O-GlcNAcylation in early stages of chronic lymphocytic leukemia: Protocol development for flow cytometry

Article type: Research Article

Authors: Temesfői, Viktória^{a; b} | Molnár, Kinga^a | Kaltenecker, Péter^c | Réger, Barbara^a | Szomor, Árpád^d | Horváth-Szalai, Zoltán^a | Alizadeh, Hussain^d | Kajtár, Béla^e | Kőszegi, Tamás^{a; b} | Miseta, Attila^a | Nagy, Tamás^{a; *} | Faust, Zsuzsanna^{a; f; *}

Affiliations: [a] Department of Laboratory Medicine, Medical School, University of Pécs, Pécs, Hungary | [b] Lab-on-a-Chip Research Group, János Szentágothai Research Center, University of Pécs, Pécs, Hungary | [c] Laboratory of Actin Cytoskeleton Regulation, Institute of Genetics, Biological Research Centre, Eötvös Loránd Research Network (ELKH), Szeged, Hungary | [d] Division of Hematology, 1st Department of Internal Medicine, Medical School, University of Pécs, Pécs, Hungary | [e] Department of Pathology, Medical School, University of Pécs, Pécs, Hungary | [f] Department of Transfusion Medicine, Medical School, University of Pécs, Pécs, Hungary

Correspondence: [*] Corresponding authors: Zsuzsanna Faust and Tamás Nagy, Department of Laboratory Medicine, Medical School, University of Pécs, H-7624 Pécs, Ifjúság útja 13, Hungary. %****␣cbm-32-cbm203049_temp.tex␣Line␣25␣**** Tel.: +36 72 536 120; E-mail: [email protected](Faust)[email protected](Nagy).

Abstract: BACKGROUND: Recent studies proved that metabolic changes in malignant disorders have an impact on protein glycosylation, however, only a few attempts have been made so far to use O-GlcNAc analysis as a prognostic tool. Glucose metabolism is reported to be altered in hematological malignancies thus, we hypothesized that monitoring intracellular O-GlcNAc levels in Rai stage 0-I (Binet A) CLL patients could give deeper insights regarding subtle metabolic changes of progression which are not completely detected by the routine follow-up procedures. OBJECTIVE: In this proof of concept study we established a flow cytometric detection method for the assessment of O-GlcNAcylation as a possible prognostic marker in CLL malignancy which was supported by fluorescence microscopy. METHODS: Healthy volunteers and CLL patients were recruited for this study. Lymphocytes were isolated, fixed and permeabilised by various methods to find the optimal experimental condition for O-GlcNAc detection by flow cytometry. O-GlcNAc levels were measured and compared to lymphocyte count and various blood parameters including plasma glucose level. RESULTS: The protocol we developed includes red blood cell lysis, formalin fixation, 0.1% Tween 20 permeabilisation and employs standardized cell number per sample and unstained controls. We have found significant correlation between O-GlcNAc levels and WBC (R2= 0.8535, p< 0.0029) and lymphocyte count (R2= 0.9225, p< 0.0006) in CLL patients. Interestingly, there was no such correlation in healthy individuals (R2= 0.05664 for O-GlcNAc vs WBC and R2= 0.04379 for O-GlcNAc vs lymphocytes). CONCLUSION: Analyzing O-GlcNAc changes in malignant disorders, specifically in malignant hematologic diseases such as CLL, could be a useful tool to monitor the progression of the disease.

Keywords: Chronic lymphocytic leukemia, O-GlcNAcylation, RL2, immunometabolism, flow cytometry

DOI: 10.3233/CBM-203049

Journal: Cancer Biomarkers, vol. 32, no. 3, pp. 353-362, 2021