Advances in cold atmospheric plasma therapy for cancer

Abstract

Cold atmospheric plasma (CAP) has emerged as a promising, non-invasive therapeutic strategy for cancer treatment, leveraging reactive species to selectively induce cancer cell death while sparing healthy tissues. This review summarizes the underlying mechanisms of CAP-mediated anti-cancer effects, including apoptosis, immunogenic cell death, pyroptosis, ferroptosis, necrosis, and modulation of the tumor immune microenvironment. Moreover, we highlight recent advances in CAP-based therapies, including direct CAP treatment and indirect approaches assisted by various delivery methods, encompassing fluids, hydrogels, tubes, and microneedles. Additionally, we survey the clinical efforts of CAP therapy for cancer. Overall, CAP represents a novel and versatile cancer treatment modality with significant translational potential. Continued research into its mechanisms, safety, and optimization strategies will be essential to harness its clinical benefits and expand its application across different cancer types.

Graphical abstract

Highlights

• •Underlying cancer cell-killing mechanisms of cold atmospheric plasma are discussed.
• •Delivery methods and applications of cold atmospheric plasma in cancer therapy are thoroughly reviewed.
• •Recent efforts in clinical translation of cold atmospheric plasma are summarized.

1. Introduction

Plasma is one of the four fundamental states of matter (i.e., plasma, gas, liquid, and solid) [1]. Generally, plasmas are fully or partly ionized gases where sufficient energy is added to a gas to strip electrons from atoms, creating a mixture of free electrons, free radicals, neutral particles, and charged species [[2], [3], [4], [5], [6]]. Based on temperature, plasma is classified as thermal and nonthermal [7]. The nonthermal plasma generated under atmospheric pressure is commonly referred to as cold atmospheric plasma (CAP).

CAP contains a mixture of reactive oxygen species (ROS), reactive nitrogen species (RNS), ultraviolet photons, charged particles, and other free radicals [[8], [10]]. These species can be categorized into long-lived species, such as hydrogen peroxide (H₂O₂), ozone (O₃), nitrite (NO₂⁻), nitrate (NO₃⁻), and peroxynitrite (ONOO⁻), and short-lived species, such as superoxide anions (O₂•⁻), hydroxyl radicals (•OH), singlet oxygen (¹O₂), and nitrogen monoxide (NO) [10,[12], [13], [14], [15], [16]]. The full composition of CAP remains unidentified, so the complexity and synergistic actions of various components endow CAP with irreplaceability, making it a unique and versatile tool for biomedical applications. For example, CAP has been extensively used for disinfection and anti-microbial applications, since it can eradicate bacteria or spores, and degrade food allergens and pesticides [[17], [18], [19]]. CAP can also promote blood coagulation [20,21]. In oral and dental treatments, CAP demonstrates effectiveness in tooth biofilm inactivation, tooth bleaching, oral disease prevention and treatment, and breath freshening [[22], [23], [24], [25], [26]]. For dermatological applications, CAP supports wound healing, scar reduction or removal, wrinkle clearance, and treatment of conditions such as actinic keratosis, psoriasis, atopic dermatitis, vitiligo [[27], [28], [29], [30], [31], [32], [33], [34], [35], [36]].

Among its various biomedical applications, CAP's potential in cancer therapy has garnered significant attention in recent years. Research has progressed through multiple stages, including in vitro cancer cell treatment, in vivo cancer therapy, and pre-clinical/clinical investigations (Fig. 1). In vitro studies have shown CAP's ability to induce tumor cell death via multiple mechanisms (e.g., immunogenic cell death (ICD), apoptosis, and necrosis) while sparing normal cells [[37], [38], [39], [40], [41]]. Many recent in vivo studies have demonstrated tumor growth inhibition and subsequent activation of anti-tumor immunity in multiple cancer types, including glioblastoma, melanoma, and breast cancer [[42], [43], [44]]. More recently, CAP has been investigated for eliminating post-surgical tumor residuals in clinical settings in Europe and the US, marking the critical advances toward clinical translation [45,46]. CAP offers several advantages in cancer therapy, including selective tumor cell targeting, multiple tumor-killing pathways, and potential synergy with other cancer treatments. Furthermore, CAP devices are relatively simple, cost-effective, and easy to operate.

Fig. 1.

Recent advances in CAP therapy for cancer treatment.

2. CAP generating devices

CAP devices are engineered to generate plasma at near-room temperature, making them suitable for biomedical applications, including sterilization, wound healing, and cancer therapy [47,48]. These devices operate under atmospheric pressure and are designed to create and sustain plasma through the ionization of various feeding gases [49].

CAP devices can be broadly categorized based on their discharge mechanisms, including dielectric barrier discharge (DBD), plasma jets, corona discharge, and arc discharge systems [50,51] (Fig. 2). Among these, DBD and plasma jet-based devices are the most commonly used for biomedical applications due to their ability to generate CAP with controlled composition and minimal thermal damage to biological tissues [52].

Fig. 2.

Scheme of CAP generating devices, including the feeding gases, CAP jet, DBD, corona discharge, and arc discharge systems.

The choice of feeding gas plays a crucial role in determining the plasma's stability, chemical composition, and therapeutic efficacy [53,54] (Fig. 2). Helium and argon are commonly used in CAP-generating devices due to their ability to generate stable and homogeneous plasmas. When ionized, helium and argon can transfer energy to air molecules (e.g., oxygen and nitrogen), generating abundant secondary ROS and RNS [[55], [56], [57]]. These noble gases have low breakdown voltages, remain stable under atmospheric conditions, and produce continuous and uniform plasma discharges [53,[58], [59], [60], [61]]. Helium and argon-based CAP have been widely applied in cancer therapy (shown in Table 1, Table 2, Table 3). Notably, kINPen, a commercially available argon-based CAP-generating device, has been used for industry, human medicine, and veterinary medicine [62]. However, their high cost drives the exploration of more economical alternatives [[55], [56], [57]]. Nitrogen and oxygen, the primary components of air, offer an abundant and cost-effective alternative (shown in Table 1, Table 2, Table 3). Nitrogen-rich CAP generates large amounts of RNS, which have been shown to promote cell death and bactericidal effects [[63], [64], [65]]. Oxygen-rich CAP produces substantial ROS, which exhibit strong oxidative properties beneficial for sterilization, antimicrobial treatments, cancer therapies, and industrial processes [[66], [67], [68]]. However, due to their polyatomic molecular structures, nitrogen and oxygen require stronger electric fields to initiate plasma, making the process less energy-efficient [69]. Additionally, filamentary discharges often occur in nitrogen-, oxygen-, or air-based CAP, leading to non-uniform plasma compared to the smoother discharges generated by noble gases [70,71].

Table 1.

Cancer cell death pathways induced by CAP in different tumor types.

Tumor	CAP device	Feeding gas	Model	Cell death pathway	Key factor and indicator	Ref.
Melanoma	Plasma system (MediPL plasma torch system, MediPL)	Argon	B16F10 melanoma in C57BL/6 mice	Apoptosis	•Upregulated cleaved caspase-3	[99]
Melanoma	CAP μ-DBD surface plasma device	Air	G-361 melanoma in Balb/c nude mice	Apoptosis	•Increased expression of apoptotic genes: Bax, Parp, Casp8, and P53 •Decreased expression of anti-apoptosis genes: Akt and Bcl-2	[100]
Melanoma	CAP device constructed using a 3D printer (LulzBot TAZ 6) at UCLA	Helium	B16F10 melanoma in C57BL/6 mice	ICD	•Calreticulin exposure	[113]
Melanoma	Low-frequency plasma jet device	Argon	B16 melanoma in Balb/c mice	Autophagy; Apoptosis	•Upregulated autophagy-related genes: Lc3 and Atg5 •Upregulated apoptotic genes: Bax and Caspase-3 •Downregulated anti-apoptosis gene: Bcl-2	[139]
Melanoma	Floating electrode dielectric barrier discharge system	Air	Melanoma cells in vitro	Necrosis; Apoptosis	•Cell necrosis	[72]
Skin cancer	CAP jet device	Helium-oxygen mixture (99:1)	A431 skin cancer in Balb/c nude mice	Ferroptosis	•Decreased GSH level •Increased malondialdehyde and Fe2+ levels •ATM/p53/SLC7A11/GPX4 cascade (ATM activation, acetyl-p53 upregulation, SLC7A11 and GPX4 expression suppression) •Lipid peroxidation	[169]
Colorectal cancer	Nanosecond-pulsed DBD plasma system	Not mentioned	CT26-GUCY2C colorectal cancer in Balb/c mice	ICD	•Calreticulin exposure •ATP secretion •HMGB1 migration translocate from nucleus to the cytoplasm and extracellular space	[114]
Ovarian cancer	DBD plasma device	Helium	Ovcar5 ovarian cancer cells in vitro	Pyroptosis	•Mitochondrial damage •Activated Caspase-9/Caspase-3 pathway, where Caspase-3 can promote GSDME cleavage for pyroptosis	[121]
Non-small cell lung cancer (NSCLC); Gastric cancer; Liver cancer	DBD CAP generator	Helium	PC9 NSCLC cells in vitro; MNK28 gastric cancer in vitro; Bel7402 liver cancer in vitro	Pyroptosis	•c-Jun N-terminal kinase and cytochrome c release •Activated Caspase-9/Caspase-3 pathway •Increased GSDME cleavage	[122]
NSCLC	Surface microdischarge plasma	Air	H358 NSCLC in Balb/c nude mice	Ferroptosis	•Facilitated HOXB9 degradation by promoted acetylation •Inhibited transcriptional expression of SLC7A11	[163]
Liver cancer	Air surface discharge plasma device	Air	Hepa1-6-Luc liver cancer in C57BL/6 mice	Pyroptosis	•Increased GSDME cleavage	[123]
Breast cancer	CAP jet source	Helium	SUM159PT with and without EGFR(Y1068F) mutation breast cancer in nude Balb/c mice	Ferroptosis	•Lipid peroxidation •The tyrosine 1068 site (Y1068) of epidermal growth factor receptor (EGFR) (EGFR(Y1068)) phosphorylation •Reduced nuclear factor erythroid 2-related factor 2 phosphorylation •Decreased GPX4 transcription	[170]

Table 2.

Representative direct CAP treatment and CAP devices for cancer therapies.

