Boosting Cancer Immunotherapy: The Immunogenic and Therapeutic Potential of Non-Thermal Plasma in Head and Neck Cancer Hanne Verswyvel
Boosting Cancer Immunotherapy: The Immunogenic and Therapeutic Potential of Non-Thermal Plasma in Head and Neck Cancer Stimulatie van Kankerimmunotherapie: De Immunogene en Therapeutische Kracht van Niet-Thermisch Plasma in Hoofd-Halskanker Proefschrift voorgelegd tot het behalen van de graad van doctor in de Medische Wetenschappen aan de Universiteit Antwerpen te verdedigen door Hanne Verswyvel Faculteit Geneeskunde en Gezondheidswetenschappen Promotoren Prof. Dr. Evelien Smits Prof. Dr. Annemie Bogaerts Dr. Abraham Lin Antwerp 2025
Members of the Jury Promotors Prof. Dr. Evelien Smits Prof. Dr. Annemie Bogaerts Dr. Abraham Lin Internal Jury Prof. Dr. Tim Van den Wyngaert, Antwerp University Hospital/University of Antwerp Prof. Dr. Eva Lion, University of Antwerp External Jury Prof. Dr. Marleen Ansems, Radboud University Medical Center, Nijmegen, NL Prof. Dr. Vandana Miller, Drexel University College of Medicine, Philadelphia, USA Funding Acknowledgment Fonds voor Wetenschappelijk Onderzoek (FWO) was the main funder of this PhD research.
Table of Content List of Abbreviations 4 Introduction CHAPTER 1 9 General Introduction, Objectives and Thesis Outline Results CHAPTER 2 33 Phototoxicity and Cell Passage A ect Intracellular Reactive Oxygen Species Levels and Sensitivity Towards Non-Thermal Plasma Treatment in Fluorescently-Labeled Cancer Cells CHAPTER 3 63 Enhancing Anti-Tumor Immunity in Head and Neck Cancer: NonThermal Plasma as a Promising Adjuvant to Cisplatin-Based Therapy CHAPTER 4 113 E ect of Plasma-Induced Oxidation on NK Cell Immune Checkpoint Ligands: A Computational-Experimental Approach Discussion and Future Perspectives CHAPTER 5 157 The pro- and anti-tumoral properties of gap junctions in cancer and their role in therapeutic strategies CHAPTER 6 201 General Discussion and Project Positioning Summary 219 Samenvatting 224 Curriculum Vitae 228 Dankwoord 237
Abbreviations │ Page 4 List of Abbreviations A APC Antigen-presenting cell Appx Appendix ATP Adenosine tri-phosphate B / C CAR Chimeric antigen receptor CDDP Cisplatin CL Cytoplasmic loop CRT Calreticulin CT Carboxyl terminus CTL Cytotoxic T-lymphocyte CTLA-4 Cytotoxic T-lymphocyte–associated antigen 4 CTOS Cancer Tissue-Originated Spheroid Cx Connexin D DAMP Damage-associated molecular pattern DBD Dielectric barrier discharge DC Dendritic cell DMEM Dulbecco's Modified Eagle Medium E EL Extracellular loop EMA European Medicines Agency EMT Epithelial-to-mesenchymal transition ER Endoplasmic reticulum F FBS Fetal bovine serum FDA U.S. Food and Drug Administration
Abbreviations │ Page 5 FMO Fluorescence minus one G GCU Green calibrated unit GFP Green fluorescent protein GJ Gap junction GJIC Gap junction intercellular communication H HMGB1 High mobility group box 1 (protein) HNSCC Head and neck squamous cell carcinoma HR Hazard ratio HSP Heat-shock protein H2O2 Hydrogen peroxide I ICD Immunogenic cell death ICI Immune checkpoint inhibition IFN Interferon IL Interleukinµ irAEs Immune-related adverse events IS Immunological synapse J / K KIR Killer immunoglobulin-like receptor L LAG-3 Lymphocyte-activation gene 3 M MD Molecular dynamics MFI Mean fluorescence intensity MHC-I Major histocompatibility complex – class I miRNA Micro-RNA MOI Multiplicity of infection N NAD Nicotinamide adenine dinucleotide
Abbreviations │ Page 6 NK Natural killer NKG2A Immune inhibitory receptor natural killer group 2 member A NSCLC Non-small cell lung cancer NTP Non-thermal plasma O / P PBS Phosphate bu ered saline PD-1 Programed death protein 1 PD-L1 Programed death ligand 1 PDO Patient-derived organoid PDT Photodynamic therapy P/S Penicillin/Streptavidin PTM Post-translation modification Q / R RF Reaction field R/M Recurrent and metastatic RMSD Root-mean-square-deviation RMSF Root-mean-square-fluctuation ROS Reactive oxygen species RT-qPCR Real time - quantitative reverse transcription polymerase chain reaction S SASA Solvent accessible surface area SEM Standard error of the mean Suppl Supplementary T TGF-β Transforming growth factor β TIGIT T cell immunoreceptor with immunoglobulin & ITIM domain
Abbreviations │ Page 7 TIM-3 T cell immunoglobulin and mucin-domain containing-3 TME Tumor microenvironment TNBC Triple negative breast cancer TNF-α Tumor necrosis factor α TU Transfection unit U ULA Ultra-low attachment plate UPR Unfolded protein response US Umbrella sampling UV Ultraviolet V VISTA V-domain immunoglobulin suppressor of T cell activation W WHAM Weighted histogram analysis method X / Y / Z / # 2D Two-dimensional 3D Three-dimensional
01 Chapter General Introduction, Objectives and Thesis Outline
Chapter 1 │ Page 10
Chapter 1 │ Page 11 1. GENERAL INTRODUCTION 1.1. The Persistent Challenge of Cancer Management and the Importance of Anti-Cancer Immunity Cancer remains one of the leading causes of death worldwide, with high mortality and quality-of-life impact, due to its aggressive nature, therapy resistance and immune evasion mechanisms[1-3]. Despite advancements in conventional therapies, including surgery, chemotherapy and radiation, many cancer cases remain incurable, especially in the advanced stages of the disease [4-6]. Cancerous growth is a highly persistent and dynamic process with complex interactions between tumor cells, immune cells and stromal components[7]. The tumor microenvironment (TME) is therefore a key determinant in disease progression and treatment outcome[8], and it is the consensus now that successful cancer treatment can not solely rely on direct tumor eradication but must also harness and restore anti-tumor immunity and disarm the TME [9, 10]. The immune system plays a critical role as the first-line defense mechanism, naturally capable of identifying and eliminating abnormal cells through immune surveillance and e ective intervention [11]. This intricate interaction is best captured in the cancer immunity cycle (Figure 1), a multi-step process in which tumor antigens must be released from dying cancer cells, presented by antigenpresenting cells (APCs), and recognized by cytotoxic T lymphocytes (CTLs) to stimulate an e ective immune response [12]. However, tumors can develop immune-evading mechanisms that suppress key aspects of this process, including damping antigen presentation, or abolish immune cell infiltration and cytotoxic activity[13, 14]. Restoring and enhancing anti-tumor immunity has thus become a major focus in oncology, leading to revolutionary breakthroughs in immunotherapy.
