This work was supported by NIH grants P50 CA174521 (to J.R. Howe and A.M. Bellizzi). P.H. Ear is a recipient of the P50 CA174521 Career Enhancement Program award.

All experiments using human neuroendocrine tumor samples have been approved by the University of Iowa Hospital and Clinics IRB committee (Protocol number 199911057). A list of all materials and equipment is described in the Table of Materials. A list of growth media and key solutions is found in Table 1.
1. Small bowel neuroendocrine tumor (SBNET) collection and cell dissociation
<ol>
	<li>Obtain resected patient SBNET samples after tumor tissues confirmation from the Surgical Pathology Core.</li>
	<li>Cut SBNETs into 5 mm&#160;cubes and store in 25 mL of DMEM/F12 medium in a conical tube for the transportation to the laboratory.</li>
	<li>Transfer the tumors in DMEM containing 1% FBS, 1% penicillin/streptomycin (Pen/Strep), 1% glutamine (Wash Medium) and incubate in this Wash Medium for 15 min.</li>
	<li>Transfer tumors to a new dish and mince tumors to less than 1 mm pieces using sterile curved scissors.</li>
	<li>Transfer the minced tissues to a new 50 mL tube containing 25 mL of Wash Medium.</li>
	<li>Centrifuge the sample at 500 x g for 15 min at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the pellets in 10 mL of Wash Medium containing collagenase (100 U/mL) and DNase (0.1 mg/mL).</li>
	<li>Allow the digestion of the minced tumors to occur in a 37 &#176;C incubator with slow shaking (50 rpm) for 1.5 h.</li>
</ol>
2. Culture of SBNETs as tumor spheroids in ECM
<ol>
	<li>After digestion is completed (step 1.8), centrifuge at 500 x g for 15 min at 4 &#176;C, discard the supernatant and resuspend the pellet in 15 mL of Wash Medium.</li>
	<li>Place a 70 &#181;m cell strainer on top of a new 50 mL tube and transfer 10 mL of the Wash Medium over the cell strainer. Swirl the Wash Medium to cover the side of the plastic tube to prevent NET cells from sticking to the side of the plastic tube.</li>
	<li>Filter the cell suspension through cell strainer.</li>
	<li>Centrifuge at 500 x g for 15 min at 4 &#176;C, discard the supernatant, resuspend the pellet in 200 &#181;L of Wash Medium (total volume is ~ 250 &#181;L) and place it on ice.</li>
	<li>Transfer 5 &#181;L of cells to 500 &#181;L of Wash Medium (1/100 dilution factor). Use the diluted cells for cell counting using a hemocytometer to obtain the number of cells per milliliter. Multiply by 100 to correct for the dilution factor.</li>
	<li>Multiply the number of cells per mL obtained in step 2.5 by the total volume of cells in suspension obtained in step 2.4 (~ 250 &#181;L).</li>
	<li>Centrifuge at 500 x g for 15 min at 4 &#176;C, discard the supernatant, and resuspend the cells in liquid ECM (1 x 106 cells/mL) and keep on ice.</li>
	<li>Transfer 5-20 &#181;L of SBNET spheroids in ECM to a 96-well plate and allow the liquid ECM to solidify by placing the plate in a 37 &#176;C incubator for 5 min.</li>
	<li>Add 200 &#181;L of SBNET culture medium (DMEM/F12 + 10% FBS + 1% PEN/STREP + 1% Glutamine + 10 mM nicotinamide + 10 &#181;g/mL insulin) to each well of the 96-well plate containing the SBNET spheroids in ECM.</li>
	<li>Alternatively, culture SBNET organoids in stem cell media similar to what has previously been described for growing either human liver or pancreatic organoids9 and listed in the Table of Materials.</li>
	<li>Change media every 5-7 days.</li>
</ol>
3. Quantification of SBNET spheroid size using ImageJ
<ol>
	<li>Take 5-10 pictures of SBNET spheroids at Day 1, 4, 7, 14, 23 and 97 of the culture using a 10x objective.</li>
	<li>Open saved images in ImageJ. Go to the Analyze tab, select the Set Measurements option and place a check on the Area option.</li>