Tumor	CAP device	CAP generation parameters	Animal model	Main findings	Ref.
Melanoma	CAP device developed by Keidar's Lab at the George Washington University	•Voltage 2–5 kV; frequency 30 kHz •Feeding gas: He •Flow rate: 11 L/min	•B16F10 melanoma in C57BL/6 mice •CAP treatment: 5 min, single treatment	•Induced tumor cell apoptosis •Inhibited tumor growth and prolonged survival	[219]
Melanoma	Nanosecond pulsed streamer discharge setup	•Voltage 24 kV; discharging duration 8 ns; frequency 30–100 pulses per second •Feeding gas: O2 or N2 •Flow rate: 0.5 L/min	•B16F10 melanoma in CD2F1 mice and in C57BL/6 mice •CAP treatment: 10 min/treatment, every day for 3–7 days	•Inhibited tumor growth •Induced an abscopal effect •Increased levels of pro-inflammatory cytokines	[220]
Melanoma	kINPen plasma jet (neoplas, Germany)	•Frequency 1 MHz; power 1 W •Feeding gas: Ar (purity 99.9999 %) •Flow rate: 3 L/min	•B16F10 melanoma in C57BL/6 mice •CAP treatment: 5 min/treatment, every 4 days for a total of 4 treatments	•Induced tumor cell apoptosis •Enhanced anti-tumor efficacy, in combination with mitochondria-targeted drugs (e.g. A-1210477, carvedilol, SBI-0206965, and navitoclax) •Elicited anti-tumor immune responses	[43]
Melanoma	kINPen plasma jet (neoplas, Germany)	•Frequency 1 MHz; power 1 W •Feeding gas: Ar (purity 99.9999 %) •Flow rate: 5 L/min	•A375 melanoma in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice •CAP treatment: 5 min/treatment, every 4 days for a total of 5 treatments	•Induced tumor cell apoptosis •Inhibited tumor growth •Elicited synergistic anti-tumor effects when combined with Sm837, a chromone-derived small molecule	[221]
Glioma	Pulsed DBD device	•Power supply voltage 300 V; transformer voltage up to 17 kV; frequency 100 Hz •Feeding gas: Air or air-Ar mixture	•U87-MG glioma in Balb/c nude mice •CAP treatment: 6 min/treatment, every day for a total of 5 treatments	•Induced tumor cell apoptosis •Decreased tumor volume	[222]
Glioblastoma	CAP device developed by Keidar's Lab at the George Washington University	•Voltage 10 V; frequency 12.5 kHz •Feeding gas: He •Flow rate: 1 L/min	•U87-MG glioblastoma in NU(NCr)-Foxn1nu athymic nude mice •CAP treatment: 1 min, single treatment	•Induced tumor cell apoptosis •Inhibited tumor growth •Sensitized glioblastoma cells to temozolomide chemotherapy	[42]
Breast cancer	FE-DBD system	•Voltage 13.2 kV; frequency 70 kHz •Feeding gas: Air	•AN3 breast cancer in albino Swiss mice •CAP treatment: 20, 40, or 50 s/treatment, every 48 h for a total of 3 treatments	•Induced tumor cell apoptosis •Enhanced anti-tumor effect	[223]
Breast cancer	InvivoPen device	•Voltage: 5 kV; frequency 8.8 kHz •Feeding gas: He •Flow rate: 0.2 L/min	•MDA-MB-231 breast cancer in Balb/c mice •CAP treatment: 5 min/treatment, every 72 h until animal death or the end of study	•Induced tumor cell apoptosis •Suppressed tumor growth and migration	[224]
Melanoma and breast cancer	Portable air-fed CAP device	•Voltage 8 kV; distance between electrodes 7 mm •Feeding gas: Air •Flow rate: 16.8 L/min	•4T1 breast cancer in Balb/c mice; B16F10 melanoma in C57BL/6 mice •CAP treatment: 1, 2, 3, or 4 min, single treatment	•Induced tumor cell ICD •Inhibited tumor recurrence and prolonged post-surgical survival •Elicited T-cell-mediated anti-tumor responses	[44]
Lung cancer	CAP device developed by Keidar's Lab at the George Washington University	•Voltage up to 8 kV; frequency 25 kHz •Feeding gas: He (industrial grade) •Flow rate: 4 L/min	•A549 lung cancer in Balb/c nude mice •CAP treatment: 2 min/treatment, every day for a total of 10 treatments	•Induced tumor cell apoptosis •Decelerated tumor growth •Improved anti-tumor effects in combination with iron oxide nanoparticles	[225]
Head and neck cancer	Piezobrush® PZ2 (Relyon Plasma Co., Regensburg, Germany)	•Frequency 50 kHz; power 0.9 mW •Feeding gas: Air	•FaDu head and neck cancer in Balb/c nude mice •CAP treatment: 1 min/treatment, 3 treatments per week until the study endpoint	•Inhibited tumor growth by tumor glycolysis inhibition at multiple enzymatic points and AKT signaling pathway suppression •Enhanced anti-tumor efficacy and prolonged survival when combined with cisplatin chemotherapy	[226]
Cholangiocarcinoma	Plasma Tesla Jet	•Voltage 9 kV; duty cycle 14 %; frequency 30 kHz •Feeding gas: He •Flow rate: 1 L/min	•EGI-1 cholangiocarcinoma in ATHYM-Foxn1 nu/nu mice •CAP treatments: 1 min/treatment, 2 treatments per week for a total of 6 treatments	•Induced tumor cell apoptosis •Inhibited tumor growth	[227]

Table 3.

Representative indirect CAP treatment and CAP devices for cancer therapies.

CAP-activating solution	CAP device	CAP generation parameters	Animal model	Major results	Ref.
Cell culture media	CAP jet device	•Voltage 10 kV; frequency 60 Hz •Feeding gas: Ar •Flow rate: 2 L/min	•NOS-2 epithelial ovarian carcinoma in Balb/c nude mice •CAP pretreatment: 4 mL culture media, 600 s •CAP treatment: 200 μL/injection, 3 injections per week	•Induced tumor cell apoptosis •Inhibited tumor growth	[234]
Cell culture media (intraperitoneal injection)	CAP jet device	•Voltage 10 kV; frequency 60 Hz •Feeding gas: Argon •Flow rate: 2 L/min	•ES-2 ovarian cancer in Balb/c nude mice •CAP pretreatment: 5.5 mL culture media, 10 min •CAP treatment: 1 injection every day for a total of 3 injections	•Induced tumor cell apoptosis •Suppressed tumor metastasis and extended survival •Inhibited the MAPK pathway and MMP-9	[235]
Cell culture media (intratumoral injection)	CAP jet device	•Voltage: 1.0–1.4 kV; frequency 8.8 kHz •Feeding gas: He •Flow rate: 1 L/min	•MDA-MB-231 breast cancer in Balb/c mice; MCF-7 breast cancer in Balb/c mice •CAP pretreatment: 2 mL culture media, 15 min •CAP treatment: 200 μL/injection, every 2 days	•Inhibited hyper-activated MAPK/JNK and NF-κB pathways in tumor cells •Inhibited tumor growth	[236]
Cell culture media	CAP jet device	•Voltage: 5 kV; frequency 25 kHz, •Feeding gas: He •Flow rate: 4 L/min	•B16F10 melanoma in C57BL/6 mice •CAP pretreatment: 1 mL culture media, 6 min •CAP treatment: 400 μL/injection, every day for a total of 25 injections	•Induced tumor cell apoptosis •Inhibited tumor growth, particularly when combined with cyclophosphamide, a chemotherapy drug	[237]
Cell culture media (intraperitoneal injection)	kINPen MED (neoplas, Germany)	•Voltage 2–6 kV; frequency 1 MHz •Feeding gas: Ar •Flow rate: 3 L/min	•6606PDA pancreatic cancer in C57BL/6 mice •CAP pretreatment: 5 mL culture media, 10 min •CAP treatment: 1 mL/injection, every day for at most 35 injections	•Induced tumor cell apoptosis •Reduced tumor mass •Promoted immune cell infiltration	[238]
Cell culture media (intraperitoneal injection)	kINPen MED (neoplas, Germany)	•Voltage 2–6 kV; frequency 1 MHz •Feeding gas: Ar •Flow rate: 5 L/min	•6606PDA pancreatic cancer in C57BL/6 mice •CAP pretreatment: 5 mL culture media, 10 min •CAP treatment: 1 mL/injection, every day for a total of 21 injections	•Induced tumor cell ICD •Increased M1 macrophages •Increased infiltration of neutrophils and T cells in treated tumors	[239]
Cell culture media	PM-10 jet (Heuermann HF-Technik GmbH, Germany)	•Frequency 2.45 GHz; power 7 W •Feeding gas: Ar •Flow rate: 1.9 L/min	•H1299 lung cancer in athymic nude mice •CAP pretreatment: 3 mL culture media, 180 s •CAP treatment: 3 injections per week for a total of 9 injections	•Induced tumor cell ferroptosis •Inhibited tumor growth	[240]
Saline (intraperitoneal injection)	kINPen MED (neoplas, Germany)	•Voltage 2–6 kV; frequency 1 MHz •Feeding gas: Ar (purity 99.9999 %) •Flow rate: 5 L/min	•CT26 colorectal cancer in in Balb/c mice •CAP pretreatment: 50 mL saline, 60 min •CAP treatment: 300 μL/injection, every 2 days for a total of 5 injections	•Induced tumor cell apoptosis and ICD •Inhibited tumor growth •Enhanced macrophage infiltration and CD4+ T cells activation	[241]
Saline (pericarcinomatous tissue injection)	Surface microdischarge array with a hexagon-shaped mesh electrode	•Voltage 8 kV; frequency 10 kHz; power 950 mW; power density 0.02 W cm−2 •Feeding gas: He	•T24 bladder cancer in Balb/c nude mice; J82 bladder cancer in Balb/c nude mice •CAP pretreatment: 5 mL saline, 20 min •CAP treatment: 200 μL/injection, 3 injections per week	•Induced tumor cell apoptosis •Reduced tumor size •Improved survival rates	[242]
Saline (pericarcinomatous tissue injection)	CAP jet device	•Frequency 21.7 kHz; power 3.8 W •Feeding gas: Ar-3 % O2 mixture (Ar purity 99.9 %, O2 purity 99.9 %) •Flow rate: 1.9 L/min	•BCR-ABL1T315I (T315I mutation) chronic myeloid leukemia in Balb/c mice •CAP pretreatment: 120 s •CAP treatment: 200 μL/injection, every 3 days for a total of 11 injections	•Induced tumor cell apoptosis, necrosis, and ferroptosis •Reduced tumor size •Improved survival rates	[214]
Water (oral gavage)	Atmospheric-pressure plasma jet	•Voltage 1.16 kV; energy 9.5 mJ/s •Feeding gas: Air	•NCI-H520 lung cancer in CAnN.Cg-Foxn1nu/CrljOri nude mice •CAP pretreatment: 5 mL double-distilled water, 30 min •CAP treatment: 2, 1, or 0.5 mL/kg mouse/administration, every day for a total of 35 administrations	•Induced tumor cell apoptosis •Suppressed tumor growth •Reduced angiogenesis	[243]
Cell culture media	CAP jet device	•Voltage 20 kV; frequency 18 kHz •Feeding gas: Ar; He-O2 mixture •Flow rate: He 4 L/min, O2 0.2 L/min, Ar 2 L/min	•4T1 breast cancer in Balb/c mice •CAP pretreatment: 5 mL culture media, 3 min	•Induced tumor cell apoptosis •Enhanced anti-tumor effect, in combination with doxorubicin, a chemotherapy drug •Mitigated hepatotoxicity and nephrotoxicity	[245]
Cell culture media (intratumoral injection)	CAP jet device	•Voltage 7 kV; frequency 10 kHz •Feeding gas: He (purity 99.999 %) •Flow rate: 3 L/min	•A549 lung cancer in Balb/c nude mice	•Induced tumor cell apoptosis •Inhibited tumor growth inhibition •Enhanced antitumor effect when combined with violet phosphorene nanosheets	[244]
Thermosensitive (poly-DL-lactide)-(poly-ethylene glycol)-(poly-DL-lactide) biogel (post-surgical cavity implantation)	CAP DBD apparatus	•Voltage 7.5 kV; frequency 10 kHz •Feeding gas: Air	•T24 bladder cancer in Balb/c nude mice •CAP pretreatment: 2 mL PLEL solution, 20 min •CAP treatment: 200 μL, single treatment	•Induced tumor cell apoptosis •Enhanced anti-tumor effect and prolonged survival	[256]
ROS-responsive on-site hydrogel (intratumoral injection)	FLA Medic+ (FLAMME, Republic of Korea)	•Voltage 15 kV •Feeding gas: Air •Flow rate: 1 L/min	•CT26 colorectal cancer in in Balb/c mice; 4T1 breast cancer in in Balb/c mice •CAP pretreatment: 0.1 mL HAT solution, 5 min •CAP treatment: 100 μL, single treatment	•Induced tumor cell ICD •Reprogramed tumor microenvironment reprogramming •Enhanced anti-tumor effects •Suppressed the growth of distant untreated tumor growth •Inhibited metastasis	[257]
Thermosensitive injectable Pluronic hydrogel (intratumoral injection)	CAP jet device	•Voltage: 6 kV •Feeding gas: He (purity 99.996 %) •Flow rate: 8.5 L/min	•4T1 breast cancer in in Balb/c mice; B16F10 melanoma in C57BL/6 mice •CAP pretreatment: 1 mL deionized water, 10 min •CAP treatment: 100 μL/injection, every 3 days for a total of 6 injections	•Induced tumor cell ICD •Elicited T cell-mediated anti-tumor immunity •Synergized with aPD-1 therapy •Induced systemic anti-tumor immunity	[207]
Thermosensitive injectable Pluronic hydrogel (intratumoral injection)	CAP jet device	•Voltage 6 V; current 3.5 A •Feeding gas: He (purity 99.996 %) •Flow rate: 8.5 L/min	•CT26 colorectal cancer in in Balb/c mice •CAP pretreatment: 1 ml distilled water, 10 min •CAP treatment: 100 μL/injection, every 2 days for a total of 6 injections	•Induced tumor cell ICD •Potentiated T cell-mediated anti-tumor immune responses when combined with aPD-1 and trehalose	[258]
Capillary tube	Micro-plasma jet device	•Voltage 4 kV; frequency 6 kHz •Feeding gas: He (purity 99.999 %) •Flow rate: 0.5 L/min	•4T1 breast cancer in Balb/c mice •CAP treatment: 3 min, single treatment	•Induced tumor cell apoptosis •Inhibited tumor growth and extended survival	[263]
Capillary tube	Plasma DBD gun with an elongated flexible capillary	•Voltage 1150 V; pulse duration 800 μs; repetition rate 487 Hz •Feeding gas: 98 % Ne-2 % Ar mixture •Flow rate: 3 L/min	•B16F10 melanoma in C57BL/6 mice •CAP treatment: 125 s/treatment, once a week; or 25 s/treatment, every day, 5 consecutive days a week	•Controlled tumor growth •Induced tumor necrosis	[264]
Endoscopic tube	Micro-sized CAP jet device	•Voltage: 8 kV; frequency 15 kHz •Feeding gas: He •Flow rate: 0.1 L/min	•U87-MG glioblastoma in NU(NCr)-Foxn1nu athymic nude mice •CAP treatment: 15 s in total (5 s/treatment for a total of 3 treatments with 15-s intervals)	•Inhibited tumor growth	[265]
Microneedle patch	CAP jet device	•Voltage 11 kV; frequency 12 kHz •Feeding gas: He (purity 99.996 %) •Flow rate: 16.5 L/min	•B16F10 melanoma in C57BL/6 mice •CAP treatment: 4 min, single treatment	•Induced tumor cell ICD •Inhibited tumor growth and prolonged survival •Enhanced tumor-specific T-cell-mediated immune responses •Amplified tumor suppression and anti-tumor immune activation in combination with Apd-1 therapy •Activated systemic anti-tumor immunity	[113]
Microneedle patch	CAP discharge apparatus	•DBD reactor: voltage 8 kV; power 15 W •Gliding arc discharge reactor: voltage rising to 1.8 kV before extinguishing; impact current 2 A; power 15 W •Feeding gas: Air •Flow rate: 6 L/min	•A375 melanoma in Balb/c nude mice •CAP pretreatment: 20 mL saline, 15 min •CAP treatment: one patch administration every day for a total of 14 administrations	•Induced tumor cell apoptosis and ferroptosis •Inhibited tumor growth and extended survival	[267]

