Atomic force microscopy for revealing micro/nanoscale mechanics in tumor metastasis: from single cells to microenvironmental cues

Abstract

Mechanics are intrinsic properties which appears throughout the formation, development, and aging processes of biological systems. Mechanics have been shown to play important roles in regulating the development and metastasis of tumors, and understanding tumor mechanics has emerged as a promising way to reveal the underlying mechanisms guiding tumor behaviors. In particular, tumors are highly complex diseases associated with multifaceted factors, including alterations in cancerous cells, tissues, and organs as well as microenvironmental cues, indicating that investigating tumor mechanics on multiple levels is significantly helpful for comprehensively understanding the effects of mechanics on tumor progression. Recently, diverse techniques have been developed for probing the mechanics of tumors, among which atomic force microscopy (AFM) has appeared as an excellent platform enabling simultaneously characterizing the structures and mechanical properties of living biological systems ranging from individual molecules and cells to tissue samples with unprecedented spatiotemporal resolution, offering novel possibilities for understanding tumor physics and contributing much to the studies of cancer. In this review, we survey the recent progress that has been achieved with the use of AFM for revealing micro/nanoscale mechanics in tumor development and metastasis. Challenges and future progress are also discussed.

Keywords: atomic force microscopy; cancerous cell; exosome; extracellular matrix; tumor mechanics; tumor microenvironment.

Fig. 1. Schematic illustration of the detailed process of tumor metastasis showing that there are significant physical processes in the process of tumor metastasis and multiple types of microenvironmental cues are involved for promoting the successful metastasis of primary cancerous cells.

Reprinted with permission from Ref. [24]. Copyright 2019 Springer Nature. Tumor metastasis is a complex multistep process. Tumor cell production of angiogenetic factors and TGFβ can activate endothelial cells and fibroblasts to remodel tissues and promote tumor cell invasion of stromal-modified spaces. Intravasation of tumor cells is promoted by binding to macrophages that cause transient permeability in the vasculature. In the circulation, platelets can bind to the circulating tumor cells (CTCs) and protect CTCs from cytotoxic immune cell recognition, escorting tumor cells to the site of extravasation [33]. Preferred colonization sites, termed premetastatic niches, can be prepared in advance of the arrival of tumor cells through the actions of extracellular vesicles such as exosomes. When tumor cells arrive the new sites, only a small subset of tumor cells initiate cell division to form micrometastases, and only a small proportion of these micrometastases persist to become vascularized metastases [34].

Fig. 2. Principles of AFM imaging.

a Schematic of AFM imaging of biological specimens (an example of cell membrane is shown) attached on the support. b Contact mode AFM imaging. In contact mode, AFM tip is scanned over the specimen surface, while the deflection of the cantilever is maintained constant. c Tapping mode AFM imaging. In tapping mode, commonly the amplitude of the oscillating cantilever is maintained constant. d SEM images showing the different shapes of AFM tips. I Pyramid tip. II Conical tip. d is reprinted with permission from Ref. [35]. Copyright 2016 Springer Nature.

Fig. 3. Stiffness changes of tumor cells during metastasis revealed by AFM indentation assays.

a Schematic diagram of an adherent mammalian cell with a summary of the mechanical properties of the cellular structures and compartments. Reprinted with permission from Ref. [29]. Copyright 2018 Springer Nature. b Different types of AFM tips for indentation assays. I Spherical tip. Zx confocal images of a cell (membrane protein is labeled with green fluorescein) indented by AFM cantilever conjugated with a fluorescent bead (blue). Reprinted with permission from Ref. [47]. Copyright 2013 Springer Nature. II Conical tip. Zx confocal images of a cell monolayer (green) grown on a soft collagen gel (black) during the indentation by an AFM cantilever with conical tip (dotted line). White arrowhead indicates an individual cell and gray arrowhead indicates the tip of the cantilever. A fluorescent dye was added to the cell culture medium (red). Reprinted with permission from Ref. [48]. Copyright 2014 The Company of Biologists Ltd. c AFM probing metastatic tumor cells and normal mesothelial cells prepared from clinical cancer patients. I Optical image of tumor cells (denoted by arrowhead) and normal mesothelial cells (denoted by arrow). The inset shows the alignment of AFM tip over the central region of a cell. II Immunofluorescence of the specimens confirming tumor cells (arrowheads) and normal cells (arrows). Stiffness histogram of tumor cells (III) and normal cells (IV). Reprinted with permission from Ref. [50]. Copyright 2007 Springer Nature. d AFM probing the stiffness changes of tumor tissues during metastasis. I Schematic of an ultrasound-guided biopsy from a patient with a suspicious lesion. II Schematic of utilizing AFM to record multiple stiffness maps (20 × 20 μm) across the biopsy specimen immobilized on the substrate. Stiffness histogram of normal breast tissues (III) and invasive breast tissues (IV). Reprinted with permission from Ref. [51]. Copyright 2012 Springer Nature.

