Profiling native pulmonary basement membrane stiffness using atomic force microscopy

ABSTRACT Mammalian cells sense and react to the mechanics of their immediate microenvironment. Therefore, the characterization of the biomechanical properties of tissues with high spatial

resolution provides valuable insights into a broad variety of developmental, homeostatic and pathological processes within living organisms. The biomechanical properties of the basement

membrane (BM), an extracellular matrix (ECM) substructure measuring only ∼100–400 nm across, are, among other things, pivotal to tumor progression and metastasis formation. Although the

precise assignment of the Young’s modulus _E_ of such a thin ECM substructure especially in between two cell layers is still challenging, biomechanical data of the BM can provide information

of eminent diagnostic potential. Here we present a detailed protocol to quantify the elastic modulus of the BM in murine and human lung tissue, which is one of the major organs prone to

metastasis. This protocol describes a streamlined workflow to determine the Young’s modulus _E_ of the BM between the endothelial and epithelial cell layers shaping the alveolar wall in lung

tissues using atomic force microscopy (AFM). Our step-by-step protocol provides instructions for murine and human lung tissue extraction, inflation of these tissues with cryogenic cutting

medium, freezing and cryosectioning of the tissue samples, and AFM force-map recording. In addition, it guides the reader through a semi-automatic data analysis procedure to identify the

pulmonary BM and extract its Young’s modulus _E_ using an in-house tailored user-friendly AFM data analysis software, the Center for Applied Tissue Engineering and Regenerative Medicine

processing toolbox, which enables automatic loading of the recorded force maps, conversion of the force versus piezo-extension curves to force versus indentation curves, calculation of

Young’s moduli and generation of Young’s modulus maps, where the pulmonary BM can be identified using a semi-automatic spatial filtering tool. The entire protocol takes 1–2 d. KEY POINTS *

The function of pulmonary alveoli is dependent on their mechanical robustness and response to external forces. The Young’s modulus _E_ (stiffness) of their basement membranes is higher than

that of the surrounding cell layers. * This protocol describes how to prepare lung sections from humans or mice and perform atomic force microscopy experiments. Challenges in data

analysis—including filtering to focus specifically on basement membrane values—are addressed using the Center for Applied Tissue Engineering and Regenerative Medicine processing toolbox.

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OTHERS SPATIAL MAPPING OF THE COLLAGEN DISTRIBUTION IN HUMAN AND MOUSE TISSUES BY FORCE VOLUME ATOMIC FORCE MICROSCOPY Article Open access 24 September 2020 INTEGRATED COMPUTATIONAL AND

EXPERIMENTAL PIPELINE FOR QUANTIFYING LOCAL CELL–MATRIX INTERACTIONS Article Open access 12 August 2021 DEVELOPMENT OF A NOVEL AIR–LIQUID INTERFACE AIRWAY TISSUE EQUIVALENT MODEL FOR IN

VITRO RESPIRATORY MODELING STUDIES Article Open access 22 June 2023 DATA AVAILABILITY All raw data and derived data used to generate graphs presented in this manuscript are available from

the corresponding authors upon reasonable request. The force maps are available at https://figshare.com/articles/journal_contribution/Force_Maps/24591198. CODE AVAILABILITY All codes used in

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human internal limiting membrane. _Invest. Ophthalmol. Vis. Sci._ 53, 2561–2570 (2012). Article  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS B.H., L.F. and H.C.-S.

acknowledge funding from the Bavarian State Ministry of Science and the Arts through the Bavarian Research Focus ‘Herstellung und biophysikalische Charakterisierung von dreidimensionalen

Geweben (CANTER)’ and the Bavarian Academic Forum (BayWISS)—Doctoral Consortium ‘Health Research’. The development of the data analysis software CANTER processing toolbox was funded by the

German Research Foundation as part of subproject 1 (CL 409/4-1/2) of the research consortium ‘Exploring articular cartilage and subchondral bone degeneration and regeneration in

osteoarthritis – ExCarBon’ (FOR2407-1/2). H.C.-S. acknowledges funding from the German Research Foundation through the major instrumentation campaign GGA-HAW (INST 99/38-1). This work was

further supported by the Danish Cancer Society (R204-A12454 (R.R.)). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Munich University of Applied Sciences, Center for Applied Tissue

