{"paper_id":"29780bcf-65ff-436c-8302-e64c3f424f34","body_text":"Advancing Precision Medicine in Esophageal Squamous Cell Carcinoma Using Patient-Derived Organoids | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Advancing Precision Medicine in Esophageal Squamous Cell Carcinoma Using Patient-Derived Organoids Suya Shen, Bing Liu, Wenyan Guan, Ziyao Liu, Yuqing Han, Yingzhe Hu, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5357253/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Dec, 2024 Read the published version in Journal of Translational Medicine → Version 1 posted 5 You are reading this latest preprint version Abstract Background & Aims: Patient-derived organoids (PDOs) represent a promising approach for replicatingthe characteristics of original tumors and facilitating drug testing for personalized treatments across diverse cancer types. However, clinical evidence regarding their application to esophageal cancer remains limited.This study aims to evaluate the efficacy of implementing PDOs in clinical practice to benefit patients with esophageal squamous cell carcinoma (ESCC). Methods: Fresh surgical biopsies were obtained from patients with esophageal cancer for the establishment of PDOs. These PDOswere subsequently characterized through histological analysis. A customized drug panel, based on standard-of-care chemotherapy regimens, was applied to the PDOs. The resulting drug sensitivity profiles were then correlated with the clinical responses observed in individual patients undergoing actual treatment. Results: A total of 34 PDOs were successfully established with a 61.8% success rate. The classification method based on chemotherapy sensitivity closely corresponded to clinical responses. The paclitaxel plus cisplatin (TP)-sensitive group demonstrated significantly longer progression-free survival (PFS) compared to the resistant groups, Hazard ratio (HR), 5.12; 95% confidence intervals (CI), 0.58-44.71; p <0.05), thus illustrating the potential of this approach for identifying personalized treatment strategies. Conclusion: Organoid biobanks wereestablished across multiple institutes to facilitate PDOs-based functional precision medicine. The findings demonstrate that this framework offers robust predictive value in clinical settings, enhances precision therapeutics, and advances drug discovery for esophageal cancer. Esophageal Squamous Cell Carcinoma Organoids Chemotherapy Precision Medicine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Surgical Relevance The high recurrence rate and varied response to chemotherapy in esophageal squamous cell carcinoma (ESCC) presents a critical challenge for surgical and oncological teams. This study demonstrates the successful application of patient-derived organoids (PDOs) in predicting drug sensitivity for ESCC, using organoids established directly from surgical biopsy samples. By correlating drug response data from PDOs with clinical outcomes, particularly progression-free survival (PFS) in patients sensitive to paclitaxel plus cisplatin, our research underscores the potential of PDOs to guide personalized treatment regimens post-surgery. Furthermore, the establishment of a PDO biobank enables reproducible, institution-wide testing and supports real-time, patient-specific decision-making, offering a promising addition to the surgical oncologist’s toolkit for precision medicine in ESCC. This advancement not only holds promise for tailored chemotherapy regimens but also enhances our ability to predict patient outcomes and support personalized postoperative management in clinical practice. Introduction Esophageal carcinoma (EC) poses a significant threat to public health due to its increasing incidence and inconspicuous symptoms, which often result in late diagnosis and poor prognosis 1 . The conventional standard-of-care for treating esophageal cancer remains surgical resection combined with chemotherapy 2 – 4 . However, many patients fail to benefit from these treatments due to tumor heterogeneity, therapeutic resistance, and substantial side effects 5 , 6 . Moreover, the abundance of available chemotherapy drugs has made it increasingly challenging for clinicians to select appropriate regimens based solely on clinical expertise and patient values. In the past decade, targeted therapies for advanced stages of esophageal adenocarcinoma (EAC) have been limited and offer minimal benefits, while progress in esophageal squamous cell carcinoma (ESCC) has been virtually non-existent 7 . Consequently, current precision medicine strategies for EC are inadequate, underscoring the need for additional preclinical models to contribute to effective treatments and facilitate personalized therapy. Numerous studies have focused on identifying significant biomarkers for predicting clinical prognosis; however, reliable preclinical models for evaluating patient responses to chemotherapeutic and targeted agents are lacking 6 , 8 , 9 . Recently, organoids derived from individual patients have emerged as a promising predictor in precision medicine due to their various advantages. Unlike patient-derived two-dimensional cell lines growing as flat monolayers, patient-derived organoids (PDOs) are cultured to form and maintain their three-dimensional structure, more accurately simulating the self-organization and cellular interactions of the tumor microenvironment 10 , 11 . Moreover, in contrast to the extended tumorigenic processes required for genetically engineered mouse models and patient-derived xenografts (PDXs), organoids can be generated directly from primary tissue within a reasonable timeframe. They accurately recapitulate the genetic and morphological characteristics of the parental tumors with a high success rate and enable long-term expansion 12 – 14 . These advantages have been demonstrated in various primary tumors, including liver 15 , pancreas 16 , breast 17 , colorectal cancer 18 , and other solid tumors. The fidelity of PDOs makes them highly valuable for predicting patient-specific responses to therapies and tailoring individualized treatment plans. However, research on the tissue characteristics of organoids derived from EC, particularly esophageal squamous cell carcinoma organoids (ESCOs), remains limited, and clinical evidence is scarce. The advancement of esophageal organoids provides significant molecular and mechanistic insights into various physiological and pathological dynamics, furthering translational research and personalized medicine 19 , 20 . A recent study by Li et al. developed a panel of EAC organoids that mirror the characteristics of primary tumors. The study found that the sensitivity of most organoid cultures corresponded to the clinical response to neoadjuvant chemotherapy, suggesting that PDOs are valuable for evaluating treatment effectiveness in a preclinical setting. 21 . While the optimization and validation of ESCOs represent significant progress, further research is necessary to conduct comprehensive profiling and assess the application of in vitro drug testing in EC. Consequently, we established a robust protocol for generating primary ESCOs culture from multiple centers, developed a standardized drug testing platform utilizing organoids, and initiated a retrospective clinical study to evaluate the potential of PDOs as predictive biomarkers for chemotherapeutic sensitivity in ESCC patients. Our case studies demonstrated that the PDO-based screening platform could effectively guide personalized treatment, given that the response to conventional anticancer regimens in PDOs correlates with the clinical response of patients. This study establishes a novel PDOs-based framework that shows promise for advancing precision medicine in EC. Materials and methods 2.1 Participant selection and study design Adults (age ≥ 18 years) with histologically and radiologically confirmed esophageal cancer were enrolled in the study based on clinical and radiologic evidence. The primary objective was to establish efficient culture methods for PDOs suitable for drug testing and to evaluate their potential in correlating patient outcomes with standard-of-care treatments for translational research. Figure 1 illustrates the study design flowchart. Esophageal cancer specimens were primarily obtained from patients undergoing surgical resection or endoscopic biopsy at Nanjing Drum Tower Hospital, Beijing Cancer Hospital, and Jiangsu Hospital of Traditional Chinese Medicine. The Institutional Ethics Committees approved all tissue donations and experiments (approval number: 2021-237-02). Two independent pathologists confirmed the histological characteristics of the samples. Following surgical tumor resection, patients received clinically verified standard-of-care chemotherapy regimens based on physician's empirical choice. Comprehensive clinical characteristics were obtained and recorded using data collection forms from the hospitals' internal electronic medical records system. Clinical responses were assessed using RECIST 1.1 criteria. In some instances, comprehensive clinical data were unavailable due to loss of follow-up or patients' unwillingness to accept the suggested adjuvant therapy. Participant enrollment occurred from September 2021 to August 2022. 2.2. Patient material processing and generation of organoids Patient specimens were washed with Hanks' Balanced Salt Solution (HBSS, Sigma) and minced into 1–2 mm³ pieces using sterile scissors. The tissue underwent enzymatic digestion at 37°C for 30 minutes with 1 mg/mL collagenase type II (Worthington), 1 mg/mL collagenase type IV (Worthington), 0.1 mg/mL DNase I (Worthington), and 100 U/mL penicillin-streptomycin (Life Technologies). The resulting fragments suspension was filtered through a 70-µm strainer, and undigested fragments were subjected to an additional 20 minutes of digestion before being re-filtered. The isolated cells were then resuspended in 7.5 mg/mL Matrigel (Corning) supplemented with ESCOs culturing media: Advanced DMEM/F12 (Gibco) was supplemented with 10 mM HEPES (Gibco), 1×GlutaMAX (Gibco), 100 U/mL penicillin-streptomycin (Gibco), 1×B27 (Gibco), 1×N2 (Gibco), 50 ng/mL EGF (Novoprotein), 10 ng/mL FGF-10 (Novoprotein), 100 ng/mL Noggin (Novoprotein), 250 nM A83-01 (Beyotime), 10 µM SB202190 (Beyotime), 100 ng/mL Wnt3a (Novoprotein), 100 ng/mL R-spondin 1 (Novoprotein), 1 mM N-Acetylcysteine (Sigma), and 10 µM Y-27632 (Beyotime). Organoids were incubated at 37°C in 5% CO₂, with the medium changed every 2–3 days. For passaging, organoids were split every 1–2 weeks at a ratio of 1:2 or 1:3. Organoids were collected and digested with TrypLE Express (Gibco) at 37°C for 5–10 min, then washed with Advanced DMEM/F12 and resuspended in Matrigel, supplemented with organoid culture medium. Organoid quantification was conducted using bright-field imaging with an IX73 microscope (Olympus, Tokyo, Japan). 