	<li>Use the oval selection tool from the tool bar to generate an ellipsoid or circle around a well-focused spheroid.</li>
	<li>Go to the Analyze tab and select the Measure option. Repeat steps 3.4 and 3.5 in order to get area measurement from 25-50 SBNET spheroids. The values are provided in pixel square.</li>
	<li>Convert the pixel area to &#181;m2 by dividing the size of each pixel with the length of each pixel to &#181;m conversion factor of the microscopy images. 
	NOTE: SBNET cells grow mainly as spheroids and sometime as ellipsoids.</li>
</ol>
4. Characterization of SBNETS spheroids by immunofluorescence
<ol>
	<li>Transfer the organoids grown in ECM to a 1.5 mL tube using a P1000 pipette.</li>
	<li>Centrifuge at 1,500 x g for 1 min and remove the supernatant.</li>
	<li>Wash the organoid culture by adding 1 mL of PBS, mix, centrifuge at 1,500 x g for 1 min and remove the supernatant.</li>
	<li>Fix the organoids by adding 500 &#181;L of 4% paraformaldehyde and incubate for 15 min.</li>
	<li>Wash the culture twice with 1 mL of PBS.</li>
	<li>Permeabilize the culture by adding 500 &#181;L of PBS + 3% BSA + 0.1% Triton X 100 for 5 min.</li>
	<li>Then wash for three times with 1 mL of PBS + 3% BSA.</li>
	<li>Incubate for 1 h with primary antibodies against synaptophysin (SYP)10 at 1/600 dilution or chromogranin A (CgA)11 and somatostatin receptor 2 (SSTR2)12 at 1/400 dilution in antibody buffer (2.5% bovine serum albumin, 0.1% sodium azide, 25 mM Tris pH 7.4, 150 mM sodium chloride). 
	NOTE: Use 300 &#181;L or more of antibody solution per tube.</li>
	<li>Wash three times with 1 mL of PBS + 3% BSA.</li>
	<li>Incubate with secondary antibodies coupled to FITC at 1/500 dilution in antibody buffer for 1 h. Use 300 &#181;L or more of antibody solution per tube.</li>
	<li>Wash 3 times with 1 mL of PBS + 3% BSA. Make sure to aspirate and discard all the supernatant.</li>
	<li>Add 5 &#181;L of mounting medium containing the nuclear stain DAPI.</li>
	<li>Use a P20 pipette to transfer 5 &#181;L of the SBNET spheroids from step 4.12 to a glass slide and seal with a cover slip.</li>
	<li>Take images using a fluorescent microscope using the 10x, 20x or 40x objectives.</li>
</ol>
5. SBNET spheroids characterization by immunohistochemistry (IHC)
<ol>
	<li>Transfer SBNET spheroids from the culture plate to a 1.5 mL tube by using a P1000 pipette.</li>
	<li>Centrifuge at 1,500 x g for 1 min and remove the supernatant.</li>
	<li>Fix SBNET spheroids by adding 500 &#181;L of 10% formalin to the tube from step 5.2 and incubate at room temperature for 2-5 days prior to paraffin embedding and cut 4 &#181;m thick spheroid sections.</li>
	<li>Deparaffinize, rehydrate, and use heat-induced epitope retrieval in Antigen Retrieval Solution at pH 9 by heating to 97 &#176;C for 20 min using the 3-in-1 automated slide processing station to prepare slides for antibody incubation.</li>
	<li>Block slides and incubate with primary antibodies against SYP10 at a 1/100 dilution for 15 min, CgA11 at a 1/800 dilution for 15 min, and SSTR212 at a 1/5,000 dilution for 30 min.</li>
	<li>Incubate with the secondary antibody detection system for 15 min for the anti-SYP and CgA staining and 30 min for anti-SSTR2 staining. Take pictures using a 400x objective.</li>
</ol>
6. Treatment of SBNET organoids with rapamycin
<ol>
	<li>Dissolve rapamycin in DMSO to obtain a 10 mM stock solution.</li>
	<li>Prepare SBNET culture medium with DMSO (e.g., 1 mL of SBNET culture medium + 1 &#181;L of DMSO) and SBNET culture medium with 10 &#181;M of rapamycin (e.g., 1 mL of SBNET culture medium + 1 &#181;L of 10 mM rapamycin stock solution).</li>