Advancements in CAP device design have led to the development of portable, handheld, and clinically adaptable plasma sources, enabling precise and localized plasma treatments. Ongoing research focuses on optimizing CAP device configurations, power supply modulation, and gas compositions to maximize therapeutic efficacy while ensuring safety and scalability for medical and industrial applications.

3. Mechanism of CAP in cancer therapy

The application of CAP in cancer therapy has been investigated about two decades ago. Fridman et al. [72] demonstrated CAP's antitumor effects by directly applying it to melanoma cells, leading to significant tumor cell death, while sparing healthy tissues within a certain CAP treatment dose, indicating CAP's potential as a selective cancer treatment strategy. Subsequent studies confirmed CAP's cytotoxic effects on various cancer cell lines. Kim et al. [73] treated SK-Hep1 (human metastatic liver cancer) and THLE-2 (human normal liver) cells with CAP, observing a greater reduction in SK-Hep1 cell viability, further demonstrating CAP's selective cytotoxicity. Similarly, Lupu et al. [74] reported that CAP effectively induced cell death in multidrug-resistant COLO320DM colon cancer cells.

Among CAP's key components, ROS and RNS are believed to play a central role in its selective anticancer effects [13,[75], [76], [77], [78], [79], [80]]. These reactive species induce oxidative stress, damaging cellular components such as lipids, proteins, and DNA, ultimately triggering multiple regulated cell death pathways, including apoptosis, ICD, pyroptosis, autophagy, necrosis, and ferroptosis in various cancer types (SeeTable 1 ) [[81], [82], [83]].

3.1. CAP-induced apoptosis

Apoptosis is a highly regulated form of programmed cell death critical for maintaining tissue homeostasis and eliminating damaged or abnormal cells [84,85]. ROS and RNS from CAP can diffuse into cells, leading to oxidative damage and disruption of redox balance. This change could result in mitochondrial membrane depolarization, the release of cytochrome c, and the formation of the apoptosome complex, ultimately activating caspase cascades [[86], [87], [88], [89]]. In parallel, CAP-induced DNA damage activates the ataxia-telangiectasia mutated (ATM)/eukaryotic translation initiation factor-2 (eIF2) signaling axis, promoting the expression of pro-apoptotic genes, such as Bax and p53-upregulated modulator of apoptosis (PUMA), while suppressing anti-apoptotic Bcl-2 family members. This transcriptional shift further sensitizes mitochondria to apoptotic stimuli [39,90,91]. CAP also triggers caspase-8 activation via the extrinsic apoptotic pathway, which can directly activate caspase-3 through Bid cleavage [92]. Additionally, CAP-induced oxidative stress can trigger endoplasmic reticulum (ER) stress, leading to the activation of the unfolded protein response. Specifically, CAP has been shown to upregulate CHOP (C/EBP homologous protein), which promotes apoptosis by repressing Bcl-2 expression and enhancing mitochondrial sensitivity to stress [[93], [94], [95], [96]]. The convergence of these pathways results in hallmark apoptotic features, including caspase activation, poly(adenosine diphosphate-ribose) polymerase (PARP) cleavage, DNA fragmentation, and membrane blebbing [97,98]. Jung et al. [99] demonstrated CAP-induced apoptosis in melanoma (B16F10, A375, A2058) and colon cancer (MC38) cells, marked by increased caspase-3 activation in a dose-dependent manner. In a xenograft melanoma model, Adhikari et al. [100] reported that CAP-treated plasma-activated medium enhanced apoptotic gene expression at the tumor site.

3.2. CAP-induced ICD

ICD is a regulated form of cell death that stimulates an adaptive immune response by exposing dead-cell-associated antigens [101]. Dying tumor cells release damage-associated molecular pattern (DAMP) signals, including the exposure of calreticulin, release of adenosine triphosphate (ATP), and high-mobility group box 1 (HMGB1), which act as danger signals to the immune system [102,103]. CAP has been shown to induce ICD in tumor cells owing to the elevated oxidative stress to the lethal limit by ROS and RNS [37]. During CAP-induced ICD, calreticulin exposed on the surface of tumor cells is a powerful “eat-me” signal for dendritic cells and macrophages [104,105]; ATP attracts immune cells and promotes their activation [106,107]; HMGB1 can bind to Toll-like receptors (TLRs) on immune cells and enhance the inflammatory response [108,109]. DAMP signals activate antigen-presenting cells (dendritic cells and macrophages), which present the tumor-associated antigens (TAAs) released from the dying tumor cells to T cells, initiating the T-cell-mediated anti-tumor immune responses [[110], [111], [112]]. For example, Chen et al. [113] demonstrated that CAP effectively induced ICD in B16F10 melanoma cells and 4T1 breast cancer cells, thereby enhancing antitumor immune responses. Similarly, Lin et al. [114] treated CT26 colorectal cancer cells with CAP and used them to vaccinate mice, successfully inducing protective immunity against subsequent tumor challenge. These findings indicate that CAP-induced ICD promotes antigen presentation and T-cell activation, reinforcing its potential in cancer immunotherapy.

3.3. CAP-induced pyroptosis

Unlike apoptosis, pyroptosis is a highly inflammatory form of cell death, often triggered by infections and immune responses [115,116]. Mechanistically, CAP leads to mitochondrial damage, activating caspase-9 and caspase-3, where Caspase-3 cleaves gasdermin (GSDM) E, which forms membrane pores that result in cell swelling, rupture, and DAMP release, thereby amplifying immune responses [[117], [118], [119], [120]]. Du et al. [121] confirmed pyroptosis in Ovcar-5 human ovarian carcinoma cells after CAP treatment, while Yang et al. [122] demonstrated dose- and time-dependent CAP-induced pyroptosis in PC9 human lung cancer cells, SGC7901 human gastric cancer cells, and Bel7402 human liver cancer cells. In an in vivo peritoneal carcinomatosis model, Qi et al. [123] enhanced CAP's pyroptotic effects using plasma-activated saline combined with hyperthermic intraperitoneal perfusion, leading to strong immune activation.

