The First Cold Atmospheric Plasma Phase I Clinical Trial for the Treatment of Advanced Solid Tumors: A Novel Treatment Arm for Cancer

Simple Summary It is estimated that 65% of solid tumor resections result in residual microscopic tumor cells at the surgical margin, which contributes to local recurrence and poor survival despite advancements in cancer therapies. Cold Atmospheric Plasma (CAP), a unique form of physical plasma, has emerged as a promising medical technology. Canady Helios Cold Plasma (CHCP) is a novel CAP device investigated in the first phase I clinical study with the primary goal of demonstrating safety. Promising findings demonstrated the device’s ability to control residual disease and improve patient survival. Ex vivo experiments on patient tissue samples showed CHCP-induced cancer cell death without harming normal cells. These results present CHCP as a safe and effective treatment in combination with surgery, providing a new avenue for controlling microscopic residual cancerous cells at the surgical margin. Abstract Local regional recurrence (LRR) remains the primary cause of treatment failure in solid tumors despite advancements in cancer therapies. Canady Helios Cold Plasma (CHCP) is a novel Cold Atmospheric Plasma device that generates an Electromagnetic Field and Reactive Oxygen and Nitrogen Species to induce cancer cell death. In the first FDA-approved Phase I trial (March 2020–April 2021), 20 patients with stage IV or recurrent solid tumors underwent surgical resection combined with intra-operative CHCP treatment. Safety was the primary endpoint; secondary endpoints were non-LRR, survival, cancer cell death, and the preservation of surrounding healthy tissue. CHCP did not impact intraoperative physiological data (p > 0.05) or cause any related adverse events. Overall response rates at 26 months for R0 and R0 with microscopic positive margin (R0-MPM) patients were 69% (95% CI, 19–40%) and 100% (95% CI, 100–100.0%), respectively. Survival rates for R0 (n = 7), R0-MPM (n = 5), R1 (n = 6), and R2 (n = 2) patients at 28 months were 86%, 40%, 67%, and 0%, respectively. The cumulative overall survival rate was 24% at 31 months (n = 20, 95% CI, 5.3–100.0). CHCP treatment combined with surgery is safe, selective towards cancer, and demonstrates exceptional LRR control in R0 and R0-MPM patients. (Clinical Trials identifier: NCT04267575).


Introduction
Cancer remains a significant global health challenge, with over 1.9 million new cases, and is the second leading cause of death in the US, with 608,570 cancer-related fatalities in 2021 [1,2]. Malignant solid tumors are characterized by high local regional recurrence (LRR) and poor five-year survival rates [3]. Despite advancements in surgery, chemotherapy, radiation, immunotherapy, and molecular-targeted therapy regimes, residual disease can persist and lead to LRR. Today, the two fundamental challenges that remain after macroscopic tumor removal are the complete eradication of residual microscopic tumor cells and the preservation of noncancerous surrounding tissue.
Surgery remains a primary treatment for most solid tumors. However, complete removal (R0) of cancerous tissue is challenging, and residual microscopic tumors (R1) or macroscopic tumors (R2) at the surgical site can lead to LRR and poor prognosis. Solid tumor LRR for microscopic positive margins at the surgical site ranges from 5% to 65% [4]. Negative pathology findings do not guarantee the absence of LRR, which often requires re-operation, radiation, chemotherapy, or biological agents [5,6].
Plasma is the fourth state of matter formed by ionizing a neutral gas, i.e., (argon, helium) with electromagnetic fields [7]. High-temperature argon plasma coagulation has been used in endoscopy and surgery for over 40 years [8,9]. Cold atmosphere plasma (CAP), a non-equilibrium ionized gas composed of many different species, has made significant progress in clinical medicine since its introduction by physicists over 10 years ago [10,11]. Three-dimensional non-contact bioelectric pulse electromagnetic fields created by CAP cause a biophysical phenomenon, "Irreversible Electroporation (IRE)", that increases cancer cell membrane permeability to CAP species such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), resulting in apoptosis [12,13].
We report the first Phase I FDA-approved IDE clinical trial for the application of CAP in combination with surgical resection for the eradication of residual microscopic tumor cells.
(RUMC), Chicago, IL, USA, and Sheba Medical Center (SMC), Tel HaShomer, Israel. Mo lecular biological studies were performed at the Jerome Canady Research Institute for Ad vanced Biological and Technological Sciences (JCRI-ABTS), Takoma Park, MD, USA. Th trial was conducted in accordance with International Council for Harmonization Goo Clinical Practice guidelines, and all patients provided written informed consent prior t enrollment. The authors vouch for the completeness and accuracy of the data and for th fidelity of the study to the protocol. pre-operative, intra-operative, and post-operative treatments. The patients included in the trial un derwent multiple rounds of chemotherapy and radiation before being considered for Canady Helio Cold Plasma (CHCP) treatment. FDA approval was specifically granted for the use of CHCP at th surgical margins after macroscopic removal of solid tumors in patients with Stage 4 metastatic o recurrent solid tumors. Patient selection was based on a comprehensive evaluation conducted by multi-disciplinary team at each institution. Eligible patients had a treatment history including chem otherapy, radiation, immunotherapy, surgery, and Hyperthermic Intraperitoneal Chemotherap (HIPEC). Notably, these patients would not traditionally have been offered surgery due to the ad vanced stage of their disease. The primary objective of the CHCP trial was to demonstrate the safet of the treatment. Achieving a complete (R0) or microscopic residual (R1) surgical resection wit CHCP treatment was to decrease the likelihood of local regional recurrence (LRR).

