The collection of human tumor samples used for the primary tumor cell cultures was performed as per institutional review board (IRB)-approved protocols at the Rabin Medical Center with written informed consent from the patients. Patients eligible for participation in the study included male and female adult and pediatric cancer patients with non-metastatic breast, colon, liver, lung, neuroendocrine, ovary, or pancreatic cancer, any pediatric cancer, or any metastatic cancer. The only exclusion criterion was the lack of capacity to provide informed consent.
1. Generation and collection of spheroids
NOTE: The isolation of primary tumor cells can be performed as described by Kodak et al.8. Importantly, primary tumor cells used for generating the spheroids can be derived directly from patient samples obtained by biopsy, resection, etc., or indirectly using tumor samples from patient-derived xenograft (PDX) models, as described by Moskovits et al.9.
<ol>
	<li>Prepare a single-cell suspension of adherent primary tumor cell cultures of 75%-100% confluence by taking a small flask (T25) with a single-cell adherent primary cell culture, removing the cell culture media, washing with PBS, and then adding 1 mL of 1x Accutase (a cell detachment solution) (see the Table of Materials) for 3 min at 37 &#176;C.</li>
	<li>Neutralize the Accutase solution by adding 5 mL of cell culture medium (RPMI-1640 culture medium supplemented with 10% FBS, 1:100 penicillin-streptomycin, 1% non-essential amino acids, and 1% L-glutamine; see the Table of Materials).</li>
	<li>Aspirate the cells with a 10 mL serological pipette, and deposit them in a 15 mL conical tube. Centrifuge the tube at 800 x g for 5 min at room temperature.</li>
	<li>Remove the cell culture medium, add 5 mL of fresh cell culture medium on top of the cell pellet, and mix gently.</li>
	<li>Count the viable cells with a hemocytometer10. To do this, take an aliquot of 50 &#956;L of the cell suspension, and mix it with 50 &#956;L of trypan blue. Count the live cells (the cells that are negative for blue color), and calculate the total number of live cells in the suspension.</li>
	<li>Prepare a &#34;3D culture medium&#34; (a cell culture medium supplemented with 5% basement membrane matrix; see the Table of Materials). 
	NOTE: The &#34;3D culture medium&#34; is liquid-like at room temperature. At 37 &#176;C, the consistency becomes more gel-like (so that the cells stay together), although it can still be pipetted (since it contains only 5% basement membrane matrix).</li>
	<li>Calculate the number of cells needed for the assay and the total volume required; each well must contain 2,000-8,000 cells in 200 &#956;L of medium.</li>
	<li>Prepare a cell suspension with the desired number of cells (e.g., 4,000 cells) in 200 &#956;L of the &#34;3D culture medium&#34; and mix gently with the pipette to ensure a homogenous distribution.</li>
	<li>Transfer the suspension to a pipetting reservoir. Using a multi-channel pipette, add 200 &#956;L of the cell suspension to each well of an ultra-low attachment 96-well plate (see the Table of Materials). Before each collection of the cells, mix the suspension well.</li>
	<li>Centrifuge the plate at 300 x g for 10 min at room temperature to enforce the clustering of the cells, thereby improving the cell aggregation, and incubate the plate at 37 &#176;C in a 5% CO2&#160;humidified incubator.</li>
	<li>Every 2-3 days, refresh the &#34;3D culture medium&#34;. Centrifuge the plate at 300 x g for 10 min at room temperature, gently remove and discard 50% of the medium (100 &#956;L), and add 100 &#956;L of fresh &#34;3D culture medium&#34; to replace the existing solution. Repeat step 1.11, and place the plate back in the 37 &#176;C, 5% CO2&#160;humidified incubator. 
	NOTE: The removal of the medium must be performed when the plate is held at 45&#176;, and the medium must be collected only from the upper part to avoid collecting the cells. A vacuum suction system should not be used. The fresh medium needs to be added slowly and gently in order not to disrupt the spheroids, which will have already started to form.</li>
	<li>Inspect the cells under a microscope every 1-2 days to monitor spheroid formation. Measure the diameter of the spheroids formed using the &#34;scale&#34; tool in the imaging software (see the Table of Materials). 