	<li>Transfer 200 &#181;L media with SBNET culture medium with DMSO or 10 &#181;M rapamycin to SBNET spheroid cultures.</li>
	<li>Incubate SBNET spheroids for 5 days in 37 &#176;C incubator.</li>
	<li>Add 1 &#181;M of ethidium homodimer and incubate for 30 min. Remove the medium containing ethidium homodimer, wash with 200 &#181;L of PBS, and transfer 200 &#181;L of PBS to each well.</li>
	<li>Take microscopy images of SBNET spheroids with and without drug treatment using the red filter cube with the 10x, 20x, or 40x objectives of a fluorescent microscope. 
	NOTE: Dead cells stained with Ethidium homodimer appears as red using the red filter cube G of a commercially available microscope (Table of Materials). Alternatively, the signal can be captured using a spectrophotometer at 535 nm excitation and 624 nm emission.</li>
</ol>
7. Splitting SBNET spheroids
NOTE: This is done for expansion and for sharing with other researchers.
<ol>
	<li>Use a P1000 pipette to mechanically break the ECM and aspirate the ECM with SBNET spheroids to a sterile 1.5 mL tube.</li>
	<li>Centrifuge at 1,000 x g at 4 &#176;C, remove all the supernatant and place the tube on ice.</li>
	<li>Add 2-4x the volume of new ECM to the pellet. Mix the new ECM with the old ECM and SBNET spheroids by pipetting up and down 10x. Avoiding introducing air bubbles.</li>
	<li>Transfer 5-20 &#181;L of the ECM and SBNET spheroids mixture to a new plate and allow ECM to solidify.</li>
	<li>Cover with new SBNET medium and transfer to the incubator. The recovery rate is approximately 95 to 100%.</li>
	<li>For shipping SBNET spheroids to another lab, transfer SBNET spheroids with new ECM to a T25 flask and allow ECM to solidify.</li>
	<li>Fill the T25 flask with SBNET spheroid medium, screw on the cap tightly, and prepare shipment package.</li>
	<li>Upon receiving an SBNET spheroid culture, remove the culture medium and perform step 7.1 to 7.5 to put SBNET spheroids back in culture.</li>
</ol>
8. Cryostorage and recovery of SBNET spheroids
<ol>
	<li>Transfer SBNET spheroids from 5-10 small wells to a 15 mL tube. Centrifuge at 500 x g for 15 min at 4 &#176;C, remove supernatant, resuspend in freezing medium (90% FBS + 10% DMSO), store in a cell freezing container, and place this at -80 &#176;C.</li>
	<li>Transfer to the liquid nitrogen for longer storage.</li>
	<li>To recover SBNET spheroids, place the frozen vial on ice and wait until the content is completely thawed. Invert the tube and re-place on ice in order to speed up the thawing process.</li>
	<li>Once the sample is thawed, placed inside a pre-chilled centrifuge and spin at 1,000 x g for 10 min.</li>
	<li>Remove the supernatant and resuspend the spheroids in ECM. Keep the tube on ice, transfer 20 &#181;L to a new plate, and wait for the ECM to solidify.</li>
	<li>Transfer 200 &#181;L of SBNET culture medium with 10 &#181;M of ROCK inhibitor (Y-27632)13 and transfer to the incubator.</li>
	<li>Allow SBNET spheroids to recover and grow for 1 week. Remove the old culture medium, replenish with 200 &#181;L of SBNET culture medium and return to the incubator.</li>
	<li>Allow SBNET spheroids to continue to grow. 
	NOTE: It takes at least 1 week for rapidly growing spheroids to start to recover after cryopreservation13. SBNET spheroids take a minimum of 2-4 weeks to start growing. Many SBNET cells will die within the first 2 weeks and the survival rate is less than 10%. Since this is a very time-consuming and low yield process, it is better to share with other researchers SBNET spheroids in culture flasks (as described in step 7). Cryostorage and recovery should only be used as a backup plan in case of a bacterial contamination occurs.</li>
</ol>

