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

Summary

The protocol describes a method for creating a patient-derived colorectal cancer (CRC) organoid model co-cultured with tumor-infiltrating lymphocytes (TILs) to study their interactions and therapeutic potential. This model provides a preclinical platform to explore immune responses in the tumor microenvironment and to predict the efficacy of TIL-based therapy for personalized CRC treatment.

Abstract

Colorectal cancer (CRC) is the third most common cancer worldwide. Tumor-infiltrating lymphocytes (TILs) have been identified as an important prognostic marker in CRC. The therapeutic application of TILs has already shown promising results in melanoma and cervical cancer. However, their use in CRC therapy remains in an exploratory phase. A suitable in vitro model to evaluate TIL efficacy is currently unavailable, hindering further advancements in this field. Patient-derived organoid (PDO) models, which closely retain the characteristics of the original tumor tissue and reflect inter-patient heterogeneity, provide an excellent platform for studying the interaction between CRC and TILs. In this study, a method is described to establish a patient-derived CRC organoid model from freshly resected tumor tissue, followed by isolation and expansion of TILs. This system allows co-culture of CRC organoids and TILs, enabling the assessment of TIL-mediated cytotoxicity and immune responses. By analyzing TIL killing efficacy on organoids, the potential outcomes of TIL-based immunotherapy for personalized CRC treatment can be predicted. Moreover, further engineering of TILs may enhance their anti-tumor efficacy, offering a promising strategy for developing more effective cellular therapies. This PDO-TIL co-culture model provides a powerful tool for preclinical evaluation of TIL therapies and personalized treatment strategies in CRC.

Introduction

Colorectal cancer (CRC) is one of the most prevalent and lethal cancers globally, representing a major public health challenge¹. Despite advances in treatment modalities, including surgery, chemotherapy, and immunotherapy, the prognosis for CRC patients-especially those with metastatic disease-remains poor. The tumor microenvironment (TME) plays a vital role in shaping cancer progression and treatment responses, with tumor-infiltrating lymphocytes (TILs) emerging as a key component influencing the immune response against cancer². TILs, a diverse population of immune cells found within tumors, have been identified as significant prognostic markers in CRC, with high levels of TIL infiltration often associated with better clinical outcomes³. Recent studies have also highlighted the therapeutic potential of TILs in other cancers, such as melanoma⁴ and cervical cancer⁵, where TIL-based immunotherapy has shown promising efficacy.

However, despite the success of TILs in some cancer types, their application in CRC remains an area of ongoing investigation. The complexity and heterogeneity of CRC, coupled with the immunosuppressive tumor microenvironment, present challenges for effective TIL-based immunotherapy⁶. CRC tumors often exhibit a highly immunosuppressive TME⁷, characterized by the presence of regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs), all of which actively inhibit TIL function. Moreover, CRC cells frequently express immune checkpoint molecules, such as PD-L1, that can further hinder TIL-mediated killing⁸. Additionally, the genetic heterogeneity of CRC tumors, coupled with an immunosuppressive stroma, complicates the ability of TILs to effectively target and eliminate tumor cells, making it more difficult to achieve the same level of success seen in melanoma and cervical cancer⁹. A critical gap in current research is the lack of suitable in vitro models that can accurately simulate the interactions between CRC and TILs. Such models are essential for evaluating the potential of TILs in CRC therapy and optimizing their application for personalized treatment strategies.

Patient-derived organoids (PDOs) have emerged as a powerful tool for modeling human cancers¹⁰. These 3D cultures, derived from patient tumor tissues, closely mimic the histological and genetic characteristics of the original tumors, providing a more accurate representation of tumor biology compared to traditional 2D cell lines. PDO models also retain inter-patient heterogeneity, making them ideal for personalized medicine approaches¹¹. In CRC, PDOs have been used for drug screening and studying tumor biology¹², but their use in evaluating immune responses-particularly in the context of TILs-remains limited. The design of a PDO-immune cell co-culture model must balance the complexity of the microenvironment with experimental controllability. Although components such as macrophages and dendritic cells can enhance biomimicry, peripheral blood mononuclear cells (PBMCs) have become the mainstream choice due to their accessibility. However, PBMCs have a low proportion of tumor-specific T cells, and the monocyte subpopulations may interfere with microenvironmental stability¹³. In contrast, TILs naturally enrich tumor-reactive clones and carry an in situ exhaustion phenotype, making them a superior model for precisely analyzing immune evasion mechanisms in colorectal cancer.

