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Method Article

Establishment of Tumor Organoids, Carcinoma-Associated Fibroblasts, and Counterpart Fibroblasts from the Same Esophageal Cancer Patient

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DOI:

10.3791/69548

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April 3rd, 2026

 ,  ,  ,  ,  ,  ,  ,  ,  ,  ,  , 
1Department of Esophageal and Gastroenterological Surgery, Graduate School of Medicine, Juntendo University, 2Department of Molecular Pathogenesis, Graduate School of Medicine, Juntendo University, 3Department of Pathology and Oncology, Graduate School of Medicine, Juntendo University, 4Faculty of Health Sciences, Graduate School of Medicine, Juntendo University

In This Article

Summary

This protocol describes a method for simultaneously isolating cancer organoids, carcinoma-associated fibroblasts (CAFs), and non-tumor-associated fibroblasts (CFs) from the same patient with esophageal squamous cell carcinoma (ESCC). Employment of these patient-matched materials could avoid issues from inter-patient variability and provide an appropriate experimental platform for ESCC research.

Abstract

In esophageal squamous cell carcinoma (ESCC), the acquisition of treatment resistance and tumor recurrence remain major clinical challenges. Carcinoma-associated fibroblasts (CAFs), key components of the tumor microenvironment, are known to influence ESCC progression and therapeutic response. However, many aspects of CAFs’ roles in tumor malignancy remain unknown, presumably due to inter-patient variability in ESCC patients. Various human ESCC cell lines available for research are thought to partially mimic the nature of human carcinoma. They are also propagated in vitro for years and decades in a Petri dish, resulting in adaptation to the culture conditions. Moreover, human ESCC cell lines harbor various genetic and epigenetic alterations distinct among the original patients, resulting in inter-patient variability. Nevertheless, long-term cultured cancer cell lines have been employed in most previous studies for experimentation with patient-mismatched CAFs. Inter-patient variability within each cancer cell line and between cancer cells and CAFs might therefore cause reduced reproducibility and reliability of results. To eliminate inter-patient variability, three distinct cell types were isolated from the same patient: cancer organoids and CAFs from a surgically dissected ESCC region, and counterpart fibroblasts (CFs) from an adjacent non-tumorous region as patient-specific controls for CAFs. A detailed protocol is presented that enables the simultaneous isolation of cancer organoids, CAFs, and CFs. The cancer organoids grew well under three-dimensional conditions. CAFs and CFs stained positively for vimentin, one of the fibroblast markers, supporting the enrichment of these cells in fibroblastic lineages. The described protocol enables us to establish ESCC organoids and patient-matched fibroblasts, providing a valuable experimental platform that excludes inter-patient variability in studying tumor-stromal interactions.

Introduction

Esophageal cancer is associated with high incidence and mortality rates worldwide1. It is broadly classified into two major histological subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC)2. ESCC is more prevalent than EAC, with a particularly higher incidence in East Asia and Africa3. Treatment strategies for ESCC are relatively well established4,5. For early-stage lesions confined to the mucosal layer, endoscopic resection is indicated as a curative and minimally invasive option. In patients with locally advanced but resectable disease, the combination of neoadjuvant chemoradiotherapy followed by esophagectomy has demonstrated clinical efficacy. For unresectable or metastatic cases, systemic chemotherapy remains the mainstay of treatment. Nevertheless, therapeutic resistance and disease recurrence continue to pose major clinical challenges.

Tumors consist not only of cancer cells, but also of a complex tumor microenvironment comprising various stromal cell types. Among them, carcinoma-associated fibroblasts (CAFs), major cellular components of the tumor microenvironment, are shown to play critical roles in tumor progression and therapeutic responsiveness6,7,8,9. In ESCC, the involvement of CAFs in tumor malignancy and treatment resistance has also been increasingly reported10,11,12. However, the precise molecular mechanisms of CAFs underlying ESCC malignancy remain largely unclear.

In most previous studies, the effects of CAFs on cancer cells were evaluated using various ESCC cell lines propagated in Petri dishes for many years, or even decades. Such cell lines would adapt to the culture conditions and bear little resemblance to the original tumors13. Furthermore, ESCC cell lines are derived from different patients and harbor various genetic and epigenetic alterations, which are evidently distinct in respective ESCC cells, serving as inter-patient variabilities. Experimental results from one cancer cell line might often be inconsistent with those from another cancer cell line. Therefore, experiments employing different ESCC cell lines and the patient-mismatched CAFs may result in difficulty in precisely addressing interactions of these cells in reproducible and reliable manners.

