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

Summary

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.

Abstract

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.

Introduction

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 s....

Protocol

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

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Results

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.......

Discussion

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.......

Materials

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