Chapter 1 │ Page 12 Figure 1: The cancer immunity cycle with potential sites of intervention discussed in this PhD dissertation. Innate NK cell immunity cycle and the traditional concept of the cancer immunity cycle (T cell-based immunity) as partners in crime. NTP and subsequent immunological e ects can stimulate several crucial steps in this process, ultimately resulting in improved ICI e icacy. Various aspect from this cycle are explained in more detail in the following text. Adapted from Bald et al. (2020)[15] and Mellman et al. (2023)[12]. 1.2. The Strengths and Weaknesses of Immunotherapy Immunotherapy has revolutionized the landscape of cancer treatment by leveraging the patient’s own immunity to eradicate malignancies. Various types of immunotherapy exist, like immune checkpoint inhibitors (ICI)[16-18], adoptive cell transfers (e.g. CAR-T or CAR-NK cell therapies) [19, 20], and cancer vaccines [21]. Among these, ICI targeting Programmed Death (Ligand) 1 (PD-1/PD-L1) and Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) represent a major breakthrough in cancer therapy, highlighted by unparalleled treatment e icacy and durability, and survival benefits across multiple cancers [16, 18]. As a result, they have been established as a first-line treatment in various cases and disease stages [22, 23]. Three main classes of ICIs have received approval from the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) for the treatment of
Chapter 1 │ Page 13 di erent cancer types: PD-1 inhibitors (e.g., Pembrolizumab), PD-L1 inhibitors (e.g., Durvalumab), and the CTLA-4 inhibitor Ipilimumab [22-25]. Immune checkpoints, like the PD-1/PD-L1 axis and CTLA-4, serve as key negative regulators of T cell function, maintaining the delicate balance between pro- and anti-inflammatory signals under homeostatic conditions [26, 27]. However, tumors exploit and overexpress these pathways to evade immune surveillance, e ectively damping the immune system [27]. By blocking these inhibitory signals with monoclonal ICI antibodies, immune tolerance can be prevented or reversed, thereby also allowing for intervention at multiple steps of the cancer-immunity cycle [12]. Herein, ICIs reinvigorate the host immune system to recognize and eliminate tumor cells by enhancing T cell activity at several critical points (Figure 1), including T cell priming and activation in the lymph nodes and during T cellmediated tumor destruction [12, 28]. As T lymphocytes attack and kill tumor cells, this leads to increased tumor cell death and release of additional tumor-associated antigens, so also fueling the principal steps in the front end of the cycle. Despite this success, ICI and other immunotherapies are not universally e ective or suitable for all patients. A major challenge is the overall limited response rate, although highly dependent on the tumor type, with only 15 to 30% of patients initially benefiting from ICI treatment [29, 30]. Additionally, some patients develop acquired resistance, a poorly characterized and highly variable phenomenon across di erent cancer types [31]. Another aspect are the immune-related adverse events (irAEs), a common consequence of ICI, occurring in approximately 90% of patients treated with anti-CTLA-4 and 70% of those receiving anti-PD-1/PD-L1 inhibitors, although in various grades[32]. Furthermore, the combination of both ICI is reported to increase the observed irAEs [32, 33]. As a result, the full potential of immunotherapy remains constrained, highlighting the urgent need to improve its e icacy while reducing toxicity.
