Tumor Mechanobiology

Focus on the role of the biophysical properties of the ECM in tumor progression

A review from the literature

Definition and scales

Cell stiffness, Cell contractility,

Cell rheology Matrix stiffness, deformability

Molecular/Cellular Mechanotransduction

Cellular Mechanobiology

Tissue Mechanobiology

ROCK-Driven Actomyosin Contractility Induces Tissue Stiffness and Tumor Growth.Kümper S and Marshall CJ, Cancer Cell. 2011

Pathak A. et al., PLoS One, 2011Discher DE. et al. Science, 2005

Matrix stiffness modulates proliferation, chemotherapeutic response, and dormancy in hepatocellular carcinoma cells.

Schrader J. et al., Hepatology. 2011

Tissue rigidity influences cell behavior

Engler JA. et al., Cell, 2006Discher DE. et al, Science 2005

Changes in the physical/mechanical environment during tumor growth

• Forces generated by the expanding tumor in a restrictive tissue space

• Denser/Stiffer tissue that forms around a tumor • Increased interstitial pressure and altered fluid flow

patterns caused by the growth of new blood vessels

Paszek MJ, et al. J Mammary Gland Biol. and Neoplasia, 2004Shieh AC. Annals of Biomedical Eng. 2011

Outline

I. Can we use tumor biophysical properties as prognostic or diagnostic tools?

II. How do tumor cells respond to physical/ mechanical changes of the environment?

III. Matrix stiffening: cause or consequence?

Outline

I. Can we use tumor biophysical properties as prognostic or diagnostic tools?

II. How do tumor cells respond to physical/ mechanical changes of the environment?

III. Matrix stiffening: cause or consequence?

Tumor mechanobiology

“The stiffer the matrix, the more aggressive the tumor”

“The denser the breast/ the liver, the worst the prognosis”

“The stiffer the matrix, the more aggressive the tumor”

“The denser the breast/the liver, the worst ”

Women who have a breast density of 75 percent or higher on a mammogram have a risk of breast cancer that is four to five times greater than that of women with little or no density, making mammographic breast density one of the strongest biomarkers of breast cancer risk.

Elastography: a non-invasive method to measure tissue stiffness

• Ultrasound or MR-based techniques for measuring stiffness by the difference in the velocity of the elastic shear wave propagation across a given tissue

• The force is generated by applying pressure.

www.breastcancer.about.com

An increased stiffness is diagnostic of diseased tissue

Fibrotic LiverNormal Liver

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Venkatesh SK, et al. A J Roentgenology. 2008

Increased tissue stiffness can assist intervention and therapeutic decisions: biopsy, treatment

Yin et al,. Clin Gastroenterol Hepatol. 2007

Assessing prognosis and candidacy for treatment in patients with chronic liver disease.

Spare patients that don’t need it the discomfort and risk of complications associated with liver biopsy

An increased stiffness is prognostic of disease outcome

An increased stiffness is diagnostic of breast tumor lesions

Invasive Lobular CarcinomaBreast Fibroadenoma

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When a mechanical compression or vibration is applied, the tumor deforms less than the surrounding tissue i.e. the strain in the tumor is less than the surrounding tissue.

Collagen density promotes mammary tumor initiation and progression

• Model system: Col1a1tmJae mouse crossed with MMTV-PyMT mouse double-transgenic tumor model with increased stromal

collagen in mouse mammary tissue.

Provenzano P .et al., BMC Medicine, 2008.

AbstractBackground: Mammographically dense breast tissue is one of the greatest risk factors for developing breast carcinoma. Despite the strong clinical correlation, breast density has not been causally linked to tumorigenesis, largely because no animal model has existed for studying breast tissue density. Importantly, regions of high breast density are associated with increased stromal collagen.

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Increased stromal collagen in mouse mammary tissue significantly increases tumor formation

10 week-old mice; Collagen staining: Picrosirius red

Provenzano P et al., BMC Medicine, 2008

Increased stromal collagen in mouse mammary tissue significantly increases metastasis formation

Provenzano P et al., BMC Medicine, 2008

Definition of tumor-associated collagen signatures

TACS-3: radially aligned collagen fibers, correlates with local invasion of tumor cells

Provenzano P et al., BMC Medicine, 2008

Can these “tumor-associated collagen signatures” be used as a prognostic marker?

Conklin, MW. Et al., AJP, 2011

The relative concentration of collagen and the orientation of fibers with respect to epithelial cells was assessed using SHG imaging on 196 biopsies. Statistical score was developed to determine : -whether TACS-3 is present in clinical histopathology samples from patients

- whether TACS3 correlated with patient survival Yes!