2.3. Histology and immunohistochemistry Surgical resection tissues were fixed in 10% formalin overnight and embedded in paraffin blocks after dehydration. Confluent organoids were collected, rinsed with ice-cold PBS to dissolve the Matrigel, and fixed in 10% formalin for 1 hour. The organoids were then settled by gravity and embedded in paraffin. All sample blocks were sectioned into 3-µm-thick slides, followed by dewaxing, rehydration, and standard hematoxylin and eosin (H&E) staining. For immunohistochemistry (IHC) staining, paraffin slides underwent deparaffinization and rehydration, then were incubated with primary antibodies specific to p53 (1:100, clone D-O7, ZSGB-Bio, ZM-0408), CK5 (1:100, clone OTI1C7, ZSGB-Bio, ZM-0313), SOX-2 (1:100, EP103, ZSGB-Bio, ZA-0571), and KLF5 (1:200, Invitrogen, PA5-27876) at 4°C overnight. Subsequently, sections were washed and incubated with secondary antibody at room temperature for 1 hour, with negative controls applied to all samples. The slides were scanned and imaged using a microscope (Nikon, Eclipse CI, Japan). The immunoreactivity of the slides was assessed independently by two senior pathologists who blinded to this study. QuPath software was utilized for digital pathology and image analysis. 2.4. Drug screening assays Drug testing was conducted with modifications to accommodate the 384-well format, as previously validated 22 . The primary organoids in good condition were collected and dissociated into single cells by incubating in TrypLE (Gibco) for 10 minutes. The single cells resuspended in the organoid culture medium with 5% Matrigel, and approximately 2000 cells were seeded per well of 384-well plates (Thermo Fisher Scientific, 242764). They were cultured for 3 days to allow cell recovery before drug treatment. Eight concentration points with 4-fold dilutions were set for each single drug or drug combination and dispensed using liquid-handling robots, with each concentration tested in triplicate. The starting concentrations for monotherapy were 100 µM for cisplatin (DDP) and 5-fluorouracil (5-FU), and 5 µM for Paclitaxel. For combination therapy, paclitaxel and cisplatin (TP) treatments, as well as vinorelbine and cisplatin (NP) treatments, were assayed in triplicate drug matrices. The starting concentrations were 100 µM for cisplatin and 5 µM for both paclitaxel and vinorelbine. For 5-FU plus cisplatin (FP) treatments, both components started at 100 µM. Similarly, for FOLFIRINOX (FFX) treatment, each component began at a concentration of 100 µM. DMSO at 0.5% (v/v) served as the negative (vehicle) control. Cell viability was assayed using Cell Counting-Lite 3D Luminescent Cell Viability Assay (Vazyme) following 5 days of drug treatment. Luminescent detection of cell viability was performed after 25 minutes of incubation with the cells at room temperature. In-vitro responses were evaluated using dose-response curves for single agents and drug combinations. IC50 and AUC (area under the curve) values were determined using Origin 2022 and GraphPad Prism 8.0. Normalized AUC values were calculated by dividing the AUC of each compound by the maximum AUC observed within the measured concentration range. 2.5 Quantification and Statistical Analysis Statistical analyses for each dataset are detailed in the corresponding figure legends. Clinical data were extracted and managed from individual electronic health records. Group comparisons were conducted using the chi-squared test (or Fisher's exact test when appropriate) for categorical variables and the student’s t-test for continuous variables. Tumor response was assessed according to RECIST guidelines (version 1.1) 23 . Data analyses were performed using Origin 2022 (OriginLab Corporation, USA) employing nonlinear regression (curve fit). Progression-free survival (PFS) differences were evaluated using Kaplan-Meier survival curves and the log-rank test with GraphPad Prism 8. p values < 0.05 were considered statistically significant. Results 3.1. Patient demographics and tumor characteristics This study involved 55 human samples from individual patients with confirmed pathological ESCC diagnoses for PDO generation. Fifty specimens were obtained through surgical resection from Nanjing Drum Tower Hospital and Peking University Cancer Hospital, while five samples were isolated from endoscopy biopsies at Jiangsu Chinese Medicine Hospital ( Supplementary Table S1 ). Figure 2A presents a consort diagram illustrating the sample distribution. Baseline characteristics included patient demographics, tumor location, histological classification, cancer stage, and primary treatment. All samples were derived from primary tumors without distant metastasis at collection time, classified as ESCC, with a median patient age of 64 years. Most tumors were moderately differentiated and located in the middle or lower esophagus, aligning with EC epidemiology 24 . Four organoids generated from esophagogastroscopy biopsy were associated with patients lost to follow-up. Of the 26 patients who underwent surgery, 14 received subsequent adjuvant chemotherapy, including six who underwent neoadjuvant chemotherapy, indicating tissue acquisition in a post-neoadjuvant condition. Among these, one patient developed a low platelet count after two cycles of paclitaxel combined with platinum treatment, preventing the continuation of subsequent regimens. Another patient received S-1 as adjuvant therapy but was lost to follow-up three months post-surgery. 3.2. Generation and histological characterization of ESCOs Fresh ESCC tissue samples were obtained from esophagectomy or esophagogastroscopy biopsy and seeded into an esophageal cancer organoid culturing medium as described in the methods section. The study design is illustrated in Fig. 2B. Organoids were cultivated in a Matrigel-supported growth medium to facilitate expansion. As shown in Fig. 2A, 17 cases failed to initiate a culture due to insufficient tumor cells or lack of expansion beyond passage two, while four organoids experienced bacterial contamination during cultivation. Overall, we achieved a ~ 62% PDO culture success rate (34 of 55 cultures) throughout the study, aligning with previous reports on gastrointestinal cancers 6 , 25 . For 4 organoids, the clinical response of the corresponding patients was not evaluable due to a short follow-up period. Ultimately, drug testing was conducted on the remaining 30 organoid lines, with the patient's demographic and clinicopathological characteristics respectively detailed in Table 1 and Supplementary Table S2 . Among the 30 drug assay reports, 24 organoids were derived from chemotherapy-naïve tumors, while 6 were derived from chemotherapy-treated tumors. We subsequently evaluated the histological profiles of the primary tissue and corresponding ESCOs to demonstrate their heterogeneous growth states (Fig. 3). Despite their diverse morphologies, the comparison demonstrated that the ESCOs replicated notable morphological similarities to the matched original tumors. The characteristic solid and compact structures of the organoids are evident in both bright-field and H&E staining, reflecting a cohesive cellular arrangement that mimics key architectural features of stratified squamous epithelium (Fig. 3A) 20 , 21 . Immunohistochemical staining of the organoids also revealed similar expression levels of the ESCC prognostic marker p53, as well as the squamous epithelial and ductal epithelial marker CK5/6 26, 27 . The expression of the tumor differentiation marker KLF5 and the tumor proliferation marker SOX2 in the organoids further substantiates their alignment with the corresponding histopathological specimens 28 , 29 . As observed in ESCO57, the specimen consisted of moderately differentiated squamous cell carcinoma cells, displaying overexpression of CK5/6, p53, and KLF5, with low expression of SOX2. The matched organoid similarly exhibited nearly negative expression for SOX2 (Fig. 3B). These findings strongly confirmed that the organoids faithfully preserved the histological organization and morphological heterogeneity of the original tumors. 3.4. Drug assay of conventional chemotherapeutics in ESCOs To evaluate the applicability of PDO lines in assessing personalized chemotherapeutic responses, therapeutic profiling was conducted on 30 ESCOs using four commonly employed standard-of-care chemotherapeutic agents: single-agent cisplatin (DDP) and combination therapies—paclitaxel plus cisplatin (TP), vinorelbine plus cisplatin (NP), and 5-fluorouracil plus cisplatin (FP). Each group underwent screening using an 8-point dilution series, with cell viability and ESCO response quantified and analyzed through dose-response curves and AUC. Detailed normalized AUC data are provided in Supplementary Table S3. The ESCO profiling revealed significant variability in response to both single and combination chemotherapeutic agents among individual patients (Fig. 4A) . Violin plots illustrate the distribution of AUC values, highlighting a spectrum of resistance and sensitivity across the samples (Fig. 4B) . Using DDP as a representative drug, individual IC50 and 95% CI from dose-response testing demonstrated that ESCOs exhibit distinct chemotherapeutic response profiles to varying doses of cisplatin (Fig. 4C). The in vitro chemosensitivity of ESCOs to DDP and the combination therapies TP, NP, and FP is presented as standardized AUC values (Fig. 4D) . The 10 representative ESCOs described in this study are dispersed throughout the whole group, indicating that they represent a range of sensitivity and resistance. Consequently, ESCOs serve as valuable tools for drug response assays, reflecting diverse responses to various conventional chemotherapeutics. Several ESCOs were also treated with FOLFIRINOX (FFX), partially shown in Fig. 5E. To further elucidate the correlation between ESCO sensitivity results and clinical outcomes, the assays were divided into three subgroups based on the AUC values for each chemotherapeutic agent: the ESCOs with their AUC ranked the top 33% were regarded as the least responsive (resistant ESCOs), those with their AUC ranked the lowest 33% as the most responsive (sensitive ESCOs), and those with their AUC ranked the middle 34% AUC as moderately sensitive ESCOs. Among the 30 ESCOs subjected to drug tests, 14 patients received chemotherapy, with detailed AUC values, sensitivities, and corresponding patient responses presented in Table 2. Given that most patients in this study were treated with TP, the correlation between clinical outcomes and PDO-based drug testing responses was emphasized. 