key: cord-0688570-ic6uz5se authors: Abramczyk, Halina; Surmacki, Jakub title: The effect of COVID-19 mRNA vaccine on human lung carcinoma cells in vitro by means of Raman spectroscopy and imaging date: 2022-05-24 journal: bioRxiv DOI: 10.1101/2022.01.24.477476 sha: aef0768d45ff09a3d1dc7f31659db0676aa76ff4 doc_id: 688570 cord_uid: ic6uz5se The effect of COVID-19 mRNA vaccine on human lung carcinoma epithelial cells (A549) in vitro as a convenient preclinical model has been studied by means of Raman spectroscopy and imaging. The paper focuses on Raman imaging as a tool to explore apoptosis and oxidative phosphorylation in mitochondrial dysfunctions. The Raman results demonstrate alterations in the oxidation-reduction pathways associated with cytochrome c. We found that the COVID-19 mRNA vaccine downregulates the concentration of cytochrome c upon incubation with tumorous lung cells. Concentration of oxidized form of cytochrome c in mitochondria of lung cells decreases upon incubation the COVID-19 mRNA vaccine. Lower concentration of oxidized cytochrome c in mitochondria illustrates lower effectiveness of oxidative phosphorylation (respiration), reduced apoptosis and lessened ATP production. Moreover, mRNA vaccine increases significantly de novo lipids synthesis in lipid droplets up to 96 hours and alterations in biochemical composition. It seems that lipid composition of cells returns to the normal level for longer incubation time (14 days). In cell nucleus the mRNA vaccine does not produce statistically significant changes. The observed alterations in biochemical profiles upon incubation with mRNA vaccine in the specific organelles of the tumorous lung cells are similar to those we observe for other types of cancers, particularly brain glial cells. The pandemic outbreak in 2019 generating acute respiratory syndrome caused 494 587 638 con-32 firmed cases of COVID-19, including 6 170 283 deaths, reported to WHO [1] . Pharmaceutical com- 33 panies including Pfizer/BioNTech in 2020 prepared vaccines based on mRNA technology. The 34 Pfizer/BioNTech vaccine (BNT162b2) was reported to be more than 90% effective against COVID-35 19 [2] . As of 5 April 2022, a total of 11 250 782 214 doses have been administered [1] . 36 The pandemics has witnessed an explosion in research examining the interplay between the 37 immune response and the intracellular metabolic pathways that mediate it. Research in the field of 38 immunometabolism has revealed that similar mechanisms regulate the host response to infection, 39 autoimmunity, and cancer. The new method of Raman imaging presented in this paper contributes 40 to better understand pathways of our immune responses, recognize metabolites that regulates these 41 pathways and suggest how to optimize mRNA technology to stimulate the adaptive immune sys-42 tem. 43 We will show that the key molecule in immunometabolism is cytochrome c. Cytochrome c 44 plays a role as a key protein that is required in maintaining life (respiration) and cell death (apop-45 tosis). Until now we do not know exactly if cytochrome c itself controls life (respiration) and death 46 (apoptosis) mechanisms or rather these are other mechanisms caused by release of cytochrome c 47 from the mitochondria to cytoplasm. 48 Dysregulation of oxidative phosphorylation and apoptosis in cells of the immune system can 49 have essential consequences, which may result in diseases, including cancer and autoimmunity. 50 This paper summarizes our current understanding of the role of cytochrome c in cancer and im-51 mune responses and aims to point out future directions of research. 52 In the paper, we study the effect of COVID-19 mRNA on the human carcinoma lung epithelial 53 cells by using a novel non-invasive tool of Raman imaging. Here we demonstrate that Raman im- 54 aging gives new insight into basic mechanisms of cancer pathology and effect of mRNA vaccines 55 on specific organelles in a single cell in vitro. 