Establishment and identification of a patient-derived organoid model for esophagogastric junction adenocarcinoma

Abstract

BACKGROUND: Traditional cell culture models have substantial limitations in accurately simulating the biological characteristics of tumors. Organoid technology, which retains key biological features of primary tumors, provides technical support for in-depth exploration of the pathogenesis of esophagogastric junction adenocarcinoma (EGJA) and for precision diagnosis and treatment research. OBJECTIVE: To construct a patient-derived organoid model of EGJA, evaluate its consistency with the biological characteristics of the primary tumor, and provide a standardized in vitro model for studying the pathogenesis and precision diagnosis and treatment of EGJA. METHODS: Surgical resection specimens from 50 patients with EGJA were collected for tissue dissociation, culture, and organoid model construction. Cell viability in organoid models was analyzed by fluorescence co-localization staining. Structural features of organoid models and primary tumor tissues were analyzed by hematoxylin-eosin staining. Mucin secretion phenotype in organoid models was detected by periodic acid-Schiff staining. Immunohistochemical staining was used to evaluate the consistency of organoid models with primary tumor histopathology. Chi-square test and univariate logistic regression were used to analyze the correlation between organoid model establishment success rate and patient clinicopathological features and tissue characteristics. RESULTS AND CONCLUSION: A patient-derived EGJA organoid model culture system was successfully established, with a primary culture success rate of 86.0% (43/50). Organoids exhibited typical three-dimensional structures, gradually developing from cell clusters into glandular-like three-dimensional solid spheres. After 7-10 days of primary culture, they could be stably passaged, and no significant phenotypic changes were observed after multiple passages. After cryopreservation and resuscitation, organoids still proliferated stably. Fluorescence co-localization staining showed that organoid models maintained high viability at different culture stages. Hematoxylin-eosin staining results showed that both organoid models and primary tumor tissues exhibited atypical glandular arrangement and high nuclear-to-cytoplasmic ratio. Periodic acid-Schiff staining showed positive mucin secretion. Immunohistochemical staining showed high consistency in the positive expression rates of Ki67, CEA, CK7, and Cadherin17 between organoid models and primary tumor tissues. Furthermore, chi-square test and logistic regression analysis showed that poorly differentiated tumor tissue (P=0.02), major pathological response to neoadjuvant chemotherapy (P=0.007), tumor tissue ex vivo time (P=0.006), and tumor mass (P=0.006) significantly affected the success rate of patient-derived EGJA organoid model establishment.

1. Introduction

Esophagogastric junction adenocarcinoma (EGJA) is a malignancy arising within 5 cm above and below the anatomical junction of the esophagus and stomach [1]. A 2018 global analysis of esophageal and gastric cancer registry data revealed that East Asia accounted for as high as 67.1% of new EGJA cases worldwide, showing a significant upward trend compared with 2012 data [2-3]. Over the past two decades, the incidence of EGJA in China has also been rising, mirroring trends observed in Western countries [4-6]. EGJA is typically characterized by strong invasiveness, early metastasis, and poor prognosis [7]. Moreover, EGJA exhibits significant tumor heterogeneity, with marked differences among patients in biological behavior, gene expression patterns, and drug sensitivity [8], indicating that standardized treatment protocols are not universally applicable and that personalized therapeutic strategies are required. However, achieving this goal faces numerous challenges, partly due to the lack of specific research models that reflect the biological characteristics of EGJA.

Current traditional models in EGJA research have notable limitations. Two-dimensional cell lines often lose key characteristics of the primary tumor, while animal models suffer from species differences and high costs, both failing to accurately simulate tumor biology. In contrast, organoids can preserve the genetic background and heterogeneity of primary tumors, reconstituting the diversity of cellular subpopulations, effectively overcoming the shortcomings of traditional two-dimensional culture models, and providing a more ideal platform for EGJA research [9-11].