The aim of this study is to develop a novel in vitro platform to assess TIL-mediated anti-tumor responses in CRC by establishing a co-culture system of CRC PDOs and TILs. This model enables direct evaluation of TIL cytotoxicity against CRC PDOs and analysis of immune responses within a patient-specific context. Utilizing this system may provide deeper insights into the potential of TIL-based immunotherapies for CRC and support the exploration of strategies to enhance TIL efficacy through engineering approaches. Previous methods were limited by the small number of TILs¹⁴. In most cases, PBMCs were first co-cultured with PDOs for two weeks, after which tumor-specific TILs could be expanded from the PBMCs and then further co-cultured with PDOs to observe cytotoxic effects¹⁵. However, this approach also suffers from extended modeling time and low overall yield. In contrast, the described method allows for the isolation and establishment of PDOs and TILs from smaller tumor tissue samples, which can then be directly co-cultured. This approach may offer greater convenience in terms of both methodology and efficiency. Ultimately, this system aims to provide a powerful tool for the preclinical evaluation of TIL therapies, paving the way for personalized treatment strategies that may improve outcomes for CRC patients.

Protocol

All experiments involving human-derived materials were conducted in accordance with the Declaration of Helsinki and were approved in advance by the Ethics Committee of Zhongshan Hospital affiliated with Dalian University (approval numbers: KY2023-206-1). Written informed consent was obtained from all patients who provided clinical specimens for this study. The reagents and equipment used in this study are listed in the Table of Materials.

Inclusion criteria included pathologically confirmed primary colorectal cancer tissue, a sufficient sample size (≥1 cm³), no history of neoadjuvant therapy (chemoradiation or targeted therapy), and signed informed consent. Exclusion criteria comprised poor sample quality with necrosis or fibrosis exceeding 50%, the presence of concurrent severe infection or autoimmune disease, a history of other malignancies, and patient refusal to participate.

1. Cell culture reagent preparation

1. Culture medium for CRC PDOs: Prepare CRC PDOs Complete Medium according to the composition described in Table 1.
2. Complete medium (CM) for TILs¹⁶: Supplement RPMI1640 medium with 10% FBS, 1% penicillin-streptomycin, HEPES, 2-mercaptoethanol, and 3000 IU/mL IL-2.
3. TILs expansion medium (EM): Supplement RPMI1640 medium with 10% FBS, 1% penicillin-streptomycin, HEPES, and 600 IU/mL IL-2.
4. AdDMEM⁺⁺⁺⁺: Supplement Advanced DMEM with 1% penicillin-streptomycin, 1% HEPES, 1% glutamine, and 5 mM Y-27632.
5. CRC digestion buffer: Add 50 µL collagenase type IV and 10 µL Penicillin-Streptomycin-Amphotericin B Solution to AdDMEM⁺⁺⁺⁺.
6. Tissue washing solution: Add 2% penicillin-streptomycin to PBS.

2. Patient-derived tissue processing

1. Resect the primary colorectal cancer tumor tissue ( to be performed by experienced surgeons). After collection, divide the tissue into two parts. Place one part in AdDMEM⁺⁺⁺⁺ in an ice box and transport it to the laboratory for tumor cell dissociation and culture. Place the other part in 4% paraformaldehyde for histopathological analysis¹⁷.
2. Thaw Matrigel slowly in a 4 °C refrigerator. Pre-warm erythrocyte lysis buffer to room temperature. Prepare tissue scissors, forceps, AdDMEM⁺⁺⁺⁺ medium, and CRC digestion buffer.
3. Transfer the tissue to a petri dish and rinse three times with 5 mL PBS. Replace with a new dish and rinse three times with 5 mL tissue wash buffer to minimize contamination.
4. Mince the tissue with scissors, transfer it to the digestion buffer, and incubate in a 37 °C water bath for 30 min until complete dissociation.
5. Cut the tissue with tissue scissors and transfer it to the tissue digestion solution. Digest the tissue in a 37 °C water bath and monitor until tissue dissociation is complete.
6. Add an equal volume of AdDMEM⁺⁺⁺⁺ to neutralize digestion. Centrifuge at 380 × g for 5 min at 25 °C.
7. Discard the supernatant using a pipette. Observe the number of erythrocytes in the pellet. Add 1-3 mL erythrocyte lysate and incubate for 10 min at 25 °C.
   1. Terminate lysis with an equal volume of AdDMEM⁺⁺⁺⁺. Centrifuge at 380 × g for 5 min at approximately 25 °C.
8. Discard the supernatant using a pipette and resuspend the cell pellet in an appropriate volume of AdDMEM⁺⁺⁺⁺.
9. Quantify viable cells using trypan blue exclusion to obtain a single-cell suspension of tumor tissue.

