Patient-derived esophageal adenocarcinoma organ chip: a physiologically relevant platform for functional precision oncology

Patient-derived esophageal adenocarcinoma organ chip: a physiologically relevant platform for functional precision oncology | 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 Patient-derived esophageal adenocarcinoma organ chip: a physiologically relevant platform for functional precision oncology Sanjima Pal, Elee Shimshoni, Salvador Flores Torres, Mingyang Kong, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6017396/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 May, 2025 Read the published version in Journal of Translational Medicine → Version 1 posted 5 You are reading this latest preprint version Abstract Background Esophageal adenocarcinoma (EAC) is the sixth most deadly cancer worldwide, with increasing incidence in North America. As no targeted therapy or immunotherapy has revolutionized the management of EAC, chemotherapy is the only standard of care. Most patients with EAC experience poor outcomes because of the inherent or acquired resistance to chemotherapy. Methods Adapting a patient-centered approach, we leveraged a microfluidic cell culture technology platform (Emulate), organoids derived from treatment-naive patient tumors or adjacent normal tissues, and patient-matched cancer-associated or normal fibroblasts respectively, to develop a novel, physiologically relevant, high-fidelity preclinical esophagus-on-a-chip model. H&E, immunofluorescence staining, live/dead assay, LDH assay, and ELISA-based detection of tumor biomarkers were used to assess treatment responses. Results Each patient-specific stroma-inclusive microfluidic esophageal adenocarcinoma on-a-chip (EAC chip) faithfully recreates the tumor-stroma interface while preserving the full diversity of two cell types (epithelia and fibroblasts), genetic landscapes and histological architecture of the source tumors. EAC chips also accurately predict the response to neoadjuvant chemotherapy (NACT) within a clinically useful timeframe (approx. 12 days). A docetaxel-based triplet chemotherapy regimen matched with the treatment of the source patient was successfully perfused through the interstitial space within this model. Therefore, EAC chips more accurately recapitulate inpatient pathological and objective responses than the corresponding static 3D-organoid-only cultures. Conclusions Overall, this model is an effective tool for predicting patients’ responses to chemotherapy and testing tumor- or stroma-targeted alternative therapies. Moreover, these high-fidelity, low-throughput EAC chips effectively complement high-throughput PDO culture-based drug testing and provide improved insights into drug efficacy before human studies. Esophageal adenocarcinoma Organ-on-a-chip Organoids Cancer-associated fibroblasts Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Esophageal adenocarcinoma (EAC) is the fastest-rising malignancy in North America [ 1 ]. The only standard-of-care (SOC) treatment for locally advanced, resectable EAC is perioperative docetaxel-based triplet chemotherapy (Table S1 )[ 2 ]. Most EAC tumors exhibit ab initio chemotherapy resistance. Meanwhile, most responders to neoadjuvant chemotherapy (NACT) exhibit high rates of progression and metastasis during the adjuvant period. Given the lack of alternative approaches, each non-responder to NACT continues to receive the SOC as adjuvant therapy. The development of accurate in vitro and in vivo preclinical models, prognostic markers for response to therapy, and alternative effective treatments for chemoresistant EAC patients all remain critical unmet clinical needs. Patient-derived organoid (PDO) culture recapitulates the heterogeneity of epithelial cell lineages more faithfully than traditional 2-dimentional (2D) cell lines do[ 3 – 6 ]. These cancer organoids have been effectively used for high-throughput (HTP) screening of monotherapies and for predicting patient outcomes [ 7 – 11 ]. There are also reports highlighting the ineffectiveness of PDOs in predicting the outcome of treatment with 5-fluorouracil (5-FU) combined with oxaliplatin [ 12 ]. Traditionally, organoids grow in complex 3-dimentional (3D)-Matrigel domes, and cultures are maintained under static conditions. Matrigel, a heterogeneous mixture of murine sarcoma-derived ECM proteins, exhibits lot-to-lot varying concentration, stiffness and influences drug diffusion [ 13 ]. Thus, PDO culture format often suffers from poor reproducibility [ 14 ]. To address this critical issue, several groups have used well-defined micro-engineered hydrogels or alginates as alternatives to Matrigel, especially for incorporating user-defined microenvironmental signals to investigate human epithelial tumors [ 15 – 18 ]. Thus the in vitro response of PDOs may diverge from the observed response in patients, especially for combination therapy where each drug may have a distinct