Establishment and Histological Analysis of Esophageal Organoids Modeling the Progression from Normal to Cancerous Tissues

Summary

The present protocol describes the establishment and histological analysis of esophageal organoid models that represent different stages of tumor progression. This method enables researchers to study changes in cellular morphology, spatial organization, and molecular marker expression patterns during the transition from normal to cancerous tissues.

Abstract

Organoids have emerged as a pivotal tool for advancing the understanding of tumorigenesis and cancer therapy. By generating human organoid models representing different tumor stages and performing histological analyses, it is possible to obtain a deeper understanding of the alterations in cellular morphology, spatial architecture, and the expression of key molecular markers as the tumor progresses. This study presents a comprehensive protocol for the establishment and culture of esophageal squamous cell organoids. Additionally, the protocol outlines methods for assessing the expression patterns and spatial organization of critical molecules within the organoids, utilizing techniques such as fixation, embedding, and staining. Through this protocol, significant changes were identified in the spatial structure of esophageal squamous epithelial cells and in the expression of various tumor biomarkers during tumorigenesis. The protocol facilitates the construction and histological analysis of organoids, enabling researchers to investigate the spatial architecture and molecular alterations of epithelial cells across different stages of tumorigenesis and therapeutic intervention.

Introduction

Tumorigenesis is a complex, multistage process characterized by progressive molecular and morphological changes in cells^1,2. Esophageal squamous cell carcinoma (ESCC), a prevalent malignancy with poor prognosis^3,4, exemplifies this stepwise progression through four distinct stages: normal mucosa, low-grade intraepithelial neoplasia (LGIN), high-grade intraepithelial neoplasia (HGIN), and invasive carcinoma⁵. Throughout these stages, epithelial cells exhibit dynamic changes in molecular expression patterns and spatial organization, accompanied by systematic alterations in tissue morphology as it advances from a normal to a malignant state^6,7. Despite advances in understanding ESCC pathogenesis, the lack of experimental models that faithfully recapitulate spatial and temporal aspects of tumor evolution-while enabling systematic histological and molecular analyses-has hindered deeper mechanistic understanding of disease progression and therapeutic development.

While 2D immortalized cancer cell lines have made significant contributions to the understanding of oncogenesis, they are inherently limited in replicating the biological complexity and pathological features of native tumors⁸. Animal models, though providing in vivo context, often poorly predict human responses due to species-specific differences⁹. In contrast, organoids have emerged as a transformative preclinical platform that faithfully preserves the cellular heterogeneity, architecture, and functionality of human tissues^10,11,12,13. As preclinical models, organoids better capture the characteristics of primary tumors, enabling detailed investigation of key molecular events and cellular changes during tumor progression¹⁴. For instance, Chen et al. utilized patient-derived esophageal organoids from different stages of ESCC to elucidate epithelial-fibroblast interactions, ultimately validating the ANXA1-FPR2 signaling axis as a critical driver of ESCC pathogenesis⁶. Similarly, Ko et al. employed genetically engineered esophageal organoids to identify key genetic determinants driving ESCC initiation and immune evasion, demonstrating how organoid models can effectively recapitulate disease features and reveal novel therapeutic targets¹⁵.

The present methodology resolves significant issues in esophageal cancer modeling by establishing a reproducible protocol for generating multistage ESCC organoids that mirror histological progression from normal epithelium to invasive carcinoma. This system integrates optimized culture conditions using an L-WRN-conditioned medium to maintain epithelial stemness, combined with standardized protocols for histological processing and multiplex immunofluorescence (mIF) analysis, providing an ideal platform to longitudinally analyze spatial and molecular changes during tumorigenesis. Compared to alternative techniques such as 2D cultures, this organoid platform uniquely preserves tissue architecture, enabling the visualization of spatially organized molecular markers, including the immune checkpoint protein PD-L1 (CD274), which mediates tumor immune evasion by inhibiting T cell responses^16,17,18, and the proliferation marker Ki-67. The protocol enables organoids from normal esophageal and precancerous tissues passage, helping researchers build a continuous organoid model from normal tissue to tumor¹⁹. By enabling detailed analysis of spatial and molecular changes during tumorigenesis, this protocol offers researchers a powerful tool for understanding the mechanisms underlying cancer development and progression, potentially leading to improved therapeutic strategies.

This methodology is particularly suited for researchers investigating epithelial carcinogenesis, tumor microenvironment interactions, or therapeutic responses in ESCC and related squamous malignancies. Its modular design allows adaptation to study other molecular markers or signaling pathways, provided appropriate validation steps are incorporated. By offering a standardized yet flexible platform, this protocol aims to advance preclinical research in tumor biology and accelerate the translation of mechanistic insights into targeted therapies.