Engineering and Regenerative Medicine – CANTER, Munich, Germany Bastian Hartmann, Lutz Fleischhauer & Hauke Clausen-Schaumann * Center for Nanoscience, Munich, Germany Bastian Hartmann, 

Lutz Fleischhauer & Hauke Clausen-Schaumann * Experimental and Clinical Pharmacology and Toxicology, Medical Faculty, University of Freiburg, Freiburg, Germany Monica Nicolau & 

Raphael Reuten * Department of Obstetrics and Gynecology, Medical Center, University of Freiburg, Freiburg, Germany Monica Nicolau, Florin-Andrei Taran & Raphael Reuten * Faculty of

Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Thomas Hartvig Lindkær Jensen * Department of Pathology, Rigshospitalet, Copenhagen, Denmark Thomas Hartvig Lindkær

Jensen Authors * Bastian Hartmann View author publications You can also search for this author inPubMed Google Scholar * Lutz Fleischhauer View author publications You can also search for

this author inPubMed Google Scholar * Monica Nicolau View author publications You can also search for this author inPubMed Google Scholar * Thomas Hartvig Lindkær Jensen View author

publications You can also search for this author inPubMed Google Scholar * Florin-Andrei Taran View author publications You can also search for this author inPubMed Google Scholar * Hauke

Clausen-Schaumann View author publications You can also search for this author inPubMed Google Scholar * Raphael Reuten View author publications You can also search for this author inPubMed 

Google Scholar CONTRIBUTIONS All authors developed experimental protocols and designed experiments. B.H., L.F. and T.H.L.J. conducted the experiments. B.H., L.F. and M.N. developed the data

analysis tools. B.H., M.N. and F.-A.T. analyzed the data. H.C.-S. and R.R. conceived the ideas and contributed to experimental interpretation. B.H., H.C.-S. and R.R. wrote the manuscript.

All authors revised the manuscript. CORRESPONDING AUTHORS Correspondence to Hauke Clausen-Schaumann or Raphael Reuten. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no

competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Protocols_ thanks the anonymous reviewer(s) for their contribution to the peer review of this work. ADDITIONAL INFORMATION

PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. RELATED LINKS Key reference using this protocol

Reuten, R. et al. _Nat. Mater_. 20, 892–903 (2021): https://doi.org/10.1038/s41563-020-00894-0 Key data used in this protocol Reuten, R. et al. _Nat. Mater_. 20, 892–903 (2021):

https://doi.org/10.1038/s41563-020-00894-0 EXTENDED DATA EXTENDED DATA FIG. 1 BM’S YOUNG’S MODULUS LEVELS OF NET4 WILD TYPE VERSUS KNOCKOUT STRATIFIED INTO FEMALE AND MALE. Figure created

with BioRender.com. Source data EXTENDED DATA FIG. 2 HUMAN TISSUES WITH SIMILAR BASEMENT MEMBRANE ANATOMY. Figure created with BioRender.com and adapted with permission from ref. 30,

Springer Nature Limited. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Suplementary discussion and Figs. 1–20. SOURCE DATA SOURCE DATA FIG. 5 Young’s modulus values histograms. SOURCE

DATA FIG. 6 Log-transformed Young’s modulus values used to generate the histograms and the QQ-plot. SOURCE DATA FIG. 7 Log-transformed Young’s modulus values of the histograms. Young’s

modulus values (individual values and summary values) of the box plots in Fig. 7d. Standard deviation values of the box plots in Fig. 7e. SOURCE DATA EXTENDED DATA FIG. 1 Young’s modulus

values from Net4 WT and KO mice splitted into female and male (individual values and summary values) of the box plots in Extended Data Fig. 1. RIGHTS AND PERMISSIONS Springer Nature or its

licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the

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Hartmann, B., Fleischhauer, L., Nicolau, M. _et al._ Profiling native pulmonary basement membrane stiffness using atomic force microscopy. _Nat Protoc_ 19, 1498–1528 (2024).

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