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><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,&amp;nbsp;</td></tr><tr><td>Cultrex&amp;nbsp;</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&amp;nbsp;ELISA reader</td><td>Biotek</td><td>Synergy H1</td><td>Gen5 3.11</td></tr><tr><td>Hydrochloric acid (HCl)&amp;nbsp;</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>&amp;nbsp;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&amp;nbsp;</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&amp;nbsp;</td><td>Gibco</td><td>11140050</td><td></td></tr><tr><td>Palbociclib&amp;nbsp;&amp;nbsp;</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&#039;s Phosphate Buffered Saline (DPBS)*Without Calcium and Magnesium</td></tr><tr><td>Penicillin&amp;ndash;streptomycin&amp;nbsp;</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&amp;nbsp;</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 ...

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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

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

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, ...

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 ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

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 ...

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines are&#160;modified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system,&#160;a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assay&#160;provides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

In vitro drug sensitivity screens are important tools for discovering anti-cancer drug combinations. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening for spheroid lines.

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens ...

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...