key: cord-0978454-yv7k673v authors: Saygideger, Yasemin; Sezan, Aycan; Candevir, Aslıhan; Demir, Burcu Saygıdeğer; Güzel, Efraim; Baydar, Oya; Derinöz, Ezgi; Kömür, Süheyla; Kuşçu, Ferit; Özyılmaz, Ezgi; Kuleci, Sedat; Hanta, İsmail; Akkız, Hikmet; Taşova, Yeşim title: COVID-19 patients’ sera induce epithelial mesenchymal transition in cancer cells date: 2021-05-25 journal: Cancer Treat Res Commun DOI: 10.1016/j.ctarc.2021.100406 sha: 964793cec8097bea1836d324a61f3cdaa1fd9c12 doc_id: 978454 cord_uid: yv7k673v Covid-19 Pneumonia of SARS-CoV-2 pandemic infection, persists to have high disease burden especially in cancer patients. Increased inflammation and thromboembolic processes are blamed to influence cancer patients more than the others but due to lack of knowledge regarding the pathophysiology of the both the virus itself and the response of the host, more basic and translational disease modeling research is needed to understand Cancer-Covid-19 interaction. In this study, serum samples from the patients, who were hospitalized due to Covid-19 pneumonia, applied to different cancer cells and cytotoxicity, motility, proliferation and gene expression analysis were performed. Serum samples derived from healthy volunteers and the fetal bovine serum that is used regularly in cell culture experiments used as controls. Hospitalized Covid-19 patients who had also cancer, were retrospectively screened, and their clinical course were recorded. Overall 12 Patient (PS) and 4 healthy serums (CS) were included in the experiments. PS applied cells showed increased motility in A549 cells as well as lost cell to cell connection in MCF7 and HCT116 cells, and induced expression of VIM, ZEB1 and SNAIL2 mRNA levels. Eight cancer diagnosed patients who were hospitalized due to Covid-19 between April and September 2020 were also reviewed retrospectively, which 5 of them were dead during SARS-CoV-2 infection. Thorax CT images of the 2 patients showed increased metastatic nodules in the lungs as of January 2021. The results of the study indicate that metastasis may be one of the prolonged consequences of COVID-19 pandemic in cancer sufferers. The COVID-19 disease caused by the SARS-CoV-2 infection, which emerged in Wuhan, China at the end of 2019 and affected the whole world throughout 2020, continues to be a multi-unknown disease by the beginning of the second year of the pandemic. As of January 2021, nearly 100 million cases and over 2 million deaths related to COVID-19 have been detected. Disruptions in the diagnosis and treatment process of many diseases, especially cancer due to the pandemic, and even the secondary morbidity and mortality burden are not included in these numbers. The disease progresses with different pictures ranging from asymptomatic carriage to respiratory failure, and the clinical course is relatively severe in patients with comorbidities 1 The patients, who were hospitalized with the laboratory-confirmed diagnose of COVID-19 pneumonia and who gave written consent to be included in the study between March and December 2020 were included in the study. Demographic data including, age, gender, medications, comorbidities, duration of hospitalization, the need of critical care and laboratory results in the first day of admission were recorded. Serum samples were collected on the first 24 hour of the admission of the patients which they all were on the first day of Favipiravir treatment and some of them had also received corticosteroids and oxygen supplementation due to their hypoxia. Collected serum samples were used immediately in the cell culture experiments or froze at -80 0 C no more than 1 week if later use is decided. The records of hospitalized patients who also had cancer as comorbidity were retrospectively screened for their clinical course and prognosis after SARS-CoV-2 infection. Frequently 10 3 cells per-well were seeded in 96 well plates and incubated for overnight in regular growth media. Next day, the old FBS containing media was removed, cells were washed with PBS and treated with increasing concentrations of patient or healthy (control) serum, diluted in serum free RPMI for 24 hours. For cytotoxicity assay, at the end of 24 hours, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) (BioFROXX), dissolved in PBS was added at 0.5mg/mL concentration to each well and incubated for another 4 hours. Then the media was carefully removed and 50µL of ethyl alcohol was added to each well to dissolve the cells. Absorbance was measured immediately at 570nm in a microplate reader (Biochrom EZ read 400). For the proliferation assays, equal amount of patient and health volunteers' serum samples were incubated for 24, 48 and 72 hours and MTT test was performed as described in cytotoxicity protocol at the given time points. A549 cells were allowed growing at 100% confluency in 3cm plates and starved overnight. Next day a scratch was made using a 10µl pipette tip in the center of the plate, rinsed with PBS and treated with indicated concentrations of FBS (as control) or patient serum diluted in serum free RPMI media. Images were obtained at time 0 and 24h. Percent closure of the scratch area was measured using ImageJ (v 1.53, NIH, USA) software. Cells were seeded to 10cm plates in 10% FBS and after they reach 70% confluency, they were overnight starved (incubated with serum-free RPMI) before adding 3% patient serum or control serum. RNA was isolated after 4 hours of incubation using RNA isolation kit (Thermo Scientific, protocol. 