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The authors have nothing to disclose.

Tumor 3D cultures have become a valuable resource for preclinical drug testing15. Various tumor organoid biobanks have recently been established from breast cancer and prostate cancer tumors16,17. In this study, we provide a detailed protocol to culture SBNET as spheroids and a simple and fast method to validate the spheroid cultures for NET markers by immunofluorescence and test drug sensitivity. From our experience, SBNET spheroids can grow in various culture media. They grow slightly faster in stem cell media for human pancreas or liver isolation that we adapted from previously published protocols9. We chose to grow the SBNETs in DMEM/F12 medium supplemented with insulin and nicotinamide because this is less expensive than stem cell media. Increasing the percentage of fetal bovine serum is another strategy to promote the growth of SBNET organoid cultures; however, this would also increase the overall cost for culture maintenance.
Performing IF staining and imaging with a fluorescent microscopy using the 10x, 20x or 40x objectives is a quick and simple method to test for the expression SBNET markers. However, it does not give a well-defined localization in comparison to IHC (Figure 2, Figure 3). For example, the IF data showed that the membrane localization of SSTR2 is difficult to detect. We mainly detect the cytosolic SSTR2, which has previously been reported18. In order to obtain a better localization of the marker proteins, we recommend using IF and confocal microscopy. Overall, IF is a useful method to rapidly confirm SBNET markers. Our antibodies (anti-SYP, anti-CgA, anti-SSTR2) did not cross-react with the negative control organoids that do not express SYP, CgA, or SSTR2 (Figure 2C) in the IF experiment. This suggestion that the fluorescent signals that we detected are specific to SBNET spheroids.
To increase the yield of SBNET spheroids, the critical steps of this protocol are during the cell filtration step (step 2.2) and mixing the SBNET with the liquid ECM for aliquoting into tissue culture plates (step 2.8). Make sure to cover the cell strainer membrane and the collection tube with media in order to prevent the SBNET cells from sticking to the plastic. Liquid ECM will rapidly solidify if not placed on ice. Make sure to have a small ice container in the tissue culture hood in order to keep the ECM and SBNET cells cold.
The limitation of this technique is the slow growth rate of the SBNET spheroids. Experiments must be planned efficiently and avoid using an excess amount of SBNET spheroids. Another limitation is the slow recovery after freeze thaw. It takes over 1 month after thawing for the SBNET spheroids to start growing. To overcome this limitation, we suggest to continuously maintain SBNET spheroids in cultures and splitting them as needed (step 7). Even after 9 months in culture, the SBNET spheroids still maintain expression of SBNET markers (Figure 2B).
Although 3D culture of SBNET spheroids is more labor intensive than traditional 2D culture of other cancer cell lines, it is an extremely valuable model for in vitro culture of SBNET because many SBNET researchers do not have access to the existing cell lines. With this protocol, scientists and clinicians can establish SBNET spheroid cultures from resected tumors and share them with other laboratories. In addition, the SBNET spheroids could potentially be used to establish SBNET patient-derived xenograft mouse models. Overall, the techniques presented here can be adapted for culturing, characterizing and performing drug testing of other NETs such as pancreatic or lung NETs. Note that the growth rate of the organoids will vary between the different types of NETs and patient samples.&#160;

Small bowel neuroendocrine tumors (SBNETs) originate from enterochromaffin cells of the small intestine. Although SBNETs are generally known to grow slowly, they commonly metastasize to the liver1. While the surgical removal or tumor ablation can be considered in many cases, recurrence is nearly universal, and, therefore, medical therapy plays an important role in management. Tremendous efforts have been invested to generate new SBNET cell lines for drug testing. However, there has been very little success. Only 6 SBNET cell lines (KRJ-I, CND2, GOT1, P-STS, L-STS, H-STS) have been reported2,3,4,5; and unfortunately one cell line no longer expresses NET markers6 and three other SBNET cell lines (KRJ-I, L-STS, H-STS) were determined to be derived from transformed lymphoblasts instead of NETs7. In order to accelerate the identification of drugs for targeting SBNETs, alternative methods for in vitro drug testing are needed.
Here, we take&#160;advantage of the availability of resected SBNETs and have established a way to culture these patient-derived SBNETs as spheroids growing in ECM. The overall goal of this manuscript is to describe a method to culture SBNET as a three-dimensional (3D) culture and outline procedures to characterize these spheroids for the retention of SBNET markers by immunofluorescence staining and immunohistochemistry.
In addition, we demonstrate how these SBNET spheroids can be used for testing the effect of rapamycin, an anti-cancer drug for NETs8. The rationale behind this protocol is to develop a new method to grow SBNET cells in vitro and use them for drug testing. The advantage of this technique over the traditional method of establishing an SBNET cell line is that 3D cultures of SBNETs can rapidly be obtained and drug testing can be done within 3 weeks. SBNET spheroids could potentially be used as a model for performing in vitro drug screens to identify new drugs for SBNET patients. Since SBNET cell lines are not widely available, 3D cultures of SBNET spheroids can serve as a new in vitro model for studying SBNETs and can be shared among scientists in the field.

