We sincerely thank Dr. Wenjin Chen for her help with the MATLAB program and the Cancer Institute of New Jersey histopathology facility and microscopy facility. We also thank the reviewers for their comments and suggestions. This study is supported by the Raymond and Beverly Sackler Research Foundation.

1. Preparation of 1% Agarose-coated 24-well Plate
<ol>
 <li>Add 1 g agarose (RPI, A20090-500) to 100 ml deionized water in a 250-500 ml bottle and autoclave to sterilize the agarose (121 &deg;C, 15 psi, 15 min in an autoclave machine). Transfer the bottle with sterilized agarose into a 70 &deg;C water bath. The agarose is ready to be poured when it's cooled to 70 &deg;C, which takes about one hour. Make sure to keep the agarose sterile at all times.</li>
 <li>Obtain the 24-well flat-bottom plates (Falcon, 353047); any kind of 24-well flat-bottom plates would work if the plates can be imaged under a phase-contrast microscope.</li>
 <li>Dispense 200 &mu;l of agarose to each well with an Eppendorf repeater pipette in a biosafety cabinet and swirl the agarose around the well to make it cover the well evenly. 200 &mu;l of agarose is appropriate to form a concave surface so that spheroids can form the consistent spherical shapes. Less than 200 &mu;l agarose may not cover the well entirely, but more than 200 &mu;l agarose may not form the concave surface and the spheroids grown on it may not form spherical shapes.</li>
 <li>The agarose-coated 24-well plates are ready to be used in about 10 min. (Optional) Add 100 &mu;l of medium to equilibrate the agarose with culture medium prior to seeding cells.</li>
 <li>Alternatively, after the agarose is fully solidified, add 100 &mu;l growth medium to each well and seal the plates with the sterile sealing tape (Nunc, 236366). These plates can be stored at 4 &deg;C for up to one week.</li>
</ol>
2. Preparation of Single Cell Suspensions
<ol>
 <li>All the experiments are done using the standard aseptic techniques in a biosafety cabinet and all the cultures are grown in a 37 &deg;C humidified incubator with 5% CO2 in air, unless specified.</li>
 <li>Human pancreatic NET BON-1 cells are maintained in DMEM/F12 (Invitrogen, 10565) with 10% fetal bovine serum (FBS, Invitrogen, 16000-044) medium in a 100 mm x 20 mm sterile tissue culture dish (Corning, 430167).</li>
 <li>When monolayer BON-1 cells reach 70-80% confluency, medium is removed. The cells are washed with PBS twice, and dislodged from the dish by adding 1 ml TrypLE (Invitrogen, 12604) with 5 min incubation at 37 &deg;C. TrypLE is similar to trypsin but is stable at 15 - 30 &deg;C as well as at 4 &deg;C. Check under microscope to make sure all cells are detached from the dish.</li>
 <li>Add 9 ml growth medium to the trypsinized cells and use a pipette to pipette cells up and down a few times to ensure an even single cell suspension.</li>
 <li>Use 70-&mu;m cell strainer (Fisher, 22363548) to filter the cells.</li>
 <li>Collect cells and perform a cell count using Guava PCA-96 Base System (Millipore).</li>
 <li>Prepare a suspension of 5,000 cells per ml in phenol-free DMEM/F12 with 10% FBS growth medium.</li>
</ol>
3. Spheroid Culture
<ol>
 <li>Overlay 200 &mu;l of the single cell suspensions to the agarose-coated 24-well plate and seal the 24-well plates with the sterile sealing tape to prevent evaporation.</li>
</ol>
Note: --The number of cells to be plated should be determined empirically based on the size of the spheroids at day 6; 300-400 &mu;m diameter of the spheroids would be the ideal size for starting the drug treatment; 1,000 BON-1 and H727 cells are plated.
-- We don't recommend starting drug treatments after day 6 because the viability of the 3D cells starts to drop at about day 10 (data not shown, manuscript in preparation).
<ol start="2">
 <li>Place the above agarose-coated 24-well plates with BON-1 cells on an orbital shaker at a very slow agitation of less than 40 rpm overnight in a 37 &deg;C humidified incubator with 5% CO2 in air. Then move these plates to a stable platform to keep the cultures growing.</li>
 <li>Add 200 &mu;l growth medium to each well of the above plates every 3 days. Do not disturb the spheroids. Try to add medium through the wall of each well by touching the pipette tip to the side of the well and letting the medium flow down into the well.</li>
 <li>Monitor the spheroid formation in the 24-well plates under a microscope.</li>
</ol>
4. Drug Treatment
<ol>
 <li>When the spheroids reach around 300-400 &mu;m in diameter at day 6, the 3D spheroids are treated with drugs. This is denoted as day 0 for drug treatment.</li>
 <li>At day 6, each well of the plate contains around 400 &mu;l of medium. To make sure the drug treatments are at 1x final concentration in each well, 3x serial dilutions of each dose of treatments are made from the drug stock in 5 ml growth medium in a 15 ml conical tube, which is enough for 8 replicates.</li>
 <li>The spheroids at each row of the 24-well plate are categorized as vehicle, dose 1, 2, 3, 4 and 5. There are 4 replicates for each dose of treatments on one 24-well plate (see Table 1). We usually treat 3D spheroids at least in 2 24-well plates, which makes at least 8 replicates for each dose of treatments.</li>
 <li>Spin down the 24-well plate at 800 rpm for 5 min at 4 &deg;C to make sure that all the condensations on the sealing tape go into the well. Due to the floating feature of the 3D spheroids, the medium in each well is not removed to avoid the disturbance of the spheroids.</li>
 <li>Carefully add 200 &mu;l of the above-made treatments of each dose into the appropriate wells along the wall of each well.</li>
