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...

<ol>
	<li>Braun, S., 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=Cytokeratin-positive+cells+in+the+bone+marrow+and+survival+of+patients+with+stage+I,+II,+or+III+breast+cancer.">Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer.</a> N Engl J Med. 342 (8), 525-533 (2000).</li><li>Benoy, I. H., 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=Relative+microvessel+area+of+the+primary+tumour,+and+not+lymph+node+status,+predicts+the+presence+of+bone+marrow+micrometastases+detected+by+reverse+transcriptase+polymerase+chain+reaction+in+patients+with+clinically+non-metastatic+breast+cancer.">Relative microvessel area of the primary tumour, and not lymph node status, predicts the presence of bone marrow micrometastases detected by reverse transcriptase polymerase chain reaction in patients with clinically non-metastatic breast cancer.</a> Breast Cancer Res. 7 (2), R210-R219 (1186).</li><li>Wapnir, I. L., Barnard, N., Wartenberg, D., Greco, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inverse+relationship+between+microvessel+counts+and+tumor+volume+in+breast+cancer.">The inverse relationship between microvessel counts and tumor volume in breast cancer.</a> Breast J. 7 (3), 184-188 (2001).</li><li>Wyckoff, J. B., 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=A+critical+step+in+metastasis:+in+vivo+analysis+of+intravasation+at+the+primary+tumor.">A critical step in metastasis: in vivo analysis of intravasation at the primary tumor.</a> Cancer Res. 60 (9), 2504-2511 (2000).</li><li>Phadke, P. A., 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=Kinetics+of+metastatic+breast+cancer+cell+trafficking+in+bone.">Kinetics of metastatic breast cancer cell trafficking in bone.</a> Clin Cancer Res. 12 (5), 1431-1440 (2006).</li><li>Braun, S., 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=Lack+of+an+effect+of+adjuvant+chemotherapy+on+the+elimination+of+single+dormant+tumor+cells+in+bone+marrow+of+high+risk+breast+cancer+patients.">Lack of an effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in bone marrow of high risk breast cancer patients.</a> J Clin Onc. 18 (1), 80-86 (2000).</li><li>Korah, R., Boots, M., 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=Integrin+&#945;5&#946;1+promotes+survival+of+growth-arrested+breast+cancer+cells:+an+in+vitro+paradigm+for+breast+cancer+dormancy+in+bone+marrow.">Integrin &#945;5&#946;1 promotes survival of growth-arrested breast cancer cells: an in vitro paradigm for breast cancer dormancy in bone marrow.</a> Cancer Res. 64 (13), 4514-4522 (2004).</li><li>Najmi, S., Korah, R., Chandra, R., Abdellatif, M., 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=Flavopiridol+blocks+integrin-mediated+survival+in+dormant+breast+cancer+cells.">Flavopiridol blocks integrin-mediated survival in dormant breast cancer cells.</a> Clin Cancer Res. 11 (5), 2038-2046 (2005).</li><li>Braun, S., 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=A+pooled+analysis+of+bone+marrow+micrometastasis+in+breast+cancer.">A pooled analysis of bone marrow micrometastasis in breast cancer.</a> N England J Med. 353 (8), 793-802 (2005).</li><li>Dent, R., 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=Triple-negative+breast+cancer:+clinical+features+and+patterns+of+recurrence.">Triple-negative breast cancer: clinical features and patterns of recurrence.</a> Clin Cancer Res. 13 (15 Part 1), 4429-4434 (2007).</li><li>Kim, R. S., 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=Dormancy+signatures+and+metastasis+in+estrogen+receptor+positive+and+negative+breast+cancer.">Dormancy signatures and metastasis in estrogen receptor positive and negative breast cancer.</a> PLoS One. 7 (4), e35569 (2012).</li><li>Hanin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+invisible:+how+mathematical+models+uncover+tumor+dormancy,+reconstruct+the+natural+history+of+cancer,+and+assess+the+effects+of+treatment.">Seeing the invisible: how mathematical models uncover tumor dormancy, reconstruct the natural history of cancer, and assess the effects of treatment.</a> Adv in Exp Med &amp; Biol. 734, 261-282 (2013).</li><li>Wilkie, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+mathematical+models+of+cancer-immune+interactions+in+the+context+of+tumor+dormancy.">A review of mathematical models of cancer-immune interactions in the context of tumor dormancy.</a> Adv in Exp Med &amp; Biol. 734, 201-234 (2013).</li><li>Barkan, D., Green, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+system+to+study+tumor+dormancy+and+the+switch+to+metastatic+growth.">An in vitro system to study tumor dormancy and the switch to metastatic growth.</a> J Vis Exp. (54), e2914 (2011).</li><li>Barrios, J., 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=Dual+FGF-2+and+intergrin+&#945;5&#946;1+signaling+mediate+GRAF-induced+RhoA+inactivation+in+a+model+of+breast+cancer+dormancy.">Dual FGF-2 and intergrin &#945;5&#946;1 signaling mediate GRAF-induced RhoA inactivation in a model of breast cancer dormancy.</a> Cancer Microenvironment. 2 (1), 33-47 (2009).</li><li>Ganapathy, V., 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=Luminal+breast+cancer+metastasis+is+dependent+on+estrogen+signaling.">Luminal breast cancer metastasis is dependent on estrogen signaling.</a> Clin &amp; Exp Metastasis. 29 (5), 493-509 (2012).