Background

Cancer is a devastating disease and the second cause of death in western societies. A particular trait of many types of cancers is their ability to evolve and become refractory to different treatment modalities, such as chemotherapy, radiotherapy and immunotherapy. To this end, novel treatment avenues are utterly needed to target tumours from multiple pathways simultaneously. Among many approaches being investigated in preclinical research, cold physical plasma has gained traction in translational research due to its inherent capacity to deposit many different ROS (reactive oxygen species) directly on the tumour tissue.1

Physical plasma is a partially ionised gas and multicomponent system. Plasmas expel ions, electrons, UV radiation, electric fields, and ROS to a different degree, depending on the type of plasma system and its settings being used.2 Plasma-derived ROS have been shown to limit tumour growth in several animal models, including, e.g. skin cancer,3,4,5 pancreatic cancer6,7,8 and colon cancer.9,5a). The integrity of the mitochondrial membrane potential was sustained. However, this effect was not seen in SK-MEL-28 cells (Fig. S4A), correlating with the findings above (Fig. 4a). The exogenous amino acid supplementation also rescued Panc-1 spheroids in 3D culture 72 h post-treatment (Fig. 5b). These results suggest that a subset of tumour cells activates the expression of genes involved in amino acid transport, thereby leading to a pro-survival phenotype following plasma exposure to attenuate ROS-mediated cytotoxicity.

Fig. 4: Metabolic activity of tumour cells following exogenous amino acid supplementation.
figure 4

Kinetic of metabolic activity and normalised area under the curve (AUC) of plasma-treated SK-MEL-28 (a), Panc-1 (b), and MeWO (c) cells with or without supplementation of exogenous glutamine, valine or tyrosine (200 μM). Data are mean ± SEM derived from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. Statistical analysis was done using one-way ANOVA with Tukey post-test for multiple comparisons. aa amino acid.

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Fig. 5: Exogenous amino acid supplementation inhibits plasma-mediated oxidative stress and viability.
figure 5

a representative images and quantification of viability (sytox green), cellular ROS (CellROX green) and mitochondrial membrane potential (TMRE) of plasma-treated Panc-1 cells supplemented with exogenous glutamine, valine or tyrosine (200 μM). Scale bar is 100 μm. b representative images and quantification of viability (sytox green) in Panc-1 derived spheroids supplemented with exogenous glutamine, valine or tyrosine (200 μM). Scale bar is 500 μm. Data are mean ± SEM derived from three independent experiments with *p < 0.05, **p < 0.01 and ***p < 0.001. Statistical analysis was done using one-way ANOVA with Tukey post-test for multiple comparisons.

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Discussion

Cold physical plasma is an evolving technology employed to target tumour cells by delivering ROS directly to the tumour tissue.24 Since tumour cells are vulnerable to exogenous ROS, physical plasma treatment modalities provide a distinct advantage in the selective elimination of tumour cells.25 However, the ability of ROS to initiate a variety of signalling mechanisms26 and the observed tumour heterogeneity27 necessitates a more detailed understanding of how tumour cells respond to plasma.

Although glucose is the principal source of energy in cells and anti-glycolytic agents augment toxic plasma effects,28 amino acids are an important class of nutrients obligatory for cell survival. While some amino acids have specific biologic functions in metabolic processes, epigenetic regulation, and GSH synthesis,20 the primary physiologic function is to serve as building blocks for protein synthesis.29 Tumour cells employ opportunistic strategies to obtain these metabolic substrates during the exponential growth phase or to bypass therapeutic interventions. Since ROS induce gene expression and modulate protein activity, we employed a two-step screening approach to understand the metabolic signatures in tumour cell lines. The substrate utilisation in tumour cells assessed 1-h post plasma treatment could be attributed to the early post-translational modifications (e.g. phosphorylation, s-glutathionylation and nitration) of various enzymes and proteins of the TCA, glycolysis fatty acid and amino acid pathways.30 However, the mRNA expression analysis was carried out at 6 h post plasma exposure, to include specific gene expression patterns induced by redox-regulated transcription factors.19 Examining these datasets from several tumour cell lines revealed amino acid substrates, and its corresponding membrane transporters play a central role in metabolic reprogramming in tumour cells.