56 Raman spectroscopy and imaging provides quantitative and non-invasive monitoring of intra-57 cellular changes without the need of exogeneous labelling. Conventional methods of molecular bi- 58 ology require destroying of cell membranes to extract intracellular components for studying the 59 3 biochemical changes inside specific organelles. In Raman imaging, we do not need to break cells to 60 learn about biochemical composition of intracellular organelles. Visualization of alterations in bio-61 chemical composition in separate organelles is extremely valuable to monitor molecular mecha-62 nisms in cancer development and mechanisms of infections. Until now, no technology has proven 63 effective for detecting concentration of specific chemical compounds in separate living cell orga-64 nelles. Therefore, existing analytical technologies cannot detect the full extent of biolocalization of 65 different chemical compounds inside and outside specific organelles. 66 We will study human cancer lung cells upon incubation with mRNA vaccine. In the view of these results it is important to monitor biodistribution and location of metabolites 88 upon mRNA vaccines in human host cells in vitro and appropriate experimental animal models. 89 Visualization of chemical alterations in single cells upon delivery of mRNA vaccines would help 90 evaluate the efficacy of candidate formulations and aid their rational design for preclinical and 91 translational studies. 92 In this paper we will study the effect of COVID-19 mRNA vaccine on the human lung epithelial 93 cancer cells in the respiratory system. Here, we will show that Raman imaging allows for quantita-94 tive, and non-invasive monitoring biochemical alterations in specific organelles in response to 95 mRNA vaccine. We will compare the effect of mRNA on cancer lung cells with the effect of cancer 96 itself (control). According to our best knowledge the mRNA vaccine has not been clinically tested 97 for cancer patients. Therefore, this contribution will help monitoring responses in host lung cells 98 similar to a viral infection, because the incubation with COVID-19 mRNA vaccine mimics some 99 mechanisms of COVID-19 infection. Of course it has to be remembered that instead of the whole 100 virus, only one protein S, essential for the immune response, is injected without COVID-19 virus 101 replications. 102 We will study human lung cancer cells in vitro (A549) incubated with the COVID-19 mRNA 103 vaccine by Raman imaging. We will monitor the effect of the mRNA vaccine on biodistribution of 104 different chemical components, particularly cytochrome c, in the specific organelles of a cell: nu-105 cleus, mitochondria, lipid droplets, cytoplasm and membrane. In this paper we will explore altera-106 tions in reduction-oxidation pathways related to cytochrome c in human cancer lung cells upon 107 incubation in vitro with COVID-19 mRNA vaccine. 108 We will demonstrate that Raman spectroscopy and Raman imaging are competitive clinical 109 diagnostics tools for cancer diseases linked to mitochondrial dysfunction and are a prerequisite for 110 successful pharmacotherapy of cancer. The strength of our approach is that data on the biology of 111 pulmonary cells is of interest. Patients recovering from COVID-19 sometimes suffer from a vague 112 syndrome of the respiratory system. 117 The human lung carcinoma cell line A549 (ATCC CCL-185) was purchased from American The statistical significance was calculated with the one-way ANOVA using the Tukey test, as-214 terisk * denotes that the differences are statistically significant, p-value ≤ 0.05. Raman spectra can be used as markers of redox status of cytochrome c. 239 The peak at 1584 cm -1 in Figure 2 represents the "redox state Raman marker" of cytochrome c. The similar mechanism may play a key role in adaptive immune system, because it is reported 270 that cytokines play an important role in adaptive immunity [20, 21] . CD4 + helper T cells (Th) can be 271 divided into two subgroups, type I helper T lymphocytes (Thl) and type II helper T cells (Th2) [20] . 272 We provided evidence that the balance between apoptosis and the oxidative phosphorylation 305 In order to check if the observed lower concentration of cytochrome c in mitochondria is related 306 to release of cytochrome c (apoptosis) to cytoplasm we studied the localization of cytochrome c in 307 cytoplasm. Figure 3 shows the effect of the COVID-19 mRNA vaccine on the Raman spectra in cy-308 toplasm of human lung carcinoma (A549) by Raman imaging compared with the control cells. COVID-19) Dashboard | WHO Coronavirus (COVID-19) Dashboard With 421 Vaccination Data Covid-19 vaccines: In the rush for regulatory approval, do we need more data? 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