3. PDO model establishment and TILs culture

1. Divide the single-cell suspension of tumor tissue into two portions: use one for PDO model establishment and the other for TILs culture.
2. After cell counting, mix 80,000 cells per well of the cell suspension with Matrigel at a 1:1 ratio for PDO model establishment.
3. Resuspend the remaining cell suspension in CM in a 24-well plate at a concentration of 1 × 10⁶ cells/mL for TILs culture.
4. Observe daily. Take photographs to record the status of PDOs and TILs. Change the medium every 2 days.

4. TILs purification and expansion

1. Culture TILs for 10-20 days before purification.
2. Collect TILs into a centrifuge tube. Rinse the well plate with fresh medium and collect any remaining cells from the well plate.
3. Centrifuge at 125 × g for 5 min at approximately 25 °C.
4. Discard the supernatant using a pipette. Wash with 2 mL PBS and centrifuge again at 125 × g for 5 min at approximately 25 °C.
5. Perform cell counting.
6. Use CD3⁺ magnetic beads for sorting to isolate CD3⁺T cells¹⁸.
7. Incubate the antibody with the cell suspension at 4 °C for 10-15 min.
8. Incubate the magnetic beads with the antibody-cell suspension at 4 °C for 10-15 min.
9. Place the sorting column into the sorting magnet. Add the cell suspension to the column and allow only the magnetic bead-labeled positive cells to be retained in the sorting column.
10. Remove the sorting column from the magnetic field. Add buffer solution to elute the positive cells retained in the column. Resuspend the cells in 2 mL of PBS and centrifuge at 195 × g for 5 min at approximately 25 °C.
11. Discard the supernatant using a pipette. Resuspend the cells in a 12-well plate with fresh EM medium at a concentration of 1 × 10⁶ cells/mL. Add 5 µg/mL CD3 antibody. Passage the cells into new culture flasks every 3 days.

5. Histology and IHC characterization of PDOs

1. Aspirate the culture medium after the organoids have grown to a size of 50 µm.
2. Fix the organoid spheres directly in 4% paraformaldehyde. Aspirate the organoids, centrifuge at 195 × g for 5 min at approximately 25 °C, embed in 2% agarose, allow to cure, and transfer to 4% paraformaldehyde for overnight fixation.
3. On the following day, perform gradient dehydration. Prepare wax blocks, cut 4 µm sections, rehydrate using a gradient, and perform immunohistochemical staining using CK7, CK20, Ki-67, and CDX-2 antibodies.

6. IF characterization of TILs

1. Purify the TILs, culture them until an adequate number is reached, and extract a portion for characterization.
2. Characterize the suspension cells using the flaking method. Set the rotational speed of the flaker to 343 × g for 4 min at approximately 25 °C.
3. Count and seed the cells at 1 × 10⁴ per slide.
4. Attach the cells firmly to the slides by centrifugation.
5. Fix the cells with 4% paraformaldehyde for 15 min and wash three times with PBS.
6. Incubate with blocking solution (PBS containing 2% FBS) for 1 h.
7. Incubate with primary antibodies (CD45, CD3, EpCAM) for 1 h at room temperature. Wash three times with PBS.
8. Prepare and incubate with the secondary antibody according to the manufacturer's recommended concentration for 1 h at room temperature. Wash three times with PBS.
   NOTE: Pay attention to antibody homology.
9. Incubate with DAPI for 15 min at approximately 25 °C.
10. Observe the stained cells under a fluorescence microscope.

