Patient-Derived Three-Dimensional Tumor Organoid Model for In Vitro Chimeric Antigen Receptor T Cell Screening

Summary

This protocol establishes an in vitro co-culture model using patient-derived tumor organoids and chimeric antigen receptor T (CAR-T) cells to evaluate the specific cytotoxic activity of CAR-T cells against solid tumor cells.

Abstract

Chimeric antigen receptor T (CAR-T) cell therapy has achieved exciting clinical efficacy in hematological malignancies, but CAR-T cell therapy for solid tumors still requires further development. Patient-derived tumor organoids are in vitro disease models that retain patient heterogeneity and have been used to test the efficacy and safety of chemotherapy and targeted drugs. This method describes an in vitro efficacy testing model of co-culturing tumor organoids with CAR-T cells. Colorectal cancer samples from patients are constructed into tumor organoids with a three-dimensional (3D) structure in a basement membrane matrix. The tumor organoids can grow stably and be passaged continuously. Mature tumor organoids are separated from the matrix by washing and centrifugation, and CAR-T cells are added at different effector-to-target (E:T) ratios to form an immune-organoid co-culture system. After 6-24 h, the morphology and intercellular structures of the organoids are observed by bright-field imaging. This experiment further performs dead cell staining in the co-culture system, which can reflect the viability of the organoids and evaluate the cytotoxic effect of CAR-T cells on tumor organoids. This experiment can effectively observe the interaction between CAR-T cells and 3D tumor organoids, and the results can be used to assess the tumor-killing activity of CAR-T cells.

Introduction

Chimeric antigen receptor T (CAR-T) cell therapy has shown remarkable clinical success in treating hematological malignancies¹. However, its application in solid tumors remains limited due to several major obstacles, including tumor heterogeneity and antigen loss, physical and chemical barriers within the tumor mass, and the immunosuppressive tumor microenvironment^2,3. These challenges contribute to the limited efficacy of CAR-T cell therapy in solid tumor settings.

In vitro evaluation of CAR-T cell efficacy is a critical step during preclinical deve....

Protocol

Colorectal cancer patient-derived organoids were used. Tumor tissue fragments were obtained from treatment-naïve patients with histologically confirmed primary colorectal adenocarcinoma (CRC). All tissues were procured from endoscopic biopsy specimens collected during standard diagnostic procedures at Shenzhen Qianhai Shekou Free Trade Zone Hospital. All donor tissues were confirmed as CRC through histopathological examination by institutional pathologists. The protocol follows institutional guidelines. Written informed consents were obtained from all patients. The study was approved by the Shenzhen Qianhai Shekou Free Trade Zone Hospital Ethical Commit....

Results

Colorectal cancer organoids were successfully derived from endoscopic biopsy samples (Figure 1), with tumor identity confirmed by pathological diagnosis (Figure 2). The tumor fragments cultured in BMM began forming 3D structures within 24 h. First-generation organoids (P0) were passaged between days 7-14. Organoids exhibited diverse morphologies, including spherical, cystic, and circular structures (Figure 2A).

Discussion

This article presents a method for evaluating the cytotoxic effect of CAR-T cells on PDTOs, combining bright-field imaging and fluorescent staining to assess organoid viability. This dual-modality approach enables robust visualization of tumor cell death in a 3D model system and provides a semi-quantitative assessment of CAR-T cytotoxicity.

A critical component of this protocol is the culture medium. Because CAR-T cells and tumor organoids have distinct nutrient requirements, the compatibility.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15-mL tube	SAINING	3030100
24-well plate	CORNING	3524
50-mL tube	SAINING	3030000
96-well plate	CORNING	3599
Advanced DMEM/F12	Gibco	C11330500BT
Alk 4/5/7 inhibitor	MCE	HY-10432
B27	Gibco	17504044
Biosafety Cabinet (Class II)	Thermo Fisher	N/A
Bovine Serum Albumin	Sigma Aldrich	A9056
Cell culture dish	BIOFIL	TCD010060
Centrifuge	Eppendorf	5810R
Click's Medium	FUJI FILM	9195
CO2 Incubator	Yamata	IP610
Collagenase II	Solarbio	C8150
Collagenase IV	Sigma Aldrich	C9407
Confocal laser microscope	Zeiss	LSM900
Cryopreservative medium	ZENOAQ	CELLBANKERTM2
Digestion buffer	Reagents prepared in-house	N/A	Composition provided in the manuscript
Digital Shaker	MIULAB	HS-25
Dispase type II	Sigma-aldrich	D4693
DMEM	Gibco	C11995500BT
Dynabeads Human T-Activator CD3/CD28	Thermo Fisher	11131D
EGF	PeproTech	AF-100-15-500UG
Fetal bovine serum	Gibco	1631389
Gastrin	GlpBio	GA20228
Gentamicin/amphoteritin B	Gibco	R0151
Glutamax	Gibco	35050061
Hanks' Balanced Salt Solution	Gibco	C14175500BT
HEPES	Gibco	15630080
Hoechst 33342	Beyotime	C1027
Hyaluronidase	Solarbio	h8030
IL-15	Peprotech	200-15-10UG
IL-2	Novoprotein	CK24-10
IL-7	Peprotech	200-07-10UG
ImageJ	NIH (open-source)	https://imagej.nih.gov/ij/
Inverted  fluorescent microscope	Mshot	MF52-N
Matrigel	Corning	356231
N2	Gibco	17502048
n-Acetylcysteine	Sigma Aldrich	A7250-5G
Niacinamide	Sigma Aldrich	N0636-100G
Noggin	PeproTech	120-10C-20
Normocin	InvivoGen	ant-nr-1
Organoid culture medium	Reagents prepared in-house	N/A	Composition provided in the manuscript
p38 inhibitor	MCE	HY-10295
Penicillin-Streptomy	Gibco	15140122
Prostaglandin E2	MCE	HY-101952
Rho-associated kinase inhibitor	Stem cell	72304
RPMI 1640	HyClone	SH30809.01B
R-spondin 1	PeproTech	120-38-20UG
S1 Pipet Fillers	Thermo Scientific	9501
Serological Pipette	BIOFIL	GSP-010-050
Single-channel pipette	Eppendorf	3123000063
Single-channel pipette	Eppendorf	3123000055
Single-channel pipette	Eppendorf	3123000098
Single-channel pipette	Eppendorf	3123000022
Sterile Disposable Scalpels	Tech-S	SSD10
SYTOX Green	Beyotime	C1181S
TexMACS Medium	Miltenyi Biotec	130-097-196
Thermostat water tank	Bluepard	BWS-10
Trypan Blue	Phygene	PH0519
TrypLE Express	Gibco	12605028
β-mercaptoethanol	Gibco	21985023