3.5. ESCOs represent chemotherapy response and clinical prognosis of ESCC patients To assess the clinical viability of utilizing PDOs platform for ESCC patients, we analyzed retrospective clinical data and compared the pharmacotyping outcomes of ESCOs with the corresponding patients' clinical responses. Among the 30 ESCOs, 14 were from patients who received chemotherapy: 6 were from patients who underwent neoadjuvant chemotherapy, while 8 were from patients who received adjuvant chemotherapy, with follow-up periods ranging from 2.8 to 22.4 months (Table 2). PFS is defined as the duration from initial treatment until signs of disease progression appear. The treatment responses of ESCC patients classified by clinicians as having a partial response (PR) or stable disease (SD) for more than one year were categorized as indicating a good response (chemotherapy-sensitive). Conversely, patients with progressive disease (PD), including tumor recurrence, metastasis, or disease progression within 1 year were classified as having a poor response (chemotherapy-resistant) as illustrated in Fig. 5A. Despite 2 patients being lost to follow-up, 9 out of the remaining 12 patients who underwent chemotherapy were classified as having a good clinical response, while the other 3 were classified as having a poor response. In the drug assay performed on the ESCOs, the organoids exhibited distinct responses to various chemotherapy regimens (Fig. 5A). As most patients received the TP regimen, we observed that 9 ESCOs demonstrated sensitivity to TP, consistent with their clinical outcomes. However, 2 stage III patients, expected to benefit clinically based on their PDO drug assays, exhibited progressive disease, indicating a lack of correlation with the ESCO responses. Lastly, one patient (ESCO25), who experienced rapid disease progression while on paclitaxel plus cisplatin, had an organoid that displayed resistance to TP. In total, 10 out of 12 (83.33%) ESCC patients exhibited consistency in drug response results with those observed in their derived PDOs. Furthermore, we compared the PFS of the two groups of patients based on their sensitivity to the TP regimen. The patients whose ESCOs were sensitive to TP in our assay exhibited significantly higher PFS, suggesting that in vitro sensitivity of ESCOs to TP is associated with a longer clinical response of the corresponding patients. The hazard ratio (HR) was 5.12 (95% CI = 0.58–44.71, log-rank test, P < 0.05; Fig. 5B ). These complementary drug assays can be performed within 3 to 4 weeks, aligning with the typical timeframe for clinical treatment decisions, which are generally made 2 to 3 months after the last treatment to evaluate alternative options. PDO-based drug testing can thus serve as a stratification tool to identify optimal therapeutic combinations for individual patients. 3.6 Case report We closely monitored the disease progression of three patients from whom we derived ESCOs, categorized as sensitive (ESCO10 and ESCO13) or resistant (ESCO25) to their respective treatments. As depicted in Fig. 5C , Patient ESCO10, diagnosed with ESCC, underwent cycles of paclitaxel plus platinum during neoadjuvant chemotherapy, achieving a confirmed PR with a reduction in primary tumor size after 2 months of treatment. The patient subsequently underwent surgery and continued TP treatment postoperatively, maintaining stable disease for over one year throughout adjuvant chemotherapy. To assess whether PDO profiling reflected the patient's response to chemotherapy, we conducted a drug assay on the ESCOs, revealing distinct sensitivities among various regimens. The AUC of TP ranked highest (AUC = 0.64), indicating that ESCO10 was responsive, aligning with the observed clinical response. A comparable drug response and consistent clinical outcome were also observed in patient ESCO13 (AUC = 0.59). Regarding the resistant case ESCO25 (AUC = 0.78), the patient was diagnosed with ESCC (pT3N2M0, stage IIIB) with perigastric lymph node metastasis, as confirmed by the postoperative pathology report. The patient received adjuvant TP treatment at a local hospital and subsequently underwent adjuvant radiotherapy. Progressive lymph node metastasis was detected 6 months post-surgery, consistent with the resistant response observed in the ESCO (AUC = 0.78). Consequently, the drug response of the PDOs demonstrated both sensitivity and resistance characteristics of their associated tumors Fig. 5D . This preclinical model shows potential value in guiding the selection of appropriate clinical chemotherapy regimens, potentially helping patients avoid ineffective treatments. Discussion EC is a severe malignancy that urgently requires clinical advancements to enhance patient outcomes. The ongoing areas of significant interest include early detection and optimal therapy for patients. Emerging research utilizing PDOs aims to verify their sensitivity and specificity as predictive biomarkers for guiding precision medicine, particularly in adjuvant chemotherapy and immunotherapy. This approach extends to liver, breast, colon, pancreatic, and lung cancers 15 – 18 , 30 – 32 . In 2011, Sato first reported organoids derived from human Barrett's epithelium, enabling long-term expansion 33 . Subsequently, the application of PDOs in EC has progressed. Li established a reliable EAC organoid biobank that accurately replicated the phenotypic and molecular characteristics of EAC, facilitating drug screening for novel therapeutic strategies 21 . Concurrently, Kijima et al. developed a robust system for culturing organoids from esophageal and oropharyngeal squamous cell carcinoma patients with high success rates, enabling therapy response evaluation and exploration of underlying mechanisms 19 . However, further studies optimizing drug panel testing for EC-derived organoids are necessary, along with validation through clinical trials. ESCC accounts for approximately 90% of all EC cases, with particularly high prevalence in Asia and parts of Africa 34 . To advance the feasibility of ESCOs-based approaches in ESCC, this study presents several novel findings from a multi-institution randomized, retrospective clinical trial. First, we demonstrate the establishment of organoids with a moderate success rate, showing that ESCOs recapitulate the morphological heterogeneity and histological characteristics of the matched original tumor tissue across various differentiation statuses and pathological types. Subsequently, we implemented a robust PDO drug screening platform, utilizing a reliable biobank to assess multi-regimen drug sensitivity, which revealed differential responses of PDOs to conventional chemotherapeutic agents. Furthermore, we tracked real-world clinical outcomes and assessed their correlation with in vitro drug screening data, indicating high consistency and supporting the integration of translational organoid technologies with personalized therapy. Our approach enables organoid growth within a moderate timeframe (3–6 weeks), with primary drug testing reports completed in an additional 7 days. This timeline is clinically relevant, considering that restaging scans are typically performed 2 to 3 months after initiating treatment, serving as criteria for evaluating whether to continue current therapy or switch to alternative treatment 35 . Collectively, these findings support the rapid advancement and significant potential of translating organoid technologies. A comprehensive analysis of drug sensitivity profiles, examining both single and combination regimens in a group of 30 PDOs, revealed significant variability in organoid responses to various chemotherapy treatments and individual drug assays across different PDOs. Notably, a strong correlation was observed between the responses in PDOs and those reported in clinical settings. This correlation aligns with recent retrospective data reported for other cancer types in real-world studies 31 , 35 . In contrast to previous studies, this multi-center study introduces a novel classification system using AUCs as the index for assessing PDOs' sensitivities to various treatment regimens, aiming to predict clinical disease control Future enrollment of additional cases will enhance the representativeness of this biobank. For chemotherapy-sensitive organoids from a patient expected to respond well to a specific regimen but with an unmatched clinical response, a review of the patient's entire inpatient record revealed an inability to continue chemotherapy cycles due to side effects. This indicates that even when PDO-based drug assays demonstrate high sensitivity to chemotherapy, patients unable to complete treatment due to adverse effects may experience poor clinical outcomes. This study presents several limitations and opportunities for future research. While achieving high consistency between organoid drug assays and clinical responses, each sample in this study originated from individual patients during surgery or endoscopic biopsy, including initial tumors and several post-NAT tissues exposed to chemotherapy. However, the absence of paired chemo-naïve and post-chemotherapy samples resulted in a missed opportunity to collect materials from the same patient at different disease stages for longitudinal organoid generation 35 . Cancer treatment is a complex, multifaceted, and ongoing process 36 . The variability in treatment effectiveness among cancer patients is intricate and associated with numerous factors, including post-operative outcomes, combination therapies, chemotherapy side effects, overall health conditions, and tumor characteristics 37 . Although the chemosensitivity of organoids may partially reflect the tumor's response at a specific point in time, it has limitations in capturing tumor evolution and changes in overall drug treatment response. An additional related challenge is the limited source of tissue; all samples in this study were acquired from patients in suitable condition for surgical resection, indicating a lack of tumor tissues of advanced stages and from metastatic sites to generate a more comprehensive biobank representing the disease at different stages. It is noteworthy that the success rate of ESCC organoid generation was lower and requires enhancement compared to other cancer types, such as colorectal cancer, breast cancer, and other adenocarcinoma cancers 10 , 12 , 30 . This disparity and associated challenges potentially stem from the inherent heterogeneity of squamous cell carcinoma and its dependence on specific stromal interactions and extracellular matrix components, which are challenging to replicate in standard organoid cultures 38 . Adenocarcinoma, originating from glandular structures in epithelial tissue, typically exhibits more predictable growth patterns, facilitating well-established organoid culture protocols 33 . Sachdeva et al. report developing reliable protocols to establish PDOs from EAC with a 80% success rate, compared to their 60% success rate in ESCC organoids generation 38 . While we did not initially control the source of samples, due to the prevalence of EC in our region, all samples included in this study were diagnosed as ESCC through professional histological analysis. Future studies should incorporate various histological subtypes, as ongoing technological advancements and refined cultural conditions will gradually mitigate the challenges associated with organoid generation. In addition to tumor organoids, the tumor microenvironment, composed of stromal cells and inflammatory cells, plays a crucial role in influencing tumor behavior and drug-resistant mechanisms 39 . Co-culture models that generate patient-derived organoids in combination with non-cancerous cells represent promising approaches to better replicate the heterogeneous features of the original tumor environment in vitro. Conclusion In conclusion, this study demonstrates that PDO-based drug assays provide significant insights into standard treatment efficacy for ESCC patients. The successful generation of PDOs from a heterogeneous cohort of ESCC patients within a clinically relevant timeframe yielded results that strongly correlate with individual clinical outcomes and drug sensitivity profiles. Further investigation is warranted to validate this approach in a larger patient cohort, including the incorporation of PDOs derived from patients with metastatic disease. Abbreviations EAC (esophageal adenocarcinoma) EC (esophageal carcinoma) ESCC (esophageal squamous cell carcinoma) ESCOs (esophageal squamous cell carcinoma organoids) PDOs (patient-derived organoids) Declarations Funding This research was funded by the National Nature Science Foundation of China (No. 82372988), the Health Technology Development Project of Nanjing City (No. YKK21077), and the Clinical Trials funding from the Affiliated Drum Tower Hospital, Medical School of Nanjing University (No. 2022-LCYJ-PY-03). Suya Shen received support from the China Scholarship Council (No. 202306190025). Acknowledgments We extend our gratitude to all patients and their families who participated in this study and generously contributed their samples. Furthermore, we acknowledge the invaluable support of our clinical and research team members for their assistance with tissue collection and processing. Author contributions Suya Shen (Conceptualization, methodology, writing original draft, data curation) Jingjing Li (Conceptualization: Lead; Funding acquisition; Investigation; Supervision; Supporting) Zheng Hongping, Bing Liu (Original draft writing; Formal analysis) Wei Ren, Yudong Qiu (Supervision; validation) Wenyan Guan (Histology analysis: Supporting; Investigation) Jian He, Jie Shen, Weifeng Tang, Pengju Zhang (Data analysis; Methodology) Yuqing Han, Yingzhe Hu (Data collection, writing – review & editing: Equal) Ziyao Liu, Yiqiang Chen, Siyuan Liu (Sample acquisition, data collection) Conflict of interest Suya Shen, Bin Liu, and Wenyan Guan contributed equally to this work. 