To address these limitations, a protocol has been developed to isolate patient-matched tumor organoids, CAFs, and non-tumor counterpart fibroblasts (CFs) simultaneously from the same ESCC surgical specimen. Patient-derived organoids generally recapitulate key architectural and phenotypic features of primary tumors while preserving molecular heterogeneity. The established organoids formed spheroids under three-dimensional (3D) culture conditions and exhibited stable proliferative capacity. Both CAFs and CFs were confirmed to be enriched for fibroblastic cell populations positive for vimentin, a fibroblast marker.

This protocol provides detailed, reproducible steps for tissue processing, enzymatic dissociation, organoid seeding, CAF/CF isolation, passaging, and cryopreservation. To help researchers assess applicability before implementation, practical information has been included: organoids typically form within 3–5 days, reach passage readiness within 7–10 days, and can be stably expanded for more than ten passages. CAFs and CFs can be passaged for at least eight generations. Organoids and fibroblasts can be cryopreserved within approximately 20 days. Establishment success rates reach approximately 70% for organoids and 80% for CAFs and CFs. Furthermore, a validated conditioned medium assay using CAF- or CF-conditioned medium is included, providing a robust approach for evaluating the paracrine effects of stromal cells on tumor organoid behavior. Collectively, this matched tumor organoid–stromal platform offers an experimentally accessible and biologically relevant model to investigate ESCC tumor–stromal interactions and support the development of precision therapeutic strategies.

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Protocol

All procedures involving human tissue must be performed under appropriate institutional biosafety approvals and in a certified biological safety cabinet. The patient provided written informed consent, and the project was approved by the Research Ethics Committee of the Juntendo University Faculty of Medicine. An overview of the procedure from protocols 2 to 6 is shown in Figure 1. The composition and concentrations of each component in the ESCC organoid medium (Eso_v1) were determined based on Kijima et al.14 and other studies15,16,17 with only minor modifications. The reagents and the equipment used are listed in the Table of Materials.

