The authors have nothing to disclose.

1. Clonogenic Assay
<ol>
	<li>Prepare a single cell suspensions of estrogen-dependent breast cancer cell lines MCF-7 and T47D cells using the steps outlined below
	<ol>
		<li>Aspirate the culture medium (DMEM/10% heat inactivated fetal calf serum/glutamine and pen/strep) from a 10 cm tissue culture dish which is no more than 50% confluent with MCF-7 or T-47D cells. Rinse with PBS. Incubate with trypsin 0.25%/2.21 mM EDTA dissolved in DMEM high glucose at 37 &#176;C for 1-4 min.</li>
		<li>Check cells at 1 min intervals under a phase contrast microscope to ensure a single cell distribution. Resuspend the cells with a 2 ml pipette by pipetting up and down several times to disrupt cell-cell contact to achieve an almost invariable, single cell status.</li>
		<li>Continue to incubate cells in trypsin at 37 &#176;C for up to 4 min if you observe clumps of cells after only 2 min of incubation. Do not use these cells for clonogenic studies if they remain adherent to each other after 4 min of trypsinization because error will be introduced in the colony number yield. 
		NOTE: If cells are clumped, the number of colonies formed will reflect the product of fewer cells than the number incubated. If cells are excessively trypsinized, their clonogenic potential may be diminished.</li>
		<li>Prepare a single cell suspension of 1,500 cells/ml culture medium for 24 well plates, or less, (+ 500 cells/ml, depending on the cell type or passage number), by serial dilutions in one master tube containing the entire volume needed for all of the variables in the experiment. 
		NOTE: The goal is a final cell density of 800 cells/cm2 (range of approximately 500 to 1,100 cells/cm2). The goal is to yield approximately 100 + 50 colonies, which permits relatively easy counting, prevents crowding and permits sufficient colonies to result in significant statistical differences when colonies are increased or decreased by experimental perturbations.</li>
	</ol>
	</li>
	<li>Incubate Cells at Clonogenic Density Using Steps Outlined Below
	<ol>
		<li>Incubate cells in quadruplicate wells on 24 well fibronectin-coated plates at a clonogenic density of 1,500 cells/well from a master single cell suspension tube of 1,500 cells/ml. Triturate the medium containing cells with a 5 ml pipette by drawing up 3 ml and dispensing 1 ml medium in each of 2 wells. 
		NOTE: Fibronectin-coated plates should be purchased pre-coated from a commercial vendor. Coating plates outside of a quality controlled, automated process results in an uneven surface unsuited for this assay.</li>
		<li>Mix the suspension by pipetting up and down with a 5 ml pipette, draw up 3 ml of cell suspension and fill 2 wells with 1 ml each. Fill only 2 wells at any one time from one pipette. Return the remaining volume in the pipette to the cell suspension in the master tube, resuspend cells again by pipetting up and down and draw up another 3 ml to fill another 2 wells with 1 ml each. 
		NOTE: Continuous mixing of the master tube is necessary because cells will continuously sediment. Drawing up sufficient volume to fill only 2 wells is necessary to add similar cell numbers to each well because cells sediment in the pipette as well.</li>
		<li>Work rapidly to distribute the large volumes of cells because allowing cells to sit in suspension at room temperature and CO2 concentration will modulate their clonogenic potential (unpublished observations).</li>
		<li>Optimize the spatial distribution of cells during the act of pipetting them into wells for colony assays. Do so by slowly pipetting the suspension containing the final cell concentration into the middle of the well. Do not subject the plate to further motion before cells settle to the bottom. Do not swirl the plate because circular mixing will effectively centrifuge the cells to the perimeter of the well creating high cell densities and uncountable confluent colonies at 6 days. 
		NOTE: Do not mix unless necessary since this is less desirable than no mixing at all after introducing the volume with the cells in suspension. If it is necessary to mix cells, do so by moving the plate back and forth in perpendicular directions while it rests on a flat surface.</li>
		<li>Incubate cells at 37 &#176;C 5% CO2 without media change for 6 days. The small number of cells in a well after 6 days will not significantly impact the nutrient or cytokine composition nor the pH of the original medium.</li>
		<li>Design the time course of the assay as follows: Incubate cells on fibronectin coated substrata on day&#160; 1. Replace existing medium with 1 ml fresh medium or fresh medium containing FGF-2 10 ng/ml on day 0. Stain cells on day 6 as below in 1.4). Conduct any experimental perturbations on day 3, as below in 1.3).&#160;</li>
	</ol>
	</li>
	<li>Set Up Experimental Perturbations of Molecular Signaling or Adhesion Molecules
	<ol>
		<li>On day 3, add 100 &#956;l of a solution containing 10x of the final intended concentration of the perturbing agent to the 1 ml medium in the wells. Do not mix. Continue to incubate the cells at 37 &#176;C, 5% CO2 for an additional 3 days. 
