Functionalised AFM Probes for the Investigation of
Integrin Distribution on the Surface of Osteosarcoma-
Federico Caneva Soumetz, Laura Pastorino, Roberto Raiteri, and Carmelina Ruggiero*, Member, IEEE
Abstract-Integrins are transmembrane glycoproteins made of the field of cell biology and tissue engineering such as for the
non-covalently bonded α and β chains involved in fundamental investigation of cell response to external stimuli.
biological processes such as growth, development and wound
repair. The expression of proper cell surface receptors plays a In this work AFM probes have been functionalised and
pivotal role to promote such phenomena. To this regard the tested for the investigation of integrins expressed on the
possibility to investigate cell membrane at the single molecule surface of a line of human osteosarcoma-derived osteoblasts
level is of high potential in the field of cell biology and tissue named MG-63. To this purpose a functionalisation protocol
engineering. In this work functionalised AFM probes have been based on the covalent immobilization of monoclonal antibodies
successfully developed and tested for the detection of integrins
expressed on the surface of a line of osteosarcoma-derived to the tip of silicon nitride AFM probes has been used. Force
osteoblasts named MG-63. The obtained results demonstrate the curves have been then collected over the surface of gently
high potential of the AFM technique for the investigation of cell fixed cells obtaining maps of the interaction between the
surface functional molecules at the nanoscale. immobilized antibodies and the respective cell membrane
Integrins are a family of glycosylated heterodimeric MATHERIALS AND METHODS
transmembrane proteins that consist of non-covalently bonded Cell culture: MG63 cells have been cultured in the presence
α and β chains. They are involved in cell-extracellular matrix of CO2 (5% v/v) at 37°C in Dulbecco’s Modified Eagle’s
and cell-cell interaction and then in fundamental processes Medium (DMEM) supplemented with fetal bovine serum (10
such as cell adhesion, growth, differentiation, tissue % v/v), 0.5% antibiotics (stock: 5000 U/mL Penicillin, 5000
development and wound repair . Until now over 17 α and 9 µg/mL Streptomycin) and 2 mM glutamine (Invitrogen). AFM
β subunits have been identified. From these subunits only 23 analysis have been carried out on gently fixed cells treated with
integrins are formed in nature, which means that not all a 2% paraphormaldehyde (PFA from Sigma-Aldrich) solution
possible combinations exist. For adhesion to ligands both in Phosphate Buffer Saline (PBS) for incubation times ranging
integin subunits and the presence of cations, which bind to from 30 seconds to 1 minute.
cation binding sites of the alpha chain, are needed [1, 2]. Fluorescence microscopy: Fluorescence microscopy analysis
Integrin expression in human osteoblast is well assessed [3-6]. have been at first carried out to identify integrin rich cell
As relates to MG-63 cells, this cell line has been shown to membrane regions in order to gain insights into integrin
express integrins of the β1 class and specifically the α3β1 distribution over the cell surface and to shorten the time
(laminin receptor), α2β1 and α5β1 integrins (respectively required to carry out the AFM analysis. Prior to be analysed by
collagen and fibronectin receptors) . It has been fluorescence microscopy, MG63 cells have been cultured in
demonstrated that the expression of such receptors can be standard conditions and then fixed with 4% PFA in PBS for 15
modulated by inflammatory mediators such as the human minutes at room temperature (RT). At this point, in order to
cytokine Transforming Growth Factor β1 (TGFβ1) and reduce non specific interactions, after a washing step in PBS,
Interleukin-1β (IL-1β) [7, 8]. In addition, it is known that the fixative aldehydic groups have been quenched by exposing
during tissues development, the presence of environmental the fixed cells to a 2% solution of glycine in PBS for 15
conditions permissive to cell differentiation is associated with minutes at RT. Subsequently, cells have been treated with a
the expression of proper cell surface receptors (e.g. integrins) solution of a primary antibody (mouse anti-human β1
. To these regards, the possibility to investigate cell monoclonal IgG - Abd Serotec) for 20 minutes at RT
membrane at the single molecule level is of high potential in (concentration: 5µg/ml). At this point cells have been labeled
with a secondary antibody (AlexaFluor®488 rabbit anti-mouse
This work was supported by the European Union within the project “Tools IgG - Invitrogen) for 40 minutes at RT (concentration: 2
and Technologies for the Analysis and Synthesis of nanostructures – Tasnano”
(contract number 516865). Roberto Raiteri is with the Biophysical and µg/ml). As final step DNA has been stained with DAPI (Sigma
Electronic Engineering Department, Via All’Opera Pia 11a, 16145 Genova, Aldrich) (incubation time: 30 minutes at RT, concentration: 1
Italy (E-mail: firstname.lastname@example.org). All other authors are with the Department of µM). Washing steps have been carried after the addition of the
Communication, Computer and System Sciences, Nanobiotechnology and
Medical Informatics Laboratory, University of Genova, Italy (*contacting
primary and secondary antibodies with a solution of 0,1% BSA
author, phone: +390103532991; fax: +390103532154; E-mail: in PBS. In order to assess the specificity of the primary
antibody, different negative control samples have been evidence the high potential of the AFM technique in the field
prepared by omitting its addition during the staining procedure. of cell biology and tissue engineering for the investigation of
AFM probes functionalisation: a functionalisation procedure cell surface functional molecules at the nanoscale.