Chapter 1 │ Page 14 1.3. Strategies to Tackle Current Limitations of Immunotherapy Both intrinsic and acquired resistance mechanisms, including tumor-driven evasion and compensatory upregulation of (alternative) inhibitory receptors, often limit the long-term e icacy of the current ICIs [29-31]. To address these hurdles, identifying novel immune checkpoint targets has become an interesting strategy to develop new combination therapies and overcome resistance. Novel targets include Lymphocyte activation gene 3 (LAG-3), with the ICI approved for treatment of advanced melanoma (2022)[34, 35], T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT), and VISTA[36-38]. Ongoing research and clinical trials explore the integration of these emerging ICIs with existing agents to enhance e icacy, or as salvage therapy to counteract resistance at disease progression [39, 40]. While these ICIs are primarily focused on harnessing the adaptive immune system, particularly CD8+ T cells, natural killer (NK) cells from the innate immunity also play a key role in anti-tumor immunological responses (Figure 1) [15]. Unlike T cells, NK cell’s activation does not rely on tumor antigen signaling via MHC-I, allowing them to act against tumors with low mutational burden or antigen presentation, two vulnerabilities of T-cell based immunology [41, 42]. On the other hand, NK cells do share considerable similarities with CD8+ T cells, expressing a diverse array of activating and inhibitory receptors (e.g. CD73, TIGIT, and NKG2A)[43, 44], mirroring the known T cell checkpoint concept. Beyond their cytotoxic ability, NK cells can promote the cancer-immunity cycle at multiple levels (Figure 1), including recruitment and maturation of dendritic cells (DCs) and blockage of regulatory T cell di erentiation [15, 45, 46]. Hence, targeting NKs o ers a promising avenue to broaden the scope of immunotherapy, either by directly enhancing NK cellmediated tumor clearance or by combining with existing ICIs to overcome resistance mechanisms.
Chapter 1 │ Page 15 Lastly, another promising, more global approach to improve immunotherapy e icacy is to enhance the immunogenicity of dying cancer cells (Figure 1) [47]. Conventional therapies, like chemotherapy and radiation, often induce immunological silent (apoptotic) cell death, especially in clinical regimes, which attenuates a strong anti-tumor response [48-50]. In contrast, immunogenic cell death (ICD) is a specialized form of regulated cell death that strongly stimulates the immune system by the release of damage-associated molecular patterns (DAMPs) and tumor antigens, leading to DC activation and T-cell priming [51]. If ICD induction could be optimized and strategically leveraged, it could amplify immune responses and improve the e icacy of ICIs and other immunotherapies. 1.4. Mechanisms Behind ICD: The role of Cellular Stress ICD is characterized by the presentation and release of numerous DAMPs, liberated in a controlled and timely manner [51, 52]. These immune-stimulating signalling molecules provide, together with novel tumor antigens, a kick-start to the front end of the cancer immunity cycle [51-53]. This process is typically induced by severe cellular stress, particularly oxidative stress and endoplasmic reticulum (ER) stress, often after treatment application [54]. Malignant growth and reactive oxygen species (ROS) have an intricate relationship, governed by a delicate balance between ROS generation and scavenging [55]. Cancer development and progression thrives under moderate elevations in ROS levels, allowing tumor cells to adapt and maintain a survival benefit via increased cellular metabolism and simultaneously adapted antioxidant system [55, 56]. In contrast, excess levels of ROS cause damage to a spectrum of intracellular structures (e.g., DNA, proteins, and lipids), which can cause cell death [56, 57]. Hence, this ROS adaptation of cancer cells compared to normal cells makes them intrinsically more vulnerable to external stimuli that further increase species production, pushing them over the tolerable threshold and leading to oxidative
Chapter 1 │ Page 16 stress-induced cell death. Moreover, this ROS overload, particularly within the mitochondria and ER, activates the unfolded protein response (UPR), a key stress response pathway that plays a pivotal role in orchestrating ICD as ROS-induced damage intracellularly reaches irreversible levels [58]. This process regulates the tra icking of key DAMPs, such as calreticulin (CRT), to the surface of cancer cells dying in an immunogenic way, thereby ultimately alarming anti-tumor immunity [59]. While the concept is promising, a major challenge lies in developing treatment strategies that can e ectively induce multiple facets of ICD without increasing toxicity. This is particularly crucial for cancer patients with advanced-stage disease and comorbidities, who often have limited tolerance for highly aggressive therapies. In this context, this study investigated non-thermal plasma (NTP) as a promising therapeutic option combining anti-cancer e ects with good tolerability, while simultaneously o ering the potential to induce immunomodulatory responses. 1.5. Non-Thermal Plasma as Novel Anti-Cancer Strategy: Selectivity, Tolerability, and Clinical Potential Non-thermal plasma (NTP), an ionized gas that is considered as the fourth state of matter, is composed of (positively and negatively charged) ions, free electrons, neutral gas molecules, and a multitude of short-lived and persistent reactive oxygen and nitrogen species (ROS/RNS) [60-62]. Unlike thermal plasma devices, such as argon coagulators or plasma scalpels, which generate high temperatures, NTP remains close to room temperature (~21°C) [63, 64]. This appealing characteristic has paved the way for biomedical applications, and initial medical applications could be found in wound healing, decontamination, and dental care [65, 66]. However, in the last two decades, reports about NTP’s potent anti-cancer properties have emerged [64, 67], creating the novel plasma-oncology research field.