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New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 2009;45:228–47. Then EO, Lopez M, Saleem S, et al. Esophageal Cancer: An Updated Surveillance Epidemiology and End Results Database Analysis. World J Oncol. 2020;11:55–64. Vlachogiannis G, Hedayat S, Vatsiou A, et al. Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science. 2018;359:920–6. Kaufmann O, Fietze E, Mengs J, et al. Value of p63 and cytokeratin 5/6 as immunohistochemical markers for the differential diagnosis of poorly differentiated and undifferentiated carcinomas. Am J Clin Pathol. 2001;116:823–30. Zhong L, Li H, Chang W, et al. TP53 Mutations in Esophageal Squamous Cell Carcinoma. Front Biosci (Landmark Ed). 2023;28:219. Zhang J, Wang Z, Zhao H, et al. The roles of the SOX2 protein in the development of esophagus and esophageal squamous cell carcinoma, and pharmacological target for therapy. Biomed Pharmacother. 2023;163:114764. Jiang YY, Jiang Y, Li CQ, et al. TP63, SOX2, and KLF5 Establish a Core Regulatory Circuitry That Controls Epigenetic and Transcription Patterns in Esophageal Squamous Cell Carcinoma Cell Lines. Gastroenterology. 2020;159:1311–27. e19. Tan T, Mouradov D, Lee M, et al. Unified framework for patient-derived, tumor-organoid-based predictive testing of standard-of-care therapies in metastatic colorectal cancer. Cell Rep Med. 2023;4:101335. Wang HM, Zhang CY, Peng KC, et al. Using patient-derived organoids to predict locally advanced or metastatic lung cancer tumor response: A real-world study. Cell Rep Med. 2023;4:100911. Takahashi N, Hoshi H, Higa A et al. An In Vitro System for Evaluating Molecular Targeted Drugs Using Lung Patient-Derived Tumor Organoids. Cells 2019;8. Sato T, Stange DE, Ferrante M, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology. 2011;141:1762–72. Abnet CC, Arnold M, Wei WQ. Epidemiology of Esophageal Squamous Cell Carcinoma. Gastroenterology. 2018;154:360–73. Demyan L, Habowski AN, Plenker D, et al. Pancreatic Cancer Patient-derived Organoids Can Predict Response to Neoadjuvant Chemotherapy. Ann Surg. 2022;276:450–62. Liu B, Zhou H, Tan L, et al. Exploring treatment options in cancer: Tumor treatment strategies. Signal Transduct Target Ther. 2024;9:175. Miller KD, Nogueira L, Devasia T, et al. Cancer treatment and survivorship statistics, 2022. CA Cancer J Clin. 2022;72:409–36. Sachdeva UM, Shimonosono M, Flashner S, et al. Understanding the cellular origin and progression of esophageal cancer using esophageal organoids. Cancer Lett. 2021;509:39–52. Zheng S, Liu B, Guan X. The Role of Tumor Microenvironment in Invasion and Metastasis of Esophageal Squamous Cell Carcinoma. Front Oncol. 2022;12:911285. Tables Table 1 and 2 are available in the Supplementary Files section. Supplementary Files Table1.pdf Table2.pdf 2.ESCCSupplementarytables.xlsx Cite Share Download PDF Status: Published Journal Publication published 31 Dec, 2024 Read the published version in Journal of Translational Medicine → Version 1 posted Editorial decision: Major revision 20 Nov, 2024 Reviewers agreed at journal 09 Nov, 2024 Reviewers invited by journal 09 Nov, 2024 Editor assigned by journal 08 Nov, 2024 First submitted to journal 29 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-5357253\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":376091747,\"identity\":\"c3a4b7dd-7a8d-4b35-9095-857e75491cab\",\"order_by\":0,\"name\":\"Suya Shen\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYDACCTBpA2QwNjAwsBGvJY10LYehDGK0yM9uPvaA4c/5xH7p5gaGD2WHGfhnJODXYnDnWLoBY9vtxJlzDjYwzjh3mEHiBiEtEjlmQF/czt1wI7GBmbcN6EJCWuRn5H+TYPhzLnc/SMtfoBZ5QloYbuSwSTCwHcjdIAHUwgjUYkDQYTfSzCQS25LrZwBtOdhzLp3H8MwDQg5Lfibx4Y+dMf+M9IcPfpRZy8kdJ+QwEICpOQDEPESoHwWjYBSMglFACAAATt5GCrzL8jkAAAAASUVORK5CYII=\",\"orcid\":\"https://orcid.org/0000-0002-8960-119X\",\"institution\":\"Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Suya\",\"middleName\":\"\",\"lastName\":\"Shen\",\"suffix\":\"\"},{\"id\":376091748,\"identity\":\"9105fa4e-e4da-4fa9-9e29-5c730d861e39\",\"order_by\":1,\"name\":\"Bing Liu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Peking University Cancer Hospital: Beijing Cancer 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Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yiqiang\",\"middleName\":\"\",\"lastName\":\"Chen\",\"suffix\":\"\"},{\"id\":376091754,\"identity\":\"dbd5ae31-8e9a-435d-8010-52482a945557\",\"order_by\":7,\"name\":\"Siyuan Liu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Siyuan\",\"middleName\":\"\",\"lastName\":\"Liu\",\"suffix\":\"\"},{\"id\":376091755,\"identity\":\"9554d0e8-5352-4172-aaa7-e7111ac43d05\",\"order_by\":8,\"name\":\"Jian He\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jian\",\"middleName\":\"\",\"lastName\":\"He\",\"suffix\":\"\"},{\"id\":376091756,\"identity\":\"4bb9e6d2-b80e-477c-93e5-58bb45baeea7\",\"order_by\":9,\"name\":\"Zhiwen Li\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Zhiwen\",\"middleName\":\"\",\"lastName\":\"Li\",\"suffix\":\"\"},{\"id\":376091757,\"identity\":\"c9f9ef0b-a508-457a-bfed-517c6c755974\",\"order_by\":10,\"name\":\"Weifeng Tang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Weifeng\",\"middleName\":\"\",\"lastName\":\"Tang\",\"suffix\":\"\"},{\"id\":376091758,\"identity\":\"bc439ddb-0421-4602-ae90-be601cfe5717\",\"order_by\":11,\"name\":\"Pengju Zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Zhejiang Honray Medical Technology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Pengju\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":376091759,\"identity\":\"3ae94659-b86a-4441-810e-bc3a74da1621\",\"order_by\":12,\"name\":\"Wei Ren\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Wei\",\"middleName\":\"\",\"lastName\":\"Ren\",\"suffix\":\"\"},{\"id\":376091760,\"identity\":\"e2348f79-4ad5-4ed4-a864-381d1924b215\",\"order_by\":13,\"name\":\"Yudong Qiu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yudong\",\"middleName\":\"\",\"lastName\":\"Qiu\",\"suffix\":\"\"},{\"id\":376091761,\"identity\":\"ed53eb1e-9fa7-45c8-a4b7-3275e369b41c\",\"order_by\":14,\"name\":\"Hongping 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16:17:13\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":4311305,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5357253/v1/7b26da20-3f86-4cc3-a15e-40d7442288ef.pdf\"},{\"id\":71723072,\"identity\":\"e692b347-45d9-49a7-be41-5620f65283c6\",\"added_by\":\"auto\",\"created_at\":\"2024-12-18 05:32:22\",\"extension\":\"pdf\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":133856,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Table1.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5357253/v1/3b8702a67bcfebef8858ce2f.pdf\"},{\"id\":71723069,\"identity\":\"500b5552-fb2a-4a59-a3ba-8df6c3348186\",\"added_by\":\"auto\",\"created_at\":\"2024-12-18 05:32:22\",\"extension\":\"pdf\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":114572,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Table2.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5357253/v1/77775aef28b854caf9245af6.pdf\"},{\"id\":71724037,\"identity\":\"6ad19b7a-e2e6-411c-b8d1-1eabe743b836\",\"added_by\":\"auto\",\"created_at\":\"2024-12-18 05:40:22\",\"extension\":\"xlsx\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":26639,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"2.ESCCSupplementarytables.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5357253/v1/571fe7bf8e7d57c667547925.xlsx\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Advancing Precision Medicine in Esophageal Squamous Cell Carcinoma Using Patient-Derived Organoids\",\"fulltext\":[{\"header\":\"Surgical Relevance\",\"content\":\"\\u003cp\\u003eThe high recurrence rate and varied response to chemotherapy in esophageal squamous cell carcinoma (ESCC) presents a critical challenge for surgical and oncological teams. This study demonstrates the successful application of patient-derived organoids (PDOs) in predicting drug sensitivity for ESCC, using organoids established directly from surgical biopsy samples. By correlating drug response data from PDOs with clinical outcomes, particularly progression-free survival (PFS) in patients sensitive to paclitaxel plus cisplatin, our research underscores the potential of PDOs to guide personalized treatment regimens post-surgery. Furthermore, the establishment of a PDO biobank enables reproducible, institution-wide testing and supports real-time, patient-specific decision-making, offering a promising addition to the surgical oncologist\\u0026rsquo;s toolkit for precision medicine in ESCC. This advancement not only holds promise for tailored chemotherapy regimens but also enhances our ability to predict patient outcomes and support personalized postoperative management in clinical practice.\\u003c/p\\u003e\"},{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eEsophageal carcinoma (EC) poses a significant threat to public health due to its increasing incidence and inconspicuous symptoms, which often result in late diagnosis and poor prognosis\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. The conventional standard-of-care for treating esophageal cancer remains surgical resection combined with chemotherapy\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR3\\\" citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e. However, many patients fail to benefit from these treatments due to tumor heterogeneity, therapeutic resistance, and substantial side effects\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e. Moreover, the abundance of available chemotherapy drugs has made it increasingly challenging for clinicians to select appropriate regimens based solely on clinical expertise and patient values. In the past decade, targeted therapies for advanced stages of esophageal adenocarcinoma (EAC) have been limited and offer minimal benefits, while progress in esophageal squamous cell carcinoma (ESCC) has been virtually non-existent\\u003csup\\u003e\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u003c/sup\\u003e. Consequently, current precision medicine strategies for EC are inadequate, underscoring the need for additional preclinical models to contribute to effective treatments and facilitate personalized therapy.