Protocol

This study was approved by the Institutional Review Board of the Cancer Hospital, Chinese Academy of Medical Sciences (Approval Nos. 20/069–2265, 22/221-3423 and 23/305-4047). Esophageal tissue samples were obtained from patients undergoing surgery or early screening for esophageal squamous cell carcinoma (ESCC) at the Cancer Hospital, Chinese Academy of Medical Sciences between 2021 and 2024, for the purpose of establishing human esophageal organoids. None of the patients included in this study had received chemotherapy or radiotherapy prior to sample collection. Informed consent was obtained from all participants, and relevant clinical information was retrieved from medical records. A complete list of reagents and equipment used in this study is provided in the Table of Materials.

1. Preparation of esophageal epithelial organoids

1. Preparation of L-WRN-conditioned medium
   1. Preparation of L-WRN cell line
      1. Thaw L-WRN cell line (storage at -80 °C) in a 37 °C water bath for 1-2 min. Mix with 5 mL pre-warmed basal culture medium (DMEM supplemented with 10% FBS).
      2. Centrifuge the mixture at 200 x g for 5 min at room temperature (RT).
      3. Discard the supernatant. Resuspend the cells with 2-3 mL of fresh pre-warmed selection medium (DMEM containing 10% FBS, 0.5 mg/mL hygromycin B, and 0.5 mg/mL G-418).
         NOTE: Keep the medium at 4 °C (maximum storage 1 month).
      4. Transfer cells to a 10 cm culture dish containing the selection medium. Culture the cells at 37 °C in a 5% CO₂ humidified incubator.
   2. Passage of L-WRN cell line
      1. Dissociate confluent cells using 0.025% trypsin-EDTA (1-2 min at 37 °C) and split at a 1:2 ratio.
   3. Collection of the L-WRN-conditioned medium
      1. Replace culture medium with basal culture medium at 80% cell confluence. Transfer the supernatant to a centrifuge tube.
      2. Filter through 0.22 µm membrane to eliminate cellular debris. Collect the filtrate as an L-WRN-conditioned medium.
         NOTE: Keep L-WRN-conditioned medium at 4 °C (1 week) or at -80 °C (6 months).
2. Human esophageal organoid culture medium (H-EOCM) preparation
   1. H-EOCM preparation
      1. Vortex the L-WRN-conditioned medium (1.5 mL volume) following 4 h of equilibration at 4 °C.
      2. Combine Advanced DMEM/F12 medium with these additives to create H-EOCM: 3% L-WRN-conditioned medium, 1× Anti-Anti, 1× L-glutamine, 1× N2 supplement, 1× B27 supplement, 0.15 mM HEPES, 40 ng/mL EGF, 10 µM Y-27632, and 50 µM A83-01.
         NOTE: Maintain frozen storage of H-EOCM at -20° C for six-month preservation.
3. Pre-experimental preparation
   1. Defrost H-EOCM by maintaining it at 4 °C for 4 h.

2. Establishment of human esophageal organoid

1. Preparation of materials
   1. Thaw the basement membrane matrix and H-EOCM at 4 °C. Sterilize surgical scissors and tweezers for the next experiment.
   2. Pre-cool the pipette tips to 4 °C. Pre-warm the 24-well plate to 37 °C.
2. Tissue processing and cell isolation
   NOTE: ESCC tumor tissue, dysplastic lesions (≤2 cm from the tumor margin), and matched normal esophageal tissue (≥5 cm from the tumor margin) were collected from the same individuals with ESCC who underwent surgical resection. In addition, multistage esophageal samples were obtained through an ESCC early detection and screening program, as described in our previous studies^6,7.
   1. Clean the sample with room temperature wash buffer (PBS containing 1×Anti-Anti and 0.15 mM HEPES) in 5 mL centrifuge tubes three times.
      NOTE: Wash the samples more than three times to reduce the risk of contamination.
   2. Mince the tissue into 1 mm³ fragments using sterile scissors. Transfer fragments to 1.5 mL centrifuge tubes.
   3. Suspend the sample with 1 mL digestion buffer and shake it at 37 °C, 50-100 rpm, for 10-20 min to digest the tissue.
   4. Centrifuge the mixture at 400 x g for 5 min at 4 °C. Discard supernatant.
   5. Resuspend the precipitate in 500 µL of 0.025% trypsin-EDTA. Incubate at 37 °C for 10 min. Add 1 mL of DMEM supplemented with 10% FBS to stop the enzymatic activity.
   6. Pass the suspension through a 70 µm sterile filter. Collect the filtrate in a 1.5 mL centrifuge tube.
   7. Centrifuge filtrate for 5 min at 400 x g at 4 °C. Discard supernatant. Resuspend the cells with 100 µL of H-EOCM.
3. Organoid seeding
   1. Determine the cell density, then prepare a fresh 1.5 mL centrifuge tube with 5,000-15,000 cells in suspension.
   2. Centrifuge for 5 min at 400 x g at 4 °C. Aspirate the supernatant carefully. Gently resuspend cells in 50-100 µL of basement membrane matrix evenly.
      NOTE: Store the basement membrane matrix at 4 °C to avoid solidification.
   3. Add 50 µL of basement membrane matrix with the mixed cells to the center of each 24-well plate. Polymerize basement membrane matrix through 37 °C incubation (30 min duration).
   4. Add 500 µL of pre-warmed H-EOCM (37 °C) to cover the basement membrane matrix. Culture the organoids in a humidified 5% CO₂ incubator at 37 °C.
4. Medium replacement
   1. Aspirate the spent medium and replenish with 500 µL of fresh H-EOCM (warmed to 37 °C).
      NOTE: Replace H-EOCM at 3-day intervals.