	NOTE: Microscopic inspection should first reveal irregular round-to-oval bodies that assume a spheroid shape as time progresses11,12.
	<ol>
		<li>When the spheroid diameter reaches 100-200 &#956;m, perform the drug efficacy experiments. 
		NOTE: The drug efficacy experiments are performed when the spheroids reach this diameter as, at that time, the majority of the cells are proliferating, which is conducive to evaluating the response to treatment. Larger diameter spheroids contain a necrotic core and a quiescent layer11,12, resulting in a smaller proportion of proliferating cells that respond to treatment.</li>
	</ol>
	</li>
	<li>For spheroid collection, use a 1,000 &#956;L pipette to collect the spheroids from each well, and deposit them into a 15 mL conical tube. 
	NOTE: Spheroids are inherently fragile and require gentle handling. When transferring the medium with the spheroids into the conical tube, hold the tube at 45&#176;, and pipette slowly on the wall of the tube. Spheroids are visible to the eye.</li>
	<li>Centrifuge the conical tube at 300 x g for 5 min at room temperature, and carefully aspirate and discard the supernatant using a pipette.</li>
	<li>Add 0.5 mL of the cell culture medium, and resuspend the pellet well but gently. Avoid making bubbles.</li>
	<li>Perform spheroid counting following the steps below.
	<ol>
		<li>Use a 96-well plate and draw a plus sign on the underside of a well to divide the well into quadrants (to help track the counting).</li>
		<li>Add 50 &#956;L of the suspension to the well, and count the spheroids manually under the microscope (using a 10x objective lens).</li>
		<li>Count the spheroids in each quadrant, be careful not to double count, and calculate the total number of spheroids in the well. 
		NOTE: For accuracy of the counting, count at least 50 spheroids in a well. If there are less than 50 spheroids, gently mix the suspension to ensure homogenous distribution, and count again using a larger volume of the suspension added to a new well. Alternatively, if there are less than 50 spheroids in a well, the spheroids can be centrifuged and resuspended gently in a volume of &#60;0.5 mL. If there are over 100 spheroids, add cell culture medium to the spheroid suspension, mix gently, and recount.</li>
		<li>Calculate the spheroid concentration (spheroid count/counting volume [&#956;L]), and then calculate the total number of spheroids in the suspension (spheroid concentration &#215; total volume).</li>
	</ol>
	</li>
</ol>
2. Drug efficacy assay (MTT assay)
NOTE: For details, please see van Meerloo et al.13. Also, for the MTT assay, only the cell culture medium and not the &#34;3D culture medium&#34; must be used (adding the basement membrane matrix is not necessary and could potentially interfere with the MTT assay).
<ol>
	<li>In a new tube, prepare a spheroid suspension at a concentration of 200 spheroids per 200 &#956;L of cell culture medium (RPMI-1640 culture medium supplemented with 10% FBS, 1:100 penicillin-streptomycin, 1% non-essential amino acids, and 1% L-glutamine).
	<ol>
		<li>For each drug treatment, prepare a stock of spheroids in a 15 mL tube. Calculate the amount needed for each drug by the number of wells needed for repeats: (5-8) &#215; 200 &#956;L. Add the drug to the tube to the final concentration needed. 
		NOTE: Please see the representative results section regarding the drugs used for the present study and their dosage. Also, the commercial details of the drugs are listed in the Table of Materials.</li>
	</ol>
	</li>
	<li>Transfer 200 &#956;L of the spheroid suspension into the wells of an ultra-low attachment 96-well plate, and incubate the plate at 37 &#176;C in a 5% CO2&#160;humidified incubator. Do not use the external rows and columns of the 96-well plate for the assay, as these wells are characterized by increased evaporation, which could lead to increased variability between the repeat experiments; instead, add PBS to these wells. 
	NOTE: It is key that the culture of the spheroids is homogeneous before the culture is aliquoted into the 96-well plate. Also, a control condition (i.e., untreated spheroids) must be included in every experiment.</li>