Chapter 1 │ Page 17 The therapeutic e ects of NTP are primarily attributed to the generation of a large number of reactive species. These ROS/RNS interact with the exposed cancer cells, triggering direct cytotoxic e ects and initiating downstream signaling events. The resulting oxidative stress activates cellular stress responses, ultimately leading to cancer cell death [68-71]. The particular vulnerability of cancer cells to NTPinduced oxidative stress is hypothesized to originate from their fundamentally altered characteristics, including a reprogrammed cellular metabolism and increased redox balance, which may render them more susceptible to further oxidative damage [56, 57]. These intrinsic di erences between malignant and healthy cells form the basis of the ongoing research e orts to delineate NTP’s potential selectivity for cancer cells. However, early studies often struggled with experimental inconsistencies, such a culturing cancerous and non-cancerous cells in di erent media or comparing cells from unrelated tissue origins [72-74]. To address this, our lab conducted a comprehensive analysis under standardized and comparable conditions, and observed a slight selectivity at lower NTP intensities in specific models (glioblastoma and melanoma) [75]. Given the fact that this e ect disappeared at higher intensities whereby normal cells were equally killed, these findings suggest that an optimal treatment window may exist, but further validation in complex models like patient-derived organoids (PDOs) and in vivo is essential to confirm true treatment selectivity. However, encouragingly, the first clinical case study in HNSCC patients reported no significant acute adverse e ects [76, 77], supporting NTP’s potential as a selective therapy. Two main types of NTP devices are commonly used in biomedical applications: (i) a dielectric barrier discharge (DBD), in which the plasma is directly generated between one electrode and the target tissue, and (ii) a plasma jet, in which plasma is generated in a pen-like applicator and a flow of feed gas directs the ROS/RNS onto the target [78]. Notably, the kINPen® MED plasma jet received clinical approval
Chapter 1 │ Page 18 and has been successfully used in therapeutic settings (CE-certified as a Class IIa medical device by Neoplas med GmbH Greifswald in 2013). This certification, indicating the device’s compliance with safety and performance standards, has primarily led to NTP’s integration into standard clinical practice for non-oncological applications, most profoundly in wound healing. In this field, NTP is already incorporated into therapeutic regimens, particularly for chronic wounds. In here, Germany is one of the forerunners, with reimbursement by healthcare systems already under consideration [79, 80]. Besides its anti-microbial actions and pro-regenerative potential, one of the most appealing aspects of NTP is its excellent safety profile; no significant acute adverse e ects were reported in 25+ clinical trials, with only occasionally a transient, mild sensation of warmth or itching reported in a minority of participants [81-83]. Transitioning from wound healing to oncological applications, early case report studies have explored NTP’s anti-cancer potential, particularly in head and neck squamous cell carcinoma (HNSCC). Pioneering in this is the work by Metelmann et al., who reported on a small cohort of advanced, locally recurrent HNSCC patients treated with the kINPen® MED device. These patients, without therapeutic options, experienced a low-impact treatment that improved quality of life, reduced pain medication needs, and partial tumor remission in some cases (2 out of 6 patients) [77, 84, 85]. Although the treatment setting di ers from wound healing, a retrospective analysis in 20 HNSCC patients with palliative NTP treatment further supported the safety profile of NTP in this set-up. Reported side-e ects were absent, mild or moderate (e.g. bad taste, fatigue); but never severe nor life-threatening [76], thus suggesting it as a well-tolerated therapy for patients with high comorbidities [76, 77, 8486]. The focus on HNSCC as a model investigate NTP is not only practical but also clinically relevant. While the anti-tumoral e ects of NTP have been tested across
Chapter 1 │ Page 19 multiple cancer types (e.g. glioblastoma[87], bladder[67], and pancreatic cancer[88]), it is not surprising that the most significant advancements have been made in tumors that are anatomically accessible, such as melanoma and HNSCC, allowing for direct NTP application. However, beyond these practical considerations, the focus on HNSCC is also driven by the pressing clinical need of advanced patients, who often face limited treatment options and poor prognosis, especially in the recurrent and refractory setting [89]. The unique anatomy of the head and neck region encompasses many delicate structures essential for breathing, speaking, and swallowing, thereby making a highly-localized therapy like NTP very appealing. The direct physical penetration of reactive NTP components is believed to be limited to the micrometer range, supporting the concept of a highly localized and tissue-sparing treatment [90]. Interestingly, biological e ects have been observed far beyond this depth, including shrinkage of much larger subcutaneous tumors after NTP exposure [64, 88]. This suggests that NTP’s therapeutic impact may involve both direct and indirect mechanisms, a research topic that remains crucial to explore for a full understanding of its e icacy and safety. While initial clinical introduction has focused on the palliative setting for patients without further treatment options, I envision a broader role for NTP as a potent therapy addition for advanced HNSCC. Indeed, standard treatments like surgery and radiotherapy are e ective in early-stage, localized disease. However, in more advanced cases, particularly when systemic approaches such as a chemoradiation, or immunotherapy with or without chemotherapy are the first-line approach and complete resection is not immediately an option, NTP could contribute to improved local tumor control, total tumor load reduction, or increased sensitivity to other treatments in both an adjuvant or neo-adjuvant setting. Nevertheless, fully realizing this potential will also require a deeper understanding of NTP’s immunomodulatory e ects, as these could play a crucial role in its integration into modern immune-based treatment strategies.
Chapter 1 │ Page 20 1.6. Non-Thermal Plasma as Novel Strategy for ICD Induction and Immune modulation NTP has been reported to also have immunomodulatory properties, and our lab was the first to identify short-lived reactive species in plasma as major inducers of ICD [62]. In addition, several studies have touched the topic of ICD and immunomodulation via NTP exposure. Illustrative for that, in osteosarcoma, injectable plasma-treated alginate hydrogels triggered ICD hallmarks [68]. In melanoma, enhanced NK cell activity could be demonstrated both in vitro [91] and in vivo [64]. Additionally, in a colorectal cancer model, NTP demonstrated both immune protection in a vaccination setting, and tumor-specific T cell immune activation after the treatment of established tumors in vivo[92]. However, this field needs to be explored more to elucidate its full immunogenic potential. First, it is essential to determine whether NTP's ability to induce ICD remains e ective when combined with less immunogenic standard treatments. Only by demonstrating its robustness and compatibility with existing therapies, NTP will realize its full clinical potential. Given that current standard treatments are wellestablished and relatively successful, I see the clinical potential of NTP in a (neo) adjuvant approach rather than as a replacement of current therapies. Other areas of interest include a more profound understanding of how NTP modulates immune checkpoints and receptors, a promising gateway, as demonstrated previously in our studies on CD47 and CD44 [93, 94]. Moreover, its impact on innate immunity, particularly in activating NK cells, is a crucial factor that needs to be incorporated more broadly to understand NTP’s global immunological profile, rather than focusing solely on adaptive immune responses. Further investigation into these aspects will help position NTP as a complementary adjuvant in cancer treatment, particularly in the context of immune-engaging therapies, establishing its clinical relevance and expanding its potential in cancer immunotherapy.
