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

Summary

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.

Abstract

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.

Introduction

Breast cancer cells metastasize to the bone marrow before the disease is detectable¹, as soon as small tumors develop blood vessels^2,3. 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 microenvironment^7,8. 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 models^12,13, in vitro models^7,8,14,15, in vivo xenograft models¹⁶, combinations of in vitro and xenograft models^17,18 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 TGFβ²², survival signaling through PI3 Kinase^7,8 and ERK⁸ and morphogenic differentiation to an epithelial phenotype, which depended on RhoA inactivation, integrin α5β1 upregulation and ligation of stromal fibronectin for survival^7,15 (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/ml^21,23. 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 systems^24-27. 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 stroma^31,32 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 α5β1 lost with malignant progression. They bind fibronectin through integrin α5β1 and activate survival signaling that render them resistant to cytotoxic therapy^7,8,15 (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.

Protocol

1. Clonogenic Assay

1. Prepare a single cell suspensions of estrogen-dependent breast cancer cell lines MCF-7 and T47D cells using the steps outlined below
   1. 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 °C for 1-4 min.
   2. 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.
   3. Continue to incubate cells in trypsin at 37 °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.
   4. 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.
2. Incubate Cells at Clonogenic Density Using Steps Outlined Below
   1. 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.
   2. 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.
   3. 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).
   4. 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.
   5. Incubate cells at 37 °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.
   6. Design the time course of the assay as follows: Incubate cells on fibronectin coated substrata on day  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). 
3. Set Up Experimental Perturbations of Molecular Signaling or Adhesion Molecules
   1. On day 3, add 100 μl 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 °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.
   2. Stain colonies on day 6, as follows. 
4. Stain Colonies
   1. 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.
   2. 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.
   3. 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.
   4. Dry plates overnight by placing them upside down on towels on the bench top adjacent to their corresponding labeled covers. 
5. Count Colonies
   1. 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 >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 1^7,15.
      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.

2. Clonogenic Incubation for Immunofluorescence Studies

1. 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.
2. 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).
3. 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 μl volumes, as described in the colony assay procedures in 1.3).
4. Stain cells on day 6 with antibodies to cell adhesion molecules such as integrins α4, α5, α6, β1, β3, for example, focal adhesion complex molecules FAK, paxillin and vinculin, for example, proteins involved in motility such as α-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.
   1. Remove slides with forceps day 6. Fix in acetone/methanol 1:1 at -20 °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.
   2. 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.
   3. Incubate overnight at 4 °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.
   4. 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.
   5. For direct immunofluorescence, carry out the BSA blocking as above in 2.4.2), incubate with fluorophor-conjugated primary antibody overnight at 4 °C. Wash 3 times with PBS. Incubate the slides with an antifade agent and Dapi and seal with nail polish.
   6. Cover slide trays with aluminum foil and store at 4 °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.
   7. 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.

3. Clonogenic Incubation for Molecular Studies

1. Western Blots
   1. 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 °C in 5% CO₂.
   2. 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.
   3. 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.
   4. Centrifuge cells at 10,000 x g for 2 min, remove media by aspiration, add 200 μl lysis buffer, sonicate the cells in lysis buffer and determine the protein concentration.
   5. 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 μg 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).
2. Flow Cytometry
   1. 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.

Results

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 μl volumes at 10x final concentrations desired. Figure 2B demonstrates the typical appearance of growing and dormant colonies. Growing ...

Discussion

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 marrow^31,32 and incubation of cells at clonogenic density where their interactions are primarily wit...

Materials

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