In our study, ASCT2 and SLC3A2 were consistently upregulated in tumour cell lines that exhibited prolonged survival following plasma treatment. The importance of these genes was further validated using a non-transformed HSMC cell line. ASCT2 belongs to the group of system ASC, transporting alanine, glutamine, serine and cysteine along with other aliphatic amino acids.31 SLC3A2 is an essential transmembrane protein critical for activating LAT1/2/3, ASC1, BAT1 and xCT involved in amino acid transport.32,33,34 Supplementation of exogenous ASCT2-specific amino acids (glutamine and valine) led to improved metabolic activity and survival in plasma-treated tumour cells in Panc-1 and MeWo but not in SK-MEL-28 cells. Glutamine is the primary amino acid that drives the TCA cycle and sustains ATP production.35 In the absence of glucose, glutamine-derived fumarate malate and citrate are increased in a glucose independent TCA cycle.36 Vice versa, the pharmacological or genetic inhibition of ASCT2 sensitised the tumour cells to plasma-induced cell death. These results validate that tumour cells are dependent on extracellular substrates for their biosynthetic machinery during stress by activation of respective transporters.

Interestingly, the LAT1-specific amino acid tyrosine also improved the metabolic activity in Panc-1 cells. The qPCR screen did not detect significant changes in LAT1 expression in Panc-1 cells, but there was a significant induction at the protein level following plasma exposure. This could be due to an observed alternate LAT1 isoform (~70 kDa) induced in these cells leading to tyrosine uptake. The increase in metabolic function due to amino acid supplementation also reduced oxidative stress and partially restored mitochondrial membrane potential and viability in Panc-1 cells. However, the comparable effect was not observed in SK-MEL-28 cells as it lacked the induction of transporter expression following plasma exposure. Our screen also identified the consumption of fatty acid and TCA substrates. It has been previously described that amino acids can supply carbon atoms for lipid biosynthesis and the TCA cycle via sustained acetyl-CoA pools.29,37 The maintenance of cellular amino acid pools is also regulated by autophagy, evoked during cellular stress, thereby providing substrates for energy production in tumour cells.38,39 It could be speculated from the current study that uptake of exogenous amino acids could be the first choice to maintain cellular amino acid pools, after which cell may commit to autophagy. The observed metabolic alterations in our study could also have a direct consequence in the tumour microenvironment (TME). Extracellular glutamine supports the differentiation of T cells into TH1 T cells.40 Targeting glutamine metabolism in tumour cells by CB-839/anti-PD-1/PD-L1 led to the depletion of glutamine in the tumour microenvironment (TME), enabling potent anti-tumour immune responses.41 However, further studies are warranted to differentiate amino acid pools derived from exogenous and de novo sources following plasma treatment and their influence in the TME. A previous study highlighted the importance of D-glutamine and D-glutamate metabolism in leukemic cells exposed to cold plasma.42 Investigating plasma-treated myeloma cells, the authors demonstrated in another study a strong enrichment of the β-alanine, propanoate, and linoleic acid metabolism, while the metabolic pathways for alanine, aspartate, glutamate, arginine, and proline were found to be enriched as well, albeit to a lesser extent.43 Our previous study determined that the cystine/glutamate antiporter xCT is induced in tumour cells following plasma exposure, leading to treatment resistance.18 In the current study, we implicate exogenous amino acids in the prolonged survival of some tumour cells. However, the involvement of other amino acid transporters (not included in this study) cannot be ruled out. Apart from this, another recent study suggested the involvement of purine metabolism and Pantothenate and CoA biosynthesis in both non-malignant and malignant cells following plasma exposure in vitro.44

Taken together, there is strong evidence that amino acids play a vital role in metabolic reprogramming in tumour cells. Due to multiple substrate specificities of the amino acid transporters,31 and rampant dosage compensation in the SLC gene family,45,46 single-transport inhibitors are unlikely to be effective against cancer therapy. Hence, combination with plasma and specific transport inhibitors could be useful in the targeting of tumour cells.