Patients
Inclusion criteria: Stage IV or recurrent solid tumors, male and female patients ≥1 years, biopsy verified histopathology or cytology diagnosis of a malignant solid tumor a defined by the World Health Organization (WHO) or by cross-sectional imaging reviewe by a board-certified radiologist, good performance status (Eastern Cooperative Oncolog Group (ECOG) ≤ 2, Karnofsky > 60% and American Society of Anesthesiology (ASA)) scor of ≤3, and scheduled for complete surgical resection. Additional eligibility criteria can b found at ClinicalTrials.gov identifier: NCT04267575.
Prior treatment evaluation: History and physical examination, baseline review, Ches X-ray, MRI or CT of the tumor site, and Chest and Abdominal CT and PET scan whe appropriate. Surgical respectability and postoperative chemotherapy, radiation, or immu notherapy were determined between the surgeon and multidisciplinary managemen team. pre-operative, intra-operative, and post-operative treatments. The patients included in the trial underwent multiple rounds of chemotherapy and radiation before being considered for Canady Helios Cold Plasma (CHCP) treatment. FDA approval was specifically granted for the use of CHCP at the surgical margins after macroscopic removal of solid tumors in patients with Stage 4 metastatic or recurrent solid tumors. Patient selection was based on a comprehensive evaluation conducted by a multi-disciplinary team at each institution. Eligible patients had a treatment history including chemotherapy, radiation, immunotherapy, surgery, and Hyperthermic Intraperitoneal Chemotherapy (HIPEC). Notably, these patients would not traditionally have been offered surgery due to the advanced stage of their disease. The primary objective of the CHCP trial was to demonstrate the safety of the treatment. Achieving a complete (R0) or microscopic residual (R1) surgical resection with CHCP treatment was to decrease the likelihood of local regional recurrence (LRR).

Patients
Inclusion criteria: Stage IV or recurrent solid tumors, male and female patients ≥18 years, biopsy verified histopathology or cytology diagnosis of a malignant solid tumor as defined by the World Health Organization (WHO) or by cross-sectional imaging reviewed by a board-certified radiologist, good performance status (Eastern Cooperative Oncology Group (ECOG) ≤ 2, Karnofsky > 60% and American Society of Anesthesiology (ASA)) score of ≤3, and scheduled for complete surgical resection. Additional eligibility criteria can be found at ClinicalTrials.gov identifier: NCT04267575.
Prior treatment evaluation: History and physical examination, baseline review, Chest X-ray, MRI or CT of the tumor site, and Chest and Abdominal CT and PET scan when appropriate. Surgical respectability and postoperative chemotherapy, radiation, or immunotherapy were determined between the surgeon and multidisciplinary management team.

