These studies were funded, in part, by grants from the Alternative Research and Development Foundation (107588), the National Institute of Health (1U54CA136465-04S1), and the California Breast Cancer Research Program (201B-0141). We thank Drs. Andrew Wilber and R. Scott McIvor for the generous donation of their <pkt2 lubig=""> transponson and pK/hUbiC-SB11 transposase plasmids. We gratefully acknowledge John Tamaresis, Ph.D. for advice on statistical methods, Timothy Brown for performing flow cytometry, Georgette Henrich for advice on migrations assays, and Nancy Bellagamba for facilitating the collection of THR specimens. </pkt2>

Femoral heads were collected from patients undergoing elective THR surgery in the Department of Orthopaedic Surgery at the Stanford University School of Medicine. All tissues were collected as de-identified specimens in accordance with regulations of the Stanford University Research Compliance Office.
&#8203;1. Selecting a Breast Cancer Cell Line(s)
<ol>
	<li>Obtain or transfect the desired breast cancer cell line(s) with a luciferase-GFP reporter construct. The following experiments were performed using the MDA-MB-231 and MCF-7 breast cancer cell lines engineered for the stable expression of firefly luciferase (fLuc) and enhanced green fluorescent protein (EGFP) by transfection with the Sleeping Beauty transposon plasmid pKT2/LuBiG and the transposase plasmid pK/hUbiC-SB1145,46. 
	NOTE: The transfected cell lines are referred to as MDA-MB-231-fLuc-EGFP, and MCF-7-fLuc-EGFP.</li>
</ol>
2. Isolating Tissue Fragments from Femoral Heads
<ol>
	<li>Following THR surgery, collect the discarded femoral head in a sterile container filled with physiological saline. Transfer the specimen to the laboratory and process it within 1-3 hr of surgery. Note: if there is a delay in processing, place the specimen at 4 &#176;C.</li>
	<li>Prepare a working area by placing the following items on an absorbent mat in a laminar flow tissue culture hood: sterile gloves, surgical rongeur, forceps, beaker with 70% isopropanol to rinse instruments, and tissue culture vessels as stipulated below for each type of experiment.</li>
	<li>Wearing a sterile surgical glove to hold the round femur head in one hand, use the rongeur in the other hand to extract trabecular bone fragments of desired size (~2-5 mm) from the exposed upper shaft of the femur. Transfer bone fragments into the culture vessel (e.g., 6-, 12-, or 24-well tissue culture plate or migration chamber), depending on the type of proliferation, colonization or migration experiment, as described in steps 3, 4 or 5. 
	NOTE: Bone tissue fragments should be harvested as described above after completing the initial steps for each specific proliferation, colonization or migration assay. Fragments will range in size as shown in Figure 2. Fragments can be weighed before or after experiments for the purpose of standardization.</li>
</ol>

