Cells grown in three-dimensional spheroids mirror in vivo metabolic response of epithelial cells

Lagies, Simon; Schlimpert, Manuel; Neumann, Simon; Wäldin, Astrid; Kammerer, Bernd; Borner, Christoph; Peintner, Lukas

doi:10.1038/s42003-020-0973-6

Cited by 105 publications

(86 citation statements)

References 48 publications

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“…Displayed data including the Gb3 profile of H1299 cells and Gb3 species of pull-down experiments were measured by targeted LC-QqQ-MS (6460, Agilent Technologies) operated in SRM/MRM mode. Chromatographic separation was achieved on a BEH C18 column (100 mm × 2.1 mm, 1.8 µm, Waters Corporation) as previously described [ 47 ]. For evaluation of the lectin preferences, cell lysates of unstimulated, LecA- and StxB-stimulated H1299 cells were split into two fractions: 10% were used to assess the Gb3 levels present in the individual sample (input).…”

Section: Methodsmentioning

confidence: 99%

The Gb3-enriched CD59/flotillin plasma membrane domain regulates host cell invasion by Pseudomonas aeruginosa

Brandel

Aigal

Lagies

et al. 2021

Cell. Mol. Life Sci.

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The opportunistic pathogen Pseudomonas aeruginosa has gained precedence over the years due to its ability to develop resistance to existing antibiotics, thereby necessitating alternative strategies to understand and combat the bacterium. Our previous work identified the interaction between the bacterial lectin LecA and its host cell glycosphingolipid receptor globotriaosylceramide (Gb3) as a crucial step for the engulfment of P. aeruginosa via the lipid zipper mechanism. In this study, we define the LecA-associated host cell membrane domain by pull-down and mass spectrometry analysis. We unraveled a predilection of LecA for binding to saturated, long fatty acyl chain-containing Gb3 species in the extracellular membrane leaflet and an induction of dynamic phosphatidylinositol (3,4,5)-trisphosphate (PIP3) clusters at the intracellular leaflet co-localizing with sites of LecA binding. We found flotillins and the GPI-anchored protein CD59 not only to be an integral part of the LecA-interacting membrane domain, but also majorly influencing bacterial invasion as depletion of either of these host cell proteins resulted in about 50% reduced invasiveness of the P. aeruginosa strain PAO1. In summary, we report that the LecA-Gb3 interaction at the extracellular leaflet induces the formation of a plasma membrane domain enriched in saturated Gb3 species, CD59, PIP3 and flotillin thereby facilitating efficient uptake of PAO1.

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Section: Methodsmentioning

confidence: 99%

The Gb3-enriched CD59/flotillin plasma membrane domain regulates host cell invasion by Pseudomonas aeruginosa

Brandel

Aigal

Lagies

et al. 2021

Cell. Mol. Life Sci.

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“…First, many cell lines were established from metastatic sites and do underrepresent characteristics found in primary tumors or pancreatic intraepithelial neoplasia (PanIN) lesions [ 39 ], and second, established cell lines grow extremely fast which could easily lead to the selection of different clones and a genetic drift as reported previously [ 40 ]. Application of 3D-grown conventional cell lines can in general overcome some issues of abnormal proliferation behavior observed in 2D-cultures in comparison to in situ situations [ 41 ]. Other research systems include patient-derived xenografts (PDX).…”

Section: Introductionmentioning

confidence: 99%

Metabolic Profiling of Early and Late Recurrent Pancreatic Ductal Adenocarcinoma Using Patient-Derived Organoid Cultures

Braun

Lagies

Klar

et al. 2020

Cancers

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Pancreatic ductal adenocarcinoma (PDAC) is associated with high mortality and will become the second most common cause of cancer-associated mortality by 2030. The poor prognosis arises from a lack of sensitive biomarkers, limited therapeutic options, and the astonishingly high recurrence rate after surgery of 60–80%. The factors driving this recurrence, however, remain enigmatic. Therefore, we generated patient-derived organoids (PDOs) from early- and late-recurrent PDAC patients. Cellular identity of PDOs was confirmed by qPCR, ddPCR, and IHC analyses. This is the first study investigating the metabolism in PDOs of different, clinically significant PDAC entities by untargeted GC/MS profiling. Partial least square discriminant analysis unveiled global alterations between the two sample groups. We identified nine metabolites to be increased in early recurrent PDOs in comparison to late recurrent PDOs. More than four-times increased were fumarate, malate, glutamate, aspartate, and glutamine. Hence, α-keto acids were elevated in PDO-conditioned medium derived from early recurrent patients. We therefore speculate that an increased anaplerotic metabolism fuels the Krebs-cycle and a corresponding higher accessibility to energy fastens the recurrence in PDAC patients. Therein, a therapeutic intervention could delay PDAC recurrence and prolong survival of affected patients or could serve as biomarker to predict recurrence in the future.

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“…Indeed, we observed apoptotic cells mainly in the core of spheroids, with a highly proliferative outer ring. Thus, it is suggested that MCTS are more representative, also in terms of metabolism [36], compared to 2D cultured cells. This makes OS MCTS an attractive model for further pre-clinical (metabolic) drug testing.…”

Section: Conclusion and Discussionmentioning

confidence: 99%

Targeting the NAD Salvage Synthesis Pathway as a Novel Therapeutic Strategy for Osteosarcomas with Low NAPRT Expression

Franceschini

Oosting

Tamsma

et al. 2021

IJMS

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For osteosarcoma (OS), the most common primary malignant bone tumor, overall survival has hardly improved over the last four decades. Especially for metastatic OS, novel therapeutic targets are urgently needed. A hallmark of cancer is aberrant metabolism, which justifies targeting metabolic pathways as a promising therapeutic strategy. One of these metabolic pathways, the NAD+ synthesis pathway, can be considered as a potential target for OS treatment. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the classical salvage pathway for NAD+ synthesis, and NAMPT is overexpressed in OS. In this study, five OS cell lines were treated with the NAMPT inhibitor FK866, which was shown to decrease nuclei count in a 2D in vitro model without inducing caspase-driven apoptosis. The reduction in cell viability by FK866 was confirmed in a 3D model of OS cell lines (n = 3). Interestingly, only OS cells with low nicotinic acid phosphoribosyltransferase domain containing 1 (NAPRT1) RNA expression were sensitive to NAMPT inhibition. Using a publicly available (Therapeutically Applicable Research to Generate Effective Treatments (TARGET)) and a previously published dataset, it was shown that in OS cell lines and primary tumors, low NAPRT1 RNA expression correlated with NAPRT1 methylation around the transcription start site. These results suggest that targeting NAMPT in osteosarcoma could be considered as a novel therapeutic strategy, where low NAPRT expression can serve as a biomarker for the selection of eligible patients.

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Cells grown in three-dimensional spheroids mirror in vivo metabolic response of epithelial cells

Cited by 105 publications

References 48 publications

The Gb3-enriched CD59/flotillin plasma membrane domain regulates host cell invasion by Pseudomonas aeruginosa

The Gb3-enriched CD59/flotillin plasma membrane domain regulates host cell invasion by Pseudomonas aeruginosa

Metabolic Profiling of Early and Late Recurrent Pancreatic Ductal Adenocarcinoma Using Patient-Derived Organoid Cultures

Targeting the NAD Salvage Synthesis Pathway as a Novel Therapeutic Strategy for Osteosarcomas with Low NAPRT Expression

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