		NOTE: Perturbing agents can include a variety of inhibitors and blocking agents of adhesion molecules, receptors or other surface proteins, inhibitors of intracellular signaling pathways, molecules, factors, cofactors or structural proteins, that may play roles in supporting the dormant state.</li>
		<li>Stain colonies on day 6, as follows.&#160;</li>
	</ol>
	</li>
	<li>Stain Colonies
	<ol>
		<li>Stain cells after 6 days in culture with a freshly made 0.1% crystal violet in 2% ethanol/10 mM sodium borate (pH 9.0) solution. Aspirate media and add one ml crystal violet solution to each well for 20 min.</li>
		<li>Wash plates by immersing them with the well openings facing down at an acute angle into an ice bucket overflowing with continuously running tap water in the sink. Tilt the plate to a horizontal angle once underwater, well opening down, and then tilt back to an acute angle when removing in one gentle flowing motion.</li>
		<li>Repeat the immersion 2 or 3 times until the water at the bottom of the wells is no longer blue. Vigorous washes may remove cells or colonies that are less adherent due to experimental intervention, adding significantly to the error in counting and the data.</li>
		<li>Dry plates overnight by placing them upside down on towels on the bench top adjacent to their corresponding labeled covers.&#160;</li>
	</ol>
	</li>
	<li>Count Colonies
	<ol>
		<li>Count the number of growing and dormant colonies in each well after 6 days of incubation, staining and drying. Count colonies optimally at 40X magnification in an inverted phase contrast microscope. Count colonies of &#62;30 cells as growing and colonies of 12 or less cells, with the morphological appearance of very large size compared to growing cells, large expanded cytoplasm with large cytoplasm to nucleus ratios, shown in Figure 17,15. 
		NOTE: Clusters of 13-29 cells are not normally counted as they are not very frequent in straightforward dormancy assays. They can be counted if perturbations shift the growth potential of either growing or dormant cells. These results will then need to be correlated with biological significance.</li>
	</ol>
	</li>
</ol>
2. Clonogenic Incubation for Immunofluorescence Studies
<ol>
	<li>Adjust cell numbers to approximately 7,500-8,000 cells/well in 6 well plates, to correspond to cell numbers/surface area analogous to 24 well experiments.</li>
	<li>Place a round, sterile, fibronectin-coated cover slip into each well base of 6 well plates for imaging studies prior to cell addition. Pipet cells in 3 ml volumes into each well 2 wells at a time at concentrations outlined, as described for the clonogenic experiments above in 1.2).</li>
	<li>Incubate cells for 6 days, as above in 1.2.4 and 1.2.5. Add perturbing factors on day 3 at 10x concentrations in 300 &#956;l volumes, as described in the colony assay procedures in 1.3).</li>
	<li>Stain cells on day 6 with antibodies to cell adhesion molecules such as integrins &#945;4, &#945;5, &#945;6, &#946;1, &#946;3, for example, focal adhesion complex molecules FAK, paxillin and vinculin, for example, proteins involved in motility such as &#945;-tubulin, for example, signaling pathway members such as phospho-Akt, phospho-ERK, phospho-p38, phospho-JNK, for example, or any other protein that is the target of investigation for its role in dormancy, using standard techniques for direct or indirect immunofluorescence staining.