3. Co-culturing Breast Cancer Cells Adjacent to Bone Fragments to Measure Breast Cell Proliferation Using BLI
<ol>
	<li>Prior to the experiment, prepare a suspension containing 2 x 105 breast cancer cells/50 &#181;l, and prepare plugs of bone wax for immobilizing bone fragments in culture. Cut a P200 micropipette tip into 3 pieces and use the narrow end of the largest piece in a &#8220;cookie cutter&#8221; manner to cut small (~35 mg) pieces of bone wax. Use the wide end of the smallest piece to eject the bone wax plugs into a Petri dish for storage.</li>
	<li>Place a piece of bone wax at the 12 o&#8217;clock position of each well and gently press down with a sterile-gloved index finger.</li>
	<li>Pipette 50 &#181;l of cell suspension containing 1 x 105 breast cancer cells in DMEM-10% FBS + Pen-Strep in the center of each well of a 6-well plate. (Alternatively, seed cells in a dispersed pattern if desired.) Place the plate in a 37 &#176;C tissue culture incubator for 45 min to promote cell attachment.</li>
	<li>Place 1 bone fragment onto 1 piece of bone wax for each experimental well and apply gentle pressure with the rongeur to secure it. Perform experiments in triplicate such that 3 bone fragments from a given THR specimen are placed into the top three wells, while the bottom three wells contain bone wax only as controls.</li>
	<li>Using a 5 ml pipette, gently and slowly deliver 5 ml of DMEM-10% FBS to the side of each well to avoid dislodging the breast cells or bone fragments. Place the plate in a 37 &#176;C tissue culture incubator for 20-24 hr.</li>
	<li>To perform bioluminescence imaging (BLI) remove the plate from the incubator and add luciferin substrate (to achieve a concentration of 300 &#181;g/ml) to each well. Image the plate immediately on an IVIS Imaging Platform using the following parameters: f-stop of 1, small binning, exposure time of 1 sec, level D. Measure the signal intensity of each well as average radiance (photons/second/square centimeter/steradian).</li>
	<li>Use imaging software to define Regions of Interest (ROI) to quantitate the average radiance for all experimental and control wells. Export average radiance values to an Excel sheet to perform statistics and generate graphs.</li>
</ol>
4. Co-culturing Breast Cancer Cells Seeded Directly Onto Bone Fragments to Study Colonization and Cell Number
<ol>
	<li>Use forceps to place bone fragments directly into the empty wells of a 24-well tissue culture plate (1 fragment/well).</li>
	<li>Pipette a 50 &#181;l cell suspension droplet of DMEM-10% FBS containing 1 X 103-6 breast cancer cells directly on top of each bone fragment. Place the plate in a 37 &#176;C tissue culture incubator for 45 min to promote cell attachment. Gently add 1-2 ml DMEM-10% FBS into the side of each well such that the fragment is entirely submerged in medium. Incubate the plate in a 37 &#176;C tissue culture incubator for 20-24 hr.</li>
	<li>After incubation, use forceps to transfer each bone tissue fragment to a fresh 24-well plate containing 1 ml DMEM-10% FBS in each well. To perform BLI, follow steps 3.6-3.7 above. Additionally, fragments may be viewed using a fluorescence microscope, or flushed to obtain the marrow population for analyses of breast cancer cell numbers and properties as described in step 6.</li>
	<li>To prepare the intact fragments for qualitative evaluation by fluorescence microscopy following BLI, pipette 5 &#181;l of fluorescent bisphosphonate labeling solution (prepared according to the manufacturer&#8217;s instructions) into each well to label the mineralized spicules of the bone fragments. Incubate the plate in the tissue culture incubator for another 24 hr.</li>
	<li>Following 24 hr of culture in fluorescent bisphosphonate labeling solution, use forceps to transfer the fragments to tissue culture plates containing phenol red-free medium and view using a fluorescence microscope configured to image GFP (485 nm) and bisphosphonate label (680 nm).</li>
</ol>
5. Measuring Migration of Breast Cancer Cells to Bone Tissue Fragments and Cultured Bone Fragment Supernatants
<ol>
	<li>Migration toward bone tissue culture supernatant. 
	NOTE: Perform experiments in triplicate such that 3 fragments from a given THR specimen are used to generate 3 supernatants. For each experiment, migration is compared across three wells containing bone supernatants vs. three wells containing control medium only.
	<ol>
		<li>Generate bone tissue supernatants by placing bone fragments into the wells of a 12-well plate containing 2.2 ml DMEM-10% FBS per well. Culture in a 37 &#176;C tissue culture incubator for 24 hr. Withdraw the medium at 24 hr and replace with 2.2 ml fresh DMEM-10% FBS. Treat control wells containing DMEM-10% FBS the same way. 
		NOTE: The medium is changed at 24 hr to remove factors secreted in response to tissue trauma resulting from surgery. Because bone tissue supernatants are generated in FBS-containing medium, wells containing FBS-containing medium only are included to control for the contributions of FBS to breast cancer cell migration.</li>