Fig. 4. Mechanical changes of cells in the process of EMT revealed by AFM.

a Schematic of EMT process. Epithelial cells displaying apical–basal polarity are held together by tight junction, adherens junctions, and desmosomes. Epithelial cells are tethered to the underlying basement membrane by hemidesmosomes. Induction of EMT results in cellular changes that include the disassembly of epithelial cell–cell junctions and the dissolution of apical–basal cell polarity. The loss of epithelial features is accompanied by acquisition of mesenchymal features. Mesenchymal cells display front-to-back polarity and have a reorganized cytoskeleton. After EMT, cells become motile and acquire invasive capabilities. Reprinted with permission from Ref. [58]. Copyright 2019 Springer Nature. b–d Cellular changes during EMT induced by TGF-β1. Cells before TGF-β1 treatment (I) and after TGF-β1 treatment (II). b Confocal fluorescent images. F-actins were stained with red fluorescein and nuclei were stained with blue fluorescein. The insets are the enlarged view of individual cells denoted by the arrows. c AFM morphological changes of cells. The insets are the enlarged view of local structures of the cells. d Statistical histograms of cellular Young’s modulus. b, c are reprinted with permission from Ref. [61]. Copyright 2012 Elsevier Inc. d is reprinted with permission from Ref. [62]. Copyright 2018 Elsevier B.V.

Fig. 5. AFM-based single-cell force spectroscopy (SCFS) revealing the changes of adhesive properties of tumor cells during metastasis.

a, b Principle of SCFS. a Schematic of performing approach–retract movement on substrate with the use of cell probe in SCFS assays. The cell-conjugated AFM cantilever is first lowered toward the substrate (I) until a preset force is reached (II). After a given contact time, the cantilever retracts from the substrate (III) until cell and substrate are completely separated (IV). b A representative force curve recorded during SCFS showing steps (I–IV) corresponding to those outlined in (a). Several unbinding events can be observed in the retraction curve (s denotes force steps, t denotes unbinding of membrane tethers, F_d denotes maximal detachment force). Reprinted with permission from Ref. [65]. Copyright 2010 Springer Nature. c–f Adhesion properties of tumor cells with different invasion potentials. In the following, breast cell lines are ranked from left to right in ascending order of their invasive character. c Cell Young’s modulus. d Cell adhesion force. e Individual membrane tether force. f Number of membrane tethers. Reprinted with permission from Ref. [71]. Copyright 2016 American Chemical Society.

Fig. 6. AFM-based single-molecule force spectroscopy (SMFS) revealing the mechanics of molecules on the surface of tumor cells.

a Principle of SMFS. I Schematic of probing specific receptors on cell surface by SMFS with the use of ligand-conjugated tip. Reprinted with permission from Ref. [77]. Copyright 2019 Springer Nature. II Schematic and III practical force curves recorded during SMFS assays. Reprinted with permission from Ref. [79]. Copyright 2018 Elsevier Ltd. b Mapping nanoscale organizations of receptors on heterogenous surface of tumor cells by AFM. Reprinted with permission from Ref. [88]. Copyright 2018 Elsevier Ltd. c Combining AFM with confocal fluorescence microscopy to reveal specific molecular interactions on cell surface. I Differential interference contrast (DIC) image and II mCherry channel superimposed with DIC channel. The functionalized probe can be seen above the cells. III AFM height image and IV adhesion image of cells recorded in the scan region denoted by the dashed square in (II). Reprinted with permission from Ref. [90]. Copyright 2017 Springer Nature. Distribution of molecular adhesion forces measured on target cells (V) and control cells (VI). Reprinted with permission from Ref. [80]. Copyright 2017 Springer Nature.

Fig. 7. Probing the mechanics of tumor cell spheroids by AFM.

a–g Visualizing the heterogeneous stiffness signature of tumor cell spheroids at different depths by analyzing the force curves obtained on spheroids. a Schematic showing that three types of nanomechanical topographies are identified during AFM indentation measurements, including collagen type I stress fibers (I), the interface of cell membrane and ECM (II) with high stiffness, and cells embedded deep inside the ECM (III). b Topography image and c–g corresponding stiffness images of a tumor spheroid at different depths. The double red arrows denote the collagen type I stress fibers, the double green arrows denote the interface of cell membrane and ECM, and the dotted red square denotes the cells embedded deep inside the ECM. Reprinted with permission from Ref. [100]. Copyright 2019 Springer Nature. h Mechanical dynamics of tumor spheroids and tumor cells regulated by the rigidity of microenvironment. I Confocal fluorescent images of the spheroids grown in hydrogels of varying stiffness. II AFM indentation assays on the whole tumor spheroids. III AFM measurements on individual tumor cells isolated from the tumor spheroids. Reprinted with permission from Ref. [102]. The insets in (II, III) show the schematic of AFM measurements on tumor spheroids or single tumor cells, respectively. Copyright 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fig. 8. Imaging and force spectroscopy measurements of individual exosomes by AFM revealing the mechanical phenotypes of exosomes in promoting tumor progression.