 <li>Seal the 24-well plates with the sealing tape and incubate the cultures in the 37 &deg;C humidified incubator with 5% CO2 in air.</li>
 <li>If necessary, retreat the spheroids at each well with the respective dose of treatments after 72-96 hr.</li>
</ol>
5. Monitor 3D Spheroid Cultures
<ol>
 <li>At each chosen time point after drug treatments (0, 24, 48, 72 hr), the spheroids at each well of the 24-well plate are imaged with a Zeiss Axiovert 200/M-based phase-contrast microscope using a 5x objective.</li>
</ol>
Note: Images can be taken on any light microscope with a calibrated scaling between &mu;m and pixel. A 5x objective is ideal because the spheroids quickly outgrow the imaging field with a 10x objective.
<ol start="2">
 <li>Spheroid analysis can be performed manually using Zeiss AxioVision 4.8.2 Software with the raw images.</li>
 <li>Alternatively, a custom-developed MATLAB program is used to estimate the size of the spheroids automatically/semi-automatically. The automatic program implements the active contour algorithm4 to accurately locate contour of the spheroid in a given image and measure its major (L) and minor (W) axes automatically. A semi-automatic version of the same algorithm allows users to help initiate the program when strong artifact is present. All the images need to be exported as TIFF or JPEG file formats from the raw images to be read by the program. The volume of the spheroid is estimated as 0.5 x L x W2.</li>
</ol>
6. HistoGel-embedding of the NET 3D Spheroids
<ol>
 <li>10-24 spheroids are collected from the 24-well plates, washed in PBS twice, fixed in 10% formalin for 2 hr, washed in 50% and 70% ethanol for 15 min twice, respectively, and are ready for HistoGel embedding. Alternatively, they can be kept in 70% ethanol at 4 &deg;C for several months.</li>
 <li>A tube of cold HistoGel (Thermo Scientific, HG-4000-012) is put in a beaker filled with water. The beaker is heated in a microwave for 1 min or until the gel is completely liquefied, then kept in the same water-filled beaker at all times.</li>
 <li>The spheroids in 70% ethanol from above are transferred into a biopsy cryomold (Tissue-Tek, Sakura Finetek, 4565) using a toothpick. Make sure all the spheroids are transferred. Let the spheroids sit for a minute to sink down to the bottom of the biopsy cryomold. Try to get rid of the liquids in the biopsy cryomold with Kimwipes and meanwhile, very gently push the spheroids to the center at the bottom of the biopsy cryomold using the disposable plastic Pasteur pipette. This step may be easier to be done under a dissecting microscope.</li>
 <li>A thin layer of warmed HistoGel is added to the biopsy cryomold to stabilize the spheroids at the bottom of the biopsy cryomold; meanwhile, the toothpick is used to keep the spheroids at the center at the bottom of the biopsy cryomold very gently. Don't add too much HistoGel but just enough for stabilizing the spheroids at the bottom of the biopsy cryomold.</li>
 <li>Let the spheroids/HistoGel sit for 1-2 min at room temperature or till the HistoGel is solidified. Then fill the rest of the biopsy cryomold with the warmed HistoGel.</li>
 <li>Allow it to solidify at room temperature for about 10 min.</li>
 <li>Just prior to popping out the spheroids/HistoGel block from the biopsy cryomold, leave it at 4 &deg;C for 1 min, which helps popping out the intact spheroids/HistoGel block. The spheroids/HistoGel block is then wrapped in a piece of Bio-wraps (Surgipath, 01090) and put into a tissue and biopsy cassette (Fisher Scientific, 15-182-701D). The tissue and biopsy cassette is stored in a container with 70% ethanol. Then the routine tissue processing procedures for paraffin embedding are followed.5</li>
 <li>The paraffin block can be sectioned into 3-&mu;m thick layer slides. The slides can be stained with hematoxylin and eosin to determine histological changes and can also be used for immunohistochemical staining.</li>
</ol>
7. Representative Results
Human pancreatic neuroendocrine tumor cell lines BON-1 (provided to Verto Institute by Kjell Oberg, Uppsala University, Sweden), QGP-1 (Japanese Health Sciences Foundation, Osaka, Japan), and human lung neuroendocrine tumor cell line H727 (ATCC) were grown on an agarose-coated 24-well plate. As seen in Figure 1, the three cell lines show different morphology. BON-1 and H727 cells formed 3D spheroids. Under the microscope, H727 cells showed tighter and more compact spheroids than BON-1 cells. We hypothesize that H727 cells have an increased capacity for cell-cell adhesion and form tighter junctions as a result. QGP-1 cells show large grape-like porous masses, which are not regarded as spheroids. Due to the rising interest of pancreatic neuroendocrine tumor research in recent years, BON-1 cells are used for the rest of the figures. After seeding BON-1 cells on the agarose-coated 24-well plate, multicellular spheroids formation typically occurs by the 3rd or 4th day and the spheroids become rounder by the 6th day. Due to the nutrient and oxygen limitation, cells in the dense core of the spheroids start to die around day 10 and by day 17, the spheroids begin to disintegrate due to the large size or in some cases the ejection of the necrotic core. The spheroids typically can grow up to 1,000 &mu;m in diameter. Figure 2 shows the sections of the formation, growth and disintegration of BON-1 3D spheroids.
