Effect of collagen I and fibronectin on the adhesion, elasticity and cytoskeletal organization of prostate cancer cells

doi:10.1016/j.bbrc.2010.10.034

Biochemical and Biophysical Research Communications

Volume 402, Issue 2, 12 November 2010, Pages 361-366

https://doi.org/10.1016/j.bbrc.2010.10.034 Get rights and content

Abstract

Despite of intensive research efforts, the precise mechanism of prostate cancer metastasis in bone is still not fully understood. Several studies have suggested that specific matrix production by the bone cells, such as collagen I, supports cancer cell invasion. The aim of this study was to investigate the effect of collagen I (COL1) and fibronectin (FN) on cell adhesion, cell elasticity and cytoskeletal organization of prostate cancer cells.

Two cell lines, bone marrow- (PC3) and lymph node-derived (LNCaP) were cultivated on COL1 and FN (control protein). By using a quantitative adhesion assay and time-lapse analysis, it was found that PC3, but not LNCaP, adhered strongly and were more spread on COL1. Next, PC3 and LNCaP were evaluated by atomic force microscopy (AFM) and flatness shape factor and cellular Young’s modulus were calculated. The shape analysis revealed that PC3 were significantly flatter when grown on COL1 in comparison to LNCaP. In general, PC3 were also significantly stiffer than LNCaP and furthermore, their stiffness increased upon interaction with COL1. Since cell stiffness is strongly dependent on actin organization, phalloidin-based actin staining was performed and revealed that, of the two cell types as well as the two different matrix proteins, only PC3 grown on COL1 formed robust actin cytoskeleton.

In conclusion, our study showed that PC3 cells have a strong affinity towards COL1. On this matrix protein, the cells adhered strongly and underwent a specific cell flattening. Moreover, with the establishment of PC3 contact to COL1 a significant increase of PC3 stiffness was observed due to a profound cytoskeletal rearrangement.

Research highlights

► Depending on the metastatic origin, prostate cancer cells differ in their affinity to COL1. ► COL1 affects specifically the F-actin and cell elasticity of bone-derived prostate cancer cells. ► Cell elasticity can be used as a biomarker for cancer cells from different metastases.

Introduction

Prostate cancer is the second most common form of cancer in U.S. male population [1]. Although the primary tumor originates in the prostate, the prostate cancer cells frequently spread to other organs, particularly the bones and the lymph nodes [2], [3]. An earlier study by Bubendorf et al. [4] reported that skeletal metastases occur in up to 90% of patients dying from prostate carcinoma.

In order to invade and form the secondary tumor in the bone, prostate cancer cells have to interact with bone-residing cells and with bone-specific extracellular matrix proteins (reviewed in [5], [6]). Collagen type I (COL1) represents the most abundant protein in bone. Kiefer et al., [7] have shown that prostate cancer cells can effectively attach and proliferate on COL1. Hall et al. [8] suggested that the interaction between the prostate cancer cells and COL1 is mediated by integrin receptors. Furthermore, the authors observed that prostate cancer cells which possess COL1-binding affinity develop a significant number of bone tumors in contrast to cancer cells which do not attach to COL1 [8].

Several studies have reported that cancer cell transformation can lead not only to a molecular drift but also to changes in the cell biophysical properties, such as cell elasticity and viscosity. These properties are directly dependent on the cytoadherance, actin architecture and intracellular tension ([9] and reviewed in [10]). The elastic properties of a live cell can be measured by atomic force microscopy (AFM). In particular, the cellular Young’s modulus can be calculated from the AFM cantilever deflection, when the AFM tip is pushed onto the cell [11]. Li et al. [12] have used AFM to characterize the elasticity of benign and malignant human breast epithelial cells and reported that the latter have significantly lower Young’s modulus than non-transformed cells. In a recent study, we have also performed AFM analysis and compared the cellular Young’s modulus of mesenchymal stem and progenitor cells, osteoblasts and osteosarcoma cells. We found that, among the different cell types, osteosarcoma cells exhibited the lowest Young’s modulus when cultivated on COL1 [13]. Hence, it has been suggested that investigating the mechanical properties of cancer cells may serve as a biomarker for early detection of cancer as well as it may help to understand the biophysical mechanisms contributing to cancer metastasis [12], [13], [14], [15].

In this study, we focused on prostate cancer cells and we implicated two different cell lines: PC3, obtained from bone marrow metastasis and LNCaP, obtained from supraclavicular lymph metastasis. Our objective was to investigate the effect of COL1 on the biophysical properties of prostate cancer cells which differ in their metastatic source. For this purpose, we first compared PC3 and LNCaP attachment and spreading on COL1 and FN. We then used AFM technology to characterize the shape and determine the Young’s modulus of the cells cultivated on COL1 and FN. Finally, we performed phalloidin-based actin staining to correlate the cell elasticity measurements to the cell’s actin content and structure.