7. PDOs and TILs co-culture system

1. Add IFN-γ to the organoid medium to enhance antigen presentation. Incubate at 37 °C with 5% CO₂ for 24 h¹⁵.
2. Dilute the anti-CD28 antibody with PBS. Coat a 96-well plate by adding 50 µL of the antibody solution to each well. Seal the plate with sealing film and incubate at 4 °C for 24 h.
3. After 24 h, dissociate the organoids into single cells using TrypLE. Count the cells and wash twice with PBS.
4. Collect the paired TILs, count the cells, and wash twice with PBS.
5. Inoculate organoids at 1 × 10⁴ cells per well and inoculate TILs at 1 × 10⁵ cells per well to achieve an effector-to-target (E:T) ratio of 10:1 for co-culture.
6. Perform co-culture in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin, 3000 U/mL IL-2, and 20 µg/mL anti-PD-1 blocking antibody. Incubate for 3 days and capture images for observation and documentation.
   NOTE: Set up organoids without TILs as a control group.

8. Functional assay of TILs by flow cytometry

1. Collect the PDOs and TILs at the end of the co-culture period.
2. Wash the cells twice with PBS by centrifugation at 300 × g for 5 min each time. Discard the supernatant using a pipette.
3. Prepare the antibody cocktail containing CD3-FITC, CD4-APC/Cyanine7, CD8-APC, CD107a-PE-Cyanine7, CD279-PE, and 7-AAD.
4. Resuspend the cells in 100 µL of the antibody cocktail and incubate at room temperature for 20 min, protected from light.
5. Add 1 mL PBS to wash the cells. Centrifuge at 300 × g for 5 min and discard the supernatant using a pipette.
6. Resuspend the cells in 500 µL PBS.
7. Perform flow cytometry analysis. Refer to Supplementary Figure 1 for the detailed analysis strategy.

Results

Establishment and characterization of CRC PDOs
Tumor specimens were obtained from CRC patients. Freshly resected CRC tumor tissue was used to establish PDOs and TILs, which were subsequently employed in PDO-TIL co-culture experiments (Figure 1).

Discussion

This study utilizes patient-derived CRC tissue to obtain both tumor cells and TILs, from which PDOs are established and TILs are expanded. The interaction between these two components is then investigated through a co-culture system.

In previous studies, TILs were extracted using complex instrumentation¹⁹ or directly from tumor tissue suspensions²⁰. However, these methods often fail to generate a sufficient number of TILs within a short time fram...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Advanced DMEM/F-12	Gibco	12634028
2-Mercaptoethanol	sigma	63689
4% paraformaldehyde	BBI	E672002-0100
7-AAD	Biolegend	420403
A83-01	Tocris	2939
B27 supplement	Gibco	17504-44
CD107a-PE-Cyanine7	Biolegend	328618
CD279-PE	Biolegend	329906
CD3 Monoclonal Antibody (OKT3)	Invitrogen	14-0037-82
CD3-FITC	Biolegend	300406
CD45 (Intracellular Domain) (D9M8I)	CST	13917S
CD4-APC/Cyanine7	Biolegend	300518
CD8-APC	Biolegend	344722
CDX-2	MXB	RMA-0631
CK20	MXB	Kit-0025
CK7	MXB	Kit-0021
Dylight 488, Goat Anti-Rabbit IgG	Abbkine	A23220
Dylight 549, Goat Anti-Mouse IgG	Abbkine	A23310
EGF	Peprotech	AF-100-15
EpCAM	CST	36746T
Fetal Bovine Serum (FBS)	Cellmax	SA211.02
FGF 10	Peprotech	100-26
Gastrin (Human)	Tocris	3006
GlutaMax 100x	Gibco	35050-061
Hepes	Sigma	H4034
HEPES	Sigma	H4034
Human CD3+ Cell Separation Kit (RUO)	RWD	K1201-10
Human IFN-gamma Recombinant Protein	PeproTech	300-02-20UG
Ki-67	CST	9449s
Matrigel	corning	354234
N-Acetylcysteine	Sigma	A9165
Nicotinamide	Sigma	N0636
Nivolumab (anti-PD-1)	Sellerk	A2002
Penicillin/Streptomycin	Sigma	V900929
penicillin-streptomycin	NCM Biotech	C100C5
Primocin	Invivogen	Ant-pm-1
RBC Lysis Buffer	thermo-invitrogen	00-4300-54
Recombinant Human IL-2 (carrier-free)	Biolegend	589104
RPMI 1640	Gibco
SB202190	Sellerk	S1077
Trypan Blue stain 0.4%	thermo-invitrogen	T10282
Ultra-LEAF Purified anti-human CD28 Antibody	Biolegend	302934
Ultra-LEAF Purified anti-human CD3 Antibody	Biolegend	317325
Y-27632	Sellerk	S1049