3.4. CAP-induced autophagy

Autophagy is a regulated cellular degradation process that maintains homeostasis by recycling damaged organelles and proteins under stress conditions [[124], [125], [126]]. Excessive autophagy can lead to autophagic cell death [127]. Mechanistically, CAP-induced oxidative stress activates the adenosine monophosphate-activated protein kinase (AMPK), which inhibits mechanistic target of rapamycin (mTOR), facilitating autophagy initiation via the UNC-51-like kinase 1 (ULK1) complex [[128], [129], [130], [131]]. Meanwhile, CAP suppresses the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mTOR pathway, further enhancing autophagy initiation [[132], [133], [134]]. Oxidative stress also induces ER stress, activating the unfolded protein response (UPR), particularly the protein kinase RNA-like ER kinase (PERK)-eIF2α-activating transcription factor 4 (ATF4) axis, which upregulates key autophagy-related (ATG) proteins including microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, and ATG5 [93,94,[135], [136], [137], [138]]. Golpour et al. [139] and Alimohammadi et al. [91] confirmed CAP-induced autophagy in melanoma-bearing mice and observed upregulation of LC3 and ATG5 in melanoma tissues.

3.5. CAP-induced necrosis

Necrosis is an uncontrolled, accidental cell death that occurs in response to extreme stress [140,141]. It is characterized by plasma membrane rupture, uncontrolled release of intracellular contents, and a strong inflammatory response [140,142,143]. CAP damages the structural integrity of cellular membranes, a hallmark of necrosis, leading to uncontrolled ion fluxes, osmotic imbalance, and eventual rupture of cell membrane [[144], [145], [146]]. Excessive calcium ion influx induces intracellular calcium overload, followed by activation of enzymes such as proteases, phospholipases, and endonucleases, which degrade cellular components and exacerbate necrotic damage [[147], [148], [149]]. Fridman et al. [72] demonstrated that high-dose CAP treatment (>15 s at 1.4 W/cm²) induced necrosis in melanoma cells while sparing healthy tissues.

3.6. CAP-induced ferroptosis

Ferroptosis is a form of iron-dependent cell death characterized by lipid peroxidation and oxidative membrane damage [[151], [152], [153], [154]]. ROS/RNS in CAP can initiate lipid peroxidation, whose accumulation can induce ferroptosis [[155], [156], [157], [158], [159]]. In addition to promoting lipid oxidation, CAP has shown to decrease intracellular glutathione (GSH), either by direct oxidation or downregulating the cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11), resulting in impaired activity of glutathione peroxidase 4 (GPX4), a key antioxidant enzyme responsible for reducing lipid hydroperoxides [152,[160], [161], [162], [163], [164]]. The inactivation of GPX4 allows lipid peroxides to accumulate, driving cells toward ferroptotic death. Furthermore, CAP-induced ROS/RNS can trigger autophagic degradation of ferritin, which increases free intracellular Fe²⁺ that promotes Fenton reaction, generating •OH that exacerbate lipid peroxidation and intensify oxidative damage, thereby further promoting ferroptosis and potentially amplifying other cell death pathways [[165], [166], [167], [168]]. Sun et al. [169] demonstrated CAP-induced ferroptosis in A431 squamous cell carcinoma cells, confirmed by suppressed GPX4 expression. Dai et al. [170] observed increased Fe²⁺ levels, decreased GSH, and reduced GPX4 expression in triple-negative breast cancer (TNBC) models following CAP treatment, and specified that •OH was a prerequisite for generating lipid peroxyl radical, leading to ferroptosis. Zhang et al. [163] reported that CAP downregulated SLC7A11 and Homeobox B9 (HOXB9), two key regulators of ferroptosis resistance, in non-small cell lung cancer, with effects reversed by ferrostatin-1, a ferroptosis inhibitor.

3.7. CAP as a multifaceted cancer therapy

CAP could induce a spectrum of cell death pathways, including apoptosis, ICD, pyroptosis, autophagy-dependent death, ferroptosis, and necrosis, which are not mutually exclusive but often mechanistically interconnected. For example, under CAP-induced stress, calreticulin exposure, ATP secretion, and HMGB1 release may occur during late apoptosis, eliciting the ICD pathway [[171], [172], [173]]. Autophagy is frequently activated as an adaptive response to CAP-induced oxidative and metabolic stress via the AMPK-mTOR-ULK1 axis [82,125,[174], [175], [176]]. When excessive or dysregulated, autophagy may shift toward autophagic cell death and promote ferroptosis by enhancing ferritinophagy-mediated iron release [127]. Moreover, apoptotic Caspase-3 activation can cleave GSDME, initiating secondary pyroptosis in GSDME-expressing cells [119,177]. In situations where oxidative damage surpasses the cell's compensatory capacity, regulated necrosis may dominate, leading to passive DAMP release and pro-inflammatory responses [178,179]. These pathways are often interdependent—for example, autophagy can sensitize cells to ferroptosis, apoptosis and pyroptosis share caspase activation cascades, and ICD can be the outcome of various regulated cell deaths depending on DAMP expression.

Tumor heterogeneity profoundly influences the type and extent of cell death elicited by CAP. Factors such as genetic mutations, epigenetic landscapes, metabolic reprogramming, and immune microenvironmental context shape how individual tumors respond to CAP-induced ROS/RNS. For example, apoptosis is more readily induced in tumors with intact mitochondrial signaling, functional p53, and a balanced expression of pro-/anti-apoptotic proteins, but may be bypassed in tumors with TP53 mutations or Bcl-2 overexpression [[180], [181], [182]]. ICD requires both DAMP release from tumor cells and an immunocompetent tumor microenvironment; thus, CAP-induced ICD is more effective in “hot” tumors with pre-existing immune infiltration compared to “cold” tumors lacking immune priming [183]. Ferroptosis susceptibility varies across tumor types and is modulated by lipid peroxidation potential, GPX4 activity, ferritinophagy capacity, and iron metabolism. CAP-induced oxidative stress can shift resistant or metabolically vulnerable tumors toward ferroptosis [[184], [185], [186]]. Autophagy can serve dual roles depending on cellular context, either protecting cells from oxidative stress or promoting cell death in synergy with other regulated cell death pathways [[187], [188], [189]]. This is influenced by oncogenic signaling pathways such as PI3K/AKT/mTOR [127,190]. Pyroptosis depends on inflammasome components and GSDM expression profiles, which are heterogeneous across tumors. [191]. Collectively, tumor heterogeneity can largely affect the dominant cell death pathways engaged, necessitating a tailored therapeutic approach to maximize efficacy across heterogeneous tumor landscapes.

Different cancer types exhibit varied susceptibility to CAP-induced cell death mechanisms due to distinct genetic profiles, metabolic states, and tumor microenvironment characteristics. For instance, melanoma, an immunogenic tumor type, tends to undergo ICD and apoptosis more readily following CAP exposure [[192], [193], [194], [195], [196]]. Breast cancer generally displays moderate immunogenicity and frequent p53 mutations, and its relatively high intracellular iron and low GSH levels make it more prone to ferroptosis [[197], [198], [199], [200], [201], [202]]. While emerging results may support these tendencies, a comprehensive, comparative analysis and mechanistic studies of CAP-induced death mechanisms across cancer types remain limited. A deeper understanding of how genetic and microenvironmental factors influence CAP-induced death pathways is essential to identify dominant mechanisms in each cancer subtype. Addressing this knowledge gap through rigorous preclinical studies will help refine the therapeutic index of CAP and optimize its integration with standard treatments such as immunotherapy, chemotherapy, or radiotherapy. Ultimately, such mechanistic insights will strengthen the rationale for patient-specific CAP-based treatment strategies and facilitate its clinical translation.

Overall, CAP employs multiple mechanisms to selectively eliminate tumor cells, primarily through oxidative stress-induced cell death pathways. Tumor cells, due to their altered metabolism, exhibit higher baseline oxidative stress than normal cells, making them more susceptible to CAP-induced damage [37,[203], [204], [205]]. This selective vulnerability not only underpins the safety profile of CAP but also highlights its promise as a therapeutic modality in oncology. Beyond its standalone efficacy, CAP activates a diverse array of molecular and cellular responses that present opportunities for synergy with other cancer therapies. For example, CAP-induced ICD stimulates T cell–mediated anti-tumor immunity by enhancing crosstalk between tumor cells, dendritic cells (DCs), and T cells [48,206]. This immune activation reprograms the immunosuppressive tumor microenvironment into one that supports effector immune responses, promoting M1 macrophage and natural killer (NK) cell activity while suppressing regulatory T cells (Tregs) and M2 macrophages [207,208]. Such immunomodulation complements immunotherapies, particularly immune checkpoint inhibitors, by enhancing antigen presentation and restoring T cell function. Similarly, CAP enhances the efficacy of chemotherapeutic agents through multiple mechanisms. It elevates intracellular ROS levels, sensitizing tumor cells to ROS-inducing drugs like doxorubicin, cisplatin, and paclitaxel [209]. CAP-induced DNA double-strand breaks amplify the cytotoxic effects of DNA-targeting agents by overwhelming tumor cells’ repair capacity [210,211]. Besides, lipid peroxidation induced by CAP may increase the permeability of cell membranes, facilitating drug uptake and lowering the effective dose required for tumor inhibition [212,213]. Meanwhile, CAP also leads to mitochondrial dysfunction, which makes it possible to synergize with drugs disrupting mitochondrial function, including cisplatin or metformin. Additionally, CAP can potentiate radiotherapy by enhancing oxidative stress and DNA damage in tumor cells, thereby amplifying radiation-induced cytotoxicity [68,[214], [215], [216], [217], [218]]. Taken together, these mechanisms support the use of CAP in rational combination regimens with immunotherapy, chemotherapy, or radiotherapy. Further investigation into the molecular basis of these interactions will be essential for optimizing CAP-based multimodal cancer therapies.

4. Direct CAP treatment for cancer therapy

4.1. Direct CAP treatment for skin cancer

CAP has been extensively investigated for skin cancer therapy due to its unique advantages, including its non-invasive nature, selective cytotoxicity toward tumor cells, and ability to induce immunogenic responses. Unlike conventional treatments, CAP generates ROS and RNS that selectively target cancer cells while sparing normal tissue. Several studies have explored the efficacy of direct CAP application for melanoma and other skin cancers, demonstrating its potential in both localized and systemic anti-tumor responses.

Keidar et al. [219] established subcutaneous melanoma mouse models and applied direct CAP treatment to the skin above tumor tissues (Fig. 3A). A single CAP treatment led to tumor ablation or reduced tumor volume, resulting in a significant improvement in survival rates, with a median survival of 33.5 days in treated mice compared to 24.5 days in untreated controls. Mizuno et al. [220] inoculated mice with two melanomas, one on each leg, and applied direct CAP treatment to one tumor. Notably, CAP treatment inhibited tumor growth not only in the treated tumor but also in the untreated tumor, suggesting systemic immune activation. Meanwhile, CAP treatment increased pro-inflammatory IFN-γ secretion from the spleen, validating the activation of the adaptive immune system.