Chapter 1 │ Page 21 2. RATIONALE, OBJECTIVES AND THESIS OUTLINE NTP represents, considering the established anti-cancer e ects, proven ICD-inducing potential and favorable safety profile, a highly localized and welltolerated therapeutic strategy with the potential to act as an e ective adjuvant to standard-of-care treatments. By enhancing treatment cytotoxicity and tumor immunogenicity, NTP could serve as an innovative approach to bridge conventional and immune-based therapies, ultimately improving treatment outcomes in cancer with high clinical need, like HNSCC. Therefore, I delineated my vision as follows: The main goal of this PhD dissertation was to investigate the immunestimulatory potential of NTP in the context of cancer therapy, with a particular focus on its future integration into combination strategies to enhance tumor immunogenicity and therapeutic e icacy. To achieve this, the following objectives were defined: 1. Emphasize the importance of proper experimental design of ROS-related studies, particularly in the context of immunology and cancer therapy research 2. Investigate the potential of NTP to enhance anti-tumor immunity in a novel NTPCDDP combination strategy for HNSCC 3. Evaluate the immunomodulatory capacities of NTP-induced oxidation on tumor-associated NK cell ligands and their role in shaping the tumor-immune landscape in a computational-experimental approach 4. Establish the importance of intercellular communications and discuss gap junction potential in cancer immunology and therapeutic strategies The chapters are designed to contribute to the central theme of this dissertation - immune stimulation via NTP to improve therapeutic outcomes - going from fundamental observations to mechanistic insights, and therapeutic applications to future opportunities and perspectives (Figure 2).
Chapter 1 │ Page 22 Figure 2: Schematic overview of the thesis outline. This figure shows the contribution and structure of the di erent aspects discussed throughout the PhD dissertation. In chapter 2 (research article), the impact of confounding ROS production in fluorescence-based live cell imaging for experimental read-outs was determined, as it is crucial to ensure accurate experimental design before evaluation of redoxbased therapies like NTP. I found out that stable incorporation of a fluorescent reporter can increase intracellular ROS levels, leading to aberrant cellular behavior and di erences in therapeutic response in the context of NTP exposure. We incorporated this knowledge, and adapted our experimental models according to this for the next chapter, chapter 3 (research article), in which I evaluated the immunogenetic benefit of NTP addition to standard-of-care HNSCC therapy. Using
Chapter 1 │ Page 23 advanced in vitro 3D models, including tumor spheroids and patient-derived organoids (PDOs), I assessed tumor kinetics, treatment cytotoxicity and synergy of the NTP-CDDP combination, followed by studying the immunological impact via DAMP and cyto/chemokine secretion. This in vitro data underlines the beneficial impact of incorporating NTP into conventional cisplatin therapy. Additionally, I demonstrated bona fide ICD induction and immune protection of NTP-CDDP via the gold standard vaccination assay in a syngeneic SCC7-C3H/HeN HNSCC mouse model. As protective immunity via vaccination is majorly driven by adaptive immunity, the goal of chapter 4 (research article) was to shed light on NTP’s immunomodulatory e ects in the context of innate immunity. By employing an unique approach, combining computational (contribution of drs. Pepijn Heirman) and experimental (my contribution) techniques, we explored the impact of NTP-induced oxidation of NK cell ligands on tumor cells. Molecular dynamics and umbrella sampling were employed to predict oxidative changes on the MHC-I complexes HLA-Cw4 and HLA-E, and showed no significant impact on the binding a inity of these markers to their corresponding NK cell receptor, which was also experimentally confirmed. Furthermore, I broadened the scope to other key ligands for NK cell reactivity, and demonstrated both rapid transient chemical interactions as well as triggering of a cascade of downstream cellular responses in NK ligands, including CD155, CD112, CD73 and stress proteins MICA/B, suggesting that NTP can reshape the tumor immune landscape to favor immune recognition. While the first three research chapters focused on several immunomodulatory aspects of NTP, the final chapter 5 (review article) broadens the perspective and describes the role of gap junctions (GJs) in cancer, immunology and therapy. As key mediators of intercellular communication, GJs are intricately linked to various biological processes, from regulating the intracellular redox balance to shaping immune reactions in the tumor microenvironment, and influencing therapeutic
Chapter 1 │ Page 24 responses, several concepts touched upon in the light of NTP in this work. Moreover, GJs have been reported in mediating bystander e ects of conventional treatments such as radiotherapy, raising the question of their potential involvement in similar e ects observed with NTP. It could also be appreciated that understanding the dual pro- vs anti-tumoral role of GJs is needed to provide additional perspectives on optimizing combination therapies and deepen our mechanistic insights into emerging redox-based cancer treatments. Finally, the results of the experimental chapters and novel insights touched upon in the review chapter are combined and discussed extensively in the light of the current literature in the last chapter 6.