1. Reagents and tissue preparation

  1. Prepare reagents and equipment.
    1. Sterilize all instruments, including scissors, forceps, and pipettes, before beginning the procedure.
    2. Prepare 10 mL of phosphate-buffered saline (PBS) in a 15 mL tube.
    3. Prepare low-attachment 15 mL tubes and label them according to sample origin (e.g., Tumor Wash Tube (A), Tumor Digestion Tube (B), Non-tumor Wash Tube (C), Non-tumor Digestion Tube (D)).
    4. Coat working pipettes by aspirating and dispensing fetal bovine serum (FBS) 2–3 times to reduce cell adhesion during processing.
      NOTE: FBS coating prevents tissue fragments from sticking to plastic surfaces during mechanical dissociation.
  2. Prepare enzyme solutions
    1. Prepare Enzyme Solution I for tumor digestion by mixing 2.7 mL of Dulbecco's Modified Eagle's Medium (DMEM) with 10% FBS and 1x penicillin–streptomycin (PS) (hereafter, DMEM/10% FBS), 0.3 mL of 10 mg/mL collagenase, 0.15 mL of 10000 PU/mL dispase, and 0.03 mL of 2.5 µg/mL Amphotericin B. Warm to 37 °C.
    2. Prepare Enzyme Solution II for non-tumor digestion by mixing 2.7 mL of DMEM/10% FBS, 0.3 mL of 10 mg/mL collagenase, and 0.03 mL of 2.5 µg/mL Amphotericin B. Warm to 37 °C.
      NOTE: Collagenase and dispase facilitate the dissociation of tumor tissue.
  3. Prepare organoid culture medium
    1. Prepare ESCC organoid medium (Eso_v1 medium) containing 60% organoid basal medium, 40% L-WRN conditioned medium18 (see step 1.4), 50 ng/mL EGF (Growth factor), 1 mM N-acetyl-L-cysteine (Antioxidant), 5 mM Nicotinamide (Water-soluble vitamin), 500 nM A83-01 (TGF-βR inhibitor (preventing the growth of fibroblasts)), 1x B-27 (Serum-free culture supplement), 1x N-2 (Serum-free culture supplement), and 10 µM Y-27632 (ROCK inhibitor).
    2. Equilibrate the medium to 37 °C before use.
      NOTE: L-WRN conditioned medium (Supernatant from L-WRN cells) provides WNT, R-spondin, and Noggin signals essential for organoid initiation. Y27632 is contained in the medium throughout the culture period.
  4. Prepare L-WRN conditioned medium
    1. Thaw a vial of L-WRN cells and seed them onto a 100-mm dish with 10 mL of DMEM/10% FBS and culture for 1–2 days.
    2. Replace the medium with 10 mL of DMEM/10% FBS containing G418 (final 500 µg/mL) and Hygromycin B (final 500 µg/mL), and culture until the cells reach 80% confluence.
    3. Passage the cells onto two 100-mm dishes with 10 mL of DMEM/10% FBS (with G418 and Hygromycin B) and culture until the cells reach 100% confluence.
    4. Passage the cells onto five 150-mm dishes with 20 mL of DMEM/10% FBS (no antibiotics) per dish and culture to 100% confluence.
    5. Aspirate and discard the medium from each 150-mm dish.
    6. Add 25 mL per dish of collection medium (organoid basal medium supplemented with 5% FBS).
    7. Incubate for 24 h.
    8. Collect the medium from all five dishes (25 mL × 5 = 125 mL) into a sterile glass bottle.
    9. Repeat steps 1.4.6–1.4.8 for a total of 8 collections (target pooled volume: ~1,000 mL).
    10. Sterile-filter the pooled CM using a bottle-top filter.
    11. Aliquot 40 mL into 25 sterile 50 mL tubes.
    12. Store aliquots at −80 °C.
  5. Prepare fibroblast culture medium
    1. Prepare fibroblast medium-1.
    2. Equilibrate the medium to 37 °C before use.
  6. Prepare tissue handling dishes
    1. Dispense several (three to five) drops of FBS with a P1000 tip onto two 100-mm dishes to create a coating (Dish T for tumor tissue, Dish N for non-tumor tissue).
      NOTE: The FBS coating promotes smoother mechanical mincing and prevents adherence of small tissue fragments to the dish surface.
  7. Tissue handling and specimen identification
    1. Confirm that tumor and non-tumor specimens are transported on ice.
    2. Place the tissues on a 60-mm dish containing a small amount of DMEM/10% FBS and store on ice until use. Begin processing within 16 h of surgical resection to maintain viability.
    3. Identify the tumor tissue by its firm texture and irregular surface morphology, which differ from those of the non-tumor tissue.

2. Tissue processing, washing, and enzymatic digestion

NOTE: This step is performed to digest minced tumor and non-tumor tissue sections with different enzyme solutions.

  1. Mince the tumor and non-tumor tissues
    1. Transfer each tissue specimen (approximately 5 mm × 5 mm) onto its respective FBS-coated dish (Dish T for tumor tissue and Dish N for non-tumor tissue).
    2. Mince each tissue into ~0.5-mm fragments using a sterile scalpel.
      NOTE: Smaller fragments increase enzymatic accessibility and improve digestion efficiency.
  2. Wash the minced tissue.
    1. Coat a 10 mL pipette with FBS to prevent tissue adherence.
    2. Add 5 mL of PBS onto each dish using the FBS-coated pipette and gently resuspend the minced tissue.
    3. Transfer the suspension to the corresponding Tumor Wash Tube (Tube A) or Non-tumor Wash Tube (Tube C).
    4. Repeat steps 2.2.2 and 2.2.3 (total volume: 10 mL).
    5. Allow the tubes to stand for 2 min at room temperature; remove the supernatant while leaving approximately 0.5–1 mL of PBS and tissue at the bottom.
    6. Add 10 mL of fresh PBS with a new pipette; after coating the pipette with FBS, pipette the suspension forcefully while avoiding bubble formation.
      NOTE: Forceful pipetting assists in mechanical dissociation and removal of blood and mucus. Avoiding bubbles prevents shear-induced cell lysis. All wash steps are intended to remove debris.
    7. Allow the tubes to stand for 2 min at room temperature; remove the supernatant while leaving approximately 0.5–1 mL of PBS and tissue at the bottom.
    8. Repeat the wash procedure (steps 2.2.6 and 2.2.7) twice for a total of three washes.
  3. Transfer tissues to digestion tubes.
    1. Add 10 mL of fresh PBS with a new pipette; after coating the pipette with FBS, transfer the tumor tissue into the Tumor Digestion Tube (Tube B) and the non-tumor tissue into the Non-tumor Digestion Tube (Tube D).
    2. Allow the tubes to stand for 2 min at room temperature, remove the supernatant while leaving approximately 0.5 mL of PBS and tissue at the bottom.
  4. Perform enzymatic digestion.
    1. Tumor tissue digestion
      1. Add the entire volume of Enzyme Solution I (collagenase + dispase) into the Tumor Digestion Tube (Tube B).
      2. Incubate at 37 °C for 2 h with gentle rotation.
    2. Non-tumor tissue digestion
    3. Add the entire volume of Enzyme Solution II (collagenase only) into the Non-tumor Digestion Tube (Tube D).
      1. Incubate at 37 °C for 4 h with gentle rotation.
    4. Continue to step 3 (Tumor sample processing) or step 6 (Counterpart fibroblast seeding), depending on the sample origin.
      NOTE: The types of enzymes used to digest tumor and non-tumor tissues, as well as the processing times, were determined based on prior experimental optimization. Tumor tissues often contain dense extracellular matrix; dispase in combination with collagenase enhances digestive capacity.