Fig. 3.

Direct CAP treatment on skin cancer. (A) Representative photos in in vivo CAP treatment process: (a) CAP device; (b) typical image of mice with three tumors before and after treatment (shown after 24 h); (c and d) typical image of mice with a single tumor before and approximately 1 week after treatment; (e) tumor before and immediately after the 2.5-min treatment with CAP jet. (f) CAP treatment effect on the growth of established tumor and mice survival in a murine melanoma model. Reproduced with permission [219]. (B) Anti-tumor effect of cold gas plasma and drug mono and combination treatment in vivo: (a) study scheme, (b) Kaplan-Meyer plot (survival) of mice left untreated or exposed to cold gas plasma and drug mono or combination treatment, (c) tumor volume development throughout the experiment for IS112 or Sm837 mono and combination treatment, (d) area under the curve of tumor volumes, and (e) apoptosis quantification. Reproduced with permission [221].

Miebach et al. [43] employed kINPen, an atmospheric pressure plasma jet which has been classified as a medical device (class IIa/IIb) in Europe since 2013 [62]. Their study explored the combination of CAP with mitochondria-targeted drugs for melanoma treatment. While CAP alone demonstrated anti-tumor efficacy comparable to these drugs, the combination therapy further enhanced tumor growth inhibition through a synergistic effect. Additionally, CAP treatment augmented the response to immune checkpoint blockade with anti-programmed cell death protein 1 (aPD-1), reshaping the tumor immune microenvironment by increasing CD11c⁺ dendritic cell and F4/80⁺ macrophage infiltration. Boeckmann et al. [221] also applied CAP to skin cancer therapy, demonstrating significant tumor growth control and increased apoptotic tumor cell death (Fig. 3B). They also identified two chromone-derived small molecules, 6-methyl-3-(2-fluorobenzoyl)chromone (Sm837) and 6-chloro-7-methyl-3-pentafluoropropanoylchromone (IS112), that can enhance CAP-induced oxidative stress. Wherein, IS112 exerted severe in vivo toxicity, while Sm837 with good tolerability exhibited strong synergistic anti-tumor effects when combined with CAP therapy.

4.2. Direct CAP treatment for glioma/glioblastoma

CAP has emerged as a promising therapeutic approach for gliomas and glioblastomas due to its ability to selectively induce tumor cell apoptosis, modulate the tumor microenvironment, and enhance the efficacy of conventional treatments. Despite the challenge of delivering CAP to brain tumors due to the protective skull barrier, several studies have demonstrated its potential in both subcutaneous and intracranial glioblastoma models.

In 2010, Vandamme et al. [222] conducted a study investigating the in vivo anti-glioma effects of direct CAP treatment. Using a floating electrode dielectric barrier discharge (FE-DBD) CAP device, they treated subcutaneous U87-MG glioma xenografts in mice. The treatment was well tolerated, and tumor volumes decreased by 33 % following CAP exposure.

Soni et al. [42] applied a CAP jet over the skin and skull surface of mice bearing intracranial glioblastoma (Fig. 4). The CAP jet demonstrated the ability to penetrate bone, resulting in significant tumor growth inhibition of approximately 40 %. Moreover, CAP treatment sensitized glioblastomas to chemotherapy. When a single CAP treatment was followed by temozolomide (TMZ) administration, glioblastoma progression was virtually prevented. Bioluminescence imaging revealed a 1.8-fold increase in tumor volume in the combination treatment group, significantly lower than the 4.8-fold increase observed in the CAP-only group.

Fig. 4.

Direct CAP treatment on glioma/glioblastoma.In vivo CAP targeting plus TMZ treatment of intracranial glioblastoma in nude mice. (A) Schematic representation of the treatment. (B) Representative bioluminescence images at baseline (day 0) and 6 and 13 days following a single non-invasive CAP treatment with or without daily TMZ or vehicle administration. Helium was used as a CAP control. (C) Schematic representation of the non-invasive modality to treat glioblastoma in vivo and (D) penetration of electromagnetic waves through the skin and skull to target glioblastoma (E) Quantitative summary of radiance (i.e., light emission). over the course of the study. “Helium” represented the control for CAP, as direct feeding gas treatment without CAP jet generation. “Vehicle” represented the control for the drug TMZ, which consisted of dimethyl sulfoxide and phosphate-buffered saline as the solvent of TMZ for intraperitoneal injections. Reproduced with permission [42].

4.3. Direct CAP treatment for breast cancer

Recent studies have explored the efficacy of direct CAP treatment in breast cancer models, demonstrating its potential for both tumor growth inhibition and prevention of recurrence. Adil et al. [223] established a breast cancer mouse model and directly exposed tumors to CAP generated from an FE-DBD device, achieving a remarkable 90 % reduction in tumor growth, however no significant survival increase. Zhou et al. [224] developed an innovative CAP delivery device, the invivoPen, designed for minimally invasive in situ treatments. The invivoPen features two co-axial needles with different calibers, allowing for direct CAP ejection into tumors while avoiding the need for an invasive gas path. This approach significantly decelerated but nor regressed tumor growth compared to controls, resulted in a 100 % survival rate in mice over 30 days and induced apoptosis in tumor cells while suppressing tumor migration.

Recently, Chen et al. [44] investigated the use of CAP in preventing tumor recurrence in a breast cancer resection model (Fig. 5). After surgically removing tumors, CAP was applied directly to residual tumor cells within the resection cavity. This treatment not only decelerated tumor recurrence but also prolonged post-surgical survival. Immunological analyses revealed enhanced T-cell-mediated anti-tumor responses, with increased infiltration of CD4⁺ and CD8⁺ T cells, as well as elevated CD8⁺ T cell proliferation and secretion of pro-inflammatory cytokines. These immune effects were strongly correlated with the duration of CAP treatment. Similar therapeutic benefits were observed in melanoma models, further supporting CAP's potential to prevent recurrence in various cancers.

Fig. 5.

Direct CAP treatment on breast cancer. Portable air-fed CAP device for postsurgical cancer treatment. (A) Portable air-fed CAP was applied on residual tumor cells within the surgical cavity and induced ICD of the residual tumor cells. TAAs released from dying cells were presented by dendritic cells to T cells in the tumor-draining lymph node, generating cytotoxic T lymphocytes to combat tumor cells. (B) Schematic of the working mechanism for the CAP device and a photograph of plasma discharge containing spark at joule energy level. (C) Quantification of CRT markers on the remaining 4T1 cells after CAP treatment. (D) Average tumor growth kinetics and (E) Kaplan-Meier survival curves of mice in experimental groups. (F) Quantification of dendritic cell maturation in vivo in the tumor-draining lymph nodes 5 days after the treatments. Quantification analyses of intratumoral (G) CD8⁺ T cells and (H) CD4⁺ T cells gating on CD3⁺ cells 5 days after the treatments. (I) Quantitative analysis of Ki67 expression in CD3⁺CD8⁺ T cells within the tumors 5 days after treatment. Reproduced with permission [44].

While all three studies focused on breast cancer treatment, they demonstrated varying therapeutic efficacies of CAP in terms of tumor growth inhibition, survival prolongation, and anti-tumor immune activation. These differences likely stem from multiple contributing factors, including tumor types, mouse strains, CAP parameters, and treatment regimens. While each study reported improved treatment outcomes, further optimization of these factors will be essential to enhance the overall efficacy of CAP therapy.

4.4. Direct CAP treatment for lung cancer

Li et al. [225] investigated the combination of direct CAP treatment with iron oxide-based magnetic nanoparticles (MNPs) for xenograft lung cancer therapy (Fig. 6A). Similar to CAP, these nanoparticles exhibited selective cytotoxicity against tumor cells while sparing normal lung epithelial cells. This selectivity was attributed to the ability of iron oxide nanoparticles to enhance intracellular ROS accumulation in tumor cells. CAP treatment alone decelerated tumor growth, reduced tumor cell proliferation, and enhanced apoptosis. The combination therapy further amplified these anti-tumor effects, demonstrating a synergistic potential for lung cancer treatment.

Fig. 6.

(A) Direct CAP treatment on lung cancer. (a) Molecular mechanisms of tumor-selective killing effect of CAP enhanced by MNPs. Attenuated tumor growth in xenograft nude mice models by CAP and iron oxide-based MNPs: (b) growth curves of xenograft non-small-cell lung carcinoma in nude mice treated by CAP and iron oxide-based MNPs, (c) photo of tumor bearing mice co-treated with MNPs and CAP, (d) weight changes of xenograft tumor. Reproduced with permission [225]. (B) Direct CAP treatment on head and neck cancer. (a) Mechanisms of glycolysis disruption and PI3K/AKT/mTOR/HIF-1α signaling pathway attenuation by CAP, as well as sensitization of chemotherapy and immunotherapy by CAP. (b) Anti-tumor effect in a xenograft mouse head and neck cancer model by CAP combined with DDP: Flowchart outlining the in vivo CAP therapy regimen, tumor growth curves throughout the entire course of CAP treatment in mice xenografts, Kaplan-Meier survival curve of mice bearing Fadu cells subcutaneous tumors treated with CAP. (c) Anti-tumor effect in a xenograft mouse squamous cell carcinoma of the head and neck model by CAP combined with aPD-1: experimental paradigm to assess the treatment effect of CAP and the combination of CAP with aPD-1 in xenograft models using SCC7 cells, growth curves of xenograft tumors derived from SCC7 cells with different drug treatments, Kaplan-Meier survival curves of mice bearing SCC7 cells subcutaneous tumors treated with different drugs. Reproduced with permission [226].

4.5. Direct CAP treatment for head and neck cancer

Wang et al. [226] employed a handheld piezoelectric CAP device to treat xenograft head and neck cancer in mice (Fig. 6B). CAP treatment effectively promoted tumor cell apoptosis and autophagy, leading to significant tumor growth suppression compared to untreated controls. Furthermore, CAP synergized with cisplatin (DDP, a chemotherapy drug) and an immune checkpoint inhibitor (aPD-1), resulting in enhanced anti-tumor efficacy and prolonged survival in both combination groups termed as “Comb”. Mechanistic studies revealed that CAP treatment inhibited tumor glycolysis at multiple enzymatic points and suppressed the AKT signaling pathway, contributing to tumor growth inhibition. Additionally, the combination of CAP with aPD-1 induced a more pronounced infiltration of CD4⁺ and CD8⁺ T cells in tumors, further supporting its immunomodulatory potential.

4.6. Direct CAP treatment for cholangiocarcinoma

Vaquero et al. [227] established a cholangiocarcinoma (CCA) xenograft model by inoculating EGI-1 cells into immunodeficient mice and directly applying CAP treatment to the tumors. CAP therapy significantly inhibited tumor growth compared to untreated controls. While its therapeutic effect was weaker than gemcitabine—one of the standard chemotherapeutic agents for CCA—CAP treatment caused no observable liver damage, highlighting its safety. Mechanistic investigations showed that CAP induced double-strand DNA breaks, resulting in cell cycle arrest and apoptosis-mediated tumor cell death.

