The nanomechanical signature of breast cancer Marija Plodinec 1,2 , Marko Loparic 1,2 , Christophe A. Monnier 1† , Ellen C. Obermann 3 , Rosanna Zanetti-Dallenbach 4 , Philipp Oertle 1 , Janne T. Hyotyla 1 , Ueli Aebi 2 , Mohamed Bentires-Alj 5 , Roderick Y. H. Lim 1 * and Cora-Ann Schoenenberger 2 * Cancer initiation and progression follow complex molecular and structural changes in the extracellular matrix and cellular architecture of living tissue. However, it remains poorly understood how the transformation from health to malignancy alters the mechanical properties of cells within the tumour microenvironment. Here, we show using an indentation-type atomic force microscope (IT-AFM) that unadulterated human breast biopsies display distinct stiffness profiles. Correlative stiffness maps obtained on normal and benign tissues show uniform stiffness profiles that are characterized by a single distinct peak. In contrast, malignant tissues have a broad distribution resulting from tissue heterogeneity, with a prominent low-stiffness peak representative of cancer cells. Similar findings are seen in specific stages of breast cancer in MMTV-PyMT transgenic mice. Further evidence obtained from the lungs of mice with late-stage tumours shows that migration and metastatic spreading is correlated to the low stiffness of hypoxia-associated cancer cells. Overall, nanomechanical profiling by IT-AFM provides quantitative indicators in the clinical diagnostics of breast cancer with translational significance. Q1 1 P hysical and chemical forces mediate the order by which 2 cells proliferate, differentiate and migrate 1 within the three- 3 dimensional microenvironment of living tissue 2 . 4 Perturbations to this intricate balance 3–5 are known to promote 5 tumorigenesis and progression to metastasis 6 . At the molecular 6 level, tumour initiation and progression are accompanied by 7 complex structural changes in the extracellular matrix (ECM) and 8 cellular cytoarchitecture, which are anticipated to develop differen- 9 tiable mechanical responses 7 . However, it has been difficult to reach 10 a consensus as to how such biomechanical heterogeneities occur and 11 what their role might be. This is due to challenges in being able to 12 discriminate between the mechanoresponses of cells and the sur- 13 rounding ECM within native tumour tissue with adequate spatial/ 14 structural resolution and force sensitivity. Not least for its 15 implications in diagnostics and treatment, being able to understand 16 the mechanobiology of tumorigenesis is paramount in revealing its 17 deterministic role in cancer development and metastasis 5 . 18 Long-standing ambiguities Q2 exist because efforts to understand 19 cancer biomechanics are largely polarized between tumour-level 20 and single-cell experimentation. In accordance with conventional 21 wisdom (breast palpation), studies on whole mouse mammary 22 glands show that breast tumours are considerably more rigid than 23 the surrounding tissue due to a relative stiffening of the peripheral 24 tumour stroma 7,8 . This notion is consistent with the increase in 25 matrix deposition and crosslinking observed in three-dimensional 26 cell cultures and mouse mammary glands during cancer pro- 27 gression 9,10 . Paradoxically, biophysical techniques 11–16 reveal that 28 single (cultured) cancer cells are more compliant (or ‘softer’) than 29 their healthy counterparts. This increase in elasticity and/or 30 deformability is accompanied by alterations in the cytoarchitecture 31 that have known associations with malignant transformation 17 . 32 Because of their softness, cancer cells have been detected by inden- 33 tation-type atomic force microscopy (IT-AFM) in tissue sections 34 from tumours that were surgically removed from patients with 35 advanced cancer 18 . Furthermore, Cross et al. found that metastatic 36 cells isolated from the pleural fluid of human patients are softer 37 than normal cells 12 , suggesting that metastasis might be promoted 38 by cell compliance. No doubt, criticism is common on both sides 39 of the length-scale divide. On the one hand, the mechanoresponse 40 of whole tumours is arguably dominated by stiff structural elements 41 in the peripheral stroma (for example, collagen 19,20 ), leaving poten- 42 tially more compliant regions in the underlying cancerous core 43 insensitive to detection. On the other hand, the relevance of single 44 cell measurements has been questioned given the lack of a proper 45 three-dimensional tissue environment 21 . 46 The diversity of biomechanical profiles underscores the impor- 47 tance of obtaining a holistic understanding of malignancy and 48 how it manifests in breast tumours. This includes correlating biome- 49 chanical and microenvironmental properties at different stages of 50 cancer progression. In this Article, we report on a comprehensive 51 effort to correlate the nanomechanical properties of native human 52 breast biopsies to specific histopathological markers in healthy 53 tissue and in benign and malignant tumours. As a standard 54 animal model, our human biopsy results are validated with systema- 55 tic experiments on MMTV-PyMT (mouse mammary tumour virus- 56 polyoma middle T antigen) transgenic mice, which we follow from 57 early cancer to metastasis 22 . In bridging across length scales, our 58 nanomechanical measurements reconcile tumour-level and single- 59 cell measurements in both humans and transgenic mice and 60 reveal unique mechano-markers that can be used to identify differ- 61 ent cancer stages. Moreover, these findings suggest close correlations 62 between cell softening, tumour hypoxia and lung metastasis. 63 Nanomechanical signature of human breast biopsies 64 To elucidate and correlate the respective nanomechanical profiles 65 to pathohistological findings in normal, benign and malignant 1 Biozentrum and the Swiss Nanoscience Institute, University of Basel, 4056 Basel, Switzerland, 2 Maurice E. Mueller Institute for Structural Biology, Biozentrum, University of Basel, 4056 Basel, Switzerland, 3 Institute of Pathology, University Hospital Basel, 4031 Basel, Switzerland, 4 Department of Gynecology and Gynecological Oncology, University Hospital Basel, University of Basel, 4031 Basel, Switzerland, 5 Mechanisms of Cancer, Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland; † These authors contributed equally to this work. *e-mail: roderick.lim@unibas.ch; cora-ann.schoenenberger@unibas.ch ARTICLES PUBLISHED ONLINE: XX XX 2012 | DOI: 10.1038/NNANO.2012.167 NATURE NANOTECHNOLOGY | ADVANCE ONLINE PUBLICATION | www.nature.com/naturenanotechnology 1