Argon gas plasma-treated physiological solutions stimulate immunogenic cell death and eradicates immunosuppressive CD47 protein in lung carcinoma

Cancer remains one of the most severe and rapidly spreading health problems worldwide. GLOBOCAN2020 revealed 10.0 million cancer deaths and 19.3 million new cancer occurrences [1]. Most of the available cancer therapeutic systems exhibit toxicity to normal tissues, including chemotherapy, radiotherapy, and hormone therapy. Furthermore, drug-induced side effects on various organs, immunosuppression, and drug resistance by cancer tissues emphasize the requirement for safe, effective, compatible, and cheap therapeutic alternatives to treat cancer [2]. Drug exposure damages normal cells while inhibiting the cancerous cells; however, non-cancerous cells can repair themselves [3]. The chemical agents are distributed to normal tissues and may cause severe side effects in patients who are vulnerable or immunocompromised [4,5]. Disease relapse and drug resistance are also major issues that require immediate resolution [4]. Despite various discoveries for efficacious cancer therapy, the urgency to minimize toxicity and develop selective anti-cancer therapy remains unchanged [6,7].

Plasma is defined as a matter consisting of ionized gas comprised of charged particles and neutrons [8,9]. Cold atmospheric plasma (CAP) is a category of plasma where all atoms, ions, and molecules have kinetic energies that are significantly lower than those of electrons, and this property contributes to its ambient temperature [[8], [9], [10]]. Generally, there are two approaches to plasma application in medical research: (1) direct treatment on cells by plasma discharge; and (2) indirect treatment involving exposure of plasma-treated liquids (PTLs) on cells, tissues, or animal models in vivo [8]. PTLs are advantageous over direct treatment for prolonging the shelf life of reactive oxygen-nitrogen species, such as hydrogen peroxide (H2O2), nitrites (NO2−), and nitrates (NO3−) [11]. Moreover, the penetration distance is limited as the plasma-producing reactive species only reach up to (a few layers) 3 mm inside the tissue whereas, PTLs can be directly injected to treat internal solid tumors and blood tumors [12]. Direct plasma treatment on cancer cells has the potential to be therapeutic, however, there is currently no approved plasma device or plasma treatment method for the treatment of cancer [13]. As mentioned above, the use of PTLs has gained interest for selective anti-cancer therapy in various tissue types with minimum toxicity toward normal cells [[11], [12], [13], [14]]. Several pieces of literature evidenced that plasma-activated medium (PAM) could promote apoptotic cell death via DNA damage in several human cancer types [[15], [16], [17]]. Nevertheless, PAM contains various complex constituents, such as fetal bovine serum (FBS) and amino acids which interfere with its efficiency [18] and cannot be utilized in a clinical atmosphere. However, it highlights the significance of indirect plasma treatment for the anticancer effect [19]. PTLs such as phosphate-buffered saline (PT-PBS), saline, and Ringer’s lactate solution (PT-RL) have advantages over cell-specific culture mediums because they can be implemented for medical applications [[18], [19], [20], [21], [22]]. H2O2 is the key species causing cancer cell death; recent research, however, showed that nitrates or nitrites may affect the anti-cancer efficacy [23,24]. Extracellular reactive oxygen and nitrogen species RONS instigate intracellular reactive oxygen species (ROS) generation, which can ultimately induce a cell death mechanism [11,24,25].

Host microevolution has allowed the development of several mechanisms to bypass immunosurveillance [[26], [27], [28]]. One of the promising approaches for effective therapy is to target immunostimulation by causing immunogenic cell death (ICD), as this kind of cell death can provide a long-lasting anti-cancerous immunological response. Optimization of plasma devices to generate exclusive PTLs to initiate ICD can be an efficient strategy to selectively inhibit cancer cells via improving anti-cancer immunity [4,21,[29], [30], [31]]. Cells undergoing ICD release soluble constituents recognized as damage-associated molecular patterns (DAMPs) on the cell surface; these elements encourage immune cells that present antigens to phagocytose cancer cells that are on the verge of death [[32], [33], [34]]. The most common DAMPs allied with ICD are calreticulin (CRT), which transfers to the cellular membrane from the endoplasmic reticulum (ER) to send an “eat me signal” to phagocytic cells [31,35]. Besides that, a molecule released from the nucleus via shuttling called high mobility group box protein 1 (HMGB1) [29]. After being released by dying cells, HMGB1 activates toll-like receptors (TLRs), which leads to immune stimulation. Furthermore, adenosine triphosphate (ATP) is another important DAMP known to send a “find me” signal which stimulates purinergic receptor P2RX7 on dendritic cells DCs [36,37]. This cross-interaction of molecules released by dying and immune cells initiates a cascade of inflammatory mechanisms resulting in protective action against tumor cells. On the surface of tumor cells, the inhibitory receptor CD47, often regarded as a "don't eat me" signal, is expressed, which enables cancer cells to evade immune cells in the TME. It has gained major interest as a target for immune therapy since it communicates with signal receptor protein-alpha (SIPR-α) of phagocytic cells [38]. Moreover, T-cell infiltration subsequently activates immunostimulatory mechanisms in the TME [39]. Macrophages play crucial roles in the TME and are known as tumor-associated macrophages (TAMs). TAMs exhibit distinct mechanisms and phenotypes depending on the signals produced by tumor- and stromal cells in the TME [40]. The generation of ICD in dying cancer cells could turn them into vaccines which further stimulates immunomodulation by enhancing the differentiation, maturation, and collection of immune cells, including cytotoxic T-cells and dendric cells (DCs) [41,42].

The selective anti-proliferative effect of simple, widely used plasma-treated physiological solutions like PT- PBS and PT-RL indicates that normal cells are less vulnerable than malignant cells [[43], [44], [45]]. Excessive ROS modulates the immune system and may inhibit both T cell growth and anticancer activity [35,46]. Thus, it is crucial to explore the collaborative effect of plasma-donated extracellular nitrogen oxide (NO) species along with ROS. Recent studies demonstrated that NO species is an excellent candidate for immunomodulation which stimulates TME by enhancing immune cell infiltration [47]. Consequently, we investigated the cytotoxic effect of plasma-treated physiological liquids; PT-PBS and PT-RL. We correspondingly investigated the effect of PTL treatment on ICD hallmarks in A549 (lung adenocarcinoma) and MDA-MB231 (breast adenocarcinoma) cell lines. Finally, we co-cultured macrophages with these cancer cell lines to assess the immunogenic response conferred by PTLs.

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