5. Indirect CAP treatment for cancer therapy

While direct CAP treatments have shown promising results in cancer therapy (See Table 2), particularly for superficial tumors, they are limited by CAP's poor penetration ability into deeper tissues. [228,]. To address this, indirect methods of CAP delivery have been developed, enhancing its potential for treating deep-tissue tumors. These methods include the use of CAP-activating liquids, hydrogels, tubes, and microneedle patches, which help preserve, guide, or deliver CAP into tumor areas. This section reviews these indirect delivery strategies (See Table 3).

5.1. Liquid-mediated CAP delivery

CAP-activated liquid represents a promising method for indirect CAP delivery. Reactive species from CAP are enriched into the liquid (Fig. 7), which retains its cytotoxic properties and has demonstrated efficacy against various cancer cell lines [[230], [231], [232], [233]]. Unlike direct CAP treatment, CAP-activated liquids can be injected or perfused into non-superficial tumors.

Fig. 7.

Liquid-mediated CAP delivery for cancer therapy. (A) Scheme of CAP-activated liquid preparation [230]. (B) (a) Experiment design with mice treated once every 3 days from day 6–40. (b) Tumor bioluminescence of untreated and PAS-treated mice. (c) Individual tumor kinetics, (d) tumor volume on day 18 and 21, and (e) survival kinetics for untreated and PAS-treated mice. Reproduced with permission [214]. (C) (a) Schematic of the combination therapy using violet phosphorene nanosheets and CAP to improve tumor cell death and inhibit tumor growth. (b) Safety and (c) ability to inhibit tumor growth by violet phosphorene nanosheets and CAP treatments. Reproduced with permission [244].

Utsumi et al. [234] established the models of NOS-2 and chronic paclitaxel-resistant NOS-2 epithelial ovarian carcinoma on mice. They treated cell culture media with CAP to produce CAP-activated solution, and intratumorally injected the solution in the tumor areas. This treatment resulted in led to a 66 % reduction in NOS-2 tumor weight and a 52 % reduction in paclitaxel-resistant NOS-2 tumors, compared to controls. Nakamura et al. [235] revealed that intraperitoneal injections of CAP-activating liquid suppressed ovarian cancer metastasis and extended survival. The mechanistic assessment suggested that CAP-activating liquid inhibited the MAPK pathway and downregulated MMP-9, a critical protein for cancer cell motility. Xiang et al. [236] also explored CAP-activating liquid on breast cancer, showing significant inhibition of TNBC growth, while non-TNBC tumors were less affected. This selective sensitivity may be due to altered redox homeostasis and inhibition of hyper-activated MAPK/JNK and NF-κB pathways in TNBC cells. Saadati et al. [237] demonstrated that CAP-activating liquid treatment, compared to direct CAP, led to greater melanoma growth inhibition and enhanced tumor cell apoptosis, particularly when combined with the chemotherapy drug cyclophosphamide.

Additional studies have explored the use of CAP-activated liquid in other cancer types. Liedtke et al. [238] injected CAP-activated liquid intraperitoneally into mice with pancreatic tumors, leading to significant tumor mass reduction and prolonged survival, while also promoting immune cell infiltration and tumor cell apoptosis. Their following report revealed a similar therapeutic effect of CAP-treated medium, accompanied by a higher ICD in tumor cells, increased M1 macrophages, and increased infiltration of neutrophils and T cells in treated tumors [239]. Jo et al. [240] injected CAP-activated liquid into nude mice bearing human NSCLC. CAP-activated liquid induced ferroptosis in H1299 NSCLC cells in vivo, with downregulation of ferroptosis suppressor protein 1 (FSP1), leading to significantly inhibiting tumor growth. Freund et al. [241] applied CAP-activating liquid for peritoneal lavage for peritoneal colon cancer metastasis therapy. Repetitive applications of CAP-activating liquid significantly decreased tumor growth, potentially by enhancing immune cell infiltration, such as macrophages, and activating CD4⁺ T cells. Zhang et al. [242] and Guo et al. [214] found that injecting plasma-activated saline into pericarcinomatous tissues or directly into tumors led to substantial reductions in tumor size and improved survival rates in bladder cancer and other cancer models (Fig. 7B). Song et al. [243] investigated the anti-tumor effect of orally administered CAP-activating liquid on NSCLC. Their study demonstrated significant inhibition of subcutaneous NSCLC xenograft growth, attributed to reduced tumor cell proliferation and angiogenesis. Notably, the treatment exhibited lower toxicity while enhancing anti-tumor immunity compared to gefitinib, suggesting its potential as a safer and immunomodulatory alternative for NSCLC therapy.

Moreover, CAP-activating liquid has been shown to enhance the effects of other therapies. Dezhpour et al. [245] demonstrated that CAP-activating liquid outperformed doxorubicin in some cases, while also mitigating the drug's side effects, such as hepatotoxicity and nephrotoxicity. In combination with violet phosphorene nanosheets (VPNS) reported by Qi et al. [244], CAP-activating liquid further inhibited tumor growth in xenograft lung cancer models, achieving a tumor inhibition rate over 61 % in the combination group (termed as group “Com”) (Fig. 7C). The combination therapy significantly downregulated AKT expression and decreased anti-apoptotic protein levels, leading to tumor cell apoptosis.

5.2. Hydrogel-mediated CAP delivery

While CAP-activated liquids have demonstrated significant anti-tumor effects, their fluid nature presents challenges such as rapid diffusion, dilution in body fluids, and limited tumor retention. To address these challenges, hydrogels have emerged as promising delivery platforms for CAP, offering localized drug retention, sustained payload release, and improved bioavailability [[246], [247], [248], [249], [250], [251], [252]]. Additionally, some injectable hydrogels can overcome physiological barriers, enabling drug delivery into deep tissues [[253], [254], [255]]. Leveraging these advantages, hydrogels have been explored for the preservation and controlled release of CAP to enhance its therapeutic potential.

Zhang et al. [256] developed a CAP-activated thermosensitive PLEL biogel (PAPB) by treating a (poly-DL-lactide)-(polyethylene glycol)-(poly-DL-lactide) (PLEL) solution with CAP, followed by heating to 35 °C to form gels. When applied at the operative incision site after tumor-reductive surgery in mice, PAPB resulted in complete tumor eradication and 100 % survival six weeks post-surgery. Byun et al. [257] developed a CAP-responsive hydrogel that can preserve CAP and deliver nano-adjuvants to enhance tumor cell elimination and tumor microenvironment remodeling (Fig. 8B). The hydrogel, composed of a hyaluronic acid (HA)-tyramine (HAT) conjugate with ROS-responsive gelation properties, was mixed with lipid nanoparticles loaded with a transforming growth factor-beta (TGF-β) receptor kinase inhibitor (TRKI) as nano-adjuvants. CAP irradiation induced phenol polymerization of tyrosine moieties in HAT, benefiting the crosslinking of HAT, which preserved ROS/RNS and entrapped TRKI-loaded lipid nanoparticles (TNL), generating CAP-induced hydrogel (CHG) loading TLN (TLN@CHG). This treatment effectively reprogrammed the tumor microenvironment, as evidenced by increased dendritic cell, CD4⁺, and CD8⁺ T cell populations, elevated expression of TNF-α, IL-6, MHC-II, and CD86 on dendritic cells, increased IFN-γ in CD4⁺ T cells, and enhanced Granzyme B expression in CD8⁺ T cells. Therapeutically, this treatment significantly inhibited tumor growth, with all treated mice achieving 100 % survival over 180 days. Additionally, TLN@CHG completely suppressed both treated and distant untreated tumors while significantly reducing breast cancer metastasis to the lungs, yielding a 60 % survival rate at 180 days.

Fig. 8.

Hydrogel-mediated CAP delivery for cancer therapy. (A) (a) Preparation of injectable aPD-1@CAP-gel and (b) illustration of injectable aPD-1@CAP-gel-mediated cancer immunotherapy. (c) Average tumor growth kinetics in different groups in the TNBC mouse model and (d) survival of TNBC-bearing mice. Reproduced with permission [207]. (B) (a) Schematic illustration of CAP-formable hydrogel with TRKI-loaded nanoparticles and the proposed mechanism of immune-inflamed micro-network. (b) Individual curves of primary and rechallenged tumors. (c) Survival of mice. Reproduced with permission [257].

Fang et al. [207] prepared an aPD-1@CAP-gel by dissolving aPD-1 antibody, an immune checkpoint inhibitor, in a CAP-activated water and Pluronic mixture (Fig. 8A). Pluronic, a commercially available thermosensitive hydrogel, remains liquid at room temperature but gels at body temperature. When injected intratumorally, CAP-gel induced robust ICD, enhanced dendritic cell maturation, increased CD4⁺ and CD8⁺ T cell infiltration, and promoted macrophage polarization from the immunosuppressive M2 to the immune-supportive M1 phenotype. Additionally, it elevated pro-inflammatory cytokines while suppressing anti-inflammatory cytokine secretion, effectively activating T cell-mediated anti-tumor immunity. CAP-gel provided moderate tumor growth control, while aPD-1@CAP-gel exhibited superior tumor inhibition, prolonged survival, and potent immunomodulatory effects. Notably, aPD-1@CAP-gel also inhibited distant, untreated tumor growth, indicating systemic immune activation. Building upon this work, Cao et al. [258] incorporated trehalose into the aPD-1@CAP-gel to enhance therapeutic efficacy. Given that CAP-derived ROS and RNS preserved in the hydrogel induce oxidative stress and eIF2α phosphorylation to trigger ICD, trehalose further amplified eIF2α phosphorylation. This synergistic effect enhanced ICD and potentiated T cell-mediated anti-tumor immune responses, demonstrating the promising potential of hydrogel-based CAP therapies in cancer treatment.

5.3. Tube-assisted CAP delivery

To enable direct contact of CAP to tumors in deep tissues, long flexible tubes have been explored to guide CAP for long-distance delivery [[259], [260], [261]]. The higher applied voltage and lower diameter profit the longer plasma delivery distance and improved reactive species density [262].

Mirpour et al. [263] developed a method for CAP treatment of breast cancer by creating a small hole in the tumor and inserting a micro-plasma probe subcutaneously. Simultaneously, a capillary tube was placed on the opposite side of the tumor to evacuate gas. This direct CAP application resulted in significant therapeutic effects, comparable to the chemotherapy drug doxorubicin, as evidenced by stable tumor volumes, extended survival, and increased apoptotic tumor cells compared to the control group. Binenbaum et al. [264] developed a CAP jet generator with an elongated flexible capillary, making it narrow and highly maneuverable. This setup allowed precise targeting of the CAP jet to the tumor site. When applied to melanoma tumors located just beneath the skin, CAP treatment effectively controlled tumor growth, as confirmed by tumor volume measurements and IVIS imaging. Post-treatment analysis revealed a necrotic tumor area extending up to 15 mm below the skin surface.

