vast array of medical fields, specifically in dental medicine, regeneration of tissues, and tumor therapy, without causing significant harm to healthy cells, as well as having antimicrobial effects and an impact on stem cells and nitric oxide levels [88].It has been explored as a new emerging medical approach for several types of cancer, including TNBC, with promising results in terms of cell death [20,44,75,77].As CAP is not a clinically approved treatment, being under preclinical research, a comparison was conducted to determine the efficacy of both treatments without associated interventions.
The chemotherapeutic drugs explored-paclitaxel, docetaxel, cyclophosphamide, doxorubicin, olaparib, carboplatin, and capecitabine-were selected based on ESMO and ASCO guidelines [11,12], ensuring a broad and complete assessment of cellular effects [52,79].Currently, chemotherapy drugs are used in clinical practice and are heavily used as a positive control in most experiments [89,90].For all treatments, the outcomes depended on the protocol definitions, including the dose of chemotherapy drugs administered and the time of exposure to CAP.Several assays, such as MTT, MTS, SRB, annexin-V/propidium iodide, trypan blue, and clonogenic assay, were performed to prove the in vitro efficacy of CAP and chemotherapy drugs.
We found a wide range of chemotherapy concentrations tested in in vitro assays.The papers showed a reduction in cell proliferation, which is supported by the mechanisms of action of the drugs on tumor cells [91][92][93][94][95].There were additive interactions between paclitaxel and carboplatin in vitro [50].In fact, in patients whose TNBC disease progressed after taxane administration, carboplatin was one of the recommended chemotherapy agents [11,12].According to the guidelines, taxane-anthracycline-based combinations are options for treatment [11,12].The results obtained corroborated our expectations about the efficacy of chemotherapy in TNBC cell lines.However, the evidence available was often insufficient to statistically compare several studies since the data were not described quantitatively, and the various strategies do not allow for a comparative evaluation.
CAP therapy is an emerging therapeutic approach in cancer although the mechanism of action remains unclear.Recent studies have shown its effects on cell proliferation in several types of cancer, leading to cell death [35,44,[96][97][98].Here, we examine two different strategies concerning the application of CAP in TNBC cell lines, namely CAP and PAM, which used different exposure times and solutions.Both strategies are also reported in preclinical studies regarding other types of cancer [17][18][19]44,[99][100][101].Different types of equipment that are able to generate CAP are described.Some authors used a flow of gases such as argon or helium [34,44,53,62,77], and the frequency and high voltage were also dependent on the equipment of each research group.Generally, the exposure times were 60 seconds or 120 seconds [19][20][21]33,35,44,[51][52][53]55,71,80,81].The reduction of cell viability in TNBC cells was time dependent in all studies.Apoptosis seemed to be the predominant type of cell death [19,20].Studies on different types of cancer demonstrated a close correlation with the abovementioned, supporting the anti-tumoral potential of CAP as a promising strategy.A vast number of cell lines representing melanoma, brain tumor and leukemia, cervical, breast, colorectal, gastric, lung, ovarian, head and neck, and pancreatic cancers have already been used for CAP studies, demonstrating its antiproliferative effect [88].Moreover, some authors reported the selectivity of CAP to tumor cell lines [22,102,103].The efficacy of olaparib or paclitaxel combined with CAP tended to improve the cytotoxicity of treatment in five distinct TNBC cell lines [51].PAM improved the chemosensitivity to the lowest concentration of paclitaxel and reduced the metabolic activity compared to isolated therapies [19].
Translating cell line models into animal models is a crucial stage in assessing the efficacy of therapy in a biologically complex organism.In the studies, the methods used were tumor growth or volume, animal weight, or survival monitoring.Cyclophosphamide was able to reduce the volume of tumor with no weight loss and no signs of toxicity [61].The combination of cyclophosphamide and capecitabine increased survival [70].Similarly, CAP and PAM inhibited tumor growth significantly, and no noticeable adverse effects were observed [52,84].CAP was applied locally to the tumor in both plasma approaches.Although we only analyzed two papers with CAP treatment in vivo, the results proved the efficacy in TNBC.In vivo studies with different types of cancer have been performed in recent years [104,105].Nevertheless, further studies should include cell lines representative of other TNBC molecular subtypes and consider other animal models to encompass as many of the characteristics observed in clinical practice as possible.Unfortunately, in vivo studies regarding CAP and chemotherapy combination are unavailable.In the future, to maximize the anti-tumor potential of CAP, combining it with chemotherapy drugs should also be studied in vivo, promoting a potential coordinated translation to the clinical setting.Furthermore, patient-derived xenograft models could be an enriched research option for evaluating CAP efficacy in human BC tissue samples.
As regards CAP, it could be a promising option for clinical practice.From the results obtained, we hypothesize a reduction of the side effects associated with chemotherapy with a lowering of the concentrations of the drugs administered.
One of the limitations observed was the heterogeneity between the studies due to methodological approaches, namely exposure, intervals and doses of drugs, voltages of equipment, times of exposure, and evaluation times.Moreover, the lack of important information, such as concentrations and detailed and quantitative results, impeded metaanalysis studies.In addition, we observed differences between the number of studies for each drug, which means that the sample included in some groups was reduced, limiting the conclusions.The bias assessment, based on a set of well-defined guidelines, proved the difficulty in obtaining all the information necessary to proceed to the planned statistical study, as illustrated in Figures 2 and 3. CAP studies provided a more detailed description of the conditions of treatment used.Table 3 summarizes key aspects of this systematic review, including the main considerations of the study and the next steps.In the future, based on our results, we suggest creating a list of standard methodologies to address the limitations found.Moreover, correlating studies with human tissue samples of TNBC after CAP treatment with clinical reports of patients will enhance the potential translational value of therapy.Nevertheless, combining chemotherapy and CAP or PAM in vivo appears to be a strategic option.
Table 3. Key aspects of this systematic review.