<table><tbody><tr><td>Anti-rabbit FITC</td><td>Jackson ImmunoResearch</td><td>11-095-152</td><td>Secondary antibody couple to a green fluorophore</td></tr><tr><td>Antigen Retrieval Solution</td><td>Agilent Dako</td><td>S2367</td><td>Solution at pH 9 for preparing slides for IHC</td></tr><tr><td>Autostainer Link 48</td><td>Agilent Dako</td><td>Not Available</td><td>Automated system for antibody staining</td></tr><tr><td>Cell freezing container</td><td>Thermo Scientific</td><td>5100-0001</td><td>Container to for freezing cells</td></tr><tr><td>CellSence</td><td>Olympus</td><td>Version 1.18</td><td>Computer software for using fluorescent microscope</td></tr><tr><td>Chromogranin A antibody</td><td>Abcam-45179</td><td>RB-9003-PO</td><td>Antibodies for IF</td></tr><tr><td>Chromogranin A antibody (clone LK2H10)</td><td>Thermo Scientific</td><td>MA5-13096</td><td>Antibodies for IHC</td></tr><tr><td>Collagenase</td><td>Sigma</td><td>C0130</td><td>Enzyme for digesting tumor tissue</td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965-092</td><td>Medium for tissue preparation</td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>11320-033</td><td>Medium for organoid cultures</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418</td><td>Solvent for dissolving drug</td></tr><tr><td>DNAse</td><td>Sigma</td><td>DN25</td><td>Enzyme for digesting tumor tissue</td></tr><tr><td>Ethidium Homodimer</td><td>Chemodex</td><td>CDX-E0012-T1E</td><td>DNA and RNA binding dye</td></tr><tr><td>FBS</td><td>Gibco</td><td>16000044</td><td>Reagent for culture media</td></tr><tr><td>Fluorescent microscope</td><td>Olympus</td><td>CKX35</td><td>Microscope for taking pictures of SBENT spheroids</td></tr><tr><td>Glutamine</td><td>Gibco</td><td>A2916801</td><td>Reagent for culture media</td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td>Version 1.51</td><td>Computer software for image analysis</td></tr><tr><td>Insulin</td><td>Sigma</td><td>I0516</td><td>Reagent for culture media</td></tr><tr><td>Matrigel</td><td>Corning</td><td>356235</td><td>Matrix to embed and anchore organoids</td></tr><tr><td>Mounting medium (VECTASHIELD)</td><td>Vector Laboratories</td><td>H-1200</td><td>Fixative for labelled-cells with a nuclear stain</td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>72340</td><td>Reagent for culture media</td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Reagent to fix cells</td></tr><tr><td>PEN/STREP</td><td>Gibco</td><td>15140-122</td><td>Reagent for culture media</td></tr><tr><td>PT Link</td><td>Agilent Dako</td><td>Not Available</td><td>Automated system to prepare slides for IHC staining</td></tr><tr><td>Rapamycin</td><td>Alfa Aesar</td><td>J62473</td><td>Drug that can inhibit NET growth</td></tr><tr><td>Secondary antibodies for IHC</td><td>Agilent Dako</td><td>K8000</td><td>Secondary antibodies for IHC using Polymer-based EnVision FLEX system</td></tr><tr><td>SSTR2 antibody</td><td>GeneScritp</td><td>A01591</td><td>Antibodies for IF</td></tr><tr><td>SSTR2 antibody (clone UMB1)</td><td>Abcam</td><td>ab134152</td><td>Antibodies for IHC</td></tr><tr><td>Synaptophysin antibody</td><td>Abcam</td><td>32127</td><td>Antibodies for IF</td></tr><tr><td>Synaptophysin antibody (clone DAK-SYNAP)</td><td>Agilent Dako</td><td>M7315</td><td>Antibodies for IHC</td></tr><tr><td>TritonX</td><td>Mallinckrodt</td><td>3555 KBGE</td><td>Reagent to permeablize cells</td></tr><tr><td>Y-2763 ROCK inhibitor</td><td>Adipogen</td><td>AG-CR1-3564-M005</td><td>To improve SBNET spheroid viability after freeze thaw</td></tr></tbody></table>

establishment and characterization of small bowel neuroendocrine tumor spheroids

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited because very few patient derived SBNET cell lines have been generated. Well-differentiated SBNET cells are slow growing and are hard to propagate. The few cell lines that have been established are not readily available, and after time in culture may not continue to express characteristics of NET cells. Generating new cell lines could take many years since SBNET cells have a long doubling time and many enrichment steps are needed in order to eliminate the rapidly dividing cancer-associated fibroblasts. To overcome these limitations, we have developed a protocol to culture SBNET cells from surgically removed tumors as spheroids in extracellular matrix (ECM). The ECM forms a 3-dimensional matrix that encapsulates SBNET cells and mimics the tumor micro-environment for allowing SBNET cells to grow. Here, we characterized the growth rate of SBNET spheroids and described methods to identify SBNET markers using immunofluorescence microscopy and immunohistochemistry to confirm that the spheroids are neuroendocrine tumor cells. In addition, we used SBNET spheroids for testing the cytotoxicity of rapamycin.