Chen et al. [265] developed a novel CAP device with micro-sized needle, designed for direct in vivo treatment of glioblastoma (Fig. 9A). An endoscopic tube was surgically implanted into brain at the tumor inoculation site, allowing CAP to be precisely delivered to intracranial glioblastoma areas. Compared to the 600 % increase in tumor volume in control mice, CAP treatment led to a 50 % reduction in tumor volume, demonstrating its potential as a targeted glioblastoma therapy.

Fig. 9.

(A) Tube-mediated CAP delivery for cancer therapy. (a)In vivo targeting of glioblastoma tumor with He μCAP: Photograph of a new μCAP device for plasma delivery through an intracranial endoscopic tube to target glioblastoma tumors in the mouse brain. (b) Representative in vivo bioluminescence images illustrating glioblastoma tumor volume at baseline and 2 days following He μCAP or vehicle (helium) treatment. (c) Tumor volumes in control and He μCAP treatment groups. Reproduced with permission [265]. (B) Microneedle patch-mediated CAP delivery for cancer delivery. (a) Schematic of the transdermal combination of CAP and ICB therapy assisted by the polymeric hollow-structured microneedle patch loaded with anti-programmed cell death ligand 1 (aPD-L1). (b)In vivo tumor bioluminescence of the untreated mice and mice treated with CAP, sMN/CAP, hMN/CAP, hMN-aPD-L1, and hMN-aPD-L1/CAP (sMN: solid microneedles; hMN: hollow-structured microneedles). (c) Average tumor growth kinetics and (d) survival curves for treated and control mice. (e) Left (distant) and right (primary) tumor growth curves for treated and untreated mice in distant tumor models. Reproduced with permission [113].

5.4. Microneedle-mediated CAP delivery

Microneedles offer a minimally invasive, controllable, and precise method for drug delivery at specific skin depths [266]. These features make microneedles a promising approach for mediating indirect CAP treatments while ensuring deeper tumor penetration and maintaining therapeutic efficiency. Given the gas nature of CAP, Chen et al. [113] designed a hollow-structured microneedle patch composed of biocompatible polymers-polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA) (Fig. 9B). The hollow microneedles functioned as microchannels, facilitating CAP delivery through the skin into tumor tissues. When applied to melanoma, CAP treatment via the hollow-structured microneedle patch resulted in significantly slower tumor growth and prolonged survival in mice compared to direct CAP treatment and CAP delivery through solid microneedles that had no hallow structures. This approach enhanced tumor-specific T-cell-mediated immune responses, evidenced by increased tumor-infiltrating lymphocytes, higher CD4⁺ and CD8⁺ T cell populations, and elevated levels of pro-inflammatory cytokines. When aPD-L1 (a checkpoint inhibitor) was incorporated into the hollow microneedles and released into tumors, the combination of CAP and immune checkpoint blockade further amplified tumor suppression and anti-tumor immune activation. Notably, this local therapy also induced regression in untreated distant tumors, demonstrating the activation of systemic anti-tumor immunity.

Zhang et al. [267] introduced a plasma-activated cryo-microneedle system, where plasma-activated saline was frozen in preformed microneedle molds. Upon insertion into tumors, the microneedle tips melted at skin temperature, releasing ROS and RNS into tumor tissues. By day 20 after tumor inoculation, tumor volumes in the control, saline cryo-microneedle, and plasma-activated cryo-microneedle groups were approximately 3.6, 3.2, and 1.01 times their initial sizes, respectively, with corresponding survival rates of 30 %, 25 %, and 100 %. These results demonstrated that plasma-activated cryo-microneedles significantly inhibited tumor growth and extended survival. Further analysis revealed that the treatment induced both apoptosis and ferroptosis in tumor cells, with no significant toxicity observed in normal tissues.

In summary, these indirect methods offer promising solutions to the limited tissue penetration of direct CAP application, thereby enabling treatment of deep-seated tumors. However, several challenges persist (See Table 4). First, indirect approaches may result in loss or attenuation of certain reactive components during the delivery process. While ROS and RNS are recognized as the primary cytotoxic agents generated by CAP, other components, such as charged particles, UV photons, and short-lived radicals, may also contribute to its therapeutic effects [268]. Furthermore, short-lived ROS and RNS are typically not well preserved in the indirect approaches. Thus, these losses may not be effectively preserved or transmitted through indirect delivery systems, potentially reducing the overall therapeutic efficacy of CAP. Second, all currently available indirect methods are inherently invasive to varying degrees, which may lead to tissue disruption and reduce patient compliance. Furthermore, each approach carries unique pharmacokinetic and engineering limitations. Specifically, CAP-activated liquids are relatively easy to prepare, store, and transport. However, their therapeutic efficacy is limited by the short half-life of reactive species and their rapid dilution in physiological fluids. CAP-activated hydrogels enable localized and sustained release, offer tunable mechanical properties, and can be co-loaded with other therapeutics [207,[269], [270], [271]]. However, potential interactions between CAP species and hydrogel matrices may compromise the stability or functionality of both. The long-term biocompatibility and safety of CAP-exposed hydrogels also remain to be fully elucidated. Furthermore, synthesis procedures could be complex and less scalable. Long-tube CAP devices allow internal or endoscopic plasma delivery, expanding treatment applicability [260]. Nevertheless, reactive species may decay during transit, with plasma intensity attenuating as a function of tube length and curvature [272]. Increased mechanical complexity and fragility of such systems also pose engineering challenges. Microneedle patches represent a minimally invasive route for transdermal CAP delivery and can facilitate co-delivery of other agents. However, their limited penetration depth (typically <1 mm), coupled with fabrication and safety concerns, restricts their broader clinical use.

Table 4.

Advantages and limitations of indirect CAP delivery methods.

Indirect CAP delivery method	Advantages	Limitations
CAP-activated liquids	•Easy preparation, storage, and transportation •Rapid diffusion into target tissues	•Short half-life of short-lived radicals •Fast dilution by body fluids, reducing therapeutic potency
CAP-activated hydrogels	•Localized, sustainable ROS/RNS release •Customizable mechanical properties (e.g., injectability and thermos-sensitivity) •Co-loading with drugs	•Potential CAP–hydrogel interactions affecting the stability of ROS/RNS, particularly for short-lived radicals •Long-term biocompatibility and biomanufacturing of hydrogels
Long tubes	•Endoscopic or internal CAP treatment	•Potential reactive species decay during endoscopic delivery •Plasma attenuation over distance •Increased device complexity and fragility •Complex applicability
Microneedle patches	•Minimal invasiveness •Supports transdermal and co-delivery applications	•Limited penetration depth (typically <1 mm) •Fabrication and scalability challenges

6. Clinical status of CAP therapy for cancer

The advancement of clinical trials employing CAP in cancer treatment represents a significant step toward its broader clinical application (See Table 5). Metelmann et al. [45] conducted a retrospective clinical study involving 12 patients with advanced squamous cell carcinoma of the head and neck together with intraoral or extraoral ulcerations who underwent CAP treatment in Germany. This trial was conducted in 2013, where patients received intraoral or extraoral CAP jet irradiation by kINPen for 1 to 9 cycles (1 min/cm², 3 times/week per cycle) after traditional cancer therapies (radical tumor resection, neck dissection, chemotherapy, radiotherapy). CAP therapy provided notable benefits, including reduced pain medication requirements, decreased microbial load-associated odor, and weight gain. Partial tumor remission in 4 cases and complete wound healing of infected ulcerations in 1 case were observed following CAP therapy, highlighting its potential therapeutic advantages beyond tumor control. In 2015, Metelmann et al. [273] also reported a clinical trial employing CAP therapy to treat six patients suffering from advanced cancer of oropharynx with contaminated tumor ulcerations, which was led in Greifswald University Medicine, Germany. CAP by kINPen was delivered in cycles of 3 single treatments within 1 week (1 min/cm²), followed by an intermittence of 1 week without CAP exposure. The CAP palliation resulted in a reduction of odor as an obvious effect of decontamination, less demand of pain medication, and very mild side effects on patients. Four patients exhibited survival longer than 7.5 months, indicating the potential of CAP palliation in improving life quality and prolonging survival of patients with advanced cancer of oropharynx.

Table 5.

CAP for cancer therapy in clinical trials.

Tumor	CAP device	Feeding gas	Place and year of clinical trials	Study design	Major results	Ref.
Advanced squamous cell carcinoma of the head and neck	kINPen MED (neoplas, Germany)	Argon	Greifswald University Medicine, Greifswald, Germany (2013)	•Twelve patients with advanced squamous cell carcinoma of the head and neck, presenting with intraoral or extraoral ulcerations. •CAP treatment: 1 min/cm2, three times per week per treatment cycle	•Reduced pain medication requirements, decreased microbial load-associated odor, and weight gain in patients •Partial tumor remission in 4 cases and complete wound healing of infected ulcerations in one case	[45]
Cancer of the oropharynx	kINPen MED (neoplas, Germany)	Argon	Greifswald University Medicine, Greifswald, Germany (2015)	•Six patients with locally advanced cancer of the oropharynx, presenting with contaminated tumor ulcerations and no lasting remission after curative-intended treatment •CAP treatment administrated in cycles: three sessions per week, followed by a one-week break (1 min/cm2 per session).	•Noticeable odor reduction, indicating decontamination, reduced pain medication demand, and very mild side effects in patients. •Strong therapeutic response to CAP in two patients. •Survival time exceeding 7.5 months in four patients.	[273]
Stage IV metastatic colon cancer	Canady Helios™ Cold Plasma (US Medical Innovations, USA)	Helium	Baton Rouge General Hospital (Bluebonnet campus), Baton Rouge, Louisiana, USA (2015)	•One patient with stage IV metastatic colon cancer and widespread peritoneal carcinomatosis Stage IVb, previously treated with right hemi-colectomy and chemotherapy •CAP treatment applied to surgical margins (diaphragm, abdominal wall, mesentery, left colic gutter, mesenteric area, and splenic bed) at 2 min per treatment area	•No evidence of tumor recurrent at the resected and CAP-treated sites three months post-treatment •No adverse events reported	[274]
Stage IV or recurrent solid tumors after surgical resections	Canady Helios™ Cold Plasma (US Medical Innovations, USA)	Helium	Rush University Medical Center, Chicago, Illinois, USA; Sheba Medical Center, Ramat Gan, Israel (2019)	•Twenty patients with stage IV or recurrent solid tumors following surgical resection •CAP treatment on resected tumor margins	•Response rates at 26 months: R0-MPM 100 %, R0 69 % •Survival rates at 28 months: R0 86 %, R0-MPM 40 %, R1 67 %, R2 0 % •Cumulative overall survival rate at 31 months: 24 %	[46]

Canady et al. [274] reported a trial on CAP treatment for one patient with stage IV metastatic colon cancer in 2015 in Baton Rouge General Hospital, USA. Following tumor resection surgery, CAP treatment was applied to the post-surgical tissues to eliminate potential residual cancer cells. Canady Helios™ Cold Plasma system was used, and this system received the USA Food and Drug Administration (FDA) clearance for the ablation of soft tissue during surgery in 2024 [275]. Encouragingly, a three-month postoperative CT scan of the abdomen and pelvis revealed no evidence of recurrent tumor at the resected and CAP-treated sites, and no adverse events were reported, underscoring the safety and potential efficacy of CAP in post-surgical cancer management.

