LETTERS

Nanomechanical analysis of cells from

cancer patients
SARAH E. CROSS1,2†, YU-SHENG JIN3†, JIANYU RAO3*† AND JAMES K. GIMZEWSKI1,2*†
1
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA
2
California NanoSystems Institute, University of California, Los Angeles, California 90095, USA
3
Department of Pathology and Laboratory Medicine, University of California, Los Angeles, California 90095, USA
†
These authors contributed equally to this work.
*e-mail: gim@chem.ucla.edu; JRao@mednet.ucla.edu

Published online: 2 December 2007; doi:10.1038/nnano.2007.388

Change in cell stiffness is a new characteristic of cancer cells that to cells with minimal disruption18,19. Although AFM has
affects the way they spread1,2. Despite several studies on previously been used to probe cellular mechanics under
architectural changes in cultured cell lines1,3, no ex vivo physiological conditions13–15,21,23,24, it has not been used for
mechanical analyses of cancer cells obtained from patients have cancer cell analysis in a clinical setting, which is the purpose
been reported. Using atomic force microscopy, we report the of this letter.
stiffness of live metastatic cancer cells taken from the body Analyses of body fluid samples, rather than primary tumour
(pleural) fluids of patients with suspected lung, breast and samples, were chosen here, because tumour cells in body fluid
pancreas cancer. Within the same sample, we find that the are all metastatic in nature and thus provide a clonal population
cell stiffness of metastatic cancer cells is more than 70% softer, of metastatic cells for analysis. Additionally, the co-existence of
with a standard deviation over five times narrower, than the both benign and metastatic cells in a single specimen provides a
benign cells that line the body cavity. Different cancer types native internal control. Body cavities, including pleural,
were found to display a common stiffness. Our work shows pericardial and peritoneal cavities, are covered by serous
that mechanical analysis can distinguish cancerous cells from membranes consisting of a single row of flat mesothelial cells on
normal ones even when they show similar shapes. These the surface and an underlying submesothelial layer, which cover a
results show that nanomechanical analysis correlates well with very large surface area in close contact with every major organ of
immunohistochemical testing currently used for detecting cancer. the body. Because of their continuity with the lymphatic system,
Recent progress in the study of cancer cell motility and invasion these cavities are commonly the seat of metastasis. Metastatic
has generated a greater understanding of the mechanical properties malignant effusions constitute an unequivocal sign of widespread
involved in malignant transformation. In particular, the dynamic cancer. Current cancer cell detection relies on qualitative
reorganization of the cytoskeleton has become a specific point of morphological analyses of shape change resulting from
interest regarding changes in cell morphology, motility, adhesion biochemical alterations, such as cytoskeletal remodelling25.
and invasion4,5. Techniques for measuring cell adhesion and However, morphological analysis of cells recovered from an
motility have been established in the laboratory for in vitro effusion is often difficult to diagnose because of the notorious
analysis of cellular phenotypic events associated with tumour reactivity of mesothelial cells in mimicking metastatic cancer cells
cells. Current cytoskeleton analysis techniques include morphologically, featuring enlarged nuclei, increased nuclear and
biochemical purifications, gene expression and polymerization cytoplasmic ratios, among other cytomorphological features.
assays, immunofluorescent labelling, and time-lapse and electron We measured the nanomechanical properties associated with
microscopies6,7. Recently, a change in the physical properties, in the observed shape changes inherent to metastatic
particular cell elasticity, of tissue cells has been recognized as an adenocarcinoma cells and benign mesothelial cells in pleural
indication of disease2,8,9 and has emerged as a marker for cellular effusions obtained from body cavity fluid samples collected from
phenotypic events associated with cell adhesion and cytoskeletal patients with suspected metastatic adenocarcinoma, using
organization2,10–12. In particular, several studies have shown a standard protocols (see Methods). Samples were centrifuged, and
reduction in stiffness with increasing metastatic efficiency in cell pellets were resuspended in culture medium for 12 h, based
human cancer cell lines using several different in vitro on time-culture experiments to establish the optimum incubation
biomechanical assays (see, for example, refs 13–15). time for cell-substrate adherence for mechanical analysis while
In this letter we report on the associated cell stiffness (elasticity) minimizing artifacts resulting from in vitro culture. The 12 h
of pathologically defined human metastatic cancer cells and benign incubation also allowed the differentiation of benign and
reactive mesothelial cells in human body (pleural) fluid samples (as malignant cells based on their ex vivo growth and morphological
approved by the Institutional Review Board; Table 1) using atomic characteristics (that is, ‘tumour’ cells display anchorage-
force microscopy (AFM)16. AFM has been used to probe a number independent growth patterns, such as rounding of cells, whereas
of properties inherent to microbial cells, mammalian cells and ‘normal’ mesothelial cells show a large, flat morphology; Fig. 1),
biomolecules10,13–15,17–22, including analysis of cellular mechanical so that AFM analysis could be performed separately on these two
strain and elasticity, due to the precise application of low forces cell populations. These analyses were later confirmed by