3. Tumor sample processing for organoid and CAF isolation

NOTE: This step is performed to isolate epithelial tumor cells for organoid formation and stromal fibroblasts (CAFs) from the same digested tumor samples.

  1. Filter and collect tumor-derived cells.
    1. Place a 100 µm cell strainer onto a 50 mL conical tube.
    2. Pre-wet the strainer by passing 3 mL of DMEM/10% FBS through it.
      NOTE: Pre-wetting reduces cell loss by preventing adherence to dry mesh fibers.
    3. Transfer all contents of the Tumor Digestion Tube (Tube B) onto the strainer using an FBS-coated P1000 tip to remove undigested tissue fragments.
    4. Add an additional 6 mL of DMEM/10% FBS to rinse residual clusters through the strainer.
    5. Remove the strainer and return the filtrate to Tube B (a new low-attachment 15 mL tube can also be used).
  2. Wash and concentrate the filtrate.
    1. Centrifuge the tube at 300 × g for 5 min at 4 °C.
    2. Remove the supernatant using a disposable pipette.
    3. Add 10 mL of DMEM/10% FBS, mix gently by inversion, and centrifuge again at 300 × g for 5 min.
    4. Carefully aspirate the supernatant while retaining the pellet.
    5. Reduce the remaining volume to 40 µL using a P200 pipette.
  3. Allocate cells for organoid and CAF culture.
    1. Allocate 30 µL for organoid seeding.
    2. Allocate 10 µL for CAF seeding.
      ​NOTE: This ratio empirically provides sufficient epithelial and fibroblast populations for parallel expansion.

4. Organoid seeding (3D culture)

NOTE: This step is performed to establish ESCC organoids from a tumor-derived cell suspension.

  1. Prepare basement membrane matrix domes.
    1. Pre-warm a standard 24-well plate to 37 °C.
      NOTE: Pre-warming the plate accelerates solidification of the Basement Membrane Matrix domes.
    2. Mix 30 µL of the tumor-derived suspension with 90 µL of ice-cold Basement Membrane Matrix (1:3 ratio).
    3. Apply 30 µL per dome onto four wells.
    4. Allow the domes to solidify for 15 min in a CO₂ incubator.
  2. Add organoid culture medium
    1. Prepare organoid medium with pre-mixed supplements (FGF10, SB202190, and antibiotics, such as 1× Amphotericin B and 100 µg/mL Primocin, if used).
      NOTE: Well 1: Eso_v1; Well 2: Eso_v1 + 10 µM SB202190; Well 3: Eso_v1 + 100 ng/mL FGF10; Well 4: Eso_v1 + 10 µM SB202190 + 100 ng/mL FGF10 (final concentrations).
    2. Gently add 1 mL of medium to each well along the wall.
    3. Incubate under standard CO₂ culture conditions.
    4. Change medium every 3–5 days.
      ​NOTE: Premixing the supplements prevents the need to pipette small volumes directly onto fragile Basement Membrane Matrix domes. Antibiotics are added only during the first 6 days after organoid establishment. Culture conditions (FGF10 +/-, SB202190 +/-) are determined based on growth rate and morphology (preferably spherical).
  3. Monitor organoid formation.
    1. Observe domes daily using an inverted microscope.
      NOTE: Expected morphology: Day 3–5 → visible spheroids; Day 7–10 → dense spheroids ready for passaging.
    2. Passage the organoids when they reach a size of approximately 100–200 µm or show dome crowding of 90% or more.