Another major milestone in CAP cancer therapy was achieved in 2019 when the FDA approved the first CAP clinical trial in the USA. Conducted between July 2019 and April 2021, with a follow-up period ranging from 21 to 32 months [46,276,277], the study involving 20 patients and utilizing Canady Helios™ Cold Plasma to treat stage IV or recurrent solid tumors after surgical resections (ClinicalTrials.gov identifier: NCT04267575) [46]. The trial reported an overall response rate of 69 % for patients who underwent complete tumor removal (R0) and an overall response rate of 100 % for those with microscopic positive margins (R0-MPM) at 26 months. At 28 months, survival rates for R0, R0-MPM, residual microscopic tumor (R1), and macroscopic tumor (R2) patients were 86 %, 40 %, 67 %, and 0 %, respectively. The cumulative overall survival rate was 24 % at 31 months. CAP treatment combined with surgery exhibited safety, selectivity towards cancer, and exceptional recurrence control in R0 and R0-MPM patients. These findings demonstrated that CAP, when combined with surgery, is a safe and selective cancer therapy, offering exceptional local-regional recurrence control, particularly in R0 and R0-MPM patients. The results further support CAP as a promising therapeutic approach to reduce the risk of recurrence, paving the way for future clinical applications in cancer treatment.

7. Outlooks

Over the past two decades, significant advancements in CAP therapy for cancer have been made. Specifically, CAP has been demonstrated to induce various tumor cell deaths in numerous preclinical models, and its close engagement with immunotherapy. A major milestone was in later 2010 when several clinical trials of CAP in cancer therapy were initiated with promising results. These breakthroughs highlight the immense potential of CAP as a novel therapeutic approach, although further investigations are necessary to optimize its clinical application.

CAP offers several distinct advantages over traditional cancer treatments, such as, chemotherapy, radiotherapy, immunotherapy, and other non-invasive modalities, such as photodynamic therapy (PDT), photothermal therapy (PTT), and focused ultrasound (FUS). Unlike these therapies, CAP relies solely on ionized gas for treatment, eliminating the need for administration of drugs, such as chemotherapeutics, radiopharmaceuticals, immunotherapeutics, and photosensitizers, and thereby reducing the risk of associated toxicities [[278], [279], [280], [281], [282], [283], [284], [285]]. CAP also shows the ability to induce selective tumor deaths in multiple types of cancer through modulating its generation parameters [[37], [38], [39], [40], [41]]. Notably, CAP often induces multiple cell death pathways, potentially overcoming resistance mechanisms that limit conventional therapies. Additionally, CAP has been shown to elicit ICD, thereby stimulating anti-tumor immune responses capable of targeting residual or undetectable cancer cells. Additionally, in contrast, other non-invasive modalities often require complex, high-cost infrastructure (e.g., laser and ultrasound inducer) and often induce damage to surrounding tissues, many CAP devices are relatively compact, safe, and easy to operate. It is also worth noting that CAP remodels the tumor microenvironment by not only arousing anti-tumor immunity, but also impacting other non-immune components such as tumor stromal cells and extracellular matrix (ECM) as well. Tumor stroma includes various cell types, such as cancer-associated fibroblasts (CAFs), endothelial cells, and mesenchymal stem cells (MSCs). CAP has been shown to upregulate p53 expression in CAFs, cells capable of promoting tumorigenesis, thus leading to CAF apoptosis or senescence [[286], [287], [288], [289]]. Moreover, CAP can modulate the CAF hierarchy and phenotypic heterogeneity, potentially restoring tumor sensitivity to therapeutic agents [289]. CAP also inhibits the differentiation of MSCs into CAFs, thereby reducing stromal support for tumor progression [289]. Furthermore, CAP impairs angiogenesis by inducing mitochondrial dysfunction in vascular endothelial cells, thereby suppressing their responsiveness to vascular endothelial growth factor (VEGF) signaling [290]. With regards to ECM, studies have shown that ECM may be degraded by reactive oxygen species, suggesting the potential of CAP in altering tumor ECM [76,[291], [292], [293], [294]]. CAP can also enhance the efficacy of chemotherapy and immunotherapy by sensitizing tumor cells to these treatments, demonstrating synergistic therapeutic effects. Its key advantages—safety, minimal invasiveness, multifunctionality, ease of use, and cost-effectiveness—underscore its potential as a transformative cancer therapy.

Despite these promising features, the clinical translation of CAP remains limited by several critical challenges. First, there is a lack of standardized CAP treatment protocols. CAP parameters, including device configuration, CAP composition, treatment duration, and application methods, vary widely across studies, complicating the interpretation and comparison of results. To address this, it is important that all studies comprehensively report relevant parameters, acknowledging that CAP is an emerging modality still undergoing extensive preclinical and clinical optimization. In the meantime, collaborative efforts across institutions and disciplines are needed to establish consensus guidelines and frameworks for comprehensively documenting these details in each study. Details include device architecture, electrode materials and purity, gas composition and flow rate, power sources, and treatment modalities. Computational modeling and machine learning approaches may assist in standardizing and predicting optimal treatment conditions. Second, CAP therapeutic efficacy appears inconsistent across different cancer types, and the underlying molecular mechanisms remain incompletely understood. Comprehensive multi-omics analyses (e.g., genomics, proteomics, metabolomics), coupled with single-cell sequencing and high-resolution imaging, could elucidate CAP's interaction with tumor cells, the microenvironment, and immune components. Improved mechanistic understanding will enable better stratification of patients and refinement of treatment regimens. Third, the long-term safety of CAP therapy requires thorough investigation. Although normal cells generally exhibit greater resistance to oxidative stress, they are not completely immune to CAP-induced damage. Tissues with high metabolic or proliferative activity (e.g., mucosa, gut lining, stem cell niches) could be particularly vulnerable. Repeated CAP exposure may cause cumulative oxidative stress and DNA damage, potentially leading to inflammation and other toxicities. Longitudinal preclinical and clinical studies are needed to monitor these off-target effects. Mitigation strategies should be pursued concurrently. These may include targeted CAP delivery systems (e.g., microneedles, endoscopic probes) to confine exposure to tumor tissues, adjunct antioxidant therapies to protect healthy cells, and biomarker-driven patient selection to personalize treatment and minimize risks—especially in patients with pre-existing inflammatory conditions or oxidative stress sensitivities. Finally, while current clinical trials primarily investigate CAP as an adjuvant therapy—often post-surgical—to prevent recurrence, its potential as a standalone or combinatory treatment remains underexplored. Synergistic use of CAP with chemotherapy, radiotherapy, or immunotherapy (e.g., immune checkpoint inhibitors) could enhance treatment outcomes by amplifying tumor cell immunogenicity. The development of clinically deployable CAP devices with precise dose control, automation, and real-time monitoring will be essential for future clinical integration.

In summary, CAP holds great promise as an innovative and versatile cancer therapy. Its unique ability to selectively target cancer cells, trigger multiple cell death pathways, and activate anti-tumor immunity positions it as a valuable addition to the current therapeutic landscape. While challenges remain, ongoing research, technological advancements, and clinical trials are expected to drive the widespread adoption of CAP in cancer treatment.

CRediT authorship contribution statement

Tianxu Fang: Writing – review & editing, Writing – original draft, Investigation, Conceptualization. Zhitong Chen: Writing – review & editing, Conceptualization. Guojun Chen: Writing – review & editing, Writing – original draft, Funding acquisition, Conceptualization.

Ethics approval and consent to participate

This review does not involve new data collection or human participants. As such, ethical approval and consent to participate are not applicable.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviations

Abbreviation	Full name
•OH	Hydroxyl radicals
1O2	Singlet oxygen
AKT	Protein kinase B
AMPK	Adenosine monophosphate -activated protein kinase
aPD-1	Anti-programmed cell death protein 1
aPD-L1	Anti-programmed cell death ligand 1
ATF4	Activating transcription factor 4
ATG	Autophagy-related
ATM	Ataxia-telangiectasia mutated
ATP	Adenosine triphosphate
CAFs	Cancer-associated fibroblasts
CAP	Cold atmospheric plasma
CCA	Cholangiocarcinoma
CHOP	C/EBP homologous protein
DAMP	Damage-associated molecular pattern
DBD	Dielectric barrier discharge
DDP	Cisplatin
ECM	Extracellular matrix
eIF2	Eukaryotic translation initiation factor-2
EGFR	Epidermal growth factor receptor
ER	Endoplasmic reticulum
FDA	The USA Food and Drug Administration
FE-DBD	Floating electrode dielectric barrier discharge
FSP1	Ferroptosis suppressor protein 1
FUS	Focused ultrasound
GPX4	Glutathione peroxidase 4
GSDM	Gasdermin
GSH	Glutathione
H2O2	Hydrogen peroxide
HA	Hyaluronic acid
HAT	HA-tyramine
HMGB1	High-mobility group box 1
HOXB9	Homeobox B9
ICD	Immunogenic cell death
IS112	6-chloro-7-methyl-3-pentafluoropropanoylchromone
LC3	Microtubule-associated protein 1 light chain 3
MMP	Matrix metalloproteinases
MNPs	Magnetic nanoparticles
MSCs	Mesenchymal stem cells
mTOR	Mechanistic target of rapamycin
NO	Nitrogen monoxide
NO2−	Nitrite
NO3−	Nitrate
NSCLC	Non-small cell lung cancer
O2•−	Superoxide anions
O3	Ozone
ONOO−	Peroxynitrite
PAPB	PLEL biogel
PARP	Poly(adenosine diphosphate-ribose) polymerase
PDT	Photodynamic therapy
PERK	Protein kinase RNA-like ER kinase
PI3K	Phosphatidylinositol 3-kinase
PLEL	(Poly-DL-lactide)-(polyethylene glycol)-(poly-DL-lactide)
PTT	Photothermal therapy
PVA	Polyvinyl alcohol
PVP	Polyvinylpyrrolidone
RNS	Reactive nitrogen species
ROS	Reactive oxygen species
PUMA	P53-upregulated modulator of apoptosis
SLC7A11	Solute carrier family 7 member 11
Sm837	6-methyl-3-(2-fluorobenzoyl)chromone
TAAs	Tumor-associated antigens
TGF-β	Transforming growth factor-beta
TLRs	Toll-like receptors
TMZ	Temozolomide
TNBC	Triple-negative breast cancer
TNL	TRKI-loaded lipid nanoparticles
TRKI	TGF-β receptor kinase inhibitor
ULK1	UNC-51-like kinase 1
VEGF	Vascular endothelial growth factor
VPNS	Violet phosphorene nanosheets
Y1068	Tyrosine 1068 site