There are currently only 2 SBNET cell lines established and published2,3,4,5 and they are not readily available to many researchers. Here, we propose to culture SBNET as spheroids in ECM and use this as an alternative model to study SBNET drug sensitivity. Patient-derived tumor from an SBNET that metastasized to the liver was collected, digested to release SBNET cells, and mixed with liquid ECM for establishing an SBNET spheroid culture (Figure 1A). The Ki-67 of this SBNET was 4.3%. Although SBNET spheroids have a slow growth rate, their growth can be monitored by microscopic imaging (Figure 1A). It takes approximately 14 days for SBNET spheroids to double in size when the culture media is changed once a week (Figure 1B). After 14 days in culture, SBNET spheroids do not increase in size. Instead, some SBNET cells will dissociate to a neighboring location and form new spheroids. To propagate the SBNET spheroids culture, harvest the ECM containing SBNET spheroids and reseed them in new culture plate with new culture medium (Step 7).
To confirm that the organoid cultures contain SBNET cells, we describe a simple and fast method to stain the spheroids for SBNET markers such as synaptophysin, chromogranin A, and the somatostatin receptor type 2 (SSTR2) using immunofluorescence (IF) microscopy (Step 4). Using antibodies specific against synaptophysin, chromogranin A and SSTR2, our IF data showed that these markers are localized in the cytoplasm and at the membrane of SBNET cells (Figure 2A, in green) after 1 or 9 months in culture (Figure 2B). To ensure the specificity of the SYP, CgA and SSTR2 antibodies, we performed the same staining procedures on an organoid line from pancreas tumor that does not express SYP, CgA or SSTR2 (Figure 2C) as no green signal was detected. The main advantage of this SBNET spheroid IF experiment is that it can be performed within 4 h and gives similar staining information as the immunohistochemistry (IHC; Figure 3). We provide a protocol for performing IHC of the SBNET spheroids in step 5.
Culturing SBNET as spheroids is a valuable technique for identifying drugs that can inhibit SBNET growth. As a proof of principle, we treated SBNET spheroids with rapamycin for 5 days, an mTOR inhibitor, a class of drugs commonly used to treat NETs8. In comparison to our control SBNET spheroid, the rapamycin-treated spheroid formed a grape-like structure and became apoptotic or necrotic (Figure 4A-D). Dying cells can be detected using Ethidium homodimer to stain DNA and RNA and generate a bright red signal14. This dye cannot penetrate the cell membrane of live cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60303/60303fig1.jpg" /> 
Figure 1: Patient-derived small bowel neuroendocrine tumors (SBNETs) grown in extracellular matrix (ECM) as spheroids. (A) Isolation of tumor cells from a resected SBNET and put in culture by mixing with ECM. Scale bar represents 100 &#181;m. (B) Surface area of SBNET spheroids with respect to the number of days in culture quantified using ImageJ. Data were obtained from SBNET spheroids of 1 patient and are represented as the mean area &#177; standard error of the mean. The surface area of 30 to 60 spheroids were measured for each time point. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60303/60303fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60303/60303fig2.jpg" /> 
Figure 2: Immunofluorescence (IF) staining of SBNET spheroids. IF staining of SBNET spheroids after (A) 1 month in culture and (B) 9 months in culture. (C) IF staining of pancreas tumor organoids that do not express SBNET markers as negative controls. Tumor spheroids were fixed in 4% paraformaldehyde and stained using antibodies against synaptophysin (SYP) at 1/600 dilution, chromogranin A (CgA) at 1/400 dilution, and somatostatin receptor 2 (SSTR2) at 1/400. IF images were taken at 100 ms, 200 ms and 400 ms exposure time for SYP, CgA and SSTR2 staining, respectively using the 10x, 20x or 40x objectives. Scale bar represents 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60303/60303fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60303/60303fig3.jpg" /> 
Figure 3: Immunohistochemistry (IHC) staining of SBNET spheroids. Formalin-fixed and paraffin-embedded SBNET spheroids sections were deparaffinized, rehydrated, blocked and stained with (A) SYP, (B) CgA, and (C) SSTR2 antibodies. Images were taken using the 400x objective. Scale bar represents 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60303/60303fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60303/60303fig4.jpg" /> 
Figure 4: Using SBNET spheroids for drug testing. (A) Bright field image of SBNET spheroids treated with DMSO for 5 days. (B) Image of SBNET spheroids treated with DMSO and stained with Ethidium homodimer (Ethidium H). (C) Bright field image of a dead SBNET spheroid forming grape-like structure after treatment with 10 &#181;M of rapamycin for 5 days. (D) Dead SBNET spheroid stained with Ethidium H appears as red dots. Images of Ethidium H staining were taken using the red filter cube at 100 ms exposure time. Scale bar represents 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60303/60303fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Growth media or solution</td>
			<td>Composition</td>
		</tr>
		<tr>
			<td>Wash Medium</td>
			<td>DMEM containing 1% FBS, 1% PEN/STREP, 1% Glutamine</td>
		</tr>
		<tr>
			<td>SBNET culture medium</td>
			<td>DMEM/F12 + 10% FBS + 1% PEN/STREP + 1% Glutamine + 10 mM nicotinamide + 10 &#181;g/mL insulin</td>
		</tr>
		<tr>
			<td>Antibody buffer</td>
			<td>2.5% bovine serum albumin, 0.1% sodium azide, 25 mM Tris pH 7.4, 150 mM sodium chloride</td>
		</tr>
		<tr>
			<td>Freezing medium</td>
			<td>90% FBS + 10% DMSO</td>
		</tr>
		<tr>
			<td>Human liver stem cell isolation medium</td>
			<td>DMEM/F12 , 1% Pen/Strep, 1% GlutaMAX, 10 mM HEPES, 1/50 B27 Supplement, 1/100 N2 Supplement, 1 mM N-acetylcysteine, 200 ng/mL Rspo 1, 50ng/mL EGF, 100 ng/mL FGF10, 10 mM nicotinamide, 10 uM forskolin, 5uM A83-01</td>
		</tr>
		<tr>
			<td>Human pancreatic stem cell isolation medium</td>
			<td>DMEM/F12 , 1% Pen/Strep, 1% GlutaMAX, 10 mM HEPES, 1/50 B27 Supplement, 1/100 N2 Supplement, 1 mM N-acetylcysteine, 200 ng/mL Rspo 1, 25 ng/mL Noggin, 50ng/mL EGF, 100 ng/mL FGF10, 10 mM nicotinamide, 10 uM forskolin, 5uM A83-01, 3 uM PGE-2</td>
		</tr>
	</tbody>
</table>
Table 1. List of growth media and solution.