	<li>After incubating the spheroids with the study drug for 24-72 h, centrifuge the plate at 300 x g for 5 min at room temperature, and gently remove 170 &#956;L of the cell culture medium, leaving 30 &#956;L (which includes the spheroids) at the bottom the well. 
	NOTE: The removal of the 170 &#956;L of cell culture medium must be done carefully, so as not to remove the spheroids. Hold the plate at 45&#176;, and place one hand (wearing a dark glove for contrast) under the wells so that the spheroids are visible (white dots).</li>
	<li>Prepare the MTT solution (0.714 mg/mL in phenol-free RPMI, see the Table of Materials).</li>
	<li>Add 70 &#956;L of MTT solution to each well to a final volume of 100 &#956;L per well (the final MTT concentration in the well will be 0.05 mg per 100 &#956;L). In addition, prepare &#34;Blank&#34; wells with MTT solution without cells. 
	NOTE: MTT is light-sensitive. Therefore, the light in the hood must be switched off, and the tube containing the MTT solution must be covered with aluminum foil.</li>
	<li>Incubate the plate at 37 &#176;C in a 5% CO2&#160;humidified incubator for 3-4 h until a change in the color of the solution in the wells (purple color represents live cells) is observed.</li>
	<li>When a change is observed, add 100 &#956;L of stop solution (0.1N HCl in isopropanol) to each well, and gently mix the content of the wells without creating bubbles.</li>
	<li>Read the absorbance of the plate in a Fluorometer-ELISA reader (see the Table of Materials) at a wavelength of 570 nm and a background wavelength of 630-690 nM. 
	NOTE: If the available Fluorometer-ELISA reader cannot read the ultra-low attachment 96-well plate, transfer the content of each well to a corresponding flat-bottom 96-well plate.</li>
	<li>Calculate the cell viability following the steps below.
	<ol>
		<li>For each well, calculate the &#34;specific signal&#34; (&#34;specific signal&#34; = the signal at 570 nm - the signal at 630-690 nm). Then, calculate the average value of the &#34;Blank&#34; wells, and subtract this value from each well.</li>
		<li>Calculate the average of the &#34;specific signals&#34; in the control wells that contain cells that were not treated with the study drug (&#34;AV-SS-unt&#34;).</li>
		<li>Calculate the viability (percentage) of cells in each well relative to the wells with untreated cells. 
		NOTE: Viability = (specific signal in each well/&#34;AV-SS-unt&#34;) &#215; 100</li>
	</ol>
	</li>
</ol>
3. Monitoring and analyzing the morphological changes in the spheroids
NOTE: As for the MTT assay, only the cell culture medium and not the &#34;3D culture medium&#34; should be used in this evaluation (adding the basement membrane matrix is not necessary and could potentially interfere with the analysis).
<ol>
	<li>After counting the spheroids, dilute the suspension in cell culture medium (RPMI-1640 culture medium supplemented with 10% FBS, 1:100 penicillin-streptomycin, 1% non-essential amino acids, and 1% L-glutamine) to approximately 1-2 spheroids per 20 &#956;L.</li>
	<li>Put 80 &#956;L of cell culture medium into the wells of an ultra-low attachment 96-well plate, and then add 20 &#956;L of the spheroid suspension to the wells. 
	NOTE: The wells will, therefore, contain 1-2 spheroids in a volume of 100 &#956;L.</li>
	<li>Carefully check the wells under the microscope, and mark the wells that contain one spheroid, as they will be used for this analysis.</li>
	<li>Prepare the study drug in a concentration that is two-fold the concentration of interest. Add 100 &#956;L of the drug solution to the relevant wells (the total concentration in the well will then be 1x).</li>
	<li>Capture an image of each well at Day 0 (before adding the study drug) and use the &#34;scale&#34; tool in the imaging software (see the Table of Materials) to determine the diameters of the spheroids.</li>
	<li>Incubate the plate at 37 &#176;C in a 5% CO2&#160;humidified incubator, and examine the morphology and measure the diameter of the spheroids (using the &#34;scale&#34; tool) daily for 3-7 days, depending on when the drug effect is observed (e.g., cell dissemination, invasive pods, structure destruction, etc.).</li>
	<li>At the end of the experiment, plot the changes in spheroid diameters (relative to Day 0) over time. 
	NOTE: Change (percentage) = (spheroid diameter at a specific day/the diameter of that spheroid at Day 0) &#215; 100</li>
</ol>