18S was used for internal control gene and 2^-(delta delta CT) calculations were performed to determine the fold differences between patient serum treated samples and controls. Prism V8 software (GraphPad, USA) was used for calculations and the design of all graphics. Normalization, logarithmic transformation of the data and non-linear regression-curve fit analysis were used for IC 50 determination. To compare the differences in serum or control treatments in scratch assays, non-parametric two-tailed Mann Whitney test was used, and significance was considered when p<0.05. Committee (2021/108-17). All the participants have given written consent to involve in the study. Serum samples from 12 Covid-19 patients and 4 healthy volunteers were obtained for the in-vitro experiments. The list of patients and control subjects and their related parameters are given in Table- I. The mean age of the patients was 61,16 (12,71), and 83% (n=10) were male, and the mean age of the control group was 42(12), and 75% (n=3) were male, therefore, age and gender matched controls used to compare the results in each individual experiment. We run cytotoxicity tests on bronchial epithelial cells, primary lung fibroblast cells and A549 lung adenocarcinoma cells using 2 different patient samples PS#1 and PS#2 to analyze cell killing activity of the derived serum samples. We have chosen these cell lines in order to evaluate cytotoxicity to cancer cells as well as non-cancerous cells and to decide a minimal study concentration for the experiments, since we would be able to collect small amounts of serum samples from each patients and controls. Concentrations applied to cells were 0%, 0.5%, 1,5%, 3%, 5%, 15%, 30% and 50% per volume, and no significant difference was observed in the results of PS#1 and PS#2, or between the different types of cells (Figure-1) . Viability of the cells were dropped to 50% in 24 hours at around 15% concentration per volume for PS#1 and around 3% concentration for PS#2. Therefore, we decided to use 3% PS as a minimal amount of study concentration for the next experiments. The accumulating data indicate that SARS-CoV-2 induces the regulation of variety of genes to comfort its replication, and one of these genes is ZEB1, which plays an important role in the epithelial to mesenchymal transition (EMT) and has an impact on cell motility and metastasis of various cancers 5 . While the virus itself might trigger this process, the need for prolonged treatment with corticosteroids due to the persistent fibrotic pattern in these patients brings in mind that the stress related mediators, cytokines, cellular components that release from the shredded cells might have contribute to the scenario. Therefore, we designed a motility test using serum sample derived from patient #3 (PS#3), who were also diagnosed with stage IV malign melanoma at the time of Covid-19 diagnose. We compared the motility effect of PS#3 with fetal bovine serum (FBS) on A549 cells and the results revealed that 2% of PS#3 significantly increased closure ratio of A549 cells comparing to 2% FBS (Figure-2) . We obtained serum samples from patients and healthy volunteers and evaluated the difference between the expression of mRNA levels of EMT related genes VIM (Vimentin), ZEB1 and SNAIL in different cancer cell lines. We used age and gender matched control serums for each experiment. increasing the expression of at least one of the EMT related genes (Figure-3) . The baseline CT values are provided in Supplementary Table-1 . We repeated the cytotoxicity tests with 3 more PS samples and no significant difference was observed in the viability of A549 cells (Figure-4) . Only one PS (PS#10) tended to increase proliferation at 20% concentration. We than run a series of proliferation tests to evaluate the prolonged effects of We retrospectively screened Covid-19 patients' files that were hospitalized between April to September 2020 and who were also had diagnosed with cancer. After excluding hematologic malignancies, we recorded 8 patients whom 5 was already death by January 2021. The characteristics of the Covid-19 Cancer patients (CP) are listed in Table-II. For the rest 3 patients, all