5. CAF seeding

NOTE: This step is performed to establish CAFs from a tumor-derived cell suspension.

  1. Seed CAFs onto a collagen-coated dish.
    1. Prepare a collagen-coated 60-mm dish containing 1.5 mL of fibroblast medium-1.
    2. Add 10 µL of the tumor filtrate allocated for CAF seeding.
    3. Add antibiotics such as 1× Amphotericin B and 100 µg/mL Primocin.
    4. Incubate under standard CO₂ culture conditions.
    5. Change medium every 3–5 days.
      NOTE: Empirically, fibroblast medium-1 is often more suitable than other media for the initial establishment and growth of CAFs and CFs. Antibiotics are added only during the first 6 days.
  2. Monitor CAF growth
    1. Monitor for the appearance of fibroblast-like spindle morphology beginning around days 3–5.
    2. Passage fibroblasts at 90%–100% confluency.
      NOTE: If epithelial cell contamination occurs, fibroblasts can be selectively isolated by adjusting the trypsinization duration, as fibroblasts detach more quickly than epithelial cells during passaging.

6. CF seeding

NOTE: This step is performed to obtain patient-matched CFs from adjacent non-tumor tissue.

  1. Wash and prepare the non-tumor digest.
    1. Centrifuge the Non-tumor Digestion Tube (Tube D) at 300 × g for 5 min.
    2. Remove the supernatant using a disposable pipette.
    3. Add 10 mL of DMEM/10% FBS, mix gently by inversion, and centrifuge again at 300 × g for 5 min.
    4. Carefully aspirate the supernatant, while retaining the pellet.
    5. Repeat steps 6.1.3 and 6.1.4 once more.
  2. Seed CFs onto a collagen-coated dish
    1. Transfer the entire suspension onto a collagen-coated 60-mm dish containing 1.5 mL of fibroblast medium-1.
    2. Add antibiotics such as 1× Amphotericin B and 100 µg/mL Primocin.
    3. Incubate under standard CO₂ culture conditions.
    4. Change medium every 3–5 days.
      NOTE: Antibiotics are added only during the first 6 days.
  3. Monitor CF growth.
    1. Observe fibroblast-like spindle morphology beginning at 3–5 days.
    2. Passage fibroblasts at 90%–100% confluency.

7. Organoid passaging and cryopreservation

  1. Organoid passaging.
    NOTE: The purpose of this step is to expand organoids once they reach an appropriate density.
    1. Aspirate the old medium and wash each well with 1 mL of PBS.
    2. Add 0.5 mL of pre-warmed trypsin for organoid to each well.
    3. Pipette vigorously with a P1000 tip, using the edge of the tip to disrupt the Basement Membrane Matrix dome.
      NOTE: Mechanical shearing helps break down the dense Basement Membrane Matrix, improving enzymatic access.
    4. Incubate for 10–15 min in a CO₂ incubator until all gel clumps dissolve. Dissociate organoids into 20–50 cell clumps.
      NOTE: Avoid full single-cell dissociation, as it reduces viability.
    5. Collect the suspension into a low-attachment 15 mL tube.
    6. Rinse the well four times with 0.5 mL of DMEM/10% FBS and transfer all rinses to the same low-attachment tube.
    7. Centrifuge the tube at 300 × g for 5 min.
    8. Remove the supernatant and resuspend the pellet in minimal DMEM/10% FBS; adjust the suspension volume to 80 µL.
    9. Mix 80 µL of the organoid suspension with 240 µL of ice-cold Basement Membrane Matrix (1:3 ratio).
    10. Plate the mixture onto a pre-warmed 6-well plate (37 °C), with 4–6 domes per well.
    11. Allow the domes to solidify for 15 min, then add 3 mL of Eso_v1 medium (the composition that was optimal in the initial culture at step 4.2) per well.
    12. Change medium every 3–5 days.
      NOTE: Organoids are passaged when they reach a diameter of approximately 100–200 µm or when dome crowding exceeds 90%. Cell counting is not performed. For routine subcultures, organoids are divided at a ratio of 1:4 to 1:10. Rapid reformation of spheroids is expected within 48–72 h.
  2. Organoid cryopreservation
    1. Wash each well with 3 mL of PBS.
    2. Add 1.5 mL of trypsin for organoid to each well, pipette vigorously, and incubate for 10 min.
    3. Collect the entire suspension into a low-attachment 15 mL tube and rinse the well four times.
    4. Centrifuge the tube at 300 × g for 5 min and remove the supernatant.
    5. Resuspend the pellet in 2 mL of 90% FBS/10% DMSO.
    6. Aliquot 0.5 mL into each cryovial.
    7. Place vials in a freezing container and store at −80 °C overnight.
    8. Transfer vials to liquid nitrogen for long-term storage.
      ​NOTE: Organoids are stocked when they reach a diameter of approximately 100–200 µm or when dome crowding exceeds 90%.
  3. Recovery of frozen organoids
    1. Thaw the cryovial rapidly in a 37 °C water bath.
    2. Transfer the contents into 5 mL of DMEM/10% FBS in a 15 mL tube.
    3. Centrifuge the tube at 300 × g for 5 min.
    4. Remove the supernatant and resuspend the pellet in 40–60 µL of DMEM/10% FBS.
    5. Mix the suspension with ice-cold Basement Membrane Matrix at a 1:3 ratio.
    6. Plate the mixture onto a pre-warmed 6-well plate (37 °C) as 4–6 domes per well.
    7. Allow the domes to solidify for 15 min, then add 3 mL of Eso_v1 medium (the composition that was optimal in the initial culture at step 4.2) per well.

