Name: establishing 3-dimensional spheroids from patient-derived tumor samples and evaluating their sensitivity to drugs
Uploaded: 2022-12-16T13:40:00
Description: Watch this Scientific Journal Video about Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs at JoVE.com

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 c...

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	<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. 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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...

Watch this Scientific Journal Video about Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs at JoVE.com

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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