Drug treatments were started when spheroids were around 300-400 &mu;m and cells retained high viability (manuscript in preparation). Day 6 3D spheroids were chosen as the start point for drug treatments. It has been reported that histone deacetylase inhibitors showed antiproliferative and proapoptotic effects on the NET cells.6 We chose the histone-deacetylase inhibitor -Trichostatin A (TSA) as an example to illustrate the effect of drug treatments on 3D NET spheroids. Figure 3A shows the morphology of the 3D spheroids upon the treatment of TSA. Figure 3B shows the volume of the 3D spheroids upon TSA (Sigma, T8552) treatment. The size of each individual spheroid was estimated automatically by a custom-developed MATLAB computer program.4A manual drawing step was added to help acquire the spheroids, which were close to the edge in a given image. At least 8 spheroids were measured per dose of TSA treatments at each time point. The volume of each 3D spheroid was calculated as 0.5 x Length x Width2. As seen in Figure 3A and 3B, relative to vehicle, all doses of TSA shown in the figures demonstrated some degree of growth inhibition of BON-1 3D spheroids. Among them, the concentrations of 125 nM and 250 nM of TSA strongly suppressed the growth of 3D spheroids and led to cell death at 96-hr time point.
To understand the histology of the NET 3D spheroids, an H &amp; E and immunohistochemical (IHC) staining were performed on 3D spheroids that had been cultured for 17 days. Figure 4A illustrates a method to process 3D spheroids for HistoGel embedding, which is the key step for paraffin embedding of 3D spheroids. Figure 4B shows the H &amp; E staining, the immunohistochemical staining of the cleaved caspase-3 (a marker for apoptotic cells) and Ki-67 (a marker for proliferating cells) antibodies on 3D spheroids, which had been cultured for 17 days. The cells within the core of the 3D spheroids are mostly necrotic/apoptotic and the outer layer cells are actively proliferating.
<img src="/files/ftp_upload/4218/4218fig1.jpg" alt="Figure 1" /> 
Figure 1. The Morphology of NET 3D Spheroids. The 3D spheroids from three human neuroendocrine tumor cell lines are formed on agarose-coated 24-well plates. 1,000 BON-1, H727 and QGP-1 cells were seeded onto agarose-coated 24-well plates and grown in a normal physiological condition as detailed in the protocol. These are the images of 3D spheroids for BON-1 and H727 or cell aggregates for QGP-1, which were cultured for 10 days. 
<img src="/files/ftp_upload/4218/4218fig2.jpg" alt="Figure 2" /> 
Figure 2. The Growth Tracking of BON-1 3D Spheroids. The growth tracking of BON-1 3D spheroids: formation, growth and disintegration of 3D spheroids. As detailed in the protocol, 1,000 BON-1 cells are plated onto an agarose-coated 24-well plate. Cells are grown in a standard DMEM/F12 medium with 10% FBS on an orbital shaker in a physiological condition overnight, then moved on a stable platform and grown in the same condition. The growth tracking of the 3D cells were followed by imaging cells every hour for the first 5 hr after plating, and then every 1-3 days with a Zeiss Axiovert 200/M-based phase-contrast microscope using a 5x objective. The cells aggregate into masses first, then form multicellular aggregates, small spheroids, bigger spheroids, and eventually the spheroids disintegrate.
<img src="/files/ftp_upload/4218/4218fig3.jpg" alt="Figure 3" /> 
Figure 3. TSA Treatment on BON-1 3D Spheroids. (A) Images of 3D spheroids upon TSA treatment. Treatment group: V - Vehicle (0.0025% DMSO); D1- dose 1 (15.625 nM TSA); D2- dose 2 (31.25 nM TSA); D3: dose 3 (62.5 nM TSA); D4: dose 4 (125 nM TSA); D5: dose 5 (250 nM TSA). Time of treatment: 0 H - Time point that treatments started on day 6 3D spheroids; 24 H, 48 H, 72 H and 96 H are the time points after the treatments started. 3D spheroids were imaged at each time point as before. (B) Growth of the 3D spheroids upon TSA treatment. The major (L) and minor (W) axes of the 3D spheroids in (A) were estimated automatically by Matlab program at each time point of treatments (0, 24, 48, 72 and 96 hr). The volume of the 3D spheroids was estimated as 0.5 x L x W2. The volume of the spheroid in the graph was the average of 8 replicates and the error bar was calculated base on the 8 replicates.
<img alt="Table 1" src="/files/ftp_upload/4218/4218table1.jpg" /> 
Table 1. Layout of the drug treatments on a 24-well plate.
<img src="/files/ftp_upload/4218/4218fig4A.jpg" alt="Figure 4A" /> 
Figure 4A. Illustration of a Method to Process 3D Spheroids for HistoGel Embedding. A method to process 3D spheroids for HistoGel embedding and immunohistochemical staining of the 3D spheroids (A) An overview of a method to process 3D spheroids for HistoGel embedding. 3D spheroids were encapsulated in a HistoGel to be at the same level in the biopsy cryomold, then the HistoGel/spheroids block was wrapped in a blue Bio-wraps and transferred into a yellow biopsy cassette for paraffin embedding. 
<img src="/files/ftp_upload/4218/4218fig4B.jpg" alt="Figure 4B" /> 
Figure 4B. H &amp; E Staining and Immunohistochemical Staining of day-17 3D Spheroids. (B) H &amp; E staining, Immunohistochemical staining of Ki-67 and Cleaved Caspase-3 of the 17-day 3D spheroids. The paraffin-embedded sectioned slides were deparaffinized and antigen retrieval was performed using CCI (Cell Conditioning Solution, Verana Medical System, #711-065-152). Rabbit polyclonal Ki-67 antibody (Abcam. #Ab833) and Cleaved Caspase 3 antibody (Cell Signaling Technology, #9661) were applied at a concentration of 1:75 and 1:200, respectively, for 1 hr at room temperature. The anti-rabbit secondary antibody (Jackson Immunolab, #711-065-152) was applied at 1:500 for 1 hr at room temperature, followed by chromogenic detection kit DABMap (Ventana Medical Systems, #760-124). Slides were counterstained with Hematoxylin and dehydrated and cleared before coverslipping from Xylene.