		<li>At 48 hr, collect 0.9 ml of each supernatant/control medium and pipette into the wells of a receiver plate. The inserts will later be placed into these wells. Incubate the receiver plate in the tissue culture incubator while seeding the inserts. Note: The remaining supernatant (~1 ml after evaporation) can be collected for analysis of secretome factors if desired44.</li>
		<li>To seed the Transwell inserts, place them into a standard, empty 24-well tissue culture plate. Pipette 350 &#181;l cell suspension containing 1 x 105 breast cancer cells in DMEM-10% FBS onto the upper, inner membrane surface of each insert as shown in Figure 1A. Place the 24-well plate containing the seeded inserts into the 37 &#176;C tissue culture incubator for 45 min to promote attachment.</li>
		<li>Once the cells have attached to the inserts, use forceps to transfer the inserts to the receiver plate according to the manufacturer&#8217;s instructions. Angle each insert when placing it into the receiver well to prevent bubble formation between the insert membrane and the receiver well supernatant. Incubate the receiver plate with inserts for 20 hr in a 37 &#176;C tissue culture incubator. 
		NOTE: Check the bottom side of the receiver plate for bubbles between the insert membrane and the receiver well supernatant. If bubbles are visible, remove the specific inserts and place them again into the receiver well until no bubbles are present.</li>
		<li>After 20 hr use forceps to remove the inserts from the receiver plate. Using the procedures in steps 3.6-3.7 perform BLI to image the cells that have migrated into and attached to the bottom of the receiver plate wells.</li>
	</ol>
	</li>
	<li>Migration toward bone tissue fragments. 
	NOTE: Perform experiments in triplicate such that 3 fragments from a given THR specimen are placed into 3 separate inserts, and 3 glass beads are placed into 3 separate inserts as controls.
	<ol>
		<li>Prepare the receiver plate by pipetting 0.9 ml DMEM-10% FBS into each well. Place it into the tissue culture incubator while preparing the inserts.</li>
		<li>To seed the inserts, invert them on the surface of a plastic microfuge tube box that has a lid. Pipette a 50 &#181;l cell suspension droplet of DMEM-10% FBS containing 1 x 105 breast cancer cells directly onto the outer, bottom surface of each insert membrane (which is facing up on the inverted inserts). Cover the microfuge tube box with the lid cracked open and transfer it to the 37 &#176;C tissue incubator for 45 min to promote cell attachment.</li>
		<li>Transfer the microfuge tube box to the tissue culture hood, and use forceps to turn the seeded inserts right side up and transfer them into the wells of the receiver plate. Pipette 0.450 ml DMEM-10% FBS into each insert cup. Using forceps, transfer one bone tissue fragment (~3 mm) into each prepared insert cup. Optionally, use glass beads as negative controls in additional wells. Place the plate in a tissue culture incubator for 20 hr.</li>
		<li>To measure migration, prepare a standard 24-well tissue culture plate containing 1 ml DMEM-10% FBS per well. Remove the receiver plate from the incubator and use forceps to transfer the bone fragments and beads from each insert into separate wells of the standard plate. Add luciferin substrate (to achieve a final concentration of 300 &#181;g/ml) to each well and perform BLI using the procedures in steps 3.6-3.7.</li>
	</ol>
	</li>
</ol>
6.&#160;Additional Pre- and Post-experimental Analyses
<ol>
	<li>Analysis of flushed marrow.
	<ol>
		<li>Transfer a bone fragment into a 70 &#181;m cell strainer placed at the top of a 50 ml conical tube. Use a 10 ml syringe with a 25 G needle to vigorously flush the fragment with 10 ml of PBS to obtain a suspension of marrow cells in the conical tube. Centrifuge the cell suspension for 3 min at 300 x g and 21 &#176;C, aspirate the supernatant and resuspend the pelleted cells in PBS or other desired solution for flow cytometry. 
		NOTE: Resuspension volumes will vary depending on the size of the cell pellet after centrifugation, and application.</li>
		<li>For viability assays ~75-300 &#181;l volumes of PBS are appropriate, and these suspensions can be analyzed using commercially available fluorescence based viability assays according to the manufacturer&#8217;s instructions, pipetted into a conventional hemocytometer, and counted using an inverted fluorescence microscope.</li>
		<li>For flow cytometric analysis or sorting based on detection of GFP-positive breast cancer cells, resuspend the cell pellet in 1 ml PBS containing 2% FBS, 2mM EDTA, and 10 mM HEPES such that the concentration is 1 x 106 cells/ml.</li>
	</ol>
	</li>
	<li>H &#38; E Staining and Immunohistochemistry.
	<ol>
		<li>To process the fragments for hematoxylin &#38; eosin (H&#38;E) staining and/or immunohistochemistry, place the fragments in plastic cassettes labeled with a number 2 pencil, and submerge in a container filled with 10% buffered formalin for 24 hr.</li>
		<li>Process tissues for paraffin embedding, sectioning and staining using routine protocols that include a decalcification step to account for the mineralized spicules within each fragment.</li>
	</ol>
	</li>
</ol>

No disclosures to report.

Mehra et al. have previously described the postmortem collection and analysis of bone specimens from metastatic prostate cancer patients harvested at the time of autopsy, revealing high quality tissues and validating the use of human bone samples to study the metastatic process47. Here we have described protocols for collecting, processing and using human bone tissue fragments obtained from discarded femoral heads following THR surgery in a short-term co-culture system to study breast cell migration, ...

The tumor microenvironment is widely recognized as a critical determinant of cancer cell behavior during breast cancer progression and metastatic spread1-3. The goal of the method presented here is to facilitate the study of breast cancer cells within the microenvironment of human bone tissue, the most frequent site of breast cancer metastasis4-6. Bone is comprised of mineralized and marrow compartments7-9, both of which harbor cells and secreted factors implicated in metastatic progression10,11. Although widely used in vivo mouse models facilitate systemic studies of the metastatic process12-15, cell interactions within the skeleton are not readily accessible for observation and direct perturbation. Model systems for studying and culturing mouse bone tissues and mouse marrow cells provide better access and have yielded many insights regarding crosstalk and mechanisms underlying breast cancer cell metastasis of the murine skeleton16-22. However, studies have suggested that there may be species-specific patterns of osteotropism for bone tissue23,24. These patterns could reflect inherent species-specific differences in bone tissue properties, differences resulting from altered bone marrow populations in immune-deficient mice11, and/or the relatively young age of mice used in experiments, all of which are likely determinants of the bone microenvironment.
In vitro approaches for studying breast cancer cells in human bone co-cultures have typically focused on specific bone cell types such as marrow-derived osteoblasts or stromal cells cultured as monolayers or within engineered 3-dimensional model systems25-35. Although 2-dimensional cell culture models have formed the mainstay of in vitro approaches for cancer research, it has long been recognized that cell behavior is fundamentally altered in monolayer culture systems18,36,37. This has led to the development of engineered microenvironments that mimic the complexity of 3-dimensional living tissues, including matrix- and scaffold-based models composed of natural materials such as collagen, or synthetic polymers seeded with specific cell types to create tissue-like microenvironments36-41. Engineered approaches have also included the use of bioreactor platforms to control and study the hormonal milieu of the microenvironment18,19,41-43. Although biomimetic models and bioreactors provide many elements of a complex tissue microenvironment in a controlled setting, and have been successfully applied to advance the study of breast cancer metastasis to bone18,19,35,42,43, engineered model systems do not generally incorporate the full spectrum of cell types and extracellular matrix components present within the native bone environment.
Until recently, breast cancer cells have not been studied within the native, intact, 3-dimensional microenvironment of human bone tissues. We recently reported the development of a co-culture model using human bone tissue fragments isolated from total hip replacement (THR) surgery specimens44. These specimens harbor both the mineralized and marrow compartments necessary to study mechanisms underlying micro-metastasis in short term culture. In previous work we established proof-of-principle for using bioluminescence imaging (BLI) to monitor the proliferation of luciferase-expressing breast cancer cells (MDA-MB-231-fLuc) co-cultured with bone fragments44. Here we present detailed experimental protocols for studying breast cell cancer proliferation, colonization, and migration within the context of the native microenvironment using human bone tissue explants.
First we present a protocol for co-culturing breast cancer cells adjacent to bone fragments to measure breast cell proliferation using BLI. In this section, breast cell suspensions are seeded as cell spots in 6-well plates adjacent to bone fragments, which are immobilized by pieces of bone wax. Control wells contain bone wax, but no bone fragment. Once cell spots attach, medium is added and the plate is cultured for 24 hr, after which bioluminescent signal intensity (associated with cell number) is measured with BLI. In the next step we present methods for co-culturing breast cancer cells seeded directly onto bone fragments to study colonization and proliferation. Here the breast cell suspensions are pipetted directly onto the bone tissue fragments, which are monitored in culture by BLI and fluorescence microscopy over time to track colonization and cell number. In this method, the marrow compartment can be flushed from the bone fragments at any time point for analysis of colonized breast cancer cells by flow cytometry, or marrow viability assays.
In addition, we describe two different approaches for measuring breast cancer cell migration. In the first method steps are outlined for measuring migration toward bone tissue culture supernatants. In this protocol the breast cancer cells are seeded onto the inner, upper surfaces of Transwell insert membranes with an 8 &#181;m pore size (as shown in Figure 1A). The inserts are then placed into a receiver plate with wells containing bone tissue supernatant or control medium. Over the course of a 20 hr incubation period, small numbers of breast cancer cells migrate through the insert membranes down into the lower tissue culture wells where they attach for detection by BLI. In the second migration method the breast cancer cells are seeded onto the lower, outer surfaces of Transwell insert membranes. Bone tissue fragments are placed into the insert cups and the breast cancer cells migrate up through the membranes to colonize the bone fragments (as shown in Figure 1B), which are then imaged using BLI.

<table><tbody><tr><td>DMEM (1X) + GlutaMAX-l</td><td>Life Technologies</td><td>10569-010</td><td>Supplement all DMEM with 10% fetal bovine serum and 1% Pen/Strep</td></tr><tr><td>McCoy&#039;s 5A Medium (1X)</td><td>Life Technologies</td><td>16600-082</td><td>Supplement all McCoy&#039;s&amp;nbsp; with 10% fetal bovine serum and 1% Pen/Strep</td></tr><tr><td>Fetal bovine serum</td><td>Life Technologies</td><td>16000-044</td><td></td></tr><tr><td>Penicillen Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Blastocidin (10 mgl/ml)</td><td>InvivoGen&amp;nbsp;</td><td>ant-bl</td><td>Supplement media used for transfected cell lines.</td></tr><tr><td>Falcon&amp;reg; Cell Culture Inserts for 24 Well Plate with Transparent PET Membrane (8.0 &amp;mu;m pore size)</td><td>Corning Incorporated</td><td>353097</td><td></td></tr><tr><td>Falcon&amp;reg; 24 Well TC-Treated Polystyrene Cell Culture Insert Companion Plate</td><td>Corning Incorporated</td><td>353504</td><td></td></tr><tr><td>LIVE/DEAD Viability/Cytotoxicity Kit *for mammalian cells*</td><td>Invitrogen Detection Technologies</td><td>L-3224</td><td></td></tr><tr><td>FluoroBrite DMEM</td><td>Life Technologies</td><td>A18967-01</td><td></td></tr><tr><td>D-luciferin firefly, potassium salt 5 g (30 mg/ml)</td><td>Life Technologies</td><td>L-8220</td><td></td></tr><tr><td>Sterile Cell Strainer 70 &amp;mu;m</td><td>Fisher Scientific</td><td>22363548</td><td></td></tr><tr><td>6 Well TC-Treated Cell Culture Cluster</td><td>Corning Incorporated</td><td>3516</td><td></td></tr><tr><td>12 Well TC-Treated Cell Culture Cluster</td><td>Corning Incorporated</td><td>3513</td><td></td></tr><tr><td>24 Well TC-Treated Cell Culture Cluster</td><td>Corning Incorporated</td><td>3526</td><td></td></tr><tr><td>Friedman-Pearson Rongeur</td><td>Fine Science Tools</td><td>16021-14</td><td></td></tr><tr><td>Bone wax</td><td>Surgical Specialties Corporation</td><td>903</td><td></td></tr><tr><td>IVIS 50 Imaging Platform</td><td>Caliper Life Sciences/PerkinElmer</td><td></td><td></td></tr><tr><td>Living Image Software Program&amp;nbsp;</td><td>Caliper Life Sciences/PerkinElmer</td><td>133026</td><td></td></tr><tr><td>EVOS FL Imaging System</td><td>Life Technologies</td><td>Version 4.2</td><td></td></tr><tr><td>OsteoSense 680 EX</td><td>PerkinElmer</td><td>NEV10020EX</td><td></td></tr><tr><td>MCF-7 breast cancer cells</td><td>ATCC</td><td>HTB-22</td><td></td></tr><tr><td>MDA-MB-231 breast cancer cells</td><td>ATCC</td><td>HTB-26</td><td></td></tr><tr><td>Monoclonal mouse anti-human cytokeratin antibody</td><td>DAKO Cytomation</td><td>Clone (AE1/AE3)</td><td></td></tr></tbody></table>

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.

Isolating Tissue Fragments from Femoral Heads
Femoral heads were collected from equal numbers of male and female patients (44-90 years of age) undergoing elective total hip replacement surgery in the Department of Orthopaedic Surgery at the Stanford University School of Medicine. Each discarded femoral head contains a small portion of the upper femur, which harbors trabecular bone tissue composed of mineralized spicules and marrow. This tissue is accessible through the surgically exposed cross...

Watch this Scientific Journal Video about Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche at JoVE.com

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

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 ...

methods-for-culturing-human-femur-tissue-explants-to-study-breast

Research

JoVE Journal

Medicine

12.4K Views.  Stanford University School of Medicine.  Protocollen worden beschreven voor het bestuderen van borstkanker celmigratie, proliferatie en kolonisatie in een menselijk botweefsel explantaatmodel systeem. 

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

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 ...

Cancer Research

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 ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Bioengineering

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

Biology

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

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

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