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 g...

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 border="0" cellpadding="0" cellspacing="0" style="width:1009px;" width="1009">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Anti-rabbit FITC</td>
			<td style="width:196px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">11-095-152</td>
			<td style="width:437px;">Secondary antibody couple to a green fluorophore</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antigen Retrieval Solution</td>
			<td>Agilent Dako</td>
			<td style="width:119px;">S2367</td>
			<td>Solution at pH 9 for preparing slides for IHC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Autostainer Link 48</td>
			<td>Agilent Dako</td>
			<td style="width:119px;">Not Available</td>
			<td>Automated system for antibody staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell freezing container</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td>5100-0001</td>
			<td>Container to for freezing cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">CellSence</td>
			<td style="width:196px;">Olympus</td>
			<td style="width:119px;">Version 1.18</td>
			<td>Computer software for using fluorescent microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromogranin A antibody</td>
			<td>Abcam-45179</td>
			<td style="width:119px;">RB-9003-PO</td>
			<td>Antibodies for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromogranin A antibody (clone LK2H10)</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td>MA5-13096</td>
			<td>Antibodies for IHC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase</td>
			<td style="width:196px;">Sigma</td>
			<td style="width:119px;">C0130</td>
			<td>Enzyme for digesting tumor tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:196px;">Gibco</td>
			<td style="width:119px;">11965-092</td>
			<td>Medium for tissue preparation</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">DMEM/F12</td>
			<td style="width:196px;">Gibco</td>
			<td style="width:119px;">11320-033</td>
			<td>Medium for organoid cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td style="width:196px;">Sigma</td>
			<td>D8418</td>
			<td>Solvent for dissolving drug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNAse</td>
			<td style="width:196px;">Sigma</td>
			<td style="width:119px;">DN25</td>
			<td>Enzyme for digesting tumor tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethidium Homodimer</td>
			<td style="width:196px;">Chemodex</td>
			<td style="width:119px;">CDX-E0012-T1E</td>
			<td>DNA and RNA binding dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td style="width:196px;">Gibco</td>
			<td style="width:119px;">16000044</td>
			<td>Reagent for culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Fluorescent microscope</td>
			<td style="width:196px;">Olympus</td>
			<td style="width:119px;">CKX35</td>
			<td>Microscope for taking pictures of SBENT spheroids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamine</td>
			<td style="width:196px;">Gibco</td>
			<td style="width:119px;">A2916801</td>
			<td>Reagent for culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">ImageJ</td>
			<td style="width:196px;">National Institutes of Health</td>
			<td style="width:119px;">Version 1.51</td>
			<td>Computer software for image analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin</td>
			<td style="width:196px;">Sigma</td>
			<td style="width:119px;">I0516</td>
			<td>Reagent for culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel</td>
			<td style="width:196px;">Corning</td>
			<td>356235</td>
			<td>Matrix to embed and anchore organoids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting medium (VECTASHIELD)</td>
			<td style="width:196px;">Vector Laboratories</td>
			<td>H-1200</td>
			<td>Fixative for labelled-cells with a nuclear stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nicotinamide</td>
			<td style="width:196px;">Sigma</td>
			<td>72340</td>
			<td>Reagent for culture media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Paraformaldehyde</td>
			<td style="width:196px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td>Reagent to fix cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PEN/STREP</td>
			<td style="width:196px;">Gibco</td>
			<td style="width:119px;">15140-122</td>
			<td>Reagent for culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PT Link</td>
			<td>Agilent Dako</td>
			<td style="width:119px;">Not Available</td>
			<td>Automated system to prepare slides for IHC staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rapamycin</td>
			<td style="width:196px;">Alfa Aesar</td>
			<td style="width:119px;">J62473</td>
			<td>Drug that can inhibit NET growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Secondary antibodies for IHC</td>
			<td style="width:196px;">Agilent Dako</td>
			<td style="width:119px;">K8000</td>
			<td>Secondary antibodies for IHC using Polymer-based EnVision FLEX system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SSTR2 antibody</td>
			<td style="width:196px;">GeneScritp</td>
			<td style="width:119px;">A01591</td>
			<td>Antibodies for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SSTR2 antibody (clone UMB1)</td>
			<td style="width:196px;">Abcam</td>
			<td>ab134152</td>
			<td>Antibodies for IHC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synaptophysin antibody</td>
			<td style="width:196px;">Abcam</td>
			<td>32127</td>
			<td>Antibodies for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synaptophysin antibody (clone DAK-SYNAP)</td>
			<td>Agilent Dako</td>
			<td style="width:119px;">M7315</td>
			<td>Antibodies for IHC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TritonX</td>
			<td style="width:196px;">Mallinckrodt</td>
			<td style="width:119px;">3555 KBGE</td>
			<td>Reagent to permeablize cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Y-2763 ROCK inhibitor</td>
			<td style="width:196px;">Adipogen</td>
			<td style="width:119px;">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...