8. Fibroblast passaging and cryopreservation

  1. Passaging CAFs and CFs
    NOTE: The purpose of this step is to expand fibroblasts once they reach an appropriate density.
    1. Wash the dish with 2 mL of PBS.
    2. Add 0.4 mL of 0.25% trypsin.
    3. Incubate for 5 min in a CO₂ incubator, monitoring detachment under a microscope.
    4. Neutralize the trypsin with DMEM/10% FBS and transfer the entire cell suspension onto a 100-mm dish containing 10 mL of medium.
      NOTE: Fibroblasts are passaged when they reach 90%–100% confluency. For routine subcultures, they are divided at a ratio of 1:3. Empirically, fibroblast proliferation slows or ceases when cell density is low.
  2. Fibroblast cryopreservation
    1. Wash the cells with 5 mL of PBS.
    2. Add 1 mL of 0.25% trypsin, then incubate for 5 min in a CO₂ incubator.
    3. Neutralize the trypsin with DMEM/10% FBS and transfer the entire cell suspension into a 15 mL tube.
    4. Centrifuge at 300 × g for 5 min.
    5. Resuspend the cell pellet in 2 mL of 90% FBS/10% DMSO.
    6. Aliquot 0.5 mL into each cryovial.
    7. Place the vials in a freezing container and store at −80 °C overnight.
    8. Transfer the vials to liquid nitrogen for long-term storage.

9. Conditioned medium assay

NOTE: The purpose of this step is to evaluate the paracrine effects of fibroblasts on ESCC tumor organoids using conditioned medium (CM). This assay provides a reproducible stromal–tumor interaction model when direct co-culture is not yet optimized.

  1. Preparation of CAF/CF conditioned medium
    1. Culture the same number of CAFs or CFs in 3 mL of DMEM/10% FBS on 60-mm dishes until 80%–90% confluency.
    2. Wash cells once with 2 mL of PBS.
    3. Add 3 mL of DMEM/2% FBS.
    4. Incubate for 48 h in a CO₂ incubator.
    5. Collect the supernatant into a 15 mL tube.
    6. Centrifuge at 2000 × g for 5 min to remove cellular debris.
    7. Collect 2.5 mL of the supernatant into a new 15 mL tube.
    8. Aliquot the conditioned medium into 100–500 µL portions and store at -80 °C.
  2. Preparation of organoid assay medium
    1. Prepare the ESCC organoid medium as described in step 1.3.
    2. Mix the organoid medium and conditioned medium at a 1:1 ratio (50% v/v).
  3. Culture of cancer organoids using the organoid assay medium.
    1. Following organoid passaging as described in step 7, seed organoids into a single 20 µL Basement Membrane Matrix dome per well on a pre-warmed 48-well plate (37 °C).
    2. After dome solidification, add 0.3 mL of organoid assay medium to each well.
    3. Replace the organoid assay medium every 3–7 days.
    4. Assess the morphology and size of the cancer organoids.
      ​NOTE: All human-derived materials are treated as potentially infectious. Appropriate personal protective equipment, including gloves, masks, and protective eyewear, is always worn. Collagenase, dispase, and dimethyl sulfoxide (DMSO) may cause skin, eye, and respiratory irritation; therefore, direct contact and inhalation are avoided. All enzymatic procedures involving human tissues are performed in accordance with institutional biosafety guidelines. Liquid waste containing human tissue is collected in sealed biohazard containers and autoclaved prior to disposal. Chemical waste, including DMSO and enzyme solutions, is disposed of in accordance with institutional hazardous waste guidelines. All reusable instruments are rinsed with 70% ethanol and autoclaved before reuse.