<ol>
	<li>Katt, M. E., Placone, A. L., Wong, A. D., Xu, Z. S., Searson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+tumor+models:+Advantages,+disadvantages,+variables,+and+selecting+the+right+platform.">In vitro tumor models: Advantages, disadvantages, variables, and selecting the right platform.</a> Frontiers in Bioengineering and Biotechnology. 4, 12 (2016).</li><li>Yoshida, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+patient-derived+tumor+xenograft+models+and+tumor+organoids.">Applications of patient-derived tumor xenograft models and tumor organoids.</a> Journal of Hematology &amp; Oncology. 13 (1), 4 (2020).</li><li>Ledur, P. F., Onzi, G. R., Zong, H., Lenz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+conditions+defining+glioblastoma+cells+behavior:+What+is+the+impact+for+novel+discoveries.">Culture conditions defining glioblastoma cells behavior: What is the impact for novel discoveries.</a> Oncotarget. 8 (40), 69185-69197 (2017).</li><li>Richter, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+donor+to+the+lab:+A+fascinating+journey+of+primary+cell+lines.">From donor to the lab: A fascinating journey of primary cell lines.</a> Frontiers in Cell and Developmental Biology. 9, 711381 (2021).</li><li>Esparza-Lopez, J., Martinez-Aguilar, J. F., Ibarra-Sanchez, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deriving+primary+cancer+cell+cultures+for+personalized+therapy.">Deriving primary cancer cell cultures for personalized therapy.</a> Revista de Investigacio&#769;n Cli&#769;nica. 71 (6), 369-380 (2019).</li><li>Choi, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+human+cancer+models+for+biomedical+applications.">In vitro human cancer models for biomedical applications.</a> Cancers. 14 (9), 2284 (2022).</li><li>Eglen, R. M., Randle, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+discovery+goes+three-dimensional:+Goodbye+to+flat+high-throughput+screening.">Drug discovery goes three-dimensional: Goodbye to flat high-throughput screening.</a> Assay and Drug Development Technologies. 13 (5), 262-265 (2015).</li><li>Kodack, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+patient-derived+cancer+cells+and+their+potential+for+personalized+cancer+patient+care.">Primary patient-derived cancer cells and their potential for personalized cancer patient care.</a> Cell Reports. 21 (11), 3298-3309 (2017).</li><li>Moskovits, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palbociclib+in+combination+with+sunitinib+exerts+a+synergistic+anti-cancer+effect+in+patient-derived+xenograft+models+of+various+human+cancers+types.">Palbociclib in combination with sunitinib exerts a synergistic anti-cancer effect in patient-derived xenograft models of various human cancers types.</a> Cancer Letters. 536, 215665 (2022).</li><li>Ricardo, R., Phelan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+and+determining+the+viability+of+cultured+cells.">Counting and determining the viability of cultured cells.</a> Journal of Visualized Experiments. (16), e752 (2008).</li><li>Braj&#353;a, K., Trzun, M., Zlatar, I., Jeli&#263;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+cultures+as+a+new+tool+in+drug+discovery.">Three-dimensional cell cultures as a new tool in drug discovery.</a> Periodicum Biologorum. 118 (1), 59-65 (2016).</li><li>Han, S. J., Kwon, S., Kim, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+of+applying+multicellular+tumor+spheroids+in+preclinical+phase.">Challenges of applying multicellular tumor spheroids in preclinical phase.</a> Cancer Cell International. 21 (1), 152 (2021).</li><li>van Meerloo, J., Kaspers, G. J., Cloos, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+sensitivity+assays:+The+MTT+assay.">Cell sensitivity assays: The MTT assay.</a> Methods in Molecular Biology. 731, 237-245 (2011).</li><li>Walzl, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+resazurin+reduction+assay+can+distinguish+cytotoxic+from+cytostatic+compounds+in+spheroid+screening+assays.">The resazurin reduction assay can distinguish cytotoxic from cytostatic compounds in spheroid screening assays.</a> Journal of Biomolecular Screening. 19 (7), 1047-1059 (2014).</li></ol>