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	<li>Taal, B. G., Visser, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+neuroendocrine+tumours.">Epidemiology of neuroendocrine tumours.</a> Neuroendocrinology. 80, 3-7 (2004).</li><li>Lee, G. Y., Kenny, P. A., Lee, E. H., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+models+of+normal+and+malignant+breast+epithelial+cells.">Three-dimensional culture models of normal and malignant breast epithelial cells.</a> Nat. Methods. 4, 359-365 (2007).</li><li>Friedrich, J., Seidel, C., Ebner, R., Kunz-Schughart, L. 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No conflicts of interest declared.

We have demonstrated a simple, reliable, and reproducible method for forming 3D multicellular spheroid from human neuroendocrine tumor cells using agarose-coated 24-well plates. The success of this technique is the ability to obtain a single spheroid with homogenous size and uniform shape by day 6 starting from a fixed number of cells. The key step is the slow agitation of the plate with NET cells for the first 16-24 hr after plating. This method can also apply to common human breast cancer cell line MCF-7, human colon adenocarcinoma cell line HT-29, human non-small cell lung carcinoma cell line H1299 and human embryonic kidney cell line HEK-293. They can all form uniform and compact 3D spheroids for therapeutic drug screen (Data not shown). Although several methods are reported on generating 3D spheroids,7-10 the method reported here does not require specialized plates or reagents and is thus simple and cost-effective. Our unpublished data demonstrate that NET 3D spheroids grown on agarose mimic the in vivo NET xenograft tumor geometrically and molecularly, when compared to monolayer 2D cells. This is similar to what has been reported for the 3D spheroids made from on other human cancer cell lines.11,12 The limitation of the 3D cell culture model is that it cannot completely replace testing the in vivo efficacy of any drugs or therapeutic treatments because the 3D spheroids are like avascular tumors, which is different from the in vivo tumor in this aspect. Nonetheless, this method will help bridge the gap and assessments of NET therapeutic drugs from in vitro to in vivo in some aspects.
Immunofluorescence staining on multicellular spheroids is very difficult to perform due to the large size and geometry of the floating 3D spheroids and the limitation of the confocal microscope. Here we illustrate a method to process HistoGel embedding for paraffin embedding, permitting immunohistochemical staining of the serial sectioned slides of the 3D spheroids. Besides, our custom-developed MATLAB program helps accurately and reproducibly estimate the size of the spheroids, monitor the drug efficacy efficiently, and could be incorporated into a high-throughput screen in the development of drugs for NET patients.