Safety Assessment
The primary endpoint was safety. Intra-operatively, continuous monitoring of blood pressure (BP), pulse, body temperature, End Tidal CO 2 , and O 2 saturation was performed while also recording the CHCP beam temperature using a Forward Looking InfraRed  Figure S1). Biopsies of CHCP-treated margins of normal tissues were sent to RUMC or SMC Pathology Department for the detection of tissue damage. Patients were evaluated post-operatively up to 30 days from discharge according to Common Terminology Criteria for Adverse Events (CTCAE, version 4.03 to 5.0).
The secondary endpoint was sustained clinical complete response 12 months after CHCP combined with surgery. Pathological complete response was defined as the absence of residual cancer on the histologic examination of surgical specimens based on Response Evaluation Criteria in Solid Tumors (RECIST) [34]: Progressive disease, stable disease, partial response, near complete response, or complete response.
Overall Response Rate (ORR) is defined as no recurrence from the time of CHCP treatment to LRR in R0 resected patients. ORR was evaluated by postoperative T2 weighted MRI, PET, or CT scans. LRR-free survival includes events of LRR or death from the time of CHCP treatment. Overall Survival (OS) was defined as the duration from the intraoperative CHCP treatment to death.

Pathology
H&E staining: Fresh tissue specimens with/without ex vivo CHCP treatment were fixed in 10% neutral buffered formalin for 24-48 h, followed by dehydration in graded ethanol solutions and clearing with xylene and embedded in paraffin wax. Thin sections (6-7 µm thick) were obtained and underwent H&E staining involving deparaffinization, hematoxylin staining, differentiation, eosin staining, dehydration, and mounting. The stained slides were analyzed under a light microscope to evaluate cellular structures, tissue architecture, and pathological changes.
Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) Assay: Formalin-Fixed Paraffin-Embedded (FFPE) sections were deparaffinized using xylene and graded ethanol solutions and rehydration with distilled water. The TUNEL reaction mixture (Abcam, Cambridge, MA, USA), composed of the TUNEL enzyme and nucleotide mixture, was applied to the tissue sections, followed by incubation at 37 • C. Chromogenic development involved washing the sections and applying enzyme-conjugated substrate solution diaminobenzidine (DAB). Sections were counterstained with a methylene green, followed by dehydration, and mounted for analysis.

Primary Culture
Ex vivo CHCP-treated tumors samples were dissociated and processed using Miltenyi Biotec GentleMACS Dissociator (Miltenyi Biotech, Gaithersburg, MD, USA), and tumor cells were isolated using the Tumor Cell Isolation Kit (Miltenyi Biotech, NO. 130-108-339, Gaithersburg, MD, USA). The tissues were sliced into small fragments (2-4 mm 3 ) in RPMI-1640 medium (Life Technologies, Grand Island, NY, USA) supplemented with Penicillin-Streptomycin (100 IU/mL) (Life Technologies, Grand Island, NY, USA) and Amphotericin B (0.25 ug/mL) (Life Technologies, Grand Island, NY, USA) followed by filtration and centrifugation. Cell pellets were plated on collagen-coated culture plates in DMEM-F12 (Life Technologies, Grand Island, NY, USA) or RPMI-1640 supplemented with 10% fetal bovine serum (Sigma 12306C, Saint Louis, MO, USA) and incubated at 37 • C with 5% CO 2 for 3 to 16 days. The cells were imaged and counted for analysis. Cell count or growth area was plotted as Mean ± Standard Error of the Mean (SEM) (n = 3).