The authors have nothing to disclose.

The present protocol describes a simple method for generating 3D primary cell cultures (spheroids) derived from human tumor samples. These spheroids can be used for various analyses, including evaluating potential drug candidates and drug combinations, identifying potential biomarkers or therapeutic targets, and investigating the mechanisms of response and resistance. The protocol uses either primary tumor cells derived directly from patient samples or tumor cells from PDX models, which can be established using patient s...

In vitro and in vivo studies represent complementary approaches for developing cancer treatments. In vitro models allow for the control of most experimental variables and facilitate quantitative analyses. They often serve as low-cost screening platforms and can also be used for mechanistic studies1. However, their biological relevance is inherently limited, as such models only partially reflect the tumor microenvironment1. In contrast, in vivo models, such as patient-derived xenografts (PDX), capture the complexity of the tumor microenvironment and are more suitable for translational studies and individualizing treatment in patients (i.e., investigating the response to drugs in a model derived from an individual patient)1. However, in vivo models are not conducive to high-throughput approaches for drug screening, as the experimental parameters cannot be controlled as tightly as in in vitro models and because their development is time-consuming, labor intensive, and costly1,2.
In vitro models have been available for over 100 years, and cell lines have been available for over 70 years3. During the last several decades, however, the complexity of the available in vitro models of solid tumors has increased dramatically. This complexity ranges from 2-dimensional (2D) culture models (monolayer structures) that are either tumor-derived established cell lines or primary cell lines to the more recent approaches involving 3-dimensional (3D) models1. Within the 2D models, a key distinction is between the established and primary cell lines4. Established cell lines are immortalized; therefore, the same cell line can be used globally over many years, which from a historical perspective, facilitates collaboration, the accumulation of data, and the development of many treatment strategies. However, genetic aberrations in these cell lines accumulate with every passage, thus compromising their biological relevance. Furthermore, the limited number of available cell lines does not reflect the heterogeneity of tumors in patients4,5. Primary cancer cell lines are derived directly from resected tumor samples obtained via biopsies, pleural effusions, or resections. Therefore, primary cancer cell lines are more biologically relevant as they preserve elements of the tumor microenvironment and tumor characteristics, such as intercellular behaviors (e.g., cross-talk between healthy and cancerous cells) and the stem-like phenotypes of cancer cells. However, the replicative capacity of primary cell lines is limited, which leads to a narrow culture time and limits the number of tumor cells that can be used for analyses4,5.
Models using 3D cultures are more biologically relevant than 2D culture models since the in vivo conditions are retained. Thus, 3D culture models preserve the natural cell shape and growth and replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion. The most commonly used 3D models in cancer research include multicellular spheroids, scaffold-based structures, and matrix-embedded cultures4,6,7.
The present protocol generates 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluates their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. The representative results presented herein are from breast and colon cancer; however, this protocol is widely applicable to other solid tumor types (e.g., cholangiocarcinoma, gastric, lung, and pancreatic cancer) 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. The multicellular spheroids generated using this approach can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.
This protocol is divided into three sections: (1) the generation, collection, and counting of the spheroids in preparation for their use as a model for testing drug efficacy; (2) MTT assay to assess drug efficacy on the spheroids; and (3) the microscopic evaluation of morphological changes following the treatment of the spheroids with drugs as another approach for evaluating drug efficacy (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5 Fluorouracil</td>
			<td>TEVA Israel</td>