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Table 1 Patient characteristics and cytological diagnosis versus mechanical measurements (Young’s modulus, E).
Case no. Age/sex Clinical history Cytological diagnosis of pleural fluid* Stiffness (kPa): ‘Tumour’ Stiffness (kPa): ‘Normal’
1 52/Female Non-small cell carcinoma of the lung Positive for metastatic malignant cells 0.56+0.09 2.10+0.79
2 60/Female Non-small cell carcinoma of the lung Positive for metastatic malignant cells 0.52+0.12 2.05+0.87
3 49/Female Breast ductal adenocarcinoma Positive for metastatic malignant cells 0.50+0.08 1.93+0.50
4 85/Male Pancreatic adenocarcinoma Positive for metastatic malignant cells 0.54+0.08 0.54+0.12
5 40/Male Liver cirrhosis Negative for malignant cells – 1.86+0.50
6 47/Male Fever and hepatic failure Negative for malignant cells – 1.75+0.61
7 92/Female Anasarca peripheral oedema Negative for malignant cells – 2.09+0.98
*Cytomorphological diagnosis was made based on morphological analysis combined with immunohistochemical analysis (see Supplementary Information). Stiffness values (E) represent mean+s.d.

each cell to determine the relative cell stiffness (Young’s modulus,

E) of individual cells, yielding values of E for each cell type per
clinical specimen (Table 1).
To confirm the selected cell populations actually represent
tumour and mesothelial cells, immunofluorescence triple
labelling assays of the samples were performed (see Methods;
Fig. 1b– d). Labelling for DNA/Ber-EP4/F-actin (Fig. 1c) and
DNA/Ber-EP4/Calretinin (Fig. 1d) both showed staining of the
small, round cells for Ber-EP4 (red), a marker for metastatic
adenocarcinoma cells, thus confirming that the round, balled
cells (shown optically in Fig. 1a) were indeed metastatic
adenocarcinoma cells. Moreover, the large, flat cells were positive
for Calretinin staining (Fig. 1d), indicative of normal mesothelial
cells and consistent with the optical morphology (Fig. 1a).
Immunofluorescence analysis showed that the small round cells
were not apoptotic, dead or mitotic, as they have intact DNA
(Fig. 1b –d, blue fluorescence).
Cell elasticity measurements (Young’s modulus, E) taken on
Figure 1 Optical and fluorescence images of a cytological sample. a, Optical cytological samples collected from patients with suspected
image demonstrating the round, balled morphology of visually assigned tumour cells metastatic adenocarcinoma are shown in Fig. 2. Data collected
(i) and the large, flat morphology of presumed benign mesothelial (normal) cells (ii). from seven different clinical samples (positive for metastatic
The inset shows alignment of an AFM tip over the central (nuclear) region of a cell. tumour, n ¼ 40; negative for metastatic tumour, n ¼ 48) yielded
Scale bar ¼ 50 mm. b–d, Immunofluorescence analysis of a clinical pleural effusion average E values (mean+s.d.) for all tumour and benign
displaying two cell populations (as shown in a): fluorescence image of the negative mesothelial cells of 0.53+0.10 kPa and 1.97+0.70 kPa,
control (omitting primary antibodies for Ber-EP4 and Calretinin) (b); corresponding respectively (Fig. 2b,c; P ¼ 8.72  10222). A two-sample
positive immunofluorescence triple labelling assay for DNA (blue), F-actin (green) and independent t-test conducted on the tumour and mesothelial
Ber-EP4 (red) (c) and for DNA, Ber-EP4 and Calretinin (DNA ¼ blue, Ber-EP4 ¼ red, cell populations showed that the population means were
Calretinin ¼ green) (d). Arrowheads ¼ tumour, arrow ¼ mesothelial cells. Scale bars significantly different from each other at the 95% confidence
(main panels a–d) ¼ 10 mm. level (P ¼ 8.72  10222). In particular, tumour cell elasticity
measurements from a single clinical sample were found to have an
average cell stiffness (mean+s.d.) of 0.56+0.09 kPa (Fig. 2e);
immunofluorescence analysis using biomarkers specific for however, the average cell stiffness for the morphologically
metastatic cancer and mesothelial cells. Immunofluorescence determined benign mesothelial cells in the sample expressed a
analysis was performed independently, although on the same significantly increased average cellular elasticity (mean+s.d.), with
population of cells from the same pleural effusion sample, to a value of 2.10+0.79 kPa (Fig. 2f; P ¼ 7.77  1025). Interestingly,
confirm that cells subjected to AFM analysis based on growth the tumour and benign mesothelial cells exhibited significantly
characteristics indeed represented either tumour or mesothelial different trends; elasticity measurements for the metastatic cancer
cells. For each sample, eight probable mesothelial (‘normal’) cells cells and the benign mesothelial cells were best fit by gaussian
and eight probable malignant (‘tumour’) cells (except in the (normal) and log-normal fits, respectively. Tumour cells displayed
negative cases where only benign cells were present) were selected a narrow, spiked peak with little spread, whereas benign
from their culture dish for ex vivo AFM analysis, which was mesothelial cells displayed a broad peak. Similarly, elasticity
conducted in an alternate fashion to ensure similar conditions measurements collected on a single negative pleural fluid sample
were applied for both populations, without prior knowledge of yielded an average cell stiffness (mean+s.d.) of 2.09+0.98 kPa
the cytomorphological analysis. Cytomorphological (with (Fig. 2g; n ¼ 8). The data clearly show that the cell stiffness of
immunohistochemical confirmatory analysis), AFM and metastatic cancer cells is 73+11% less stiff than the benign
immunofluorescence analyses were performed independently. mesothelial cells in the same sample and when compared to
Cytomorphological analysis was performed on its own population samples collected from other patients.
of cells, but the immunofluorescence and AFM analyses were Figure 2h,i shows gaussian fits of Young’s modulus data
made on the same population of cells; however, all of the cell collected on an individual clinical sample (Table 1, Case 4)
populations were obtained from the same pleural effusion. containing only one population of cells, which were difficult to
Mechanical measurements were obtained at 37 8C at a rate of determine as either benign mesothelial cells or metastatic
1 Hz (see Methods). Force–displacement curves were recorded on malignant cells, based on cytomorphology. However, following

