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><tbody><tr><td>MCF-7 cells</td><td>ATCC</td><td>HTB-22</td><td></td></tr><tr><td>T47D cells&amp;nbsp;</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&amp;nbsp;basic</td><td>R&amp;amp;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&amp;nbsp;</td><td>CalBiochem</td><td>19-142</td><td>Chenical PI3K inhibitor</td></tr><tr><td>C3 transferase&amp;nbsp;</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&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td>129830-28-2</td><td></td></tr><tr><td>BODIPY FL-Phallacidin (green)&amp;nbsp;</td><td>Molecular Probes</td><td>B607</td><td>Fluorochrome for fibrillar actin staining</td></tr><tr><td>BODIPY FL-Rhodamine phalloidin (red)&amp;nbsp;</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&amp;nbsp;</td><td>Molecular Probes</td><td>R37114</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant with DAPI&amp;nbsp;</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

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Cancer Research

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 ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

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 ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

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

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