</li><li>Barkan, D., 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=Inhibition+of+metastatic+outgrowth+from+single+dormant+tumor+cells+by+targeting+the+cytoskeleton.">Inhibition of metastatic outgrowth from single dormant tumor cells by targeting the cytoskeleton.</a> Cancer Res. 68 (15), 6241-6250 (2008).</li><li>Ghajar, C. M., 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=The+perivascular+niche+regulates+breast+tumour+dormancy.">The perivascular niche regulates breast tumour dormancy.</a> Nat Cell Biology. 15 (7), 807-817 (2013).</li><li>Marshall, J. C., 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=Effect+of+inhibition+of+the+lysophosphatidic+acid+receptor+1+on+metastasis+and+metastatic+dormancy+in+breast+cancer.">Effect of inhibition of the lysophosphatidic acid receptor 1 on metastasis and metastatic dormancy in breast cancer.</a> J Nat Cancer Inst. 104 (17), 1306-1319 (2012).</li><li>Almog, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+regulatory+pathways+involved+in+persistence+of+dormant+micro-tumors.">Genes and regulatory pathways involved in persistence of dormant micro-tumors.</a> Adv in Exp Med &amp; Biol. 734, 3-17 (2013).</li><li>Wang, H., 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=Basic+FGF+causes+growth+arrest+in+MCF-7+human+breast+cancer+cells+while+inducing+both+mitogenic+and+inhibitory+G1+events.">Basic FGF causes growth arrest in MCF-7 human breast cancer cells while inducing both mitogenic and inhibitory G1 events.</a> Cancer Res. 57 (9), 1750-1757 (1997).</li><li>Fenig, E., 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=Role+of+transforming+growth+factor+beta+in+the+growth+inhibition+of+human+breast+cancer+cells+by+basic+fibroblast+growth+factor.">Role of transforming growth factor beta in the growth inhibition of human breast cancer cells by basic fibroblast growth factor.</a> Breast Cancer Res Treat. 70 (1), 27-37 (2001).</li><li>Fenig, E., 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=Basic+fibroblast+growth+factor+confers+growth+inhibition+and+Mitogen-activated+Protein+Kinase+activation+in+human+breast+cancer+cells.">Basic fibroblast growth factor confers growth inhibition and Mitogen-activated Protein Kinase activation in human breast cancer cells.</a> Clinical Cancer Research. 3 (1), 135-142 (1997).</li><li>Coleman-Krnacik, S., Rosen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+temporal+and+spatial+gene+expression+of+fibroblast+growth+factor+family+members+during+mouse+mammary+gland+development.">Differential temporal and spatial gene expression of fibroblast growth factor family members during mouse mammary gland development.</a> Molecular Endocrinology. 8 (2), 218-229 (1994).</li><li>Plath, A., 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=Expression+and+localization+of+members+of+the+fibroblast+growth+factor+family+in+the+bovine+mammary+gland.">Expression and localization of members of the fibroblast growth factor family in the bovine mammary gland.</a> J Dairy Science. 81 (10), 2604-2613 (1998).</li><li>Lavandero, S., Chappuzeau, A., Sapag-Hagar, M., Oka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+in+vitro+evidence+of+basic+fibroblast+growth+factor+action+in+mouse+mammary+gland+development.">In vivo and in vitro evidence of basic fibroblast growth factor action in mouse mammary gland development.</a> FEBS letters. 439 (3), 351-356 (1998).</li><li>Sinowatz, F., Schams, D., Plath, A., Kolle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+localization+of+growth+factors+during+mammary+gland+development.">Expression and localization of growth factors during mammary gland development.</a> Adv in Exp Med &amp; Biol. 480, 19-27 (2000).</li><li>Korah, R., Sysounthone, V., Scheff, E., 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=Intracellular+FGF-2+promotes+differentiation+in+T47-D+breast+cancer+cells.">Intracellular FGF-2 promotes differentiation in T47-D breast cancer cells.</a> Biochem Biophys Res Comm. 277 (1), 255-260 (2000).</li><li>Korah, R., Das, K., Lindy, M. E., Hameed, M., 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=Co-ordinate+loss+of+FGF-2+and+laminin+5+expression+during+neoplastic+progression+of+mammary+duct+epithelium.">Co-ordinate loss of FGF-2 and laminin 5 expression during neoplastic progression of mammary duct epithelium.</a> Human Pathology. 38 (1), 154-160 (2007).</li><li>Wieder, R., 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=Overexpression+of+basic+fibroblast+growth+factor+in+MCF-7+human+breast+cancer+cells:+lack+of+correlation+between+inhibition+of+cell+growth+and+MAP+kinase+activation.">Overexpression of basic fibroblast growth factor in MCF-7 human breast cancer cells: lack of correlation between inhibition of cell growth and MAP kinase activation.</a> J. Cellular Physiology. 177, 411-425 (1998).</li><li>Brunner, G., Gabrilove, J., Rifkin, D. B., Wilson, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipase+C+release+of+basic+fibroblast+growth+factor+from+human+bone+marrow+cultures+as+a+biologically+active+complex+with+a+phosphatidylinositol-anchored+heparan+sulfate+proteoglycan.">Phospholipase C release of basic fibroblast growth factor from human bone marrow cultures as a biologically active complex with a phosphatidylinositol-anchored heparan sulfate proteoglycan.</a> J Cell Biol. 114 (6), 1275-1283 (1991).</li><li>Wilson, E. L., Rifkin, D. B., Kelly, F., Hannocks, M. J., Gabrilove, J. 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 vistas.  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