based on the covalent immobilization of thiolated biomolecules
to a 8 nm long bifunctional PEG molecule (pyridyl dithio-
Polyethyleneglycol-succinimidylpropionate, Polypure)  RO. Hynes, “Integrins: a family of cell surface receptors,” Cell, vol. 48,
pp. 549-554, 1987.
previously bound on the surface of aminated cantilevers has  K. Anselme, “Osteoblast adhesion on biomaterials,” Biomaterials, vol.
been used. Specifically, the AFM tips have been at first 21, pp. 667-681, 2000.
aminofunctionalized with ethanolamine. Subsequently the  JH. Bennett, DH. Carter, AL. Alavi, JN. Beresford, and S. Walsh,
“Patterns of integrin expression in a human mandibular explants model of
introduced amino groups have been reacted with the functional osteoblast differentiation,” Arch Oral Biol, vol. 46, pp. 229-238, 2001.
succinimide group of the bifunctional linker. At this point  DE. Hughes., DM. Salter., and S. Dehar, “Integrin expression in human
protected sulfhydryl groups have been introduced in the bone,” Journal of bone and mineral research, vol. 8(5), pp. 527-533,
antibody molecules using SATP (N-Succinimidyl S-  MC. Siebers, PJ. Brugge, XF. Walboomers, and JA. Jansen, “Integrins as
acetylthioproprionate). Subsequently, the protected sulfhydryl linker proteins between osteoblasts and bone replacing materials. A
groups have been deacetylated in order to deprotect them. The critical review,” Biomaterials, vol. 26, pp. 137-146, 2005.
 M. Pistone, C. Sanguineti, A. Federici, F. Sanguineti, P. Defilippi, F.
modified antibodies have been then coupled to the thiol Santolini, G. Querze, PC. Marchisio, and P. Manduca, “Integrin synthesis
reactive group of the linker molecules previously immobilized and utilization in cultured human osteoblasts,” Cell Biol Int, vol. 20, pp.
on the surface of aminofunctionalised cantilevers. By this 471-479, 1996.
 J. Heino, and J. Massaguè, “Transforming growth factor beta switches
procedure monoclonal antibodies specific to the β1 integrin the pattern of integrins expressed in MG-63 human osteosarcoma cells
subunit (mouse anti-human β1 monoclonal IgG from Abd and causes a selective loss of cell adhesion to laminin,” The Journal of
Serotec) have been immobilized on the surface of silicon Biological Chemistry, vol. 264(36), pp. 21806-21811, 1989.
 P. Santala, J. Heino, “Regulation of integrin-type cell adhesion receptors
nitride AFM cantilevers (MSCT Microlever Probes from by cytokines,” The Journal of Biological Chemistry, vol. 266(34), pp.
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AFM analysis: Force curves have been collected in PBS  F. Rosso, A. Giordano, M. Barbarisi, and A. Barbarisi, “From Cell–ECM
Interactions to Tissue Engineering,” Journal of Cell Physiology, vol. 199,
supplemented with Ca2+ ions on a regular grid (64x64 or 174–180, 2004.
128x128) over the cell surface. Simultaneous maps of the cell
topography and of the interaction between the immobilized
antibodies and the respective cell membrane receptors have
been acquired by means of a PicoPlus System from Molecular
Imaging. Functionalised AFM probes characterized by a
Spring Constant of 0.01 N/m have been used. Blocking has
been carried out to assess the specificity of the detected
interaction by adding a 10 µg/ml solution of free monoclonal
antibodies over the sample during the analysis.
Fig. 1. 64 x 64 force map taken on a 4 x 4 µm area. (a) topography
The expression of integrins of the β1 class on the surface of (b) max adhesion (nN)
MG63 cells has been successfully assessed by fluorescence
Fig. 2. Frequency of the maxima adhesion events before and
microscopy using the described double staining procedure. after blocking the AFM tip/sample interaction
Integrins resulted to be slightly distributed all over the cell
surface. Integrin rich regions have been identified close to cell
boundaries and within regions of contact with other cells. AFM
analysis has been carried out on such regions by means of
functionalised AFM probes. In Fig. 1 maps of cell topography
and of maxima adhesion events (nN) (white spots) detected
between the antibodies immobilized on the AFM tip and the
respective cell membrane receptors are reported. As shown in
Fig. 2, the injection of free antibodies over the sample during
the analysis resulted in a reduction of the force of the maxima
adhesion events thus demonstrating their specificity. AFM tips
have been then successfully functionalised and tested for the
investigation of integrins of the β1 class on the surface of
MG63 cells. The obtained results show the possibility to map
the presence and the distribution of cell surface receptors by
means of functionalised AFM tips. In addition, results put in