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 LETTERS

30 6 3

20 4 2

Counts

Counts

Counts
10 2 1

0 0 0

30 6 9

20 4 6
Counts

Counts
Counts
10 2 3

0 0 0

15
3 6

10 2 4
Counts

Counts

Counts
5 1 2

0 0 0
0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6
Young's modulus, E (kPa) Young's modulus, E (kPa) Young's modulus, E (kPa)

Figure 2 Histograms of the associated Young’s modulus E for cytological samples collected from patients with suspected metastatic cancer. a, Histogram
of E for all data collected from seven different clinical samples (tumour, n ¼ 40; normal, n ¼ 48). b, Gaussian fit for all tumour data from the seven samples.
c, Log-normal fit for all normal data from the samples. d – f, Data from a single clinical sample: values of E for all cells (d), gaussian fit of tumour cells (e), and log-
normal fit of normal cells (f) for this sample. g, Histogram of E with log-normal fit for a single negative sample. h,i, Gaussian fits of visually diagnosed metastatic
(tumour, h) and mesothelial (normal, i) cells in an individual clinical sample (Table 1, Case 4).

ex vivo culture there still appeared to be two populations of cells, which yielded morphologically indistinguishable cell types. The
one cell type that was larger and more flat, suggestive of benign resulting Young’s modulus values for tumour and benign
mesothelial cells, and the other type, which was more round and mesothelial cells were analogous to those obtained using the 12 h
balled up, suggestive of tumour cells. Despite the difference of ex vivo culture method (see Supplementary Information for more
morphology in ex vivo culture, nanomechanical analysis details). This correlation of cell stiffness for the tumour and
concluded that these were all tumour cells. Average values normal cells in the pleural effusions experimentally demonstrates
(mean+s.d.) for both the tumour and morphologically classified that the mechanical analysis is representative of cell type, even
benign mesothelial cells were 0.54+0.08 kPa and 0.54+0.12 kPa, when considering different preparation methods and the use of
respectively (Fig. 2 h,i; n ¼ 8). These population means were not different substrates.
statistically different, and indicated the presence of a single The need for biomarkers for cancer detection and analysis is
population of malignant cells. Subsequent immunohistochemical critical due to the complexity of the disease. This study shows
staining (IHC) with a battery of markers including Calretinin, important implications for the combined use of imaging analysis
B72.3 and Ber-EP4 (see Supplementary Information) confirmed with nanomechanical measurements as a novel biomarker for
that the sample contained predominantly one cell population, evaluating and sensing changes in tumour cells, and which is
which was indeed malignant based on IHC findings (negative for easily translated into clinical settings. Our results suggest that a
Calretinin and positive for Ber-EP4 and B72.3). Additionally, this shift in biologically driven biomechanical properties towards a
particular case demonstrates no correlation between ex vivo decrease in cell stiffness correlates with an increase in metastatic
cultured cell morphology and expected measured stiffness. This potential. Under analogous conditions, cell stiffness of metastatic
lack of correlation suggests that it is unlikely that the ex vivo cancer cells is .70% lower than normal reactive mesothelial
cultured morphological differences influence measured elasticity. cells in the same sample, when compared to other pleural
For each individual patient effusion a new cantilever was used, effusions, or even for patients with different clinical histories
to avoid any contribution of possible artifacts. The similarity in the (Table 1). This finding is consistent with previous studies, which