TNBC
(1) Due to its aggressive nature and poor prognosis compared to other molecular subtypes, TNBC requires a broader range of treatment options.
(2) Chemotherapy is a well-established treatment for TNBC, but it comes with associated adverse effects.The selection of chemotherapy drugs, particularly paclitaxel, docetaxel, cyclophosphamide, doxorubicin, olaparib, carboplatin, and capecitabine, was based on ESMO and ASCO guidelines.

Plasma treatment
Cold plasma, investigated across various medical fields, including tumor therapy, shows promise in cancer treatment, including TNBC, with results indicating cell death.

Search
(1) Initial retrieval of 19,364 studies from four databases.
(2) After screening, 59 articles were included in the systematic review.Articles were published between 1986 and 2023, with focus on the efficacy of chemotherapy drugs and CAP treatment.
In vitro studies (1) In vitro studies demonstrated a reduction in cell proliferation with various chemotherapy concentrations tested, and additive interactions were observed between paclitaxel and carboplatin.
(2) Different strategies, including CAP and PAM, exhibited a time-dependent reduction in TNBC cell viability, with apoptosis being the predominant type of cell death.
(3) Combination therapies involving cold plasma and chemotherapy drugs tended to improve cytotoxicity in TNBC cell lines.
In vivo studies Animal models are crucial for assessing therapy efficacy.Studies demonstrated inhibition of tumor growth with no noticeable adverse effects in animal models regarding CAP treatment.

Limitations
Heterogeneity between studies, including methodological approaches and lack of detailed information, poses limitations to meta-analysis studies and conclusive interpretations.This emphasizes the need for standardized methodologies.
Future directions (1) This systematic review underscores the need for further research using standardized methodologies to address current limitations and advance clinical translation.
(2) Studies directly comparing CAP, PAM, and standard chemotherapy regimens should be performed, including the evaluation of cell death and associated mechanisms of action.
(3) Conducting studies to unravel specific protein alterations after CAP and PAM treatment might be a strategy for establishing combinations with drugs used in clinical practice.
(4) Future studies should consider other animal models and explore combination therapies.Cold plasma therapy could potentially reduce chemotherapy-associated side effects by lowering drug concentrations, necessitating further investigation in this field.
(5) The use of patient-derived xenografts must be considered as a key approach to verifying the effects of CAP.The findings should be correlated with patients' clinical data.

Conclusions
Chemotherapy agents effectively reduced cell proliferation in most TNBC cell lines, depending on a wide range of concentrations and experimenting conditions.CAP treatment successfully treated TNBC cell lines, with apoptosis being the most prevalent type of cell death.Paclitaxel or olaparib combined with CAP in vitro should be further investigated.Our results suggest that other combinations should be considered and evaluated.In the in vivo studies, the selection of different chemotherapeutic regimens influenced the obtained results, with both CAP and PAM effectively reducing tumor volume without visible side effects.However, the lack of information on both treatments and the diversity of experimental conditions underscore the need for more research.Despite the weaknesses observed, our results indicate that CAP is as effective as chemotherapy in TNBC.Innovative studies should be performed to increase the knowledge of CAP treatment and enhance future medical options on TNBC.

Figure 1 .
Figure 1.PRISMA flow diagram summarizing study selection in this systematic review.

Figure 1 .
Figure 1.PRISMA flow diagram summarizing study selection in this systematic review.

Figure 2 .
Figure 2. Summary of the quality assessment of the in vivo studies included in the systematic review completed with the SYRCLE tool.

Figure 2 .
Figure 2. Summary of the quality assessment of the in vivo studies included in the systematic review completed with the SYRCLE tool.

Figure 2 .
Figure 2. Summary of the quality assessment of the in vivo studies included in the systematic review completed with the SYRCLE tool.

Table 1 .
Population, intervention, comparison, and outcome (PICO) research strategy used in this systematic review.

Table 2 .
Studies' details and outcomes.

Table 2 .
Cont. Cell viability was reduced in almost all CAP exposure.Generally, better results were obtained using He + 0.5% O 2 than pure gas.The viability of MDA-MB-231 cells decreased by more than 60% after 5 min of treatment.PAM: Similar results were observed despite no significant differences between He + 0.5% O 2 and He.Cell viability decreased to about 50% after 5 min.