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	Katharina LEITHNER, MD, PhD Gluconeogenesis in tumor-promoting macrophages
Division of Pumonology, Department of Internal Medicine; Medical University of Graz, Auenbruggerplatz 15, A-8036 Graz phone: +43-316-385 80631, fax: +43-316-385 13578, ✉ e-mail
websites: [RESPImmun] [MUG]
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Katharina Leithner completed her MD in 2000 and PhD in Molecular Medicine in 2014. The main aim of her newly founded research group is to explore why aberrant activation of this metabolic pathway enables cancer cells to grow in their harsh microenvironment and how cancer cell metabolic adaptation can be therapeutically exploited. Furthermore, the group is interested in mechanisms of metabolic co-operation of malignant and non-malignant cells (e. g. leukocytes) in lung tumors in collaboration with Julia Kargl (gene expression profiling) and Ákos Heinemann (characterization of immune cells) and Grażyna Kwapiszewska (molecular signaling).

Project

Project 10: Gluconeogenesis in tumor-promoting macrophages
Co-PI: Jörg Lindenmann

Background

Macrophages are recruited and re-programmed by tumor cells to acquire a pro-tumorigenic phenotype, the so-called M2 phenotype. Cancer cells as well as accompanying non-neoplastic cells, like macrophages, reside in a largely nutrient-poor microenvironment. Adaptation of cancer cells to a decline in glucose concentrations involves the activation of the reverse pathway of glycolysis, gluconeogenesis, to generate crucial biosynthetic intermediates from non-carbohydrate precursors, like lactate, as shown by our group and subsequently by others. The initial gluconeogenesis enzyme, phosphoenolpyruvate carboxykinase (PEPCK, PCK2, the mitochondrial isoform) is functionally expressed in lung cancer cells, enhances tumor cell survival under glucose deprivation and is necessary for growth of lung cancer xenografts beyond a microscopic size. We found abundant PCK2 also in lung cancer-associated macrophages as well as normal, alveolar macrophages, however its function in these cells is unknown.

Hypothesis and objectives

We hypothesize that PCK2 mediates a yet undescribed pathway in M2 polarized macrophages that allows the flexible use of tumor cell-derived lactate and other precursors for biosynthetic purposes. PCK2-dependent biosynthetic activity might be critical for the tumor-promoting phenotype of macrophages in a nutrient-deprived environment and might be required to support glucose starved cancer cells. Our specific aims to clarify the role of PEPCK (PCK2) in tumor-promoting macrophages are as follows. To interfere with adaptive processes of cancer cells and their accompanying tumor-promoting normal cells under nutrient deprivation might be an effective therapeutic approach in poorly nourished cancers. The present study will shed light on the role PEPCK (PCK2), a key mediator of biosynthetic flexibility, in tumor-promoting macrophages and answer the question, whether this enzyme is needed for the exchange of metabolites between macrophages and tumor cells.

Methodology

Year 1– 2: The expression of gluconeogenesis enzymes will be assessed in tumor-associated macrophages, freshly isolated from human NSCLC, as well as in macrophages differentiated from human blood monocytes and in murine bone marrow-derived macrophages isolated from WT or PCK2 knockout mice and primed to the M2 versus the M1 phenotype. We will silence PCK2 in macrophages or utilize macrophages from WT or PCK2 knockout mice and infer expression of M2/M1-specific markers. Year 3 – 4: The impact of macrophages on tumor cell migration and angiogenesis under glucose-deprived or glucose-replete conditions will be assessed in vitro. Stable isotopic labelling experiments will be performed to determine the fate of gluconeogenic precursors, lactate and glutamine, in glucose-starved macrophages and in conditioned medium. The metabolic pathways mediating the uptake of gluconeogenic precursors, as well as PCK1- or PCK2-dependent downstream production and export of metabolites will be characterized in detail. The fate of ¹³C-labelled lactate and glutamine in macrophages and the possible utilization of macrophage-derived factors in tumor cells will be assessed using stable-isotope-resolved metabolomics (SIRM) using NMR spectroscopy and / or mass soectrometry in collaboration with Tobias Madl and Harald Köfeler, Medical University of Graz.

Input from collaborations within the RESPImmun programme

Julia Kargl will contribute to the project with her expertise in tumor immunology and isolation of macrophages,
Ákos Heinemann will assist with in vitro differentiation of monocyte-derived macrophages and their phenotyping under glucose starvation,
Leigh Marsh will provide help with PCK2 knockout mice breeding.