\\u003c/p\\u003e \\u003cp\\u003eNumerous studies have focused on identifying significant biomarkers for predicting clinical prognosis; however, reliable preclinical models for evaluating patient responses to chemotherapeutic and targeted agents are lacking\\u003csup\\u003e\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u003c/sup\\u003e. Recently, organoids derived from individual patients have emerged as a promising predictor in precision medicine due to their various advantages. Unlike patient-derived two-dimensional cell lines growing as flat monolayers, patient-derived organoids (PDOs) are cultured to form and maintain their three-dimensional structure, more accurately simulating the self-organization and cellular interactions of the tumor microenvironment\\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e. Moreover, in contrast to the extended tumorigenic processes required for genetically engineered mouse models and patient-derived xenografts (PDXs), organoids can be generated directly from primary tissue within a reasonable timeframe. They accurately recapitulate the genetic and morphological characteristics of the parental tumors with a high success rate and enable long-term expansion\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR13\\\" citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u003c/sup\\u003e. These advantages have been demonstrated in various primary tumors, including liver\\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003e, pancreas\\u003csup\\u003e\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u003c/sup\\u003e, breast\\u003csup\\u003e\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u003c/sup\\u003e, colorectal cancer\\u003csup\\u003e\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u003c/sup\\u003e, and other solid tumors. The fidelity of PDOs makes them highly valuable for predicting patient-specific responses to therapies and tailoring individualized treatment plans. However, research on the tissue characteristics of organoids derived from EC, particularly esophageal squamous cell carcinoma organoids (ESCOs), remains limited, and clinical evidence is scarce. The advancement of esophageal organoids provides significant molecular and mechanistic insights into various physiological and pathological dynamics, furthering translational research and personalized medicine\\u003csup\\u003e\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u003c/sup\\u003e. A recent study by Li et al. developed a panel of EAC organoids that mirror the characteristics of primary tumors. The study found that the sensitivity of most organoid cultures corresponded to the clinical response to neoadjuvant chemotherapy, suggesting that PDOs are valuable for evaluating treatment effectiveness in a preclinical setting.\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eWhile the optimization and validation of ESCOs represent significant progress, further research is necessary to conduct comprehensive profiling and assess the application of in vitro drug testing in EC. Consequently, we established a robust protocol for generating primary ESCOs culture from multiple centers, developed a standardized drug testing platform utilizing organoids, and initiated a retrospective clinical study to evaluate the potential of PDOs as predictive biomarkers for chemotherapeutic sensitivity in ESCC patients. Our case studies demonstrated that the PDO-based screening platform could effectively guide personalized treatment, given that the response to conventional anticancer regimens in PDOs correlates with the clinical response of patients. This study establishes a novel PDOs-based framework that shows promise for advancing precision medicine in EC.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1 Participant selection and study design\\u003c/h2\\u003e \\u003cp\\u003eAdults (age ≥ 18 years) with histologically and radiologically confirmed esophageal cancer were enrolled in the study based on clinical and radiologic evidence. The primary objective was to establish efficient culture methods for PDOs suitable for drug testing and to evaluate their potential in correlating patient outcomes with standard-of-care treatments for translational research. Figure\\u0026nbsp;1 illustrates the study design flowchart. Esophageal cancer specimens were primarily obtained from patients undergoing surgical resection or endoscopic biopsy at Nanjing Drum Tower Hospital, Beijing Cancer Hospital, and Jiangsu Hospital of Traditional Chinese Medicine. The Institutional Ethics Committees approved all tissue donations and experiments (approval number: 2021-237-02). Two independent pathologists confirmed the histological characteristics of the samples. Following surgical tumor resection, patients received clinically verified standard-of-care chemotherapy regimens based on physician's empirical choice. Comprehensive clinical characteristics were obtained and recorded using data collection forms from the hospitals' internal electronic medical records system. Clinical responses were assessed using RECIST 1.1 criteria. In some instances, comprehensive clinical data were unavailable due to loss of follow-up or patients' unwillingness to accept the suggested adjuvant therapy. Participant enrollment occurred from September 2021 to August 2022.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003e2.2. Patient material processing and generation of organoids\\u003c/h3\\u003e\\n\\u003cp\\u003ePatient specimens were washed with Hanks' Balanced Salt Solution (HBSS, Sigma) and minced into 1–2 mm³ pieces using sterile scissors. The tissue underwent enzymatic digestion at 37°C for 30 minutes with 1 mg/mL collagenase type II (Worthington), 1 mg/mL collagenase type IV (Worthington), 0.1 mg/mL DNase I (Worthington), and 100 U/mL penicillin-streptomycin (Life Technologies). The resulting fragments suspension was filtered through a 70-µm strainer, and undigested fragments were subjected to an additional 20 minutes of digestion before being re-filtered. The isolated cells were then resuspended in 7.5 mg/mL Matrigel (Corning) supplemented with ESCOs culturing media: Advanced DMEM/F12 (Gibco) was supplemented with 10 mM HEPES (Gibco), 1×GlutaMAX (Gibco), 100 U/mL penicillin-streptomycin (Gibco), 1×B27 (Gibco), 1×N2 (Gibco), 50 ng/mL EGF (Novoprotein), 10 ng/mL FGF-10 (Novoprotein), 100 ng/mL Noggin (Novoprotein), 250 nM A83-01 (Beyotime), 10 µM SB202190 (Beyotime), 100 ng/mL Wnt3a (Novoprotein), 100 ng/mL R-spondin 1 (Novoprotein), 1 mM N-Acetylcysteine (Sigma), and 10 µM Y-27632 (Beyotime). Organoids were incubated at 37°C in 5% CO₂, with the medium changed every 2–3 days. For passaging, organoids were split every 1–2 weeks at a ratio of 1:2 or 1:3. Organoids were collected and digested with TrypLE Express (Gibco) at 37°C for 5–10 min, then washed with Advanced DMEM/F12 and resuspended in Matrigel, supplemented with organoid culture medium. Organoid quantification was conducted using bright-field imaging with an IX73 microscope (Olympus, Tokyo, Japan).\\u003c/p\\u003e\\n\\u003ch3\\u003e2.3. Histology and immunohistochemistry\\u003c/h3\\u003e\\n\\u003cp\\u003eSurgical resection tissues were fixed in 10% formalin overnight and embedded in paraffin blocks after dehydration. Confluent organoids were collected, rinsed with ice-cold PBS to dissolve the Matrigel, and fixed in 10% formalin for 1 hour. The organoids were then settled by gravity and embedded in paraffin. All sample blocks were sectioned into 3-µm-thick slides, followed by dewaxing, rehydration, and standard hematoxylin and eosin (H\\u0026amp;E) staining. For immunohistochemistry (IHC) staining, paraffin slides underwent deparaffinization and rehydration, then were incubated with primary antibodies specific to p53 (1:100, clone D-O7, ZSGB-Bio, ZM-0408), CK5 (1:100, clone OTI1C7, ZSGB-Bio, ZM-0313), SOX-2 (1:100, EP103, ZSGB-Bio, ZA-0571), and KLF5 (1:200, Invitrogen, PA5-27876) at 4°C overnight. Subsequently, sections were washed and incubated with secondary antibody at room temperature for 1 hour, with negative controls applied to all samples. The slides were scanned and imaged using a microscope (Nikon, Eclipse CI, Japan). The immunoreactivity of the slides was assessed independently by two senior pathologists who blinded to this study. QuPath software was utilized for digital pathology and image analysis.\\u003c/p\\u003e\\n\\u003ch3\\u003e2.4. Drug screening assays\\u003c/h3\\u003e\\n\\u003cp\\u003eDrug testing was conducted with modifications to accommodate the 384-well format, as previously validated\\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e\\u003c/sup\\u003e. The primary organoids in good condition were collected and dissociated into single cells by incubating in TrypLE (Gibco) for 10 minutes. The single cells resuspended in the organoid culture medium with 5% Matrigel, and approximately 2000 cells were seeded per well of 384-well plates (Thermo Fisher Scientific, 242764). They were cultured for 3 days to allow cell recovery before drug treatment. Eight concentration points with 4-fold dilutions were set for each single drug or drug combination and dispensed using liquid-handling robots, with each concentration tested in triplicate. The starting concentrations for monotherapy were 100 µM for cisplatin (DDP) and 5-fluorouracil (5-FU), and 5 µM for Paclitaxel. For combination therapy, paclitaxel and cisplatin (TP) treatments, as well as vinorelbine and cisplatin (NP) treatments, were assayed in triplicate drug matrices. The starting concentrations were 100 µM for cisplatin and 5 µM for both paclitaxel and vinorelbine. For 5-FU plus cisplatin (FP) treatments, both components started at 100 µM. Similarly, for FOLFIRINOX (FFX) treatment, each component began at a concentration of 100 µM. DMSO at 0.5% (v/v) served as the negative (vehicle) control. Cell viability was assayed using Cell Counting-Lite 3D Luminescent Cell Viability Assay (Vazyme) following 5 days of drug treatment. Luminescent detection of cell viability was performed after 25 minutes of incubation with the cells at room temperature. In-vitro responses were evaluated using dose-response curves for single agents and drug combinations. IC50 and AUC (area under the curve) values were determined using Origin 2022 and GraphPad Prism 8.0. Normalized AUC values were calculated by dividing the AUC of each compound by the maximum AUC observed within the measured concentration range.\\u003c/p\\u003e\\n\\u003ch3\\u003e2.5 Quantification and Statistical Analysis\\u003c/h3\\u003e\\n\\u003cp\\u003eStatistical analyses for each dataset are detailed in the corresponding figure legends. Clinical data were extracted and managed from individual electronic health records. Group comparisons were conducted using the chi-squared test (or Fisher's exact test when appropriate) for categorical variables and the student’s t-test for continuous variables. Tumor response was assessed according to RECIST guidelines (version 1.1)\\u003csup\\u003e\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u003c/sup\\u003e. Data analyses were performed using Origin 2022 (OriginLab Corporation, USA) employing nonlinear regression (curve fit). Progression-free survival (PFS) differences were evaluated using Kaplan-Meier survival curves and the log-rank test with GraphPad Prism 8. \\u003cem\\u003ep\\u003c/em\\u003e values \\u0026lt; 0.05 were considered statistically significant.