diagnosed with Covid-19 in August 2020, we had 6 th -month computed tomography (CT) images for two of them with increased pulmonary metastatic nodules (Figure-5) , which one of them is the same patient that serum samples were used in Figure-2 . We could not obtain control CT images of the last CP. In The prolonged effect of the sera on EMT related gene expression is not evaluated in this research which can be counted as another restriction. The blood parameters regarding the inflammation or the clinical courses of the patients, whose sera were used, were not similar in this study suggesting that the cancer patients might become vulnerable to disease progression after having Covid-19 regardless of the severity of the infection. Still, it is hard to generalize these findings to the whole cancer types that every one of them should be considered as different diseases. In this study, we also provided CT images of 2 patients who had advanced stage cancer and infected with SARS-CoV-2 in August 2020 and had increased pulmonary metastasis in early January 2021. Obviously, it is hard to conclude that Covid-19 infection is purely responsible from these increased metastases, but it indicates the need to evaluate a cohort group of cancer patients regarding the increased metastasis. While the diagnosis and treatments are mostly interrupted during the outbreak, cancer patients may suffer more and need urgent attention in the means of routine controls and prevention of therapy delays. SARS-CoV-2 infected cancer patients are suggested to have more severe forms of the disease and more vulnerable to death comparing to the non-cancer patients mostly due to not only the immune suppressive phenotype regarding with cancer itself or chemotherapy treatments, but also the increased age, addition of other comorbid diseases, immobility, poor performance and degreased nutrition 8 . The severity of the Covid-19 disease has been attributed to the pre-existing, pro-inflammatory and immunosuppressing course seen in cancer patients with the influence of immunosenescence, metabolic syndrome and immune suppressing status such as lymphopenia which diminish or cause abnormalities in the immune response [9] [10] . A recent multi-omics study has evaluated more than 120 000 immune biomolecules in different stages of Covid-19 disease and healthy subjects and pictured how immune cells had multifunctional effects on the disease course even without any comorbidity and therefore cancer 11 . Even though it is expected that viral infections are coupled with inflammation and immune response, the increased signaling events, diverse metabolite and cellular particles in the serum and/or plasma of the Covid-19 patients as well as genotype differences between individuals and populations, requires more focus to further understand and overcome the morbidity and mortality of the current pandemics. The results of this study indicate that the integrated approach should be considered to understand the underlying mechanisms in SARS-CoV-2 infected cancer patients. Cancer patients and risk of mortality for COVID-19 Clinical characteristics and risk factors associated with COVID-19 disease severity in patients with cancer in Wuhan, China: a multicentre, retrospective, cohort study Covid-19 and Lung cancer: A greater fatality rate? Lung Cancer Cancer progression in COVID-19: Integrating the roles of reninangiotensin andesterone system, angiopoetin-2, heat shock protein-27 and epithelial mesenchymal transition COVID-19: a potential driver of immunemediated breast cancer recurrence SARS-CoV-2 infection induces EMT-like molecular changes, including ZEB1 mediated repression of the viral receptor ACE2, in lung cancer models The impact of acute inflammation on progression and metastasis in pancreatic cancer animal model Risk factors for Coronavirus Disease 2019 (COVID-19) severity and mortality among solid cancer patients and impact of the disease on anticancer treatment: A French nationwide cohort study (GCO-002 CACOVID-19) The immuno-oncological challenge of COVID-19 Cancer patients and COVID-19: Mortality, serious complications, biomarkers and ways forward Multi-omics resolves a sharp disease-state shift between mild and moderate Covid-19 We thank to CUMERLAB for providing laboratory space for the researchers. All authors declare no support, financial or otherwise, from any organization for the submitted work. YS Involved in the conceptualization of the study, supervised students and wrote the paper, AS, BSDand ED contribute to laboratory data acquisition, EG, OB, FK, SKo, SKu and IH involved in the acquisition of the patient data. AC and EO provided statistical analysis, HA and YT were involved in critical revision of the manuscript. All authors approved the final version of the manuscript. ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: