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 border="0" cellpadding="0" cellspacing="0">
	<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&#39;s 5A Medium (1X)</td>
			<td>Life Technologies</td>
			<td>16600-082</td>
			<td>Supplement all McCoy&#39;s&#160; 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&#160;</td>
			<td>ant-bl</td>
			<td>Supplement media used for transfected cell lines.</td>
		</tr>
		<tr>
			<td>Falcon&#174; Cell Culture Inserts for 24 Well Plate with Transparent PET Membrane (8.0 &#956;m pore size)</td>
			<td>Corning Incorporated</td>
			<td>353097</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon&#174; 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 grams (30 mg/mL)</td>
			<td>Life Technologies</td>
			<td>L-8220</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Cell Strainer 70 &#956;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&#160;</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. Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

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

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

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

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

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

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

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

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

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

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

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

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

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

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

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

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

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

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...