<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>DMEM/F12 Media</td>
 <td>Gibco</td>
 <td>10565-018</td>
 <td></td>
 </tr>
 <tr>
 <td>TrypLE</td>
 <td>Gibco</td>
 <td>12604</td>
 <td></td>
 </tr>
 <tr>
 <td>24-well plates</td>
 <td>Falcon</td>
 <td>353047</td>
 <td></td>
 </tr>
 <tr>
 <td>70 mM 			cell strainer</td>
 <td>Fisher Scientific</td>
 <td>22363548</td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile sealing tape</td>
 <td>Nunc</td>
 <td>236366</td>
 <td></td>
 </tr>
 <tr>
 <td>Trichostatin A</td>
 <td>Sigma</td>
 <td>T8552</td>
 <td></td>
 </tr>
 <tr>
 <td>Orbital Shaker</td>
 <td>Fisher Scientific</td>
 <td>11-402-12</td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

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

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 ...

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28.5K Views.  Raymond and Beverly Sackler Foundation. 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.

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

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

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 ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

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 ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Dynamic Lung Tumor Tracking for Stereotactic Ablative Body Radiation Therapy

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable risks for normal tissue injury and the hazards of physical tumor miss. A first-of-its-kind SBRT platform achieves high-precision ablative radiation treatment through a combination of versatile real-time imaging solutions and sophisticated tumor tracking capabilities. It uses dual-diagnostic kV x-ray units for stereoscopic open-loop feedback of cancer target intrafraction movement occurring as a consequence of respiratory motions and heartbeat. Image-guided feedback drives a gimbaled radiation accelerator (maximum 15 x 15 cm field size) capable of real-time &#177;4 cm pan-and-tilt action. Robot-driven &#177;60&#176; pivots of an integrated &#177;185&#176; rotational gantry allow for coplanar and non-coplanar accelerator beam set-up angles, ultimately permitting unique treatment degrees of freedom. State-of-the-art software aids real-time six dimensional positioning, ensuring irradiation of cancer targets with sub-millimeter accuracy (0.4 mm at isocenter). Use of these features enables treating physicians to steer radiation dose to cancer tumor targets while simultaneously reducing radiation dose to normal tissues. By adding respiration correlated computed tomography (CT) and 2-[18F] fluoro-2-deoxy-&#7429;-glucose (18F-FDG) positron emission tomography (PET) images into the planning system for enhanced tumor target contouring, the likelihood of physical tumor miss becomes substantially less1. In this article, we describe new radiation plans for the treatment of moving lung tumors.

Stereotactic ablative body radiation therapy (SBRT) involves the precise delivery of high-dose radiation to cancer tumor targets. A novel SBRT platform offers a first-of-its-kind gimbaled radiation accelerator mounted within a pivoting O-ring gantry capable of dynamic image-guided tumor tracking. Here, we describe dynamic tumor tracking for lung targets.

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...