Medium-Throughput Drug- and Radiotherapy Screening Assay using Patient-Derived Organoids

Summary

We describe detailed protocols to use patient-derived organoids for medium-throughput therapy sensitivity screenings. Therapies tested include chemotherapy, radiotherapy, and chemo-radiotherapy. Adenosine triphosphate levels are used as a functional readout.

Abstract

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a near-native state. When derived from resected or biopsied tumor tissue, organoids closely recapitulate in vivo tumor morphology and can be used to study therapy response in vitro. Biobanks of tumor organoids reflect the vast variety of clinical tumors and patients and therefore hold great promise for preclinical and clinical applications. This paper presents a method for medium-throughput drug screening using head and neck squamous cell carcinoma and colorectal adenocarcinoma organoids. This approach can easily be adopted for use with any tissue-derived organoid model, both normal and diseased. Methods are described for in vitro exposure of organoids to chemo- and radiotherapy (either as single-treatment modality or in combination). Cell survival after 5 days of drug exposure is assessed by measuring adenosine triphosphate (ATP) levels. Drug sensitivity is measured by the half-maximal inhibitory concentration (IC₅₀), area under the curve (AUC), and growth rate (GR) metrics. These parameters can provide insight into whether an organoid culture is deemed sensitive or resistant to a particular treatment.

Introduction

Organoid models established from adult stem cells and grown in a three-dimensional (3D) extracellular matrix (ECM) and a specific growth factor cocktail (also known as HUB Organoids) are gaining traction as preclinical oncological screening platforms. Patient-derived organoid (PDO) cultures can be established from both normal and diseased tissue biopsies within 1-2 weeks and can be expanded for a minimum of 1-2 months up to unlimited timespans. Cryopreservation allows for long-term usage of well-characterized cultures. Unlike traditional two-dimensional cell line models that are clonally derived, PDO models closely recapitulate the original tumor tissue, both phenotypically and genetically, and preserve tumor heterogeneity. Medium-throughput drug screens on PDOs, testing a wide range of therapies, provide a unique platform for personalized medicine.

Previous studies have described the use of organoid models for therapy screening, specifically drugs and radiotherapy, in models established from different types of tumors and show the predictive potential of organoids to guide clinical decision-making^{1,2,3,4,5,6,7,8,9,10,11}. This paper describes the methods of oncological therapy screening using PDOs in a medium-throughput capacity (Figure 1A). This protocol is set up in a 384-well plate format with semi-automation, allowing therapy testing for up to eight organoid models, 16 compounds, and up to eight 384-well plates. Besides compound drug screens, this paper also describes methods to assay radiotherapy sensitivity and sensitization. Moreover, the use of high-throughput robotics to upscale the drug screen to full-automation is discussed. Importantly, organoids from different tissues may require different media and different handling.

Here, a general drug screening assay protocol is described, which may need adaptation depending on the organoid of interest. Starting points and suggestions for optimization are included in the discussion, as well as general recommendations regarding experimental setup and organoid practice. Examples are given using head and neck squamous cell carcinoma (HNSCC) organoids, which typically have a dense morphology, and colorectal cancer (CRC) organoids which can have either a cystic or dense morphology. Please note that primary organoid establishment and expansion culture methods are not covered in this protocol; for basic organoid techniques, the reader should refer to other protocols (e.g.,¹²). This visual protocol will provide insight into the process of medium-throughput drug screening using organoid models.

Protocol

NOTE: Before using this protocol, please ensure that the guidelines of the institution's human research ethics committee are followed. Collection of patient tissue and data described in this protocol has been performed following EUREC guidelines and following European, national and local law. All organoids were derived from consenting patients, and consent can be withdrawn at any time. 1. Prior to screening Confirm the identity of newly established models (e.g., by his…

Representative Results

The aim of this experiment was to examine the sensitivity of HNSCC organoids to chemotherapy and radiotherapy as single agents. We also tested the reproducibility of the results by executing the experiment multiple times with a week's interval, resulting in several biological replicates (experiments 1-3) (Figure 2). Following the protocol, on day 0, HNSCC PDOs were harvested from 6 wells of a 6-well plate and enzymatically and mechanically sheared to single cells (or small organoids &#60…

Discussion

This article and video describe how to perform medium-throughput drug screening using PDOs. This protocol can, with optimization, be adopted to screen organoids derived from different tissue types from those described here. Determining the ideal passage timeframe prior to the screen is important as this will vary for individual organoid cultures and depend on the tissue type. The density and size of organoids seeded per well is an important factor to optimize as faster growing models will require more space within the we…

Materials

Required equipment
384-well bioluminescence platereader; e.g. Tecan Spark 10M plate reader	Tecan
Brightfield microscope with large field of view lens (2.5x)
Digital dispenser; e.g. Tecan D300e	Tecan		Drug dispensing
6 MV photon beam irradiator	Elekta model Synergy, Elekta Sweden
Liquid handler with large nozzle (“standard tube”) cassettes;
e.g. Multidrop Combi Reagent Dispenser	Thermo Scientific
Plastic container with plate holder insert for radiotherapy	Home-made
Spark control method editor software
Standard tissue culturing equipment (LAF cabinet, incubator, centrifuge, waterbath, etc)
Required materials
1.5 mL plastic tubes
15- and 50-mL plastic tubes
5, 10- and 25-mL sterile plastic pipets
6-well cell culture plates
Black 384-well ultra-low-attachment clear-bottom plate; e.g.. Corning 384 flat black	Corning	4588
Breathe-Easy sealing membrane	Merck		pre-cut polyurethane medical-grade membrane with acrylic adhesive
Glasstic slide			10-chambered slide with hemocytometer grid
Multidrop Combi Reagent Dispenser standard tube dispensing cassette	Thermo Scientific
Plugged Pasteur’s pipet of which the tip has been tightened in a flame
Reversible 20/40/70/100 µm filters: PluriStrainer	Pluriselect	e.g. 43-50020-03
Sterile P1000, P200, P20 and P2 pipet tips and low-retention filter tips ( e.g. Sapphire tips)	Greiner	750266
T8 Plus and D4 Plus casettes	HP/Tecan
Required reagents
100 x Glutamax			L-glutamine substitute
1 M HEPES
30% (v/v) Tween-20 diluted in PBS
70% EtOH
Advanced-DMEM/F12	Thermo Scientific	12634-010
CellTiter-Glo 3D cell viability assay	Promega	G9681
Compounds to test screen, including Staurosporin or other positive control
Dispase II	Sigma-Aldrich	D4693
DMSO
ECM for CRC: growth-factor reduced Matrigel, phenol-free	Corning	356231
ECM for HNSCC PDOs: BME, Cultrex RGF Basement membrane extract, Type R1	R&D Systems	3433-005-R1
Expansion growth medium (specific for each organoid type)
Organoid growth factors (specific for each organoid type)
PBS
Pen/Strep (100 U/mL)
ROCK inhibitor: Y-27632	Abmole	M1817
TrypLE
Required Software Packages:
GraphPad Prism
Microsoft Excel