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

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

Operating Procedures of the Electrochemotherapy for Treatment of Tumor in Dogs and Cats

Electrochemotherapy (ECT) is a local approach which is used for treating solid tumors of different histologies. Its mechanism is based on cell membrane permeabilization by means of &#34;electroporation&#34;. To achieve the &#34;electroporation&#34; of the cells, electric pulses are generated by a generator and delivered to the target tissue by the use of electrodes. Electroporation is a physical method which is used to introduce molecules, like cytostatic drugs, into the cells that could not pass the cell membrane on their own. In electrochemotherapy, currently, cisplatin and bleomycin are clinically used. Electrochemotherapy antitumor effectiveness is high, for example up to 100% complete response of canine mast cell tumors smaller than 2 cm3 was achieved. Additionally, electrochemotherapy can be used for the treatment of inoperable tumors. One of the important characteristics of electrochemotherapy is that it can be effective as a one-time treatment only. However, in the case of failure or partial tumor response it can be repeated several times with equal or improved effectiveness. Electrochemotherapy is already a standard treatment for cutaneous and subcutaneous tumors of various histologies in human and veterinary oncology. Furthermore, several clinical studies exploiting electrochemotherapy for deep-seated tumors are on-going.

Electrochemotherapy is the local treatment of solid tumors. The main mechanism is electroporation-mediated permeabilization of the tumor cells' membrane which enables increased internalization of cytostatics like bleomycin and cisplatin. Thus, antitumor effectiveness is potentiated at the site of the application of electric pulses to the tumors.

Electrochemotherapy (ECT) is a local approach which is used for treating solid tumors of different histologies. Its mechanism is based on cell membrane ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the spheroid at the cellular level is important for analysis, yet existing techniques are often expensive, laborious, use non-standard equipment, cause significant size distortion, or are limited to relatively small spheroids. This protocol presents a new method of mounting and clearing spheroids that address these issues while allowing for confocal analysis of the inner structure of spheroids. In contrast to existing approaches, this protocol provides for rapid mounting and clearing of a large number of spheroids using standard equipment and laboratory supplies. Mounting spheroids in a pH-neutral agarose-PBS gel solution before introducing a refractive-index-matched clearing solution minimizes size distortion common to other similar techniques. This allows for detailed quantitative and statistical analysis where the accuracy of size measurements is paramount. Furthermore, compared to liquid clearing solutions, the agarose gel technique keeps spheroids fixed in place, allowing for the collection of three-dimensional (3D) confocal images. The present article elaborates how the method yields high-quality two- and 3D images that provide information about inter-cell variability and inner spheroid structure.

Tumor spheroids are becoming increasingly utilized to assess tumor cell-microenvironment interactions and therapy response. The present protocol describes a robust but simple method for semi-high-throughput imaging of 3D tumor spheroids using rapid optical clearing.

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the ...

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and progression. Complexity within the tumor microenvironment, including tumor secretome, low-grade inflammation, hypoxia, and redox imbalance, fosters heterotypic interaction and allows the transformation of inactive resident fibroblasts to become active CAFs. CAFs are metabolically distinguished from normal fibroblasts (NFs) as they are more glycolytically active, produce higher levels of reactive oxygen species (ROS), and overexpress lactate exporter MCT-4, leading to the opening of the mitochondrial permeability transition pore (MPTP). Here a method has been described to analyze the mitochondrial health of activated CAFs isolated from the multicellular 3D tumor spheroids comprising of human lung adenocarcinoma cells (A549), human monocytes (THP-1), and human lung fibroblast cells (MRC5). Tumor spheroids were disintegrated at different time intervals and through magnetic-activated cell sorting, CAFs were isolated. The mitochondrial membrane potential of CAFs was assessed using JC-1 dye, ROS production by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining, and enzyme activity in the isolated CAFs. Analyzing the mitochondrial health of isolated CAFs provides a better understanding of the reverse Warburg effect and can also be applied to study the consequences of CAF mitochondrial changes, such as metabolic fluxes and the corresponding regulatory mechanisms on lung cancer heterogeneity. Thus, the present study advocates an understanding of tumor-stroma interactions on mitochondrial health. It would provide a platform to check mitochondrial-specific drug candidates for their efficacies against CAFs as potential therapeutics in the tumor microenvironment, thereby preventing CAF involvement in lung cancer progression.

Multicellular 3D tumor spheroids were prepared with lung adenocarcinoma cells, fibroblasts, and monocytes, followed by the isolation of cancer-associated fibroblasts (CAFs) from these spheroids. Isolated CAFs were compared with normal fibroblasts to assess mitochondrial health by studying the mitochondrial transmembrane potential, reactive oxygen species, and enzymatic activities.

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and ...

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 ...