Monitoring extracellular ion and metabolite dynamics with recombinant nanobody-fused biosensors

Sandra Burgstaller; Teresa R Wagner; Helmut Bischof; Sarah Bueckle; Aman Padamsey; Desiree Frecot; Philipp D Kaiser; David Skrabak; Roland Malli; Robert Lukowski; Ulrich Rothbauer

doi:10.1016/j.isci.2022.104907

Monitoring extracellular ion and metabolite dynamics with recombinant nanobody-fused biosensors

iScience. 2022 Aug 10;25(9):104907. doi: 10.1016/j.isci.2022.104907. eCollection 2022 Sep 16.

Authors

Sandra Burgstaller^{1

2}, Teresa R Wagner^{1

3}, Helmut Bischof², Sarah Bueckle¹, Aman Padamsey¹, Desiree Frecot^{1

3}, Philipp D Kaiser³, David Skrabak², Roland Malli^{4

5}, Robert Lukowski², Ulrich Rothbauer^{1

3}

Affiliations

¹ Pharmaceutical Biotechnology, Eberhard Karls University Tübingen, Markwiesenstrasse 55, 72770 Reutlingen, Germany.
² Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, University of Tübingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany.
³ NMI Natural and Medical Sciences Institute at the University of Tübingen, Markwiesenstrasse 55, 72770 Reutlingen, Germany.
⁴ Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria.
⁵ BioTechMed-Graz, Mozartgasse 12/II, 8010 Graz, Austria.

Abstract

Ion and analyte changes in the tumor microenvironment (TME) alter the metabolic activity of cancer cells, promote tumor cell growth, and impair anti-tumor immunity. Consequently, accurate determination and visualization of extracellular changes of analytes in real time is desired. In this study, we genetically combined FRET-based biosensors with nanobodies (Nbs) to specifically visualize and monitor extracellular changes in K⁺, pH, and glucose on cell surfaces. We demonstrated that these Nb-fused biosensors quantitatively visualized K⁺ alterations on cancer and non-cancer cell lines and primary neurons. By implementing a HER2-specific Nb, we generated functional K⁺ and pH sensors, which specifically stained HER2-positive breast cancer cells. Based on the successful development of several Nb-fused biosensor combinations, we anticipate that this approach can be readily extended to other biosensors and will open new opportunities for the study of extracellular analytes in advanced experimental settings.

Keywords: Biochemistry; Optical imaging; Sensor.