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Results

Tissue samples were obtained from the tumor region and an adjacent non-tumor region located 3 cm away from the tumor, surgically excised from a patient with ESCC who had not received neoadjuvant chemotherapy. Cancer organoids and CAFs were isolated from the tumor region, whereas CFs were isolated from the non-tumor region. Surgical tissues were minced into pieces approximately 0.5 mm in size (Figure 2A), then enzymatically treated, after which cancer organoids and fibroblasts were isolated s...

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Discussion

The previous reports employed cancer organoids and the patient-matched CAFs to investigate the role of CAFs in the proliferation and drug-resistance in human colon, breast, pancreatic, and head and neck cancers20,21,22,23,24. These studies demonstrated that CAFs influence proliferation and drug resistance. On the other hand, to the best of current knowledge, t...

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Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

This work was supported by the Juntendo University Young Investigator Award (2024 and 2025) to P. Z. and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (23K07362, 21K08693). We would like to thank all members of the Departments of Pathology and Oncology, and of Esophageal and Gastroenterological Surgery.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
100 mm dishTPP93100
100x Penicillin-Streptomycin (PS)Nacalai26253-84
15 ml tubeFastGeneFG2001
24-well cell culture plateTPP92024
48-well cell culture plateTPP92448
50 ml tubeFastGeneFG2004
60 mm dishTPP93060
6-well cell culture plateTPP92006
A83-01Cayman9001799
Advanced DMEM/F12 Gibco12634010Organoid basal medium = Advanced DMEM/F12 + GlutaMAX + 1x PS
Alexa488 anti-mouse IgGInvitrogenA11029
Amphotericin BThermoFisher15290018
Anti-Vimentin antibody (V9)Santa Cruzsc-62601/500 dilution for use
B-27ThermoFisher17504044
Bottle-top filterSartorius180F15
Cell strainer, 100 µm poreBD Falcon352360
Collagenase, Type ISIGMAC0130
Collagen-coated 60 mm disheIWAKI4010-010
Dimethyl Sulfoxide (DMSO)Wako043-07216
Dispase IGODO SHUSEI386-02271
Dulbecco's Modified Eagle Medium (DMEM)Nacalai08459-64
ECLIPSE Ti2Nicon
Fetal Bovine Serum (FBS)SERANAS-FBS-CO-015
Fibroblast Growth Medium 2PromoCellC-23020Fibroblast medium-1 = Fibroblast Growth Medium 2
G418Invivogenant-gn-1
GlutaMAX Gibco35050061
HFDM-1KSK87-160Fibroblast medium-2 = HFDM-1 + 1% FBS + 1 x PS
Hoechst33342 Nacalai04929-82
HygromycinInvivogenant-hg-1
IncuCyte systemEssen BioScience
L-WRN cellsATCCCRL-3276
MatrigelCorning354234Basement Membrane Matrix = Matrigel
N2ThermoFisher17502048
N-acetyl-L-CysteineWako017-05131
NicotinamideTCIN0078
Phosphate Buffered Saline (PBS)Nacalai14249-24
PrimocinInvivogenant-pm-05
Recombinant human EGFPEPROTECHAF-100-15
Recombinant human FGF-10PEPROTECHPEP-100-26
SB202190SelleckS1077
Scalpel, No 11Feather8-3086-02
STEMFULLSumitomo bakeliteMS-90150Low-attachment 15 mL tube = STEMFULL
TrypLE ExpressGibco12605028Trypsin for organoid = TrypLE Express
TrypsinNacalai35556-44
Y-27632SelleckS1049