	<ol>
		<li>Remove slides with forceps day 6. Fix in acetone/methanol 1:1 at -20 &#176;C for 20 min and air dry. An alternative fixative, such as paraformaldehyde, may be used, if necessary. Permeabilize cells with 0.1% Triton X-100, 0.1% sodium citrate for 2 min for detection of intracellular antigens. Wash cells with PBS.</li>
		<li>For indirect immunofluorecence staining, block slides for 1 hr at room temperature with 5% BSA or with 10% preimmune serum from the species in which the secondary antibody was generated.</li>
		<li>Incubate overnight at 4 &#176;C with primary antibodies to cell adhesion molecules, focal complex molecules, signaling pathway members or any other protein that is the target of investigation for its role in dormancy diluted to specific dilutions recommended by the manufacturer in PBS 0.1% TRITON X-100.</li>
		<li>Wash 3 times with PBS. Incubate cover slips with fluorophor-conjugated antibodies at room temperature for 2 hr. As an example, Alexa Fluor 488 Donkey anti-Mouse IgG Antibody can be used to detect murine monoclonal primary antibodies. Mount coverslips cell side down on glass slides using an antifade agent with Dapi. Seal the perimeters with nail polish.</li>
		<li>For direct immunofluorescence, carry out the BSA blocking as above in 2.4.2), incubate with fluorophor-conjugated primary antibody overnight at 4 &#176;C. Wash 3 times with PBS. Incubate the slides with an antifade agent and Dapi and seal with nail polish.</li>
		<li>Cover slide trays with aluminum foil and store at 4 &#176;C for imaging and photography anytime up to several weeks later. View and photograph cells using any fluorescence microscopic imaging systems equipped with a camera at 1,000x magnification.</li>
		<li>For fibrillar actin staining, block slides in 1% bovine serum albumin (BSA) for 30 min and incubate in BODIPY FL-Phallacidin (green) or Rhodamine phalloidin (red) at room temperature for 20 min. Add an antifade agent and seal as above.</li>
	</ol>
	</li>
</ol>
3. Clonogenic Incubation for Molecular Studies
<ol>
	<li>Western Blots
	<ol>
		<li>Incubate ER+ breast cancer cells MCF-7 or T47D at clonogenic densities of 20,000 cells/60 mm plate and 50,000 cells/100 mm plate on fibronectin-coated plates at 37 &#176;C in 5% CO2.</li>
		<li>Incubate cells at slightly higher densities of 75,000 cells/100 mm plate for molecular studies requiring mg amounts for protein from lysates. Use up to ten plates per experimental point to collect sufficient protein for molecular studies using Western blots or for RNA isolation for Northern blots.</li>
		<li>Cell number based gel loading is a necessary adjunct for comparing protein expression in vastly different-sized growing and dormant cells. Collect cells by trypsinizing and count an aliquot in a hemocytometer in 0.2% Trypan Blue for preparation of lysates for Western blots instead of scraping off the cells with a single edge blade.</li>
		<li>Centrifuge cells at 10,000 x g for 2 min, remove media by aspiration, add 200 &#956;l lysis buffer, sonicate the cells in lysis buffer and determine the protein concentration.</li>
		<li>Calculate the amount of protein per cell by dividing the protein yield by the number of cells that generated that amount. Load the lysate into each well of separate polyacrylamide gels that represents both a) equivalent amounts of protein and b) protein quantities representing equivalent cell numbers. At least 25 &#956;g protein should be loaded into each well. 
		NOTE: This will permit comparisons between growing and dormant cells, which are significantly different in size and protein content (Figure 1).</li>
	</ol>
	</li>
	<li>Flow Cytometry
	<ol>
		<li>Collect cells from 100 mm plates by trypsinization, as in 3.1 and analyze by standard fluorescence activated cell sorting (FACS) protocols using primary or secondary immunofluorescence staining for either intracellular or extracellular antigens, as outlined in 2.4. 
		NOTE: Detaching cells from tissue culture plates by trypsinization does not affect the concentration of membrane proteins as determined by antibody labeling.</li>
	</ol>
	</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+stimulates+myelopoiesis+in+long-term+human+bone+marrow+cultures.">Basic fibroblast growth factor stimulates myelopoiesis in long-term human bone marrow cultures.</a> Blood. 77 (5), 954-960 (1991).</li><li>Gupta, P., McCarthy, J. B., Verfaillie, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblast+heparan+sulfate+is+required+for+cytokine-mediated+ex+vivo+maintenance+of+human+long-term+culture-initiating+cells.">Stromal fibroblast heparan sulfate is required for cytokine-mediated ex vivo maintenance of human long-term culture-initiating cells.</a> Blood. 87 (8), 3229-3236 (1996).</li><li>Chatterjee, M., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Farnesyl+transferase+inhibitor+treatment+of+breast+cancer+cells+leads+to+altered+RhoA+and+RhoC+GTPase+activity+and+induces+a+dormant+phenotype.">Farnesyl transferase inhibitor treatment of breast cancer cells leads to altered RhoA and RhoC GTPase activity and induces a dormant phenotype.</a> Int J Cancer. 129 (1), 61-69 (2011).</li><li>Korah, R., Sysounthone, V., Golowa, Y., Wieder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+confers+a+more+differentiated+phenotype+in+MDA-MB-231+human+breast+cancer+cells.">Basic fibroblast growth factor confers a more differentiated phenotype in MDA-MB-231 human breast cancer cells.</a> Cancer Res. 60 (3), 733-740 (2000).</li><li>Dontu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+propagation+and+transcriptional+profiling+of+human+mammary+stem/progenitor+cells.">In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells.</a> Genes &amp; Dev. 17 (10), 1253-1270 (2003).</li><li>Tivari, S., Lu, H., Dasgupta, T., Wieder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivation+of+dormant+estrogen-dependent+breast+cancer+micrometastases+by+bone+marrow+stromal+injury+in+an+in+vitro+model+of+dormancy.">Reactivation of dormant estrogen-dependent breast cancer micrometastases by bone marrow stromal injury in an in vitro model of dormancy.</a> Proceedings of the 105th Annual Meeting of the American Association for Cancer Research. , (2014).</li></ol>

Supported by the Department of Defense Grants DAMD17-01-C-0343 and DAMD17-03-1-0524, the New Jersey State Commission on Cancer Research 02-1140-CCR-E0 and the Ruth Estrin Goldberg Memorial for Cancer Research (RW)

Our model is comprised of several key elements of dormancy in the bone marrow. It consists of estrogen sensitive cells, which are the type likely to remain dormant in the marrow for extended periods10, it consists of fibronectin, a key structural element of the marrow, FGF-2, a growth factor abundantly synthesized by the bone marrow stroma and heavily deposited in the extracellular matrix of the bone marrow31,32 and incubation of cells at clonogenic density where their interactions are primarily wit...