3. Passaging of organoids

1. Preparation of materials
   1. Thaw the basement membrane matrix and H-EOCM at 4 °C. Pre-cool pipette tips to 4 °C. Pre-warm the 24-well plate to 37 °C.
2. Digestion of organoid
   1. Remove the H-EOCM medium gently. Add 500 µL of pre-cooled passage buffer (Advanced DMEM/F12 containing 1× Anti-Anti and 0.15 mM HEPES, pre-cooled to 4 °C) in the well to melt the basement membrane matrix.
   2. Combine the basement membrane matrix with passage buffer in a 1.5 mL centrifuge tube. Wash the well with 500 µL of pre-cooled passage buffer and collect the buffer to retrieve any remaining organoids.
   3. Centrifuge the buffer for 5 min at 400 x g at 4 °C. Aspirate the supernatant gently. Add 500 µL of recombinant trypsin into the tube and incubate for enzymatic digestion (37 °C, 15 min). Resuspend at 5-min intervals.
   4. Centrifuge the digested sample (400 x g, 5 min, 4 °C). Discard the supernatant. Repeat steps 3.2.6 and 3.2.7. Resuspend the cells in 100 µL of H-EOCM.
3. Organoid seeding
   1. For organoid seeding, repeat step 2.3.

4. Organoid freezing and recovery

1. Organoid freezing
   1. For materials preparation, repeat step 2.1.
   2. For the digestion of the organoids, repeat step 3.2.
   3. Organoid freezing
      1. Determine the cell density, then prepare a fresh 1.5 mL centrifuge tube with 10,000 cells in suspension.
      2. Centrifuge the samples at 400 x g for 5 min at RT. Aspirate the supernatant carefully.
      3. Resuspend the cells with 500-1000 µL of cryopreservation medium. Transfer to a new cryotube. Freeze the cryotube at -80 °C for 24 h. Archive in a liquid nitrogen storage system for extended preservation.
2. Thawing of freezing organoid
   1. For the preparation of the materials, repeat step 2.1.
   2. Thawing of freezing organoid
      1. Rapidly thaw the cryotube in a 37 °C water bath for 2-3 min. Transfer the frozen material to 9 mL of pre-warmed basal culture (37 °C) and resuspend the cells evenly.
      2. Centrifuge the cell suspension for 5 min at 400 x g (at RT). Aspirate the supernatant carefully.
   3. Organoid seeding
      1. For seeding the organoids, repeat step 2.3.