References

  1. Sung, H., et al. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 71 (3), 209-249 (2021).
  2. Abbas, G., Krasna, M. Overview of esophageal cancer. Ann Cardiothorac Surg. 6 (2), 131-136 (2017).
  3. Morgan, E., et al. Global burden of colorectal cancer in 2020 and 2040: Incidence and mortality estimates from GLOBOCAN. Gut. 72 (2), 338-344 (2023).
  4. Lagergren, J., Smyth, E., Cunningham, D., Lagergren, P. Oesophageal cancer. Lancet. 390 (10110), 2383-2396 (2017).
  5. Lordick, F., et al. Oesophageal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 27 (suppl 5), v50-v57 (2016).
  6. Chhabra, Y., Weeraratna, A. T. Fibroblasts in cancer: Unity in heterogeneity. Cell. 186 (8), 1580-1609 (2023).
  7. Caligiuri, G., Tuveson, D. A. Activated fibroblasts in cancer: Perspectives and challenges. Cancer Cell. 41 (3), 434-449 (2023).
  8. Mezawa, Y., Orimo, A. The roles of tumor- and metastasis-promoting carcinoma-associated fibroblasts in human carcinomas. Cell Tissue Res. 365 (3), 675-689 (2016).
  9. Mezawa, Y., Orimo, A. Phenotypic heterogeneity, stability and plasticity in tumor-promoting carcinoma-associated fibroblasts. FEBS J. 289 (9), 2429-2447 (2022).
  10. Qiu, L., et al. Cancer-associated fibroblasts: An emerging target against esophageal squamous cell carcinoma. Cancer Lett. 546, 215860(2022).
  11. Xue, M., Tong, Y., Xiong, Y., Yu, C. Role of cancer-associated fibroblasts in the progression, therapeutic resistance and targeted therapy of oesophageal squamous cell carcinoma. Front Oncol. 13, 1257266(2023).
  12. Dunbar, K. J., Wong, K. K., Rustgi, A. K. Cancer-associated fibroblasts in esophageal cancer. Cell Mol Gastroenterol Hepatol. 17 (5), 687-695 (2024).
  13. Pimkhaokham, A., et al. Nonrandom chromosomal imbalances in esophageal squamous cell carcinoma cell lines: Possible involvement of the ATF3 and CENPF genes in the 1q32 amplicon. Jpn J Cancer Res. 91 (11), 1126-1133 (2000).
  14. Kijima, T., et al. Three-dimensional organoids reveal therapy resistance of esophageal and oropharyngeal squamous cell carcinoma cells. Cell Mol Gastroenterol Hepatol. 7 (1), 73-91 (2019).
  15. Fuchino, T., et al. Characterization of residual cancer by comparison of a pair of organoids established from a patient with esophageal squamous cell carcinoma before and after neoadjuvant chemotherapy. Hum Cell. 37 (2), 491-501 (2024).
  16. Shen, S., et al. Advancing precision medicine in esophageal squamous cell carcinoma using patient-derived organoids. J Transl Med. 22 (1), 1168(2024).
  17. Zhang, S., Wang, C. Protocol to establish patient-derived xenograft and organoid for radiosensitivity of esophageal squamous cell carcinoma. STAR Protoc. 5 (4), 103273(2024).
  18. Miyoshi, H., Stappenbeck, T. S. In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nat Protoc. 8 (12), 2471-2482 (2013).
  19. Mashima, Y., et al. KIF24 depletion induces clustering of supernumerary centrosomes in PDAC cells. Life Sci Alliance. 5 (11), e202201470(2022).
  20. Hogstrom, J. M., et al. Simultaneous isolation of hormone receptor-positive breast cancer organoids and fibroblasts reveals stroma-mediated resistance mechanisms. J Biol Chem. 299 (8), 105021(2023).
  21. Schuth, S., et al. Patient-specific modeling of stroma-mediated chemoresistance of pancreatic cancer using a three-dimensional organoid-fibroblast co-culture system. J Exp Clin Cancer Res. 41 (1), 312(2022).
  22. Farin, H. F., et al. Colorectal cancer organoid-stroma biobank allows subtype-specific assessment of individualized therapy responses. Cancer Discov. 13 (10), 2192-2211 (2023).
  23. Zapatero, M. R., et al. Trellis tree-based analysis reveals stromal regulation of patient-derived organoid drug responses. Cell. 187 (25), 7335-7349 (2024).
  24. Rademaekers, M., Johansson, E. O., Johansson, E., Roberg, K., Wiechec, E. Tumor-matched and unmatched cancer associated fibroblasts exhibit differential effect on proliferation and FMOD and MMP9 gene expression in head and neck squamous cell carcinoma cells when co-cultured in spheroids. Cancer Cell Int. 24 (1), 190(2024).

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