We gratefully acknowledge the open-source sharing of MATLAB particle-tracking code provided by Maria Kilfoil (<a href="http://people.umass.edu/kilfoil/">http://people.umass.edu/kilfoil/</a>), along with the earlier IDL code and extensive documentation provided by John C. Crocker and Eric R. Weeks. This work was made possible by funding from the National Cancer Institute (NCI/NIH), K99CA155045 and R00CA155045 (PI: JPC).

1. Culturing Tumor Spheroids
<ol>
	<li>Mix 10 ml of cell culture grade water with 0.1 g of agarose to obtain a 1% agarose solution.</li>
	<li>Heat agarose solution to above 70 &#176;C (roughly 14 sec&#160;in a standard microwave or by using a heating plate) before aliquoting 40 &#956;l&#160;of agarose solution into a well on a 96-well plate.</li>
	<li>Incubate plate at 37 &#176;C for at least 1 hr while harvesting cells using standard techniques.</li>
	<li>Use a hemacytometer to determine the concentration of cells in ...

The authors declare that they have no competing financial interests.

In this protocol we introduce a robust and widely applicable strategy for longitudinally tracking local changes in ECM rigidity in 3D tumor models. We envision that this methodology could be adopted by cancer biologists and biophysicists interested in mechanosensitive behavior implicated in matrix remodeling during tumor growth and invasion processes. Precise quantification of matrix degradation kinetics could be particularly valuable to those studying the activity of matrix metalloproteases, lysyl oxidase or other relev...

It is clear from a growing body of evidence in the literature that cancer cells, as with non-malignant mammalian epithelial cells, are highly sensitive to the mechanical&#160;and biophysical properties of the surrounding extracellular matrix (ECM) and other microenvironment components1-9. Elegant mechanistic studies have provided insights into the role of extracellular rigidity as a complex mechanosensitive signaling partner that regulates malignant growth behavior and morphogenesis2,3,10,11. This work has been facilitated in particular by the development of 3D in vitro tumor models that restore biologically relevant tissue architecture and can be grown in scaffold materials with tunable mechanics and imaged by optical microscopy12-19. However, the other side of this mechanoregulatory dialog between tumor and microenvironment, through which cancer cells in turn modify the rheology of their surroundings, remains somewhat more difficult to study. For example, during invasion processes, cells at the periphery of a tumor may undergo epithelial to mesenchymal transition (EMT) and increase expression of matrix metalloproteases (MMPs) that cause local degradation of ECM20-22, which in turn influences mechanosensitive growth behavior of other proximal tumor cells. Through a variety of biochemical processes, cancer cells continually dial the local rigidity of their environment up and down to suit different processes at different times. The methodology described here is motivated by the need for analytical tools that report local changes in the rigidity and compliance of the ECM during growth, that can be integrated with 3D tumor models and correlated longitudinally with biochemical and phenotypic changes without terminating the culture.
In search of an appropriate technique to implement in this context, particle-tracking microrheology (PTM) emerges as a strong candidate. This method, pioneered originally by Mason and Weitz23,24, uses the motion of tracer probes embedded in a complex fluid to report the frequency-dependent complex viscoelastic shear modulus, G*(&#969;) at micron length scales. This general approach has been developed with multiple variations suited for different applications in soft-condensed matter, colloids, biophysics and polymer physics25-31. PTM has certain advantages relative to other methods, since readouts of local viscoelasticity are provided by non-destructive video imaging of biochemically inactive tracer probes that are incorporated at the time of culture preparation and remain in place over extended periods of growth. This is in contrast to gold standard measurements with an oscillatory shear bulk rheometer, which necessarily requires termination of the culture and reports the bulk macroscopic rheology of the sample rather than point measurements within the complex 3D tumor microenvironment. Indeed a number of studies have illustrated the utility of interpreting measurements of tracer probe movements in or around cancer or non-cancer cells to measure deformations associated with cell migration32, mechanical stress induced by an expanding spheroid33, intracellular rheology34,35, and to map mechanical stresses and strains in engineered tissues36, and relation between pore size and invasion speed37. Other techniques suitable for microrheology, such as atomic force microscopy (AFM) can be implemented, but primarily for probing points at the sample surface and also may pose culture sterility issues that complicate longitudinal measurements38.
Here, we describe a comprehensive protocol encompassing methods for growth of 3D tumor spheroids suitable for transfer into ECM with embedded fluorescent probes for video particle-tracking and analysis methods for reliably mapping spatial changes in microrheology over time in culture. In the present implementation, 3D tumor models are grown in multiwell format with a view towards incorporation of microrheology measurements with other traditional assays (e.g., cytotoxicity) which this format is conducive to. In this representative illustration of this methodology we culture in vitro 3D spheroids using PANC-1 cells, an established pancreatic cancer cell line known to form spheroids39, but all measurements described herein are broadly applicable to study of solid tumors using a variety of cell lines suitable for 3D culture. Because this method is inherently imaging-based it is ideally suited for co-registration of high-resolution microrheology data with large transmitted-light fields of view that report changes in cell growth, migration and phenotype. The implementation of PTM integrated with transmitted light microscopy in this manner assumes reproducible positioning of the microscope stage which is typically available on motorized commercial widefield epifluorescence biological microscopes. The protocol developed below can be implemented with any reasonably equipped automated fluorescence biological microscope. This is an inherently data-intensive method, which requires acquisition of gigabytes of digital video microscopy data for offline processing.
In the following protocol, Protocol&#160;1 pertains to the initial preparation of tumor spheroids which is described here using overlay on agarose but could be substituted with a variety of other methods such as hanging drop40, or rotary culture41 techniques. Protocol&#160;2 describes the process of embedding spheroids in a collagen scaffold though alternatively, in vitro 3D tumors could be grown by encapsulation or embedding of resuspended cells in ECM12,15, rather than single pre-formed non-adherent spheroids. Subsequent protocols describe procedures for obtaining time-resolved microrheology measurements by acquiring and processing video microscopy data, respectively. Data processing is described using MATLAB, making use of open source routines for PTM built on algorithms originally described by Crocker and Grier42, which have also been extensively developed for different software platforms&#160;(see&#160;http://www.physics.emory.edu/~weeks/idl/).