5. Histological analysis of organoid

1. Preparation of paraffin-embedded sections
   1. Preparation of materials
      1. Sterilize 50 mL of embedding matrix (aqueous solution: 2% agar + 2.5% gelatin) in a 250 mL flask via autoclave. Aliquot 5 mL into 15 mL centrifuge tubes. Store at room temperature.
   2. Organoid fixing
      1. Repeat steps 3.2.1-3.2.2. Resuspend the precipitate with 1 mL of passage buffer in a fresh 1.5 mL tube.
      2. Centrifuge at 400 x g at 4 °C for 5 min. Remove the supernatant. Resuspend the organoids in 500 µL of 4% paraformaldehyde (PFA). Incubate at room temperature for 1 h or at 4 °C for at least 6 h.
   3. Organoid embedding
      1. Centrifuge the mixture (400 x g, 5 min, RT). Remove the supernatant. Add 1 mL of the passage buffer in the tube and resuspend the precipitation.
      2. Centrifuge the mixture (400 x g, 5 min, RT). Remove the supernatant.
      3. Place one tube (15 mL) with embedding medium (5 mL) into the water bath (100 mL, 150 mL container). Microwave at maximum power until the water begins to boil.
         NOTE: Unscrew the cap of the 15 mL tube before microwaving.
      4. Resuspend the organoids with 50 µL of embedding gel in a 1.5 mL centrifuge tube. Cool the tube to 4 °C until the gel hardens completely.
      5. Transfer the solidified gel into 70% ethanol. Store at 4 °C.
   4. Paraffin block preparation
      1. Dehydrate the gel through ethanol series (30% → 50% → 70% → 80% → 95% → 100%), 30 min per concentration.
      2. Put the solidified gel in clear xylene for 30 min. Immerse the solidified gel in paraffin and embed it using a heated paraffin station.
      3. Cut 4 µm thick slices with a microtome. Mount on slides and dry at 65 °C for 1 h. Store in dry conditions.
2. Immunohistochemistry (IHC) staining of organoid slices
   1. Dewaxing and hydration
      1. Heat slices in tissue clearing agent at 65 °C for 40 min. Immerse the slide in fresh tissue-clearing agent at room temperature for 20 min.
      2. Process the slides through glass trays containing ethanol series: immerse in 100% ethanol for 10 min and repeat once, then transfer to 95% ethanol for 5 min, followed by 85% ethanol for 5 min, then 75% ethanol for 5 min, and finally place in 4% PFA for 10 min.
      3. Add sterilized water to the thermostable chamber and keep 5 min on the shaker at 80 rpm. Repeat this step twice.
   2. Antigen retrieval
      1. Add Tris-EDTA antigen (pH 9.0) retrieval solution to the thermostable chamber to immerse the slides. Microwave the thermostable chamber containing the slides at maximum intensity (700 W, 3 min) until boiling. Keep microwaving at a low-power setting (70 W, 15 min) and cool down to room temperature.
      2. Wash in distilled water for 2 min on a shaker at 80 rpm. Repeat twice. Circle the location of organoids with a histological pen.
   3. Peroxidase blocking
      1. Drop peroxidase blocking solution to cover the sample area. Incubate in a humidity chamber at room temperature for 20 min.
      2. Transfer the slides to a thermostable chamber. Wash in PBST (PBS + 0.1% Tween 20) for 2 min at 80 rpm. Repeat twice.
   4. Primary antibody incubation
      1. Gently wipe away the excess PBST droplets on the slides.
         NOTE: Avoid touching organoids on slides.
      2. Drop diluted primary antibody to cover the organoid area. Incubate in a humidity chamber for 2 h at room temperature, or 8-14 h at 4 °C. Repeat this step.
   5. Secondary antibody incubation
      1. Repeat step 5.2.4.1. Drop diluted HRP secondary antibody to cover the organoid area. Incubate in a humidity chamber at room temperature for 20 min. Repeat this step.
   6. DAB (3,3′-Diaminobenzidine) staining
      1. Repeat step 5.2.4.1. Drop 1× DAB to cover the organoid area. Incubate in a humidity chamber at room temperature until the color turns brown.
      2. Immerse the slides in distilled water for 5-10 s to terminate the color change. Repeat the step.
   7. Hematoxylin staining
      1. Stain in hematoxylin for 5-8 min. Differentiate in 1% acid alcohol (1% HCl in 70% alcohol) for 5-10 s.
      2. Immerse the slides in distilled water for 5-10 s. Repeat this step.
   8. Dehydration
      1. Process the slides through glass trays in sequential order: immerse in 75% ethanol for 5 min, transfer to 85% ethanol for 5 min, move to 95% ethanol for 5 min, followed by two separate immersions in 100% ethanol for 10 min each, and finally place in xylene for two successive 20-min periods.
   9. Slide mounting
      1. Add xylene to neutral gum until it turns transparent. Air-dry slightly. Then, drop gum with xylene to cover the organoid area.
      2. Apply coverslip. Scan and analyze the slide.
3. Multiplex immunofluorescence (mIF) staining
   1. For dewaxing and hydration, repeat step 5.2.1.
   2. For peroxidase blocking, repeat step 5.2.3.
   3. For antigen retrieval, repeat step 5.2.3.
   4. Sheep serum blocking.
      1. Repeat step 5.2.4.1. Add sheep serum-blocking solution to cover the organoid area. Incubate in a humidity chamber at room temperature for 30 min.
      2. Wash in PBST for 2 min. Repeat twice.
   5. For primary antibody incubation, repeat step 5.2.4.
   6. For secondary antibody incubation, repeat step 5.2.4.1.
      1. Drop specific secondary antibody solution to cover the organoid area. Incubate in a humidity chamber at room temperature for 20 min. Repeat step 5.2.3.2.
         NOTE: Protect slides from light from this step until the end of the experiment.
   7. Fluorescent dye staining
      1. Repeat step 5.3.4.1. Drop fluorescent dye solution to cover the organoid area.
      2. Incubate in a humidity chamber at room temperature for 10-20 min. Repeat step 5.2.3.2.
   8. For multi-staining, repeat steps 5.3.3-5.3.7.
   9. DAPI staining
      1. Repeat step 5.2.4.1. Drop DAPI solution to cover the organoid area. Repeat step 5.2.3.2. Next, repeat step 5.3.4.2.
      2. Immerse the slides in sterilized water for 2 min.
   10. Slide mounting
       1. Drop antifade mounting medium to cover the organoid area. Apply coverslip.
   11. Acquire digital images using a slide scanner and analyze them.