Yes!

Outline

I. Can we use tumor biophysical properties as prognostic or diagnostic tools?

II. How do tumor cells respond to physical/ mechanical changes of the environment?

III. Matrix stiffening: cause or consequence?

Yes!

Outline

I. Can we use tumor biophysical properties as prognostic or diagnostic tools?

II. How do tumor cells respond to physical/ mechanical changes of the environment?

III. Matrix stiffening: cause or consequence?

The stiffer the matrix, the more aggressive/invasive the tumor?

Tumor cells but nor normal cells stiffen in response to stiffer environment

• Technique: Particle-tracking microrheology (PTMR) • Tracking the Brownian motions of individual tracer beads embedded

within the cells using ballistic particle delivery system• The particles motion reflect the intracellular viscoelasticity of the cells.

MCF10A possessing ErbB2 transforming potential stiffen (G’p) in response to elevated matrix stiffness (G’c), whereas non-transformed MECs don’t Increased intracellular stiffening correlated with a more motile phenotype

Baker EL. et al., Biophys. J., 2010

Matrix rigidity modulates tumor cell proliferation, morphology and migration

• Technique: 96-well plate system that arrays colI-conjugated PA gels that increase in stiffness by at least 50-fold across the plate

Tilghman R. et al., PLoS One, 2010

Matrix rigidity modulates tumor cell proliferation, morphology and migration (2)

A549 cells

Tilghman R. et al., PLoS One, 2010

Stiffness and adhesivness modulate tumor cell mechanotransduction and migration

• On stiff ECMs, more protrusive adhesions are stabilized due to higher Rho and Rac activation levels, leading to maximal stress fiber contractility, and rear adhesions progress through the initiation-rupture cycle rapidly. Both of these promote higher migration speeds

Pathak A. and Kumar S., PLoS One, 2011

Stiffness and adhesivness modulate tumor cell mechanotransduction and migration (2)

• Lower ligand-density surfaces require higher ECM elasticities to reach the maximum migration speed, whereas higher ligand-density surfaces enhance migration speed on relatively soft ECMs.

Pathak A. and Kumar S., PLoS One, 2011

Matrix rigidity governs the formation of invadopodia

On hard PA, invasive MCF10ACA1d produced more invadopodia and degraded more ECM than on soft PA.

Alexander NR. et al., Current Biol. 2008

Cell, 2009

Model: MMTV-Neu

Mammary glands conditioned with LOX expressing fibroblasts were stiffer and promoted tumor growth

1) LOX conditioning of the mammary gland results in more fibrillar collagen and more linearized collagen.

2) LOX-conditioned mammary gland favors tumor progression

Signaling: -Integrin Clustering -Enhancement of PI3K Signaling

Levental KR et al., Cell, 2009

Cancer patients expressing high levels of LOX have poor outcome

Erler J et al., Nature, 2006

Breast Cancer Patients (ER neg.)

H&N Cancer Patients

Inhibition of LOX decreased metastasis formation in vivo

Erler J et al., Nature, 2006

Model: Orthotopic injection of MDA-MB-231 in the mammary fat pad of nude mice.

The stiffer the matrix, the more aggressive/invasive the tumor?

Yes!

Outline

I. Can we use tumor biophysical properties as prognostic or diagnostic tools?

II. How do tumor cells respond to physical/ mechanical changes of the environment?

III. Matrix stiffening: cause or consequence?

Can the physical properties of the 3D environment act as an initiating event of tumorigenesis?• Question: What is the impact of mechanical stimulation on

the initiation of colon cancer. • Model System:

• Colon explants• The deformation of the tissue induced by compression was observed

by two-photon excitation (2PEF) microscopy of the endogenous tissue fluorescence

Whitehead J. et al., HFSP Journal, 2008

Myc and Twist1 expression is induced by mechanical stimulation of APC deficient but not WT colon

Whitehead J. et al., HFSP Journal, 2008

b-catenin nuclear translocation is induced by mechanical stimulation of APC1638N/+ but not WT colon

When APC is limiting, mechanical strain (intestinal transit or tumor growth), can be interpreted by cells of preneoplastic colon tissue as a

signal to initiate a b-catenin dependent transcriptional program characteristic of cancer.

Whitehead J. et al., HFSP Journal, 2008

Remodeling of the ECM is in part induced by the tumor cells

2. Secretion of ECM remodeling enzymes: transglutaminases, lysyl oxidases, etc.

Matrix remodeling

Cell mechano-

transduction

1.

3. Role of the stromal cells in environment remodeling. Before, after or in synergy with the tumor cell-induced remodeling