Breast cancer cells metastasize to the bone marrow before the disease is detectable1, as soon as small tumors develop blood vessels2,3. The metastatic process is rapid but inefficient. Cells enter the new blood vessels rapidly, at millions per day4 but few survive the trip to distant organs5. Nevertheless, some micrometastases survive in the bone and can be found as single cells or small cell clumps in bone marrow aspirates from newly diagnosed patients1. These cells resist adjuvant chemotherapy, which is administered for the very purpose of eliminating them6. This resistance is endowed, substantially, by survival signaling initiated by interactions with the bone marrow microenvironment7,8. Micrometastases can be found in about one third of women with localized breast cancer and represent an independent indicator of survival when analyzed by univariate analysis9. Some micrometastases are growth initiated, but recurrence patterns depend on cell type. Patients with triple negative breast cancer tend to recur between 1 to 4 years, suggesting poor control over the dormant state. Other cell types, including ER/PR+ cells, can remain dormant for up to 20 years, with a steady, continuous rate of recurrence10. While differences in dormancy gene expression signatures between ER+ and ER- breast cell lines and tumors reflect different dormancy potentials11, interactions with bone marrow stroma likely represent a significant contribution to dormancy.
The study of dormancy in vivo is exceptionally difficult because micrometastases are rare and are outnumbered by hematopoietic cells by more than 106-fold. Hence, relevant models must be generated that provide in vitro data that can suggest mechanisms and generate testable hypotheses in vivo. A number of dormancy models, including mathematical models12,13, in vitro models7,8,14,15, in vivo xenograft models16, combinations of in vitro and xenograft models17,18 and spontaneous tumor and metastasis models19, have yielded some insight into cancer cell dormancy20. Each of these models have their own limitations and are of themselves primarily useful for generating hypotheses regarding molecular signaling and interactions that govern dormancy to be tested in more biologically relevant models.
With the overall goal of defining the molecular mechanisms of dormancy, the interactions with the microenvironment that results in cycle arrest, redifferentiation and therapeutic resistance and mechanisms that result in recurrence in ER+ cells, we developed an in vitro model that provides selected relevant elements of the stromal microenvironment7. This model, while relatively sparse in its components, is sufficiently robust to permit investigators to derive specific molecular mechanisms that affect significant functions of dormancy. These experiments generate hypotheses that can be directly tested in vivo. The model relies on a few key elements that we demonstrated to be relevant in dormancy. They include the use of estrogen-dependent breast cancer cells, culture of cells at a clonogenic density where their interaction is primarily with the substratum and soluble components of the medium, a fibronectin substratum and the presence of basic fibroblast growth factor (FGF-2) in the medium.