Quantitative Confocal Immunofluorescence Analysis
FFPE sections were deparaffinized and permeabilized with permeabilization buffer (0.2% Triton X-100, Milwaukee, WI, USA in PBS) and blocked with a blocking (5% bovine serum albumin (BSA) (Sigma 12306C, Saint Louis, MO, USA)). Fluorescently labeled primary antibodies (specific CD44 and BID) (Cell Signalling, Danvers, MA, USA) at dilution of 1:100 in dilution buffer were incubated with the samples overnight. After washing thoroughly, the samples were then mounted with DAPI mounting media (Vector Laboratories, Inc., Newark, CA, USA) The stained sections were imaged under confocal microscope, high-resolution images were acquired, and fluorescence intensity was quantified.

Patient Demographics and Characteristics
Five patients were treated at SMC, and 15 patients at RUMC. Patient details are shown in Table 1.

Post-Operative Adverse Events
There were no adverse events related to CHCP treatment. Two patients had surgical complications (Clavien Dindo Grade III and IIIb); refer to Table 1.

Histopathology Evaluation
RUMC and SMC Pathology Department reported no thermal damage or histological changes in 19/21 (90%) samples of ex vivo CHCP-treated normal tissue. Two samples demonstrated thermal damage, but a Bovie electrocautery device was used to dissect the tissue prior to CHCP treatment.

Histopathology Findings
A total of 12 patients (60%) achieved R0 status. Tissue specimens were collected as illustrated in Figure 3A: Tumor (resected tumor tissue), Zone 0 (surgical margin), Zone 1 (1 cm away from the surgical margin), and Normal (surrounding normal tissue of the tumor). Resected tissues were CHCP treated ex vivo. The electroporation and cellular mechanism of CHCP in cancer cells is illustrated in Figure 3B,C and reported in our previous studies [30,33]. H&E stained tissue samples demonstrated CHCP-induced tumor cell

Histopathology Evaluation
RUMC and SMC Pathology Department reported no thermal damage or histological changes in 19/21 (90%) samples of ex vivo CHCP-treated normal tissue. Two samples demonstrated thermal damage, but a Bovie electrocautery device was used to dissect the tissue prior to CHCP treatment.

Apoptotic DNA Damage Analysis by TUNEL Assay
Ex vivo CHCP-treated samples were analyzed for apoptosis by TUNEL assay, and positive cells were measured (Figure 4). Less than 1% of the untreated and treated normal cells showed TUNEL staining (p > 0.05). CHCP-treated tumor tissue showed a significant increase in TUNEL-positive cells compared to untreated tumor tissue (54-88%), demonstrating CHCP-induced DNA damage and apoptosis in cancer cells (p < 0.001). A complete comparison table demonstrating all patient tissue samples with TUNEL staining of CHCP-treated and untreated tumor samples is available in Supplementary Figure S6.

Confocal Immunofluorescence Analysis
Quantitative confocal immunofluorescence analysis showed that tumor stem cell marker CD44 was significantly decreased, and the apoptotic marker BID protein

Apoptotic DNA Damage Analysis by TUNEL Assay
Ex vivo CHCP-treated samples were analyzed for apoptosis by TUNEL assay, and positive cells were measured (Figure 4). Less than 1% of the untreated and treated normal cells showed TUNEL staining (p > 0.05). CHCP-treated tumor tissue showed a significant increase in TUNEL-positive cells compared to untreated tumor tissue (54-88%), demonstrating CHCP-induced DNA damage and apoptosis in cancer cells (p < 0.001). A complete comparison table demonstrating all patient tissue samples with TUNEL staining of CHCPtreated and untreated tumor samples is available in Supplementary Figure S6.