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Chapter 1 │ Page 27 37. Walsh, R.J., R. Sundar, and J.S.J. Lim, Immune checkpoint inhibitor combinations - current and emerging strategies. British Journal of Cancer, 2023. 128(8): p. 14151417. 38. Borgeaud, M., et al., Novel targets for immune-checkpoint inhibition in cancer. Cancer Treatment Reviews, 2023. 120: p. 102614. 39. Long, G.V., et al., Overall Survival and Response with Nivolumab and Relatlimab in Advanced Melanoma. NEJM Evidence, 2023. 2(4): p. EVIDoa2200239. 40. Nishizaki, D., et al., Viewing the immune checkpoint VISTA: landscape and outcomes across cancers. ESMO Open, 2024. 9(4): p. 102942. 41. Dhatchinamoorthy, K., J.D. Colbert, and K.L. Rock, Cancer Immune Evasion Through Loss of MHC Class I Antigen Presentation. Front Immunol, 2021. 12: p. 636568. 42. Chen, S., H. Zhu, and Y. Jounaidi, Comprehensive snapshots of natural killer cells functions, signaling, molecular mechanisms and clinical utilization. Signal Transduction and Targeted Therapy, 2024. 9(1): p. 302. 43. Zhang, Q., et al., Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity. Nature Immunology, 2018. 19(7): p. 723-732. 44. Neo, S.Y., et al., CD73 immune checkpoint defines regulatory NK cells within the tumor microenvironment. J Clin Invest, 2020. 130(3): p. 1185-1198. 45. Böttcher, J.P., et al., NK Cells Stimulate Recruitment of cDC1 into the Tumor Microenvironment Promoting Cancer Immune Control. Cell, 2018. 172(5): p. 10221037.e14. 46. Schuster, I.S., et al., “Natural Regulators”: NK Cells as Modulators of T Cell Immunity. Frontiers in Immunology, 2016. 7. 47. Kepp, O., L. Zitvogel, and G. Kroemer, Clinical evidence that immunogenic cell death sensitizes to PD-1/PD-L1 blockade. OncoImmunology, 2019. 8(10): p. e1637188. 48. Asadzadeh, Z., et al., Current Approaches for Combination Therapy of Cancer: The Role of Immunogenic Cell Death. Cancers (Basel), 2020. 12(4). 49. Jiao, Y., F. Cao, and H. Liu, Radiation-induced Cell Death and Its Mechanisms. Health Phys, 2022. 123(5): p. 376-386. 50. van Schaik, T.A., K.S. Chen, and K. Shah, Therapy-Induced Tumor Cell Death: Friend or Foe of Immunotherapy? Front Oncol, 2021. 11: p. 678562. 51. Fucikova, J., et al., Detection of immunogenic cell death and its relevance for cancer therapy. Cell Death & Disease, 2020. 11(11): p. 1013. 52. Martins, I., et al., Molecular mechanisms of ATP secretion during immunogenic cell death. Cell Death & Di erentiation, 2014. 21(1): p. 79-91. 53. Obeid, M., et al., Calreticulin exposure dictates the immunogenicity of cancer cell death. Nature Medicine, 2007. 13(1): p. 54-61. 54. Cao, S.S. and R.J. Kaufman, Endoplasmic reticulum stress and oxidative stress in cell fate decision and human disease. Antioxid Redox Signal, 2014. 21(3): p. 396413. 55. Perillo, B., et al., ROS in cancer therapy: the bright side of the moon. Experimental & Molecular Medicine, 2020. 52(2): p. 192-203.
Chapter 1 │ Page 28 56. Kumari, S., et al., Reactive Oxygen Species: A Key Constituent in Cancer Survival. Biomark Insights, 2018. 13: p. 1177271918755391. 57. Zhao, Y., et al., Cancer Metabolism: The Role of ROS in DNA Damage and Induction of Apoptosis in Cancer Cells. Metabolites, 2023. 13(7). 58. Rufo, N., et al. The “Yin and Yang” of Unfolded Protein Response in Cancer and Immunogenic Cell Death. Cells, 2022. 11, DOI: 10.3390/cells11182899. 59. Rufo, N., et al., The unfolded protein response in immunogenic cell death and cancer immunotherapy. Trends Cancer. 2017;3(9):643–58. 60. Yan, D., J.H. Sherman, and M. Keidar, Cold atmospheric plasma, a novel promising anti-cancer treatment modality. Oncotarget, 2016. 8(9): p. 15977-15995. 61. Lin, A., et al., Toward defining plasma treatment dose: The role of plasma treatment energy of pulsed-dielectric barrier discharge in dictating in vitro biological responses. Plasma Processes and Polymers, 2022. 19(3): p. e2100151. 62. Lin, A., et al., Non-Thermal Plasma as a Unique Delivery System of Short-Lived Reactive Oxygen and Nitrogen Species for Immunogenic Cell Death in Melanoma Cells. Advanced Science, 2019. 0(0): p. 1802062. 63. Moszczyńska, J., K. Roszek, and M. Wiśniewski, Non-Thermal Plasma Application in Medicine-Focus on Reactive Species Involvement. Int J Mol Sci, 2023. 24(16). 64. Lin, A., et al., The e ect of local non-thermal plasma therapy on the cancerimmunity cycle in a melanoma mouse model. Bioengineering & Translational Medicine, 2022. 7(3): p. e10314. 65. Khalaf, A.T., et al., Cold atmospheric plasma (CAP): a revolutionary approach in dermatology and skincare. European Journal of Medical Research, 2024. 29(1): p. 487. 66. Wang, X.-F., et al., Potential e ect of non-thermal plasma for the inhibition of scar formation: a preliminary report. Scientific Reports, 2020. 10(1): p. 1064. 67. Stoof, J., et al., Non-thermal plasma as promising anti-cancer therapy against bladder cancer by inducing DNA damage and cell cycle arrest. Scientific Reports, 2025. 15(1): p. 2334. 68. Živanić, M., et al., Injectable Plasma-Treated Alginate Hydrogel for Oxidative Stress Delivery to Induce Immunogenic Cell Death in Osteosarcoma. Advanced Functional Materials, 2024. 34(14): p. 2312005. 69. Yun, J.H., et al., Non-thermal atmospheric pressure plasma induces selective cancer cell apoptosis by modulating redox homeostasis. Cell Communication and Signaling, 2024. 22(1): p. 452. 70. Babajani, A., et al., Reactive oxygen species from non-thermal gas plasma (CAP): implication for targeting cancer stem cells. Cancer Cell International, 2024. 24(1): p. 344. 71. Mohamed, H., et al. Di erential E ect of Non-Thermal Plasma RONS on Two Human Leukemic Cell Populations. Cancers, 2021. 13, DOI: 10.3390/cancers13102437. 72. Zucker, S.N., et al., Preferential induction of apoptotic cell death in melanoma cells as compared with normal keratinocytes using a non-thermal plasma torch. Cancer Biol Ther, 2012. 13(13): p. 1299-306.