We characterized mechanisms that govern the system in vitro, including the induction of cell cycle arrest by FGF-221, mediated through TGF&#946;22, survival signaling through PI3 Kinase7,8 and ERK8 and morphogenic differentiation to an epithelial phenotype, which depended on RhoA inactivation, integrin &#945;5&#946;1 upregulation and ligation of stromal fibronectin for survival7,15 (Figure 1). The in vitro cell cycle effects of FGF-2 on MCF-7 cells begin at concentrations at least one log below 10 ng/ml21,23. The rationale was based on the temporal control of FGF-2 expression governing mammary ductal morphogenesis, cyclic expansion and recession in a number of mammalian systems24-27. We demonstrated that FGF-2 induces differentiation, including ductal morphogenesis in 3D culture28, and that FGF-2 expression are generally lost with malignant transformation of human tumors29. The expression of FGR1 remained intact in breast carcinomas surveyed29 and MCF-7 cells continue to express all 4 FGF receptors30. In the context of dormancy, FGF-2 is exported by and heavily deposited on bone marrow stroma31,32 where it functions in the preservation of hematopoietic stem cells33. We demonstrated that FGF-2 induces a dormant state in ER+ breast cancer cells cultured on fibronectin substrata, also abundant in the marrow, where it induces morphogenic differentiation7. In the model, breast cancer cells are growth inhibited, inactivate Rho A through the RhoGap GRAF, redifferentiate to an epithelial phenotype and re-express integrins &#945;5&#946;1 &#61472;lost with malignant progression. They bind fibronectin through integrin &#945;5&#946;1 and activate survival signaling that render them resistant to cytotoxic therapy7,8,15 (Figure 1). Inhibition of Rho class GTPases has been demonstrated previously to induce a dormant phenotype34.
Here we will outline the specific procedures that will permit investigators to establish the model and study specific molecular and cellular mechanisms governing dormancy of ER+ breast cancer cells. In the experiments presented here to illustrate the use of the model, we targeted the PI3K pathway (Figure 1B) with an Akt inhibitor and a PI3K inhibitor and all members of the Rho family (Figure 1B) with a pan-Rho inhibitor and a Rho Kinase (ROCK) inhibitor.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>MCF-7 cells</td>
			<td>ATCC</td>
			<td>HTB-22</td>
			<td></td>
		</tr>
		<tr>
			<td>T47D cells&#160;</td>
			<td>ATCC</td>
			<td>HTB-123</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat Fibronectin 24 Well Clear Flat Bottom TC-Treated Multiwell Plate</td>
			<td>Corning</td>
			<td>354411</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat Fibronectin 60 mm Culture Dishes</td>
			<td>Corning</td>
			<td>354403</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat Fibronectin 100 mm Culture Dishes</td>
			<td>Corning</td>
			<td>354451</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat 22x22mm #1 Glass Coverslip with a uniform application of human fibronectin</td>
			<td>Corning</td>
			<td>354088</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well tissue culture plate</td>
			<td>CellTreat</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco Modified Eagle Medium High Glucose 10X Powder</td>
			<td>Corning Life Sciences</td>
			<td>50-013-PB</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated, Fetal Bovine Serum</td>
			<td>Serum Source International</td>
			<td>FB02-500HI</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin/2.21 mM EDTA</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution, 100X</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine, 100x, Liquid</td>
			<td>Corning</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF&#160;basic</td>
			<td>R&#38;D Systems</td>
			<td>234-FSE-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Akt Inhibitor (1L6-Hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate)</td>
			<td>CalBiochem</td>
			<td>124005</td>
			<td></td>
		</tr>
		<tr>
			<td>LY294002&#160;</td>
			<td>CalBiochem</td>
			<td>19-142</td>
			<td>Chenical PI3K inhibitor</td>
		</tr>
		<tr>
			<td>C3 transferase&#160;</td>
			<td>Cytoskeleton</td>
			<td>CTO3</td>
			<td>Inhibits RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1</td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>129830-28-2</td>
			<td></td>
		</tr>
		<tr>
			<td>BODIPY FL-Phallacidin (green)&#160;</td>
			<td>Molecular Probes</td>
			<td>B607</td>
			<td>Fluorochrome for fibrillar actin staining</td>
		</tr>
		<tr>
			<td>BODIPY FL-Rhodamine phalloidin (red)&#160;</td>
			<td>Molecular Probes</td>
			<td>R415</td>
			<td>Fluorochrome for fibrillar actin staining</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Donkey anti-Mouse IgG Antibody, ReadyProbes Reagent&#160;</td>
			<td>Molecular Probes</td>
			<td>R37114</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI&#160;</td>
			<td>Molecular Probes</td>
			<td>P-36931</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro dormancy model of estrogen-sensitive breast cancer in the bone marrow: a tool for molecular mechanism studies and hypothesis generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

Experiments were conducted to recapitulate the assay. The time course of the experiment is shown in Figure 2A. Cells are incubated at clonogenic density on day -1, FGF-2 in fresh medium is added on day 0 and cells are cultured until day 6 when they are stained and colonies are counted. Any perturbations to the system are administered on day 3 in 100 &#956;l volumes at 10x final concentrations desired. Figure 2B demonstrates the typical appearance of growing and dormant colonies. Growing ...

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An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

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8.2K Views.  Rutgers New Jersey Medical School. We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo. 

Video: An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...