Confocal Immunofluorescence Analysis
Quantitative confocal immunofluorescence analysis showed that tumor stem cell marker CD44 was significantly decreased, and the apoptotic marker BID protein expression was increased in CHCP-treated tumor samples compared to untreated tumor samples ( Figure 5). Significant morphological alterations such as shrinkage or fragmentation in the nuclear morphology of cells in the CHCP-treated tumor tissues were observed, suggesting chromatin destabilization during apoptosis. expression was increased in CHCP-treated tumor samples compared to untreated tumor samples ( Figure 5). Significant morphological alterations such as shrinkage or fragmentation in the nuclear morphology of cells in the CHCP-treated tumor tissues were observed, suggesting chromatin destabilization during apoptosis.

Gene Expression Analysis
CHCP-treated tumor samples were analyzed for gene expression using real-time qPCR to evaluate 111 genes related to apoptosis, oxidative stress, immune checkpoint, Tcell regulation, and tumor promotion. Complete details of the study, including a table of differentially regulated genes, are provided in Supplementary Table S1.

Regulation of Protein Expression
CHCP-treated and control tissue samples were analyzed using Western blot for the expression of apoptotic protein markers (BAD, BCL2, PUMA, and BID). Some cancer types exhibited significant up-regulation of pro-apoptotic proteins and down-regulation of the anti-apoptotic marker BCL2 following CHCP treatment. Supplementary Figure S7 provides detailed data and analysis.

Discussion
The LRR rate for solid tumors remains as high as 65%, even in the absence of distant disease [4]. Historically, stage IV solid tumors are typically not resected due to the high risk of palliative surgery [35]. We demonstrated that CHCP treatment in combination with surgery is a viable option for controlling LRR and possibly improving overall survival.
CHCP is a safe and potentially effective treatment option for a wide range of Stage IV or Recurrent solid tumors. Although R0 resection was achieved in 60% of patients (n = 12), microscopic tumor cells were identified at the surgical margin site (Zone 0) in 5 patients (R0-MPM, 42%) (R0004, R0005, R0009, R0011, and R0012). Despite the OS rate of 40% at 28 months observed in R0-MPM patients, none of the patients exhibited any signs of recurrence (LRR) during the follow-up period. The absence of LRR in these five R0-MPM patients following the CHCP therapeutic approach could completely eradicate undetectable microscopic tumor cells at the surgical margin site, independent of the cancer type.
Ex vivo observations of H&E and TUNEL staining showed significant cell death at Zone 0. Molecular and cell biology analyses demonstrated that CHCP treatment induced

Gene Expression Analysis
CHCP-treated tumor samples were analyzed for gene expression using real-time qPCR to evaluate 111 genes related to apoptosis, oxidative stress, immune checkpoint, T-cell regulation, and tumor promotion. Complete details of the study, including a table of differentially regulated genes, are provided in Supplementary Table S1.

Regulation of Protein Expression
CHCP-treated and control tissue samples were analyzed using Western blot for the expression of apoptotic protein markers (BAD, BCL2, PUMA, and BID). Some cancer types exhibited significant up-regulation of pro-apoptotic proteins and down-regulation of the anti-apoptotic marker BCL2 following CHCP treatment. Supplementary Figure S7 provides detailed data and analysis.