Section snippets

Cell lines

PC3 and LNCaP cell lines were obtained from the ATCC (Wesel, Germany). PC3 were maintained in RPMI-1640 cell culture media (PAA, Cölbe, Germany) supplemented with 10% FBS (Sigma–Aldrich, Munich, Germany). LNCaP cells were cultured in MEM Alpha GlutaMAX culture media (Invitrogen, Karlsruhe, Germany) and 10% FBS. During routine cell culture, both cell types were maintained at 60–80% confluence in T-75 culture flasks (Nunc, Wiesbaden, Germany) at 37 °C in 5% humidified CO₂. The culture medium was

Appearance, adhesion and spreading of PC3 and LNCaP on COL1 and FN

Fig. 1 A shows the morphological appearance of PC3 and LNCaP when cultivated on COL1 or FN. At low confluence, both cell types remained as single cells, apart of LNCaP on FN, where the cells tended to form cell clusters. PC3 and LNCaP affinity to attach onto COL1 and FN was examined by a quantitative adhesion assay (Fig. 1B). On COL1, at 60 min approx. 90% of the PC3 cells were already attached and at 120 min, virtually all PC3 cells have adhered. In contrast, the remaining samples, PC3 on FN and

Discussion

The frequent metastasis of prostate cancer cells to bone is suggested to reflect the ability of the metastatic cells to adhere to bone matrix [2], [17], [18]. COL1 is the most abundant scaffolding protein in the bone. Interestingly, it has been shown that high expression of COL1 is frequently associated with an elevated incidence of cancer metastasis [19]. A recent study by Levental et al. [20] convincingly demonstrated that the tumorigenic process in breast cancer is accompanied by COL1

Acknowledgments

The authors D.D. and M.S. acknowledge Prof. Wolf Mutschler (Head of Surgery Clinic and Policlinic, LMU) for his constant support of the research laboratory. The authors D.P. and H.C.S. gratefully acknowledge the financial support of the German Excellence Initiative via the “Nanosystems Initiative Munich (NIM)”.

References (30)

P. Msaouel et al.
Mechanisms of bone metastasis in prostate cancer: clinical implications
Best Pract. Res. Clin. Endocrinol. Metab
(2008)
L. Bubendorf et al.
Metastatic patterns of prostate cancer: an autopsy study of 1, 589 patients
Hum. Pathol.
(2000)
J.A. Kiefer et al.
Type I collagen-mediated proliferation of PC3 prostate carcinoma cell line: implications for enhanced growth in the bone microenvironment
Matrix Biol.
(2001)
A.R. Bausch et al.
Local measurements of viscoelastic parameters of adherent cell surfaces by magnetic bead microrheometry
Biophys. J.
(1998)
S. Suresh
Biomechanics and biophysics of cancer cells
Acta Biomater.
(2007)
Q.S. Li et al.
AFM indentation study of breast cancer cells
Biochem. Biophys. Res. Commun.
(2008)
J. Guck et al.
Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence
Biophys. J.
(2005)
E.M. Darling et al.
A thin-layer model for viscoelastic, stress-relaxation testing of cells using atomic force microscopy: do cell properties reflect metastatic potential?
Biophys. J.
(2007)
J. Domke et al.
Substrate dependent differences in morphology and elasticity of living osteoblasts investigated by atomic force microscopy
Colloids Surf. B Biointerfaces
(2000)
K.R. Levental et al.
Matrix crosslinking forces tumor progression by enhancing integrin signaling
Cell
(2009)

C. Rotsch et al.

Drug-induced changes of cytoskeletal structure and mechanics in fibroblasts: an atomic force microscopy study

Biophys. J.

(2000)

W.A. Lam et al.

Chemotherapy exposure increases leukemia cell stiffness

Blood

(2007)

S.Y. Tee et al.

The mechanical cell

Curr. Biol.

(2009)

C.L. Hall et al.

Type I collagen receptor (alpha2beta1) signaling promotes prostate cancer invasion through RhoC GTPase

Neoplasia.

(2008)

C.D. Nobes et al.

Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, Lamellipodia, and filopodia,

Cell

(1995)

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Effect of collagen I and fibronectin on the adhesion, elasticity and cytoskeletal organization of prostate cancer cells

Abstract

Research highlights

Introduction

Section snippets

Cell lines

Appearance, adhesion and spreading of PC3 and LNCaP on COL1 and FN

Discussion

Acknowledgments

Best Pract. Res. Clin. Endocrinol. Metab

Hum. Pathol.

Matrix Biol.

Biophys. J.

Acta Biomater.

Biochem. Biophys. Res. Commun.

Biophys. J.

Biophys. J.

Colloids Surf. B Biointerfaces

Cell

Biophys. J.

Blood

Curr. Biol.

Neoplasia.

Cell