Chapter 1 │ Page 29 73. Guerrero-Preston, R., et al., Cold atmospheric plasma treatment selectively targets head and neck squamous cell carcinoma cells. Int J Mol Med, 2014. 34(4): p. 941-6. 74. Kim, S.J. and T.H. Chung, Cold atmospheric plasma jet-generated RONS and their selective e ects on normal and carcinoma cells. Sci Rep, 2016. 6: p. 20332. 75. Biscop, E., et al., The Influence of Cell Type and Culture Medium on Determining Cancer Selectivity of Cold Atmospheric Plasma Treatment. Cancers, 2019. 11(9): p. 1287. 76. Schuster, M., et al., Side e ects in cold plasma treatment of advanced oral cancer—Clinical data and biological interpretation. Clinical Plasma Medicine, 2018. 10: p. 9-15. 77. Metelmann, H.-R., et al., Clinical experience with cold plasma in the treatment of locally advanced head and neck cancer. Clinical Plasma Medicine, 2018. 9: p. 613. 78. Ho mann, C., C. Berganza, and J. Zhang, Cold Atmospheric Plasma: methods of production and application in dentistry and oncology. Med Gas Res, 2013. 3(1): p. 21. 79. Neoplas med GmbH. Wound treatment with cold plasma: neoplas med achieves milestone for future reimbursement. 2021. Available from: https://www.neoplasmed.com/newsreader/wound-treatment-with-cold-plasma.html 80. Gemeinsamer Bundesausschuss (G-BA). Beschluss über einen Antrag auf Erprobung gemäß § 137e Absatz 7 SGB V. Berlin: G-BA; 2021. Available: https://www.g-ba.de/downloads/39-261-5163/2021-12-02_Antrag-SIRT.pdf 81. Apelqvist J, et al. Cold plasma: an emerging technology for clinical use in wound healing. J Wound Manag. 2024;25(3) 82. Bakker, O., et al., Improved Wound Healing by Direct Cold Atmospheric Plasma Once or Twice a Week: A Randomized Controlled Trial on Chronic Venous Leg Ulcers. Adv Wound Care (New Rochelle), 2025. 14(1): p. 1-13. 83. Abu Rached, N., et al., Cold Plasma Therapy in Chronic Wounds-A Multicenter, Randomized Controlled Clinical Trial (Plasma on Chronic Wounds for Epidermal Regeneration Study): Preliminary Results. J Clin Med, 2023. 12(15). 84. Metelmann, H.-R., et al., Treating cancer with cold physical plasma: On the way to evidence-based medicine. Contributions to Plasma Physics, 2018. 58. 85. Schuster, M., et al., Visible tumor surface response to physical plasma and apoptotic cell kill in head and neck cancer. Journal of Cranio-Maxillofacial Surgery, 2016. 44(9): p. 1445-1452. 86. Smolková, B., et al., Critical Analysis of Non-Thermal Plasma-Driven Modulation of Immune Cells from Clinical Perspective. Int J Mol Sci, 2020. 21(17). 87. Shaw, P., et al., Cold Atmospheric Plasma Increases Temozolomide Sensitivity of Three-Dimensional Glioblastoma Spheroids via Oxidative Stress-Mediated DNA Damage. Cancers (Basel), 2021. 13(8). 88. Brullé, L., et al., E ects of a non thermal plasma treatment alone or in combination with gemcitabine in a MIA PaCa2-luc orthotopic pancreatic carcinoma model. PLoS One, 2012. 7(12): p. e52653. 89. Brockstein BE, Song S. Overview of treatment for head and neck cancer. UpToDate; 2025. Available: https://www.uptodate.com
Chapter 1 │ Page 30 90. Szili, E.J., et al., Tracking the Penetration of Plasma Reactive Species in Tissue Models. Trends in Biotechnology, 2018. 36(6): p. 594-602. 91. Clemen, R., et al. Physical Plasma-Treated Skin Cancer Cells Amplify Tumor Cytotoxicity of Human Natural Killer (NK) Cells. Cancers, 2020. 12, (12) 92. Lin, A.G., et al., Non-thermal plasma induces immunogenic cell death in vivo in murine CT26 colorectal tumors. Oncoimmunology, 2018. 7(9): p. e1484978. 93. Lin, A., et al., Oxidation of Innate Immune Checkpoint CD47 on Cancer Cells with Non-Thermal Plasma. 2021. 13(3): p. 579. 94. Yusupov, M., et al., Oxidative damage to hyaluronan–CD44 interactions as an underlying mechanism of action of oxidative stress-inducing cancer therapy. Redox Biology, 2021: p. 101968.
