Volatile profiling above AML-derived cell culture supernatants and of AML patients’ breath using an eNose as a refined diagnostic and biomonitoring strategy ex vivo and in vivo

Baudrexler, Tobias Georg (2024): Volatile profiling above AML-derived cell culture supernatants and of AML patients’ breath using an eNose as a refined diagnostic and biomonitoring strategy ex vivo and in vivo. Dissertation, LMU München: Medizinische Fakultät

Abstract

Hydrocarbon molecules found in volatile organic compounds (VOCs) are indicative of pathological states and metabolism in both healthy individuals and patients and can be measured by an eNose. VOC analysis can be used to detect disease associated markers. Their role in tumor and immune activation could contribute to monitor antileukemic processes and might offer a refined diagnostic- and biomonitoring tool. VOCs were collected from serum, DC- and MLC culture su- pernatants or from breath masks (directly derived from patients’ exhaled breath). To prove immu- nological (tumor associated) changes in AML patients’ and healthy probands’ VOC samples we correlated cell biological analyzes with the VOC results. After we demonstrated the antileukemic effect of Kit M on whole blood and bone marrow (publication I), we first conducted an ex vivo study (publication II) and then an in vivo study (publication III). 1) To create DC/DCleu, we established dendritic cell cultures (DC cultures) from leukemic and healthy whole blood (WB) either without (Control) or with immunomodulatory Kit M (which con- sists of prostaglandin E1 (PGE1) and granulocyte macrophage colony stimulating factor (GM- CSF). In mixed lymphocyte cultures (MLC cultures), T cell-enriched immunoreactive cells were stimulated using Kit M-pretreated/not pretreated WB including DC/DCleu. Using an intracellular cytokine test (InCyt) and a degranulation assay (Deg), leukemia-specific adaptive and innate im- mune cells were identified. Using a cytotoxicity fluorescence test (CTX), anti-leukemic cytotoxicity was assessed. 2) An eNose was used to assess volatile organic compounds (VOCs) extracted from serum or DC- and MLC culture supernatants (with vs. without Kit M pretreatment and before vs. after cul- ture). Kit M-pretreated leukemic and healthy WB resulted in increased frequencies of (leukemia- derived) DC subtypes of activated and (memory) T cells after MLC compared to the Control (with- out treatment). Additionally, blast-lysing cells were produced by inducing antigen (leukemia)-spe- cific cells of several lines (innate and adaptive immune cells). Between healthy and leukemic patients’ serum, DC and MLC culture supernatant-derived volatile phases, and several superna- tants (with vs. without Kit M treatment, cultured vs. uncultured)-derived VOCs within subgroups (healthy DC or leukemic DC, or healthy MLC or leukemic MLC supernatants) could all be signifi- cantly distinguished by the eNose. Moreover, the eNose could indicate a culture-associated and Kit M-effect. 3) Throughout the course of the disease, we monitored three refractory AML patients receiving various treatments: P1511, who had chemotherapy, acted as a control. Kit M (GM-CSF and PGE1) was used to treat P1482 and P1601. During treatment period these patients were kept under close clinical, hematological and immunological observation. In the course of the disease blood samples were collected and analyzed to monitor (leukemia specific) immune cells using flow cytometry, CSA, and InCyt. VOCs were measured using an eNose after being collected in earloop masks (containing exhaled air from healthy probands and AML patients) in the course of the disease. Treatment with Kit M in vivo was demonstrated to be safe, to increase clinical pa- rameters (neutrophils, thrombocytes, and a reduction of blasts), and to stimulate (leukemia spe- cific) immunological activation; The effects subsided when the Kit M treatment was stopped. It was possible to distinguish between leukemic and healthy VOC profiles from P1511, P1482, and P1601 over the whole observation period. Additionally, there were significant differences in the VOC profiles obtained from healthy vs. AML breath donors and the VOC prints obtained during chemotherapy vs. Kit M therapy. This proof-of-concept-study, based on ex vivo and in vivo results, confirms, that VOCs derived from cell culture supernatants and breath masks using an eNose contribute to be a prospective option for a refined diagnostic tool (healthy vs. leukemia) and help determine a disease-related monitoring strategy.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 16. Dezember 2024
• 1. Bericht­erstatter:in: Helga Maria, Schmetzer