Establishment of a Co-culture System of Patient-Derived Colorectal Tumor Organoids and Tumor-Infiltrating Lymphocytes (TILs)

Zeshuo Feng; Yanjiao Li; Ju Wu; Jiajun Yin; Ruoyu Wang; Shanshan Liang

doi:10.3791/68346

Mise en place d’un système de co-culture d’organoïdes de tumeurs colorectales dérivées de patient...

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Cancer Research

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JoVE Journal Cancer Research

Establishment of a Co-culture System of Patient-Derived Colorectal Tumor Organoids and Tumor-Infiltrating Lymphocytes (TILs)

Mise en place d’un système de co-culture d’organoïdes de tumeurs colorectales dérivées de patients et de lymphocytes infiltrant la tumeur (TIL)

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08:42 min

June 27, 2025

DOI: 10.3791/68346-v

Zeshuo Feng¹, Yanjiao Li¹, Ju Wu², Jiajun Yin², Ruoyu Wang¹, Shanshan Liang¹

¹The Key Laboratory of biomarker high throughput screening and target translation of breast and gastrointestinal tumor,Affiliated Zhongshan Hospital of Dalian University, ²Department of General Surgery,Affiliated Zhongshan Hospital of Dalian University

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Overview

This protocol outlines a method for creating a patient-derived colorectal cancer organoid model co-cultured with tumor-infiltrating lymphocytes (TILs). It aims to study their interactions and the therapeutic potential of TIL-based therapy for personalized colorectal cancer treatment.

Key Study Components

Area of Science

Colorectal cancer research
Immunotherapy
Organoid models

Background

Colorectal cancer is a leading cause of cancer-related deaths.
TILs play a crucial role in the immune response against tumors.
Current therapies often have limited efficacy and high morbidity.
Patient-derived organoid models can better mimic the tumor microenvironment.

Purpose of Study

To explore the interactions between TILs and colorectal cancer organoids.
To evaluate the therapeutic potential of TIL-based therapies.
To develop a preclinical platform for personalized treatment strategies.

Methods Used

Preparation of experimental reagents and lab wear.
Minced colorectal cancer tissue is digested and dissociated.
Co-culture of TILs with organoids to study interactions.
Quantification of viable cells using trypan blue.

Main Results

The protocol successfully isolates TILs for co-culture.
Interactions between TILs and organoids can be studied effectively.
The model provides insights into immune responses in colorectal cancer.
Potential for predicting efficacy of TIL-based therapies.

Conclusions

This organoid model is a valuable tool for colorectal cancer research.
It enhances understanding of TIL interactions in the tumor microenvironment.
The findings may lead to improved personalized treatment options.

Frequently Asked Questions

What are tumor-infiltrating lymphocytes?

Tumor-infiltrating lymphocytes (TILs) are immune cells that have migrated into a tumor and are involved in the body's immune response against cancer.

How are organoids created from colorectal cancer tissue?

Organoids are created by digesting and dissociating colorectal cancer tissue, followed by culturing the cells in a specific growth medium.

What is the significance of co-culturing TILs with organoids?

Co-culturing TILs with organoids allows researchers to study the interactions between immune cells and cancer cells in a controlled environment, mimicking the tumor microenvironment.

What are the potential applications of this research?

This research can lead to better understanding of immune responses in colorectal cancer and help develop personalized therapies based on TIL efficacy.

How does this model improve upon traditional cancer research methods?

This model provides a more accurate representation of the tumor microenvironment compared to traditional 2D cell cultures, allowing for more relevant experimental results.

Le protocole décrit une méthode de création d’un modèle d’organoïde de cancer colorectal (CRC) dérivé d’un patient co-cultivé avec des lymphocytes infiltrant la tumeur (TIL) afin d’étudier leurs interactions et leur potentiel thérapeutique. Ce modèle fournit une plate-forme préclinique pour explorer les réponses immunitaires dans le microenvironnement tumoral et pour prédire l’efficacité de la thérapie basée sur le TIL pour le traitement personnalisé du CCR.

La recherche vise à explorer les interactions des lymphocytes infiltrant la tumeur avec les organoïdes du cancer colorectal, et le potentiel thérapeutique de la thérapie basée sur le TIL pour les traitements personnalisés du cancer colorectal. Notre protocole a simplifié la prévention des lymphocytes infiltrant la tumeur à partir des lymphocytes et permet leur co-culture avec des organoïdes de cancer colorectal, surmontant ainsi une efficacité inférieure et une morbidité courante chez les patients.

[Narrateur] Pour commencer, préparez les réactifs expérimentaux et les vêtements de laboratoire pour l’utilisation. Ensuite, rincez trois fois le tissu tumoral primaire du cancer colorectal avec cinq millilitres de PBS suivi d’un tampon de lavage des tissus. À l’aide de ciseaux à tissus, coupez le tissu en petits fragments. Transférez le tissu haché dans le tampon de digestion CRC. Incuber l’échantillon dans un bain-marie réglé à 37 degrés Celsius pendant 30 minutes jusqu’à ce que le tissu soit complètement dissocié. Ajoutez ensuite un volume égal d’Ad DMEM à l’échantillon digéré pour neutraliser la réaction. Centrifugez la suspension à 380 G pendant cinq minutes à 25 degrés Celsius. Jetez maintenant le surnageant de l’échantillon centrifugé à l’aide d’une pipette. Pipetez un à trois millilitres de tampon de lyse érythrocytaire préchauffé sur la pastille et incubez l’échantillon à 25 degrés Celsius pendant 10 minutes. Ajoutez un volume égal d’Ad DMEM pour terminer la lyse. Centrifugez le tube à 380 G pendant cinq minutes à environ 25 degrés Celsius. Après avoir jeté le surnageant, mettez en suspension la pastille cellulaire dans un volume approprié d’Ad DMEM. Ajoutez du bleu de trypan à la suspension pour quantifier les cellules viables et obtenir une suspension cellulaire unique du tissu tumoral.

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