Live Imaging to Quantify Cellular Radiosensitivity in Patient-Derived Tumor Organoids

Summary

Here, we detail a straightforward live imaging approach for quantifying the sensitivity of patient-derived tumor organoids to ionizing radiation.

Abstract

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, often (but not always) because of differences in the ability of malignant cells to repair oxidative DNA damage as elicited by ionizing rays. Clonogenic assays have been employed for decades to assess the sensitivity of cultured cancer cells to ionizing irradiation, largely because irradiated cancer cells often die in a delayed manner that is difficult to quantify with short-term flow cytometry- or microscopy-assisted techniques. Unfortunately, clonogenic assays cannot be employed as such for more complex tumor models, such as patient-derived tumor organoids (PDTOs). Indeed, irradiating established PDTOs may not necessarily abrogate their growth as multicellular units, unless their stem-like compartment is completely eradicated. Moreover, irradiating PDTO-derived single-cell suspensions may not properly recapitulate the sensitivity of malignant cells to RT in the context of established PDTOs. Here, we detail an adaptation of conventional clonogenic assays that involves exposure of established PDTOs to ionizing radiation, followed by single-cell dissociation, replating in suitable culture conditions and live imaging. Non-irradiated (control) PDTO-derived stem-like cells reform growing PDTOs with a PDTO-specific efficiency, which is negatively influenced by irradiation in a dose-dependent manner. In these conditions, PDTO-forming efficiency and growth rate can be quantified as a measure of radiosensitivity on time-lapse images collected until control PDTOs achieve a predefined space occupancy.

Introduction

External beam radiation therapy (RT) is one of the mainstays of modern oncology, reflecting not only a pronounced anticancer activity associated with a well-defined spectrum of generally manageable side effects¹, but also an exceptionally widespread clinical availability (most cancer centers in developed countries are equipped with modern linear accelerators for external beam RT)². In line with this notion, RT is globally employed with success for both curative purposes, generally in the context of early-stage disease^3,4, and palliative applications, to contain s....

Protocol

The reagents and equipment used in the study are listed in the Table of Materials.

1. Organoid culture

NOTE: TNBC#1 PDTOs were established in our lab based on tumor tissue surgically removed from a patient with triple-negative breast cancer (TNBC) who provided informed consent to participate in a biobanking protocol (IRB21-06023682). After validation by histology and RNA sequencing (RNAseq), TNBC#1 PDTOs are cultured in 66% matrigel d.......

Discussion

Here, we describe an adaptation of conventional clonogenic assays that harnesses breast cancer PDTOs and live imaging to quantify PDTO radiosensitivity based on (1) the persistence of PTDO-forming stem-like cells upon PDTO irradiation in vitro, and (2) the growth rate of the PDTOs these cells (may) generate. Critical steps of this protocol include (1) the establishment of PDTOs to a dome occupancy enabling good viability, (2) PDTO exposure to ionizing irradiation at different doses, including mock irradiated con.......

Materials

Name	Company	Catalog Number	Comments
40 µm mesh filter	Thomas Scientific	1164H35
B27	Invitrogen	17504-044
Cellometer Auto T4 Bright Field Cell Counter	Nexcelom
DMEM F/12	Corning	12634-010
Epidermal Growth Factor hEGF	Peprotech	AF-100-15
EVOS FL Digital Inverted Fluorescence Microscope	Thermo Fisher Scientific	12-563-460
FGF10	Peprotech	100-26
FGF7	Peprotech	100-19
GlutaMax	Invitrogen	35050061
Hepes	Invitrogen	15630-080
IncuCyte software 2021A	Sartorius		version: 2021A
Incucyte SX1	Sartorius		model SX1
Incucyte validated 48 well plate	Corning	3548
Matrigel	Discovery Labware	354230
nAc	Sigma Aldrich	A9165-5G
Nicotinamide	Sigma-Aldrich	N0636
Noggin			Purchased from the Englander Institute for Precision Medicine, Weill Cornell, NY, USA
Non-treated 6 well plate	Cellstar	657 185
NR (Heregulin)	Peprotech	100-03
p38 MAP inhibitor p38i SB202190	Sigma Aldrich	S7067
PBS	Corning	21-040-CV
PenStrep	Invitrogen	15140-122
Primocin	Invivogen	ant-pm-1
Rspondin Media			Purchased from the Englander Institute for Precision Medicine, Weill Cornell, NY, USA
Small Animal Radiation Research Platform (SARRP)	Xstrahl Ltd
TGFbeta Receptor Inhibitor A83-01	Tocris	2939
Trypan blue Stain (0.4%)	Gibco	15250-61
TrypLE	Gibco	112605-028
Y-27632 (RhoKi)	Selleck	S1049