data from patient to patient and cell to cell is strongly suggestive of found a decrease in stiffness of human cancer cell lines with
a parallel between patient effusions and cell type. To further increasing metastatic efficiency using different single-cell in vitro
confirm that the measured differences between tumour and biomechanical assays13–15. Despite morphological overlap between
benign cells were not an artifact of ex vitro culturing, we used a tumour and normal cell types, which often poses a significant
modified cytocentrifugation procedure without ex vivo culturing, problem for cytomorphological and immunohistochemical

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LETTERS
diagnosis of malignant cells, mechanical analysis provides the followed by 1:10,000 DAPI for 5 min. All incubations were performed at room
ability to distinguish cancer cells. These findings clearly temperature and there were three PBS washing steps in between. Cells were
demonstrate the ability of nanomechanical-based functional covered with mounting medium for fluorescence microscopic examination.
Images were taken using an Olympus BX-40 microscope with a 40 objective.
analysis to detect metastatic tumour cells in body fluid. Likewise,
the distribution of measured cell stiffness for tumour cells is over STATISTICAL ANALYSIS
five times narrower than the corresponding distribution for Data were expressed as mean+s.d., and the statistical significance of differences in
benign mesothelial cells. We found that tumour cell stiffness fits mean values was assessed using a two-sample independent Student’s t-test at the
with a normal distribution, whereas the measured benign 95% confidence level. Differences among means are reported using exact P values.
mesothelial cell stiffness values better fit a log-normal
distribution. The finding that no quantitative overlap was Received 9 September 2007; accepted 19 October 2007;
observed in the measured cell elasticity values collected for the published 2 December 2007.
metastatic tumour and reactive mesothelial cells is of major
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mouse anti-human Ber-EP4 (DAKO) at 1:300 dilution for 1 h, followed by Acknowledgements
Cy3-conjugated AffiniPure goat anti-mouse IgG(H þ L) (Jackson S.E.C. and J.K.G. acknowledge partial support from National Institutes of Health research grant no. 5 R21
GM074509 and from the Institute for Cell Mimetic Space Exploration, a National Aeronautics and Space
ImmunoResearch Lab) at 1:200 dilution for 30 min, then with BODIPY FL
Administration University Research Engineering Technology Institute. J.R. and Y.J. acknowledge partial
phallacidin F-actin (Molecular Probes) at 1:40 dilution for 30 min. Finally, cells support from National Institutes of Health research grant no. U01CA96116 and Alper grant, Johnsson
were incubated with 1:10,000 DAPI for 5 min. For DNA/Calretinin/Ber-EP4 Comprehensive Cancer Center.
labelling, cells were first incubated with mouse anti-human Ber-EP4 (DAKO) at Correspondence and requests for materials should be addressed to J.K.G. and J.R.
Supplementary information accompanies this paper on www.nature.com/naturenanotechnology.
1:300 dilution for 1 h, followed by Cy3-conjugated AffiniPure goat anti-mouse
IgG(H þ L) (Jackson ImmunoResearch Lab) at 1:200 dilution for 30 min. Cells
were then further incubated with 1:600 diluted rabbit anti-human Calretinin Author contributions
S.E.C., Y.J., J.R. and J.K.G. contributed equally to this work.
antibody (Zymed) for 1 h then with FITC-conjugated AffiniPure goat anti-rabbit
IgG(H þ L) (Jackson ImmunoResearch Lab) at 1:50 dilution for 30 min, Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/

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