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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