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003ch2\\u003e3.1. Patient demographics and tumor characteristics\\u003c/h2\\u003e\\u003cp\\u003eThis study involved 55 human samples from individual patients with confirmed pathological ESCC diagnoses for PDO generation. Fifty specimens were obtained through surgical resection from Nanjing Drum Tower Hospital and Peking University Cancer Hospital, while five samples were isolated from endoscopy biopsies at Jiangsu Chinese Medicine Hospital (\\u003cb\\u003eSupplementary Table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e\\u003c/b\\u003e). Figure\\u0026nbsp;2A presents a consort diagram illustrating the sample distribution. Baseline characteristics included patient demographics, tumor location, histological classification, cancer stage, and primary treatment. All samples were derived from primary tumors without distant metastasis at collection time, classified as ESCC, with a median patient age of 64 years. Most tumors were moderately differentiated and located in the middle or lower esophagus, aligning with EC epidemiology\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e. Four organoids generated from esophagogastroscopy biopsy were associated with patients lost to follow-up. Of the 26 patients who underwent surgery, 14 received subsequent adjuvant chemotherapy, including six who underwent neoadjuvant chemotherapy, indicating tissue acquisition in a post-neoadjuvant condition. Among these, one patient developed a low platelet count after two cycles of paclitaxel combined with platinum treatment, preventing the continuation of subsequent regimens. Another patient received S-1 as adjuvant therapy but was lost to follow-up three months post-surgery.\\u003c/p\\u003e\\u003ch3\\u003e3.2. Generation and histological characterization of ESCOs\\u003c/h3\\u003e\\u003cp\\u003eFresh ESCC tissue samples were obtained from esophagectomy or esophagogastroscopy biopsy and seeded into an esophageal cancer organoid culturing medium as described in the methods section. The study design is illustrated in \\u003cb\\u003eFig.\\u0026nbsp;2B.\\u003c/b\\u003e Organoids were cultivated in a Matrigel-supported growth medium to facilitate expansion. As shown in \\u003cb\\u003eFig.\\u0026nbsp;2A, 17\\u003c/b\\u003e cases failed to initiate a culture due to insufficient tumor cells or lack of expansion beyond passage two, while four organoids experienced bacterial contamination during cultivation. Overall, we achieved a ~ 62% PDO culture success rate (34 of 55 cultures) throughout the study, aligning with previous reports on gastrointestinal cancers\\u003csup\\u003e\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u003c/sup\\u003e. For 4 organoids, the clinical response of the corresponding patients was not evaluable due to a short follow-up period. Ultimately, drug testing was conducted on the remaining 30 organoid lines, with the patient's demographic and clinicopathological characteristics respectively detailed in \\u003cb\\u003eTable\\u0026nbsp;1\\u003c/b\\u003e and \\u003cb\\u003eSupplementary Table S2\\u003c/b\\u003e. Among the 30 drug assay reports, 24 organoids were derived from chemotherapy-naïve tumors, while 6 were derived from chemotherapy-treated tumors.\\u003c/p\\u003e\\u003cp\\u003eWe subsequently evaluated the histological profiles of the primary tissue and corresponding ESCOs to demonstrate their heterogeneous growth states (Fig.\\u0026nbsp;3). Despite their diverse morphologies, the comparison demonstrated that the ESCOs replicated notable morphological similarities to the matched original tumors. The characteristic solid and compact structures of the organoids are evident in both bright-field and H\\u0026amp;E staining, reflecting a cohesive cellular arrangement that mimics key architectural features of stratified squamous epithelium (Fig.\\u0026nbsp;3A)\\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e. Immunohistochemical staining of the organoids also revealed similar expression levels of the ESCC prognostic marker p53, as well as the squamous epithelial and ductal epithelial marker CK5/6\\u003csup\\u003e26, 27\\u003c/sup\\u003e. The expression of the tumor differentiation marker KLF5 and the tumor proliferation marker SOX2 in the organoids further substantiates their alignment with the corresponding histopathological specimens\\u003csup\\u003e\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e. As observed in ESCO57, the specimen consisted of moderately differentiated squamous cell carcinoma cells, displaying overexpression of CK5/6, p53, and KLF5, with low expression of SOX2. The matched organoid similarly exhibited nearly negative expression for SOX2 (Fig.\\u0026nbsp;3B). These findings strongly confirmed that the organoids faithfully preserved the histological organization and morphological heterogeneity of the original tumors.\\u003c/p\\u003e\\u003ch2\\u003e3.4. Drug assay of conventional chemotherapeutics in ESCOs\\u003c/h2\\u003e\\u003cp\\u003eTo evaluate the applicability of PDO lines in assessing personalized chemotherapeutic responses, therapeutic profiling was conducted on 30 ESCOs using four commonly employed standard-of-care chemotherapeutic agents: single-agent cisplatin (DDP) and combination therapies—paclitaxel plus cisplatin (TP), vinorelbine plus cisplatin (NP), and 5-fluorouracil plus cisplatin (FP). Each group underwent screening using an 8-point dilution series, with cell viability and ESCO response quantified and analyzed through dose-response curves and AUC. Detailed normalized AUC data are provided in Supplementary Table S3. The ESCO profiling revealed significant variability in response to both single and combination chemotherapeutic agents among individual patients \\u003cb\\u003e(Fig.\\u0026nbsp;4A)\\u003c/b\\u003e. Violin plots illustrate the distribution of AUC values, highlighting a spectrum of resistance and sensitivity across the samples \\u003cb\\u003e(Fig.\\u0026nbsp;4B)\\u003c/b\\u003e. Using DDP as a representative drug, individual IC50 and 95% CI from dose-response testing demonstrated that ESCOs exhibit distinct chemotherapeutic response profiles to varying doses of cisplatin (Fig.\\u0026nbsp;4C). The in vitro chemosensitivity of ESCOs to DDP and the combination therapies TP, NP, and FP is presented as standardized AUC values \\u003cb\\u003e(Fig.\\u0026nbsp;4D)\\u003c/b\\u003e. The 10 representative ESCOs described in this study are dispersed throughout the whole group, indicating that they represent a range of sensitivity and resistance. Consequently, ESCOs serve as valuable tools for drug response assays, reflecting diverse responses to various conventional chemotherapeutics. Several ESCOs were also treated with FOLFIRINOX (FFX), partially shown in Fig.\\u0026nbsp;5E. To further elucidate the correlation between ESCO sensitivity results and clinical outcomes, the assays were divided into three subgroups based on the AUC values for each chemotherapeutic agent: the ESCOs with their AUC ranked the top 33% were regarded as the least responsive (resistant ESCOs), those with their AUC ranked the lowest 33% as the most responsive (sensitive ESCOs), and those with their AUC ranked the middle 34% AUC as moderately sensitive ESCOs. Among the 30 ESCOs subjected to drug tests, 14 patients received chemotherapy, with detailed AUC values, sensitivities, and corresponding patient responses presented in \\u003cb\\u003eTable\\u0026nbsp;2.\\u003c/b\\u003e Given that most patients in this study were treated with TP, the correlation between clinical outcomes and PDO-based drug testing responses was emphasized.\\u003c/p\\u003e\\u003ch2\\u003e3.5. ESCOs represent chemotherapy response and clinical prognosis of ESCC patients\\u003c/h2\\u003e\\u003cp\\u003eTo assess the clinical viability of utilizing PDOs platform for ESCC patients, we analyzed retrospective clinical data and compared the pharmacotyping outcomes of ESCOs with the corresponding patients' clinical responses. Among the 30 ESCOs, 14 were from patients who received chemotherapy: 6 were from patients who underwent neoadjuvant chemotherapy, while 8 were from patients who received adjuvant chemotherapy, with follow-up periods ranging from 2.8 to 22.4 months (Table\\u0026nbsp;2). PFS is defined as the duration from initial treatment until signs of disease progression appear. The treatment responses of ESCC patients classified by clinicians as having a partial response (PR) or stable disease (SD) for more than one year were categorized as indicating a good response (chemotherapy-sensitive). Conversely, patients with progressive disease (PD), including tumor recurrence, metastasis, or disease progression within 1 year were classified as having a poor response (chemotherapy-resistant) as illustrated in Fig.\\u0026nbsp;5A. Despite 2 patients being lost to follow-up, 9 out of the remaining 12 patients who underwent chemotherapy were classified as having a good clinical response, while the other 3 were classified as having a poor response. In the drug assay performed on the ESCOs, the organoids exhibited distinct responses to various chemotherapy regimens (Fig.\\u0026nbsp;5A). As most patients received the TP regimen, we observed that 9 ESCOs demonstrated sensitivity to TP, consistent with their clinical outcomes. However, 2 stage III patients, expected to benefit clinically based on their PDO drug assays, exhibited progressive disease, indicating a lack of correlation with the ESCO responses. Lastly, one patient (ESCO25), who experienced rapid disease progression while on paclitaxel plus cisplatin, had an organoid that displayed resistance to TP. In total, 10 out of 12 (83.33%) ESCC patients exhibited consistency in drug response results with those observed in their derived PDOs. Furthermore, we compared the PFS of the two groups of patients based on their sensitivity to the TP regimen. The patients whose ESCOs were sensitive to TP in our assay exhibited significantly higher PFS, suggesting that in vitro sensitivity of ESCOs to TP is associated with a longer clinical response of the corresponding patients. The hazard ratio (HR) was 5.12 (95% CI = 0.58–44.71, log-rank test, P \\u0026lt; 0.05; \\u003cb\\u003eFig.\\u0026nbsp;5B\\u003c/b\\u003e). These complementary drug assays can be performed within 3 to 4 weeks, aligning with the typical timeframe for clinical treatment decisions, which are generally made 2 to 3 months after the last treatment to evaluate alternative options. PDO-based drug testing can thus serve as a stratification tool to identify optimal therapeutic combinations for individual patients.\\u003c/p\\u003e\\u003ch2\\u003e3.6 Case report\\u003c/h2\\u003e\\u003cp\\u003eWe closely monitored the disease progression of three patients from whom we derived ESCOs, categorized as sensitive (ESCO10 and ESCO13) or resistant (ESCO25) to their respective treatments. As depicted in \\u003cb\\u003eFig.