Watch this Scientific Journal Video about Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids at JoVE.com

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

Neuroendocrine tumors (NETs) originate from neuroendocrine cells of the neural crest. They are slow growing and challenging to culture. We present an alternative strategy to grow NETs from the small bowel by culturing them as spheroids. These spheroids have small bowel NET markers and can be used for drug testing.

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

establishment-characterization-small-bowel-neuroendocrine-tumor

Nanyang Technological University

Research

JoVE Journal

Cancer Research

6.4K Views.  University of Iowa Carver College of Medicine. Neuroendocrine tumors (NETs) originate from neuroendocrine cells of the neural crest. They are slow growing and challenging to culture. We present an alternative strategy to grow NETs from the small bowel by culturing them as spheroids. These spheroids have small bowel NET markers and can be used for drug testing.

Video: Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

Bioengineering

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 ...

Medicine

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the evaluation of anticancer drugs. However, the conventional culture methods lack the ability to manipulate the tumor spheroids in a homogeneous manner at the three-dimensional level. To address this limitation, in this paper, we present a convenient and effective method of constructing average-sized tumor spheroids. Additionally, we describe a method of image-based analysis using artificial intelligence-based analysis software that can scan the whole plate and obtain data on three-dimensional spheroids. Several parameters were studied. By using a standard method of tumor spheroid construction and a high-throughput imaging and analysis system, the effectiveness and accuracy of drug tests performed on three-dimensional spheroids can be dramatically increased.

This article demonstrates a standardized method for constructing three-dimensional tumor spheroids. A strategy for spheroid observation and image-based deep-learning analysis using an automated imaging system is also described.