			<td>lot 16c22NA</td>
			<td>Fluorouracil, Adrucil</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Gibco</td>
			<td>A1110501</td>
			<td>StemPro Accutase Cell Dissociation</td>
		</tr>
		<tr>
			<td>Cisplatin</td>
			<td>TEVA Israel</td>
			<td>20B06LA</td>
			<td>Abiplatin,&#160;</td>
		</tr>
		<tr>
			<td>Cultrex&#160;</td>
			<td>Trevigen</td>
			<td>3632-010-02</td>
			<td>Basement membrane matrix, type 3</td>
		</tr>
		<tr>
			<td>DMSO (dimethyl sulfoxide)</td>
			<td>Sigma Aldrich</td>
			<td>D2650-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>2391595</td>
			<td></td>
		</tr>
		<tr>
			<td>Flurometer&#160;ELISA reader</td>
			<td>Biotek</td>
			<td>Synergy H1</td>
			<td>Gen5 3.11</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)&#160;</td>
			<td>Sigma Aldrich</td>
			<td>320331</td>
			<td>for stop solution</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health, Bethesda, MD, USA</td>
			<td>&#160;Version 1.52a</td>
			<td>Open-source software ImageJ</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Gadot</td>
			<td>P180008215</td>
			<td>for stop solution</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>1843977</td>
			<td></td>
		</tr>
		<tr>
			<td>MTT&#160;</td>
			<td>Sigma Aldrich</td>
			<td>M5655-1G</td>
			<td>3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide</td>
		</tr>
		<tr>
			<td>Non-essential amino acids&#160;</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Palbociclib&#160;&#160;</td>
			<td>Med Chem Express</td>
			<td>CAS # 571190-30-2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190094</td>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (DPBS)*Without Calcium and Magnesium</td>
		</tr>
		<tr>
			<td>Penicillin&#8211;streptomycin&#160;</td>
			<td>Invitrogen</td>
			<td>2119399</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol-free RPMI 1640</td>
			<td>Biological industries, Israel</td>
			<td>01-103-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippeting reservoir</td>
			<td>Alexred</td>
			<td>RED LTT012025</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 culture medium&#160;</td>
			<td>Gibco</td>
			<td>11530586</td>
			<td></td>
		</tr>
		<tr>
			<td>Sunitinib</td>
			<td>Med Chem Express</td>
			<td>CAS # 341031-54-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Trastuzumab</td>
			<td>F. Hoffmann - La Roche Ltd, Basel, Switherland</td>
			<td>10172154 IL</td>
			<td>Herceptin</td>
		</tr>
		<tr>
			<td>Trypan blue 0.5% solution</td>
			<td>Biological industries, Israel</td>
			<td>03-102-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-low attachment 96 well plate</td>
			<td>Greiner Bio-one</td>
			<td>650970</td>
			<td></td>
		</tr>
		<tr>
			<td>Vinorelbine</td>
			<td>Ebewe</td>
			<td>11733027-03</td>
			<td>Navelbine</td>
		</tr>
	</tbody>
</table>

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.

This protocol presents procedures for generating a homogenous culture of spheroids from primary tumor cells, quantitatively evaluating drug efficacy on spheroid culture (MTT assay), and determining the effect of study drugs on spheroid morphology. Data from the representative experiments in spheroids generated from colon and breast cancer cell cultures are presented. Similar experiments were performed using other tumor types, including cholangiocarcinoma, gastric, lung, and pancreatic cancer (data not shown). All the exp...

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

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 ...

establishing-3-dimensional-spheroids-from-patient-derived-tumor

Research

JoVE Journal

Cancer Research

2.0K Views.  Felsenstein Medical Research Center. 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. 

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

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

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 ...

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited.
We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2R&#947;null (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.

Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

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.

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 ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

A highly accurate in vitro high-throughput assay system was developed to evaluate anticancer drugs using patient-derived tumor organoids (PDOs), similar to cancer tissues but are unsuitable for in vitro high-throughput assay systems with 96-well and 384-well plates.

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...