\\u0026nbsp;5C\\u003c/b\\u003e, Patient ESCO10, diagnosed with ESCC, underwent cycles of paclitaxel plus platinum during neoadjuvant chemotherapy, achieving a confirmed PR with a reduction in primary tumor size after 2 months of treatment. The patient subsequently underwent surgery and continued TP treatment postoperatively, maintaining stable disease for over one year throughout adjuvant chemotherapy. To assess whether PDO profiling reflected the patient's response to chemotherapy, we conducted a drug assay on the ESCOs, revealing distinct sensitivities among various regimens. The AUC of TP ranked highest (AUC = 0.64), indicating that ESCO10 was responsive, aligning with the observed clinical response. A comparable drug response and consistent clinical outcome were also observed in patient ESCO13 (AUC = 0.59). Regarding the resistant case ESCO25 (AUC = 0.78), the patient was diagnosed with ESCC (pT3N2M0, stage IIIB) with perigastric lymph node metastasis, as confirmed by the postoperative pathology report. The patient received adjuvant TP treatment at a local hospital and subsequently underwent adjuvant radiotherapy. Progressive lymph node metastasis was detected 6 months post-surgery, consistent with the resistant response observed in the ESCO (AUC = 0.78). Consequently, the drug response of the PDOs demonstrated both sensitivity and resistance characteristics of their associated tumors \\u003cb\\u003eFig.\\u0026nbsp;5D\\u003c/b\\u003e. This preclinical model shows potential value in guiding the selection of appropriate clinical chemotherapy regimens, potentially helping patients avoid ineffective treatments.\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eEC is a severe malignancy that urgently requires clinical advancements to enhance patient outcomes. The ongoing areas of significant interest include early detection and optimal therapy for patients. Emerging research utilizing PDOs aims to verify their sensitivity and specificity as predictive biomarkers for guiding precision medicine, particularly in adjuvant chemotherapy and immunotherapy. This approach extends to liver, breast, colon, pancreatic, and lung cancers\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR16 CR17\\\" citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e–\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e, \\u003cspan additionalcitationids=\\\"CR31\\\" citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e–\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e\\u003c/sup\\u003e. In 2011, Sato first reported organoids derived from human Barrett's epithelium, enabling long-term expansion\\u003csup\\u003e\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e. Subsequently, the application of PDOs in EC has progressed. Li established a reliable EAC organoid biobank that accurately replicated the phenotypic and molecular characteristics of EAC, facilitating drug screening for novel therapeutic strategies\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e. Concurrently, Kijima et al. developed a robust system for culturing organoids from esophageal and oropharyngeal squamous cell carcinoma patients with high success rates, enabling therapy response evaluation and exploration of underlying mechanisms\\u003csup\\u003e\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e. However, further studies optimizing drug panel testing for EC-derived organoids are necessary, along with validation through clinical trials.\\u003c/p\\u003e\\u003cp\\u003eESCC accounts for approximately 90% of all EC cases, with particularly high prevalence in Asia and parts of Africa\\u003csup\\u003e\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e\\u003c/sup\\u003e. To advance the feasibility of ESCOs-based approaches in ESCC, this study presents several novel findings from a multi-institution randomized, retrospective clinical trial. First, we demonstrate the establishment of organoids with a moderate success rate, showing that ESCOs recapitulate the morphological heterogeneity and histological characteristics of the matched original tumor tissue across various differentiation statuses and pathological types. Subsequently, we implemented a robust PDO drug screening platform, utilizing a reliable biobank to assess multi-regimen drug sensitivity, which revealed differential responses of PDOs to conventional chemotherapeutic agents. Furthermore, we tracked real-world clinical outcomes and assessed their correlation with in vitro drug screening data, indicating high consistency and supporting the integration of translational organoid technologies with personalized therapy. Our approach enables organoid growth within a moderate timeframe (3–6 weeks), with primary drug testing reports completed in an additional 7 days. This timeline is clinically relevant, considering that restaging scans are typically performed 2 to 3 months after initiating treatment, serving as criteria for evaluating whether to continue current therapy or switch to alternative treatment\\u003csup\\u003e\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u003c/sup\\u003e. Collectively, these findings support the rapid advancement and significant potential of translating organoid technologies.\\u003c/p\\u003e\\u003cp\\u003eA comprehensive analysis of drug sensitivity profiles, examining both single and combination regimens in a group of 30 PDOs, revealed significant variability in organoid responses to various chemotherapy treatments and individual drug assays across different PDOs. Notably, a strong correlation was observed between the responses in PDOs and those reported in clinical settings. This correlation aligns with recent retrospective data reported for other cancer types in real-world studies\\u003csup\\u003e\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u003c/sup\\u003e. In contrast to previous studies, this multi-center study introduces a novel classification system using AUCs as the index for assessing PDOs' sensitivities to various treatment regimens, aiming to predict clinical disease control Future enrollment of additional cases will enhance the representativeness of this biobank. For chemotherapy-sensitive organoids from a patient expected to respond well to a specific regimen but with an unmatched clinical response, a review of the patient's entire inpatient record revealed an inability to continue chemotherapy cycles due to side effects. This indicates that even when PDO-based drug assays demonstrate high sensitivity to chemotherapy, patients unable to complete treatment due to adverse effects may experience poor clinical outcomes.\\u003c/p\\u003e\\u003cp\\u003eThis study presents several limitations and opportunities for future research. While achieving high consistency between organoid drug assays and clinical responses, each sample in this study originated from individual patients during surgery or endoscopic biopsy, including initial tumors and several post-NAT tissues exposed to chemotherapy. However, the absence of paired chemo-naïve and post-chemotherapy samples resulted in a missed opportunity to collect materials from the same patient at different disease stages for longitudinal organoid generation\\u003csup\\u003e\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u003c/sup\\u003e. Cancer treatment is a complex, multifaceted, and ongoing process\\u003csup\\u003e\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e\\u003c/sup\\u003e. The variability in treatment effectiveness among cancer patients is intricate and associated with numerous factors, including post-operative outcomes, combination therapies, chemotherapy side effects, overall health conditions, and tumor characteristics\\u003csup\\u003e\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e\\u003c/sup\\u003e. Although the chemosensitivity of organoids may partially reflect the tumor's response at a specific point in time, it has limitations in capturing tumor evolution and changes in overall drug treatment response. An additional related challenge is the limited source of tissue; all samples in this study were acquired from patients in suitable condition for surgical resection, indicating a lack of tumor tissues of advanced stages and from metastatic sites to generate a more comprehensive biobank representing the disease at different stages.\\u003c/p\\u003e\\u003cp\\u003eIt is noteworthy that the success rate of ESCC organoid generation was lower and requires enhancement compared to other cancer types, such as colorectal cancer, breast cancer, and other adenocarcinoma cancers\\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003e. This disparity and associated challenges potentially stem from the inherent heterogeneity of squamous cell carcinoma and its dependence on specific stromal interactions and extracellular matrix components, which are challenging to replicate in standard organoid cultures\\u003csup\\u003e\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e\\u003c/sup\\u003e. Adenocarcinoma, originating from glandular structures in epithelial tissue, typically exhibits more predictable growth patterns, facilitating well-established organoid culture protocols\\u003csup\\u003e\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e. Sachdeva et al. report developing reliable protocols to establish PDOs from EAC with a 80% success rate, compared to their 60% success rate in ESCC organoids generation\\u003csup\\u003e\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e\\u003c/sup\\u003e. While we did not initially control the source of samples, due to the prevalence of EC in our region, all samples included in this study were diagnosed as ESCC through professional histological analysis. Future studies should incorporate various histological subtypes, as ongoing technological advancements and refined cultural conditions will gradually mitigate the challenges associated with organoid generation. In addition to tumor organoids, the tumor microenvironment, composed of stromal cells and inflammatory cells, plays a crucial role in influencing tumor behavior and drug-resistant mechanisms\\u003csup\\u003e\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e\\u003c/sup\\u003e. Co-culture models that generate patient-derived organoids in combination with non-cancerous cells represent promising approaches to better replicate the heterogeneous features of the original tumor environment in vitro.\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eIn conclusion, this study demonstrates that PDO-based drug assays provide significant insights into standard treatment efficacy for ESCC patients. The successful generation of PDOs from a heterogeneous cohort of ESCC patients within a clinically relevant timeframe yielded results that strongly correlate with individual clinical outcomes and drug sensitivity profiles. Further investigation is warranted to validate this approach in a larger patient cohort, including the incorporation of PDOs derived from patients with metastatic disease.