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...

3D-PDAC Model Capturing Desmoplasia for Therapeutic Evaluation

Growing Desmoplastic Three-Dimensional Pancreatic Cancer Spheroids from Co-Culture

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of &#60;12%. The biggest barrier to therapy is the dense desmoplastic extracellular matrix (ECM) that surrounds the tumor and reduces vascularization, generally termed desmoplasia. A variety of drug combinations and formulations have been tested to treat the cancer, and although many of them show success pre-clinically, they fail clinically. It, therefore, becomes important to have a clinically relevant model available that can predict the response of the tumor to therapy. This model has been previously validated against resected clinical tumors. Here a simple protocol to grow desmoplastic three-dimensional (3D)-coculture spheroids is described that can naturally generating a robust ECM and do not require any external matrix sources or scaffold to support their growth.
Briefly human pancreatic stellate cells (HPaSteC) and PANC-1 cells are used to prepare a suspension containing the cells in a 1:2 ratio, respectively. The cells are plated in a poly-HEMA coated, 96-well low attachment U-well plate. The plate is centrifuged to allow the cells to form an initial pellet. The plate is stored in the incubator at 37 &#176;C with 5% CO2, and media is replaced every 3 days. Plates can be imaged at designated intervals to measure spheroid volume. Following 14 days of culture, mature desmoplastic spheroids are formed (i.e. average volume of 0.048 + 0.012 mm3&#160;(451 &#181;m x 462.84 &#181;m)) and can be utilized for experimental therapy assessment. Mature ECM components include collagen-I, hyaluronic acid, fibronectin, and laminin.

Author Spotlight: Validating Cancer Therapy Responses with Desmoplastic Spheroid Models

Pancreatic cancer remains one of the toughest cancers to treat. Therefore, it is critical that pre-clinical models evaluating treatment efficacy are reproducible and clinically relevant. This protocol describes a simple co-culture procedure to generate reproducible, clinically relevant desmoplastic spheroids.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of &#60;12%. The biggest barrier to therapy is 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 ...

Fabrication Followed by 3D Encapsulation of Tumor Spheroids in PEG Hydrogels

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth. Here, we designed an in vitro 3D glioblastoma model for spheroid encapsulation to mimic the tumor extracellular microenvironment. First, we formed square pyramidal microwell molds using polydimethylsiloxane. These microwell molds were then used to fabricate tumor spheroids with tightly controlled sizes from 50-500 &#956;m. Once spheroids were formed, they were harvested and encapsulated in polyethylene glycol (PEG)-based hydrogels. PEG hydrogels are a versatile platform for spheroid encapsulation, as hydrogel properties such as stiffness, degradability, and cell adhesiveness can be tuned independently. Here, we used a representative soft (~8 kPa) hydrogel to encapsulate glioblastoma spheroids. Finally, a method to stain and image spheroids was developed to obtain high-quality images via confocal microscopy. Due to the dense spheroid core and relatively sparse periphery, imaging can be difficult, but using a clearing solution and confocal optical sectioning helps alleviate these imaging difficulties. In summary, we show a method to fabricate uniform spheroids, encapsulate them in PEG hydrogels and perform confocal microscopy on the encapsulated spheroids to study spheroid growth and various cell-matrix interactions.

Author Spotlight: In Vitro Hydrogel Model for Glioblastoma Microenvironment Study

Here, we present a protocol that enables fast, robust, and cheap fabrication of tumor spheroids followed by hydrogel encapsulation. It is widely applicable as it does not require specialized equipment. It would be particularly useful for exploring spheroid-matrix interactions and building in vitro tissue physiology or pathology models.

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth.  Here, we ...

Splitting Spheroids for Sub-culturing and Shipping: A Procedure for Propagating and Transferring Spheroids from Small Bowel Neuroendocrine Tumor

Source: Ear, P. H. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/60303" target="_blank">Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids.</a> J. Vis. Exp. (2019)
This video demonstrates a strategy to split and propagate spheroids developed from small bowel neuroendocrine tumor cells. These spheroids have small bowel neuroendocrine tumor markers and can be propagated and further used to test anti-cancer drugs.

Dzielenie sferoid w celu hodowli i wysyłki: procedura rozmnażania i przenoszenia sferoid z guza neuroendokrynnego jelita cienkiego

Source: Ear, P. H. et al. Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids. J. Vis. Exp. (2019)
This video demonstrates a ...