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02 Chapter Phototoxicity and Cell Passage Affect Intracellular Reactive Oxygen Species Levels and Sensitivity Towards Non-Thermal Plasma Treatment in Fluorescently-Labeled Cancer Cells This chapter has been adapted from my publication in the Journal of Physics: Applied Physics. Hanne Verswyvel, Christophe Deben, An Wouters, Filip Lardon, Annemie Bogaerts, Evelien Smits, and Abraham Lin. “Phototoxicity and Cell Passage Affect Intracellular Reactive Oxygen Species Levels and Sensitivity Towards Non-Thermal Plasma Treatment in Fluorescently-Labeled Cancer Cells.” Journal of Physics D: Applied Physics, 2023. 56(29): p. 294001
Chapter 2 │ Page 34
Chapter 2 │ Page 35 Graphical Abstract
Chapter 2 │ Page 36 Abstract Live-cell imaging with fluorescence microscopy is a powerful tool, especially in cancer research, widely-used for capturing dynamic cellular processes over time. However, light-induced toxicity (phototoxicity) can be incurred from this method, via disruption of intracellular redox balance and an overload of reactive oxygen species (ROS). This can introduce confounding e ects in an experiment, especially in the context of evaluating and screening novel therapies. Here, we aimed to unravel whether phototoxicity can impact cellular homeostasis and response to non-thermal plasma (NTP), a therapeutic strategy which specifically targets the intracellular redox balance. We demonstrate that cells incorporated with a fluorescent reporter for live-cell imaging have increased sensitivity to NTP, when exposed to ambient light or fluorescence excitation, likely through altered proliferation rates and baseline intracellular ROS levels. These changes become even more pronounced the longer the cells stayed in culture. Therefore, our results have important implications for research implementing this analysis technique and are particularly important for designing experiments and evaluating redoxbased therapies like NTP.
Chapter 2 │ Page 37 1. INTRODUCTION Live-cell imaging with fluorescence microscopy is a powerful and convenient method for real-time, high-throughput capturing of dynamic cellular processes and treatment responses. This type of imaging uses in situ time-lapse microscopy to provide spatiotemporal visualization and quantification of living biological samples via light excitation of specific fluorophores [1]. By removing the need for fixation or dissociation of the cell culture, fluorescence live-cell imaging has advantages over other standard read-out and detection methods, including high sensitivity and selectivity, improved resolution and reproducibility, and automation and high-throughput capacities [2]. Furthermore, a wide range of fluorescence labelling techniques (e.g. chemical, enzymatic, protein/peptide tagging) are currently available, enabling the tagging of practically all subcellular compartments and structures [3, 4]. Based on application goals and biological targets, researchers can choose between transient or stable labelling of the sample. While transient labelling is ideal for rapid read-outs of monocultures, permanent incorporation of a reporter (e.g. via viral transduction or transfection), allows for the establishment of a stable fluorescent clone, thus enabling long-term analysis, screening assays, and cell tracking in relevant co-culture models [5, 6]. These appealing properties have facilitated the adoption of this versatile analytical tool in various research fields, including developmental biology, neurology, and oncology [7]. Nevertheless, a major drawback of this technique is the potential for light-induced toxicity, known as phototoxicity, which has often been drastically underestimated [8-12]. Cell cultures maintained in vitro are typically not adapted to coping with repetitive excitation of incorporated fluorophores by common sources of light, thus resulting in additional cellular stress, damage, or even cell death [9, 11]. Disruption of homeostasis by phototoxicity can be a major confounding factor in test samples,
Chapter 2 │ Page 38 which negatively a ects experimental read-outs and data interpretation. Even before visible indications of photodamage have occurred (e.g. membrane blebbing, mitochondrial enlargement, cell detachment), physiological behavior can be subtly altered in response to the induced cellular stress [9]. A key factor in phototoxicity is the generation of reactive oxygen species (ROS) upon energy transfer from the light-excited fluorophore to nearby oxygen molecules [8]. In particular, recent research has shown that blue light, at intensities used to excite common fluorophores like green fluorescence protein (GFP), negatively influenced cell motility, which was directly linked to the production of the ROS, hydrogen peroxide (H2O2) [12]. Furthermore, quantitative real-time polymerase chain reaction (RT-qPCR) analysis demonstrated an upregulation of several antioxidant genes upon illumination [12]. Other studies concordantly reported the link between phototoxicity and redox disturbance with e ects on the cell cycle, mitochondrial fragmentation, and cell migration in di erent cellular models [8, 10]. Therefore, acknowledging the considerable impact of phototoxicity on cellular physiology is critically needed when fluorescence live-cell imaging analysis is employed for various research applications, particularly when it is used to investigate experimental therapies, such as non-thermal plasma (NTP). Live-cell fluorescence imaging has been a valuable tool in the field of plasma medicine for investigating cellular and subcellular NTP treatment e ects for di erent biomedical applications, including wound healing and cancer therapy [1316]. Mounting evidence has demonstrated that the versatile medicinal properties of NTP arise from the diverse short-lived and persistent reactive species (e.g. •OH, •NO, O/O3, H2O2) that are generated [17-19]. ROS can strongly influence cellular physiology via (i) direct interaction with the plasma membrane and biochemical molecules (e.g. proteins, lipids) and/or (ii) interference with the endogenous antioxidant systems responsible for normal redox balance within the cell [20]. Moreover, the concentration of delivered ROS is determinative for biological
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