<table border="0" cellpadding="0" cellspacing="0" width="779">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:177px;">Bovine type 1 collagen</td>
			<td style="width:235px;">BD Biosciences, San Jose, California</td>
			<td style="width:100px;">354231</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PANC-1</td>
			<td>American Type Cell Culture, Manassas, VA</td>
			<td>CRL1469</td>
			<td>or other appropriate cell type</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescent Microspheres</td>
			<td>Life Technologies, Carlsbad, California</td>
			<td>906906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel</td>
			<td>BD Biosciences, Bedford MA</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose</td>
			<td>Fisher Bioreagents, Waltham, MA</td>
			<td>C12H18O9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaOH</td>
			<td>Fisher Bioreagents, Waltham, MA</td>
			<td>NC0480985</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-well Imaging plates</td>
			<td>Corning Inc., Corning, NY</td>
			<td>3904</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Hyclone, Waltham, MA</td>
			<td>SH30243.01</td>
			<td>or appropriate&#160; cell culture media</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Zeiss AxioObsever Microscope</td>
			<td>Zeiss, Oberkochen, Germany</td>
			<td></td>
			<td>includes high-speed camera and imaging software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MATLAB software</td>
			<td>The Mathworks, Natick, MA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

longitudinal measurement of extracellular matrix rigidity in 3d tumor models using particle-tracking microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

To verify the validity of G*(&#969;) measurements at localized positions within a complex model tumor microenvironment, two initial validation experiments were conducted. First, we sought to validate our measurements against the "gold standard" of bulk oscillatory shear rheometry. We prepared identical samples of collagen matrix (without cells) at a concentration of 1.0 mg/ml collagen. These samples were probed with a bulk rheometer (TA Instruments AR-G2, using 40 mm parallel plate geometry) and by PTM (using th...

Watch this Scientific Journal Video about Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology at JoVE.com

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

longitudinal-measurement-extracellular-matrix-rigidity-3d-tumor

Research

JoVE Journal

Bioengineering

11.4K 조회수.  University of Massachusetts Boston.  입자 추적 microrheology은 비파괴 정량 및 공간적 차원 종양 모델에서 세포 외 기질 (extracellular matrix) 기계적 성질의 변화를 매핑하는 데 사용할 수 있습니다. 