a Schematic of exosomes in the communication of cells. I Release of exosomes by donor cells can be induced by diverse signaling pathways and occurs in a reversed budding event. The uptake of exosome is accomplished via endocytosis, receptor-ligand interaction or by direct fusion. II Exosome content. Reprinted with permission from Ref. [106]. Copyright 2017 Elsevier Ltd. b AFM is able to probe the structure and properties of single exosomes. I Imaging the topography of single exosomes. Reprinted with permission from Ref. [116]. Copyright 2014 Elsevier Inc. II Indenting single exosomes. Reprinted with permission from Ref. [118]. Copyright 2010 American Chemical Society. III, IV Probing single receptors on exosomes. III Schematic of using antibody-modified tip to recognize specific receptors on exosomes. IV Force curves obtained during AFM force spectroscopy assays discriminating the specific molecular interactions and nonspecific molecular interactions. Reprinted with permission from Ref. [120]. Copyright 2018 IOP Publishing Ltd.

Fig. 9. Imaging the morphology and measuring the mechanics of basement membranes by AFM.

a Basement membrane localization and composition. I Basement membrane underlies or surround most tissues such as epithelial. II The self-assembling polymeric networks of type IV collagen and laminin provide basement membranes with their core structure and these networks associate with each other through interactions (denoted by arrows) with bridging adapter proteins, such as perlecan and nidogen. The laminin network is closely associated with cell surface through interactions with integrins and dystroglycan receptors as well as sulfated glycolipids. Reprinted with permission from Ref. [132]. Copyright 2017 Elsevier Inc. b AFM revealing the dynamic mechanics of basement membranes during the invasion process of tumor cells. AFM height images of the basement membranes without growing cells (I) and basement membranes treated by cancer cells (CCs) and cancer-associated fibroblasts (CAFs) (II). Statistical histograms of the changes in roughness (III) and stiffness (IV) of basement membranes after growing cells. Reprinted with permission from Ref. [136]. Copyright 2017 Springer Nature.

Fig. 10. AFM assays on decellularized matrix.

a Types of decellularized matrices. I Tissue-/organ-derived decellularized matrices. II Cultured cell-derived decellularized matrices. III Various forms of decellularized matrices. Reprinted with permission from Ref. [145]. Copyright 2017 Royal Society of Chemistry. b AFM assays on decellularized matrix prepared from organ. Photographic image of a mouse lung before (I) and after (II) decellularization by simultaneous tracheal instillation and arterial perfusion of a solution of triton X-100 (0.1%) and sodium dodecyl sulfate (1%). III A slice (12 μm thick) of decellularized mouse lung probed with an AFM placed on the stage of an inverted optical microscope. Reprinted with permission from Ref. [147]. Copyright 2017 Wiley Periodicals, Inc. c Diagram showing the typical steps of AFM force spectroscopy experiments on cultured cell-derived decellularized matrix for measuring matrix stiffness. Reprinted with permission from Ref. [152]. Copyright 2016 Elsevier Inc.

Fig. 11. AFM for visualizing the fine structures and measuring the mechanics of single ECM nanofibrils.

a Schematic illustrating the effect of fibrillar structures on mechanotransduction. Cells can exert forces on the fibers through cell-surface proteins and the cytoskeleton, which are both mechanically coupled to the ECM, initiating signaling pathways via mitogen-activated protein kinase (MAPK) and the RHO family of GTPases (RHO). Reprinted with permission from Ref. [156]. Copyright 2019 Springer Nature. b SEM images of collagen (I) and fibrin (II) gels, respectively. Reprinted with permission from Ref. [159]. Copyright 2015 Authors. c Imaging the fine structures of ECM and measuring the adhesion force of ECM by AFM [160, 161]. I Porous network structures of ECM visualized by large-size AFM scanning. II, III Single nanofibrils visualized by small-size AFM scanning. IV A typical force curve obtained on ECM for evaluating the adhesive capabilities of ECM. d Measuring the stiffness of single nanofibrils by AFM. I AFM image of tripeptide fibers. Statistical histogram of the Young’s modulus of the nanofibrils formed by Pro-Phe-Phe (II) or Hyp-Phe-Phe (III). Reprinted with permission from Ref. [164]. Copyright 2019 Springer Nature.