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cp\\u003eEAC (esophageal\\u0026nbsp;adenocarcinoma)\\u003c/p\\u003e\\n\\u003cp\\u003eEC (esophageal carcinoma)\\u003c/p\\u003e\\n\\u003cp\\u003eESCC (esophageal squamous cell carcinoma)\\u003c/p\\u003e\\n\\u003cp\\u003eESCOs (esophageal squamous cell carcinoma organoids)\\u003c/p\\u003e\\n\\u003cp\\u003ePDOs (patient-derived organoids)\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003ch2\\u003eFunding\\u003c/h2\\u003e \\u003cp\\u003eThis research was funded by the National Nature Science Foundation of China (No. 82372988), the Health Technology Development Project of Nanjing City (No. YKK21077), and the Clinical Trials funding from the Affiliated Drum Tower Hospital, Medical School of Nanjing University (No. 2022-LCYJ-PY-03). Suya Shen received support from the China Scholarship Council (No. 202306190025).\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe extend our gratitude to all patients and their families who participated in this study and generously contributed their samples. Furthermore, we acknowledge the\\u0026nbsp;invaluable support of our clinical and research team members for\\u0026nbsp;their assistance with tissue collection and processing.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eSuya Shen (Conceptualization, methodology, writing original draft,\\u0026nbsp;data curation)\\u003c/p\\u003e\\n\\u003cp\\u003eJingjing Li (Conceptualization: Lead; Funding acquisition; Investigation; Supervision; Supporting)\\u003c/p\\u003e\\n\\u003cp\\u003eZheng Hongping, Bing Liu (Original draft writing; Formal analysis)\\u003c/p\\u003e\\n\\u003cp\\u003eWei Ren, Yudong Qiu (Supervision; validation)\\u003c/p\\u003e\\n\\u003cp\\u003eWenyan Guan (Histology analysis: Supporting; Investigation)\\u003c/p\\u003e\\n\\u003cp\\u003eJian He, Jie Shen, Weifeng Tang, Pengju Zhang (Data analysis;\\u0026nbsp;Methodology)\\u003c/p\\u003e\\n\\u003cp\\u003eYuqing Han, Yingzhe Hu\\u0026nbsp;(Data collection, writing \\u0026ndash; review \\u0026amp; editing: Equal)\\u003c/p\\u003e\\n\\u003cp\\u003eZiyao Liu, Yiqiang Chen, Siyuan Liu (Sample acquisition, data collection)\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of interest\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eSuya Shen, Bin Liu, and Wenyan Guan contributed equally to this work. The authors declare that no members of the research team were involved in any commercial or financial relationships that could be construed as a potential conflict of interest.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eRustgi AK, El-Serag HB. Esophageal carcinoma. N Engl J Med. 2014;371:2499\\u0026ndash;509.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eIshihara R, Arima M, Iizuka T, et al. Endoscopic submucosal dissection/endoscopic mucosal resection guidelines for esophageal cancer. Dig Endosc. 2020;32:452\\u0026ndash;93.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKitagawa Y, Ishihara R, Ishikawa H, et al. Esophageal cancer practice guidelines 2022 edited by the Japan Esophageal Society: part 2. Esophagus. 2023;20:373\\u0026ndash;89.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eIshihara R, Tani Y, Okubo Y, et al. 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Eur J Cancer. 2009;45:228\\u0026ndash;47.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eThen EO, Lopez M, Saleem S, et al. Esophageal Cancer: An Updated Surveillance Epidemiology and End Results Database Analysis. World J Oncol. 2020;11:55\\u0026ndash;64.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eVlachogiannis G, Hedayat S, Vatsiou A, et al. Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science. 2018;359:920\\u0026ndash;6.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKaufmann O, Fietze E, Mengs J, et al. Value of p63 and cytokeratin 5/6 as immunohistochemical markers for the differential diagnosis of poorly differentiated and undifferentiated carcinomas. Am J Clin Pathol. 2001;116:823\\u0026ndash;30.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhong L, Li H, Chang W, et al. TP53 Mutations in Esophageal Squamous Cell Carcinoma. Front Biosci (Landmark Ed). 2023;28:219.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhang J, Wang Z, Zhao H, et al. The roles of the SOX2 protein in the development of esophagus and esophageal squamous cell carcinoma, and pharmacological target for therapy. Biomed Pharmacother. 2023;163:114764.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJiang YY, Jiang Y, Li CQ, et al. TP63, SOX2, and KLF5 Establish a Core Regulatory Circuitry That Controls Epigenetic and Transcription Patterns in Esophageal Squamous Cell Carcinoma Cell Lines. Gastroenterology. 2020;159:1311\\u0026ndash;27. e19.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTan T, Mouradov D, Lee M, et al. Unified framework for patient-derived, tumor-organoid-based predictive testing of standard-of-care therapies in metastatic colorectal cancer. Cell Rep Med. 2023;4:101335.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWang HM, Zhang CY, Peng KC, et al. Using patient-derived organoids to predict locally advanced or metastatic lung cancer tumor response: A real-world study. Cell Rep Med. 2023;4:100911.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTakahashi N, Hoshi H, Higa A et al. An In Vitro System for Evaluating Molecular Targeted Drugs Using Lung Patient-Derived Tumor Organoids. Cells 2019;8.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSato T, Stange DE, Ferrante M, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology. 2011;141:1762\\u0026ndash;72.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAbnet CC, Arnold M, Wei WQ. Epidemiology of Esophageal Squamous Cell Carcinoma. Gastroenterology. 2018;154:360\\u0026ndash;73.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDemyan L, Habowski AN, Plenker D, et al. Pancreatic Cancer Patient-derived Organoids Can Predict Response to Neoadjuvant Chemotherapy. Ann Surg. 2022;276:450\\u0026ndash;62.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLiu B, Zhou H, Tan L, et al. Exploring treatment options in cancer: Tumor treatment strategies. Signal Transduct Target Ther. 2024;9:175.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMiller KD, Nogueira L, Devasia T, et al. Cancer treatment and survivorship statistics, 2022. CA Cancer J Clin. 2022;72:409\\u0026ndash;36.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSachdeva UM, Shimonosono M, Flashner S, et al. Understanding the cellular origin and progression of esophageal cancer using esophageal organoids. Cancer Lett. 2021;509:39\\u0026ndash;52.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZheng S, Liu B, Guan X. The Role of Tumor Microenvironment in Invasion and Metastasis of Esophageal Squamous Cell Carcinoma. Front Oncol. 2022;12:911285.\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"},{\"header\":\"Tables\",\"content\":\"\\u003cp\\u003eTable 1 and 2 are available in the Supplementary Files section.\\u003c/p\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"journal-of-translational-medicine\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"jtrm\",\"sideBox\":\"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/jtrm/default.aspx\",\"title\":\"Journal of Translational Medicine\",\"twitterHandle\":\"@BioMedCentral\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Esophageal Squamous Cell Carcinoma, Organoids, Chemotherapy, Precision Medicine\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-5357253/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-5357253/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cstrong\\u003eBackground \\u0026amp; Aims:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ePatient-derived organoids (PDOs) represent a promising approach for replicatingthe characteristics of original tumors and facilitating drug testing for personalized treatments across diverse cancer types. However, clinical evidence regarding their application to esophageal cancer remains limited.This study aims to evaluate the efficacy of implementing PDOs in clinical practice to benefit patients with esophageal squamous cell carcinoma (ESCC).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMethods:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFresh surgical biopsies were obtained from patients with esophageal cancer for the establishment of PDOs. These PDOswere subsequently characterized through histological analysis. A customized drug panel, based on standard-of-care chemotherapy regimens, was applied to the PDOs. The resulting drug sensitivity profiles were then correlated with the clinical responses observed in individual patients undergoing actual treatment.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eResults:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA total of 34 PDOs were successfully established with a 61.8% success rate. The classification method based on chemotherapy sensitivity closely corresponded to clinical responses. The paclitaxel plus cisplatin (TP)-sensitive group demonstrated significantly longer progression-free survival (PFS) compared to the resistant groups, Hazard ratio (HR), 5.12; 95% confidence intervals (CI), 0.58-44.71; p \\u0026lt;0.05), thus illustrating the potential of this approach for identifying personalized treatment strategies.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConclusion:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eOrganoid biobanks wereestablished across multiple institutes to facilitate PDOs-based functional precision medicine. The findings demonstrate that this framework offers robust predictive value in clinical settings, enhances precision therapeutics, and advances drug discovery for esophageal cancer.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Advancing Precision Medicine in Esophageal Squamous Cell Carcinoma Using Patient-Derived Organoids\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-12-18 05:32:17\",\"doi\":\"10.21203/rs.3.rs-5357253/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Major revision\",\"date\":\"2024-11-21T03:41:40+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"\",\"date\":\"2024-11-10T01:29:10+00:00\",\"index\":0,\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-11-09T15:58:53+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-11-08T12:53:10+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Journal of Translational Medicine\",\"date\":\"2024-10-29T20:08:59+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"journal-of-translational-medicine\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"jtrm\",\"sideBox\":\"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/jtrm/default.aspx\",\"title\":\"Journal of Translational Medicine\",\"twitterHandle\":\"@BioMedCentral\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"698b3132-dfa7-4e59-b6ca-027e95257cd9\",\"owner\":[],\"postedDate\":\"December 18th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-01-06T16:03:49+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-5357253\",\"link\":\"https://doi.org/10.1186/s12967-024-05967-1\",\"journal\":{\"identity\":\"journal-of-translational-medicine\",\"isVorOnly\":false,\"title\":\"Journal of Translational Medicine\"},\"publishedOn\":\"2024-12-31 15:57:51\",\"publishedOnDateReadable\":\"December 31st, 2024\"},\"versionCreatedAt\":\"2024-12-18 05:32:17\",\"video\":\"\",\"vorDoi\":\"10.1186/s12967-024-05967-1\",\"vorDoiUrl\":\"https://doi.org/10.1186/s12967-024-05967-1\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-5357253\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-5357253\",\"identity\":\"rs-5357253\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}