Discussion
The LRR rate for solid tumors remains as high as 65%, even in the absence of distant disease [4]. Historically, stage IV solid tumors are typically not resected due to the high risk of palliative surgery [35]. We demonstrated that CHCP treatment in combination with surgery is a viable option for controlling LRR and possibly improving overall survival.
CHCP is a safe and potentially effective treatment option for a wide range of Stage IV or Recurrent solid tumors. Although R0 resection was achieved in 60% of patients (n = 12), microscopic tumor cells were identified at the surgical margin site (Zone 0) in 5 patients (R0-MPM, 42%) (R0004, R0005, R0009, R0011, and R0012). Despite the OS rate of 40% at 28 months observed in R0-MPM patients, none of the patients exhibited any signs of recurrence (LRR) during the follow-up period. The absence of LRR in these five R0-MPM patients following the CHCP therapeutic approach could completely eradicate undetectable microscopic tumor cells at the surgical margin site, independent of the cancer type.
Ex vivo observations of H&E and TUNEL staining showed significant cell death at Zone 0. Molecular and cell biology analyses demonstrated that CHCP treatment induced differential expression of pro-apoptotic and anti-apoptotic genes. These findings provide compelling evidence of CHCP-induced apoptosis in cancer cells, underscoring its potential to eliminate microscopic tumor cells at the surgical margin.
The 26-month non-LRR rate of R0 and R0-MPM patients reached 57% and 80%, respectively. There was no significant difference in OS rate between R0, R0-MPM, and R1 patients at 28 months. Furthermore, overwhelming significance in OS was observed when comparing R0, R0-MPM, R1, and R2 resection, with rates of 86%, 40%, 67%, and 0% at 28 months, respectively. These data underscore the heightened therapeutic efficacy of CHCP treatment when combined with R0, R0-MPM, and R1 surgical resection. Consequently, our study highlights the potential of expanding CHCP treatment to encompass less invasive solid tumors, as it offers promise for enhancing local control.
CHCP is a non-invasive and non-contact electroporation device that creates PTEF™, ROS, RNS, and other species which can be personalized to the patient's cancer or subtype. CHCP treatment takes only 5-7 min intra-operatively and preserves the surrounding noncancerous tissue with no side effects in contrast to contact-based electroporation devices such as Optune (Novocure), NanoKnife (Angiodynamics), and Aliya System (Galvanize Therapeutics). CHCP seamlessly integrates into existing standard-of-care protocols without adding extra burden or disrupting the patient's overall treatment regimen.
The JCRI-ABTS laboratory discovered two mechanisms underlying the effects of CHCP-generated electromagnetic fields on membrane permeability: electroporation and alteration of membrane lipid structure [33], consistent with previous studies on CAP [12,13]. We provide valuable insight into the biological effects of PTEF™ and the mechanisms underlying its ability to induce membrane permeability in living cells [36][37][38].
Furthermore, our laboratory discovered that CHCP-treated cancer cells undergo histone mRNA oxidation and degradation, leading to the upregulation of DNA damage response genes and apoptosis [30]. Compared to normal cells, cancer cells have frequent cell cycles and a greater percentage of cells in the S-phase, and higher levels of intracellular ROS and RNS [39]. Additional CAP-generated species may overwhelm the system and switch the ROS and RNS effect from tumor-promoting to tumor-suppressing [40].
Studies on CAP treatment of various cancer types have demonstrated the abscopal effect in tumors, suggesting its potential to induce an immune response by releasing cytokines and DAMPs from apoptotic cancer cells [41]. However, immunosuppressants may hinder this effect, possibly contributing to early LRR in patient R0013 with a history of immunosuppressant use.
We acknowledge the study limitation of 20 patients, and a phase II study will be conducted with a larger racially, ethnically diverse cohort of patients to determine CHCP efficacy on different cancer molecular subtypes.

Conclusions
This clinical trial opens an important window into the potential use of this technology following Stage IV solid tumor cytoreduction and primary tumorectomy. These results highlight the safety, selectivity, and significant reduction of local regional recurrence achieved through the synergistic approach of CHCP treatment and surgery. Our comprehensive findings underscore the remarkable capacity of CHCP to eradicate the possibility of recurrence, even when microscopic tumor cells are detected at the surgical margin site in R0 patients. The combination of CHCP potency on cancer cells and the benign interaction with normal tissue renders it an exceptionally enticing therapeutic approach for addressing surgical margins, thereby mitigating the risk of recurrence.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cancers15143688/s1, Figure S1: CHCP Treatment Temperature Measurements; Figure S2: Patient vital signs during surgery; Figure S3: Patient vital signs during surgery; Figure S4: Histopathology of Patient Tissue Samples; Figure S5: Primary Cell Culture of Patient Tumor Samples; Figure S6: TUNEL Assay Analysis; Figure S7: Western blot analysis for apoptotic markers after CHCP treatment on patient tumor samples; Figure S8: Tumor Cell Death at the surgical margin; Table S1  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available in this article (and supplementary material).