Results

This protocol describes organoid sampling and histological analysis at different stages of ESCC tumorigenesis (Figure 1). By sampling normal esophageal mucosa, low-grade intraepithelial neoplasia (LGIN), high-grade intraepithelial neoplasia (HGIN), and tumor tissues from ESCC patients, organoids representing different stages of tumorigenesis can be constructed. Furthermore, paraffin embedding and sectioning of these organoids were performed, followed by immunofluorescence staining.

Discussion

The establishment and histological analysis of organoids represent a significant advancement in modeling tumor progression. The protocol offers notable advantages over existing methods for studying tumorigenesis²⁰. Unlike traditional 2D cell culture systems, organoids maintain complex three-dimensional architecture and cellular heterogeneity that better reflect in vivo conditions. Compared to animal models, organoids derived from human tissue more accurately represent human disease charac...

Materials

Name	Company	Catalog Number	Comments
0.22 μm filter	Merck	Cat#SLGPR33RB
24-well plate	Corning	Cat#3524
4% Paraformaldehyde	Beyotime	Cat# P0099
70 μm sterile strainer	Falcon	Cat#352350
A83-01	Tocris Bioscience	Cat# 2939
Advanced DMEM/F12	Gibco	Cat# 12634028
Agar	Solarbio	Cat# A8190
Anti-Anti (Antibiotic-Antimycotic)	Gibco	Cat# 15240062
B-27 supplement	Gibco	Cat# 17504044
CO2 incubator	Thermo	Cat#371GPCN
Collagenase IV	Gibco	Cat# 17104019
Cryostor	STEMCELL	Cat# 07930
DMEM	Corning	Cat# 10-013-CV
EGF	Gibco	Cat# PHG0313
Fetal bovine serum	Cell Technologies	Cat# 30070
G-418	Sigma	Cat# A1720
Gelatin	Solarbio	Cat# G8061
GlutaMAX	Gibco	Cat# 35050061
Growth factor-reduced Matrigel	Corning	Cat# 354230
HE staining kit	Beijing Yili Fine Chemicals Co., Ltd	NA
HEPES	Gibco	Cat# 15630080
Histological pen	Zsbio	Cat#ZLI-9305
Hygromycin B	Sigma	Cat# 400050
Immunohistochemical staining kit	ZSGB-BIO	PV-8000
L-WRN	ATCC	CRL-3276; RRID:CVCL_DA06
N-2 supplement	Gibco	Cat# 17502048
Neutral gum	Zsbio	Cat#ZLI-9555
Opal 5-Color Manual IHC Kit	PANOVUE	Cat# 10144100100
PBS	MeilunBio	Cat#MA0015
Rabbit Monoclone anti-PD-L1	CST	Cat# 13684; RRID:AB_2687655
Rabbit Polyclonal anti-Ki67	Abcam	Cat# ab16667; RRID:AB_302459
Rabbit Polyclonal anti-KRT6A	Proteintech	Cat# 10590-1-AP; RRID: AB_2134306
Sheep serum	Zsbio	Cat#ZLI-9056
TrypLE Express	Gibco	Cat# 12604021
TrypLE-EDTA	Gibco	Cat#15400-054
Whole slide image scanner	Hamamatsu	Cat#C13210
Y-27632	Selleck Chemicals	Cat# S1049