pharmacokinetic profile and toxicity (e.g. docetaxel). Since organoids lack various cellular (stromal and immune) and physiological factors (oncotic pressure and interstitial fluid movement) of the tumor microenvironment (TME), they often fail to fully capture the intricacies of drug efficacy and distribution observed in actual tumors [ 18 – 23 ]. There is thus significant room to improve on cancer organoid technology by combining organoids and stromal cells to develop a higher-fidelity platform that more effectively recapitulates human-relevant esophageal organ structure, cellular/tissue heterogeneity, physiology, pathogenesis, and drug distribution and is more likely to accurately predict in patient response to therapy [ 8 ]. Although still a low-throughput system, microfluidic organ-chip technology offers multiple advanced features that are lacking in static organoid culture technology, including recapitulation of the organ-level TME [ 24 – 26 ]. Organ Chips provide dynamic fluid flow and thus provide a unique opportunity to mimic human relevant pharmacokinetic and pharmacodynamic (PK/PD) profiles of drugs in vitro to recapitulate treatment regimen-specific drug efficacy and toxicity [ 27 – 32 ]. Additionally, organ chips also demonstrated some translatable endpoints similar to those observed for human pathophysiology [ 28 , 33 ]. Here, we highlight a key limitation of 3D PDO culture technology and propose an improved approach by integrating PDO and cancer-associated fibroblast (CAF) co-cultures with microfluidic organ-on-a-chip technology. We describe a first-generation, syngeneic human EAC-Chip platform containing PDO-derived EAC cells separated by a porous membrane from patient-matched CAFs), co-cultured under continuous flows of nutrient media. Furthermore, we also demonstrate that the EAC-chip platform achieves clinical mimicry when a triplet chemotherapy regimen is perfused through the bottom (stromal) channel lined with CAFs. Methods Study cohort Consented patients with locally advanced resectable EAC (n = 8) who received triplet NACT were selected for the current study (Fig. 1 B). Patients were divided into two groups, (i) Chemosensitive (SENS) (n = 4) patients who demonstrated either complete or partial objective response (OR) to NACT. (ii) Chemoresistant (RES) (n = 4) patients were either clinically assessed for stable disease (SD) or progressive disease (PD) following NACT. Isolation and propagation of a syngeneic patient-derived epithelial/tumor niche and associated fibroblasts Tissue specimens were collected from the adenocarcinoma and adjacent normal regions at the time of diagnostic endoscopic biopsy. Organoids and regional fibroblasts were established from the specimens and propagated using the protocol below. Briefly, the tissue was minced and enzymatically digested with tissue digestion buffer in a GentleMACSTM Octo Dissociator with heaters (Milltenyi Biotec.). The cell pellets were obtained and trypsinized to obtain single cells. The resulting cells were resuspended and were allowed to stand undisturbed for 10min. Later a fraction was transferred to a new tube for fibroblast culture and centrifuged. a. For fibroblast propagation : Cell pellets were resuspended in 1ml fibroblast propagation medium and seeded in Type I collagen precoated plates. b. For organoid propagation : Cell pellet was resuspended in ice cold Matrigel and plated. Organoids in Matrigel domes were maintained in expansion medium . Mature PDOs were passaged after 10–12 days. Single cells were obtained through enzymatic digestion and trypsinization. Both PDOs and associated fibroblasts were cultured under hypoxic conditions, i.e., 3% O 2 . All consumables used for each method are listed in Table S4 . For analysis of organoid histology, Histogel blocks were prepared following an existing protocol ( https://ccr.cancer.gov/sites/default/files/2022-11/Histogel_Protocol_0.pdf ). Culture of stroma-exclusive, fragmented and dispersed PDOs under static conditions Fragmentated PDOs were obtained with TrypLE™ (Invitrogen 12605010) at 37°C for 1 minute. 5000 fragmented PDOs were seeded on ECM precoated 96 well plates and maintained in organoid expansion medium. On the sixth day post-seeding, the expansion medium (EM) was replaced with differentiation medium (DM) which was maintained throughout the experiment. Cells were maintained for 12 days and harvested for measuring cell viability and microscopy. Establishment of syngeneic esophageal tissue-on-a-chip The complete organ-on-a-chip setup (Chip-S1™ stretchable organ chips, POD™ portable modules, Zoe-CM-1™ culture module and Orb-HM1™ Hub Module) was procured from Emulate Inc. The chip design comprises two parallel microfluidic channels, separated by an optically transparent 50 µm PDMS porous membrane (7 µm diameter). On day 0, chip surfaces were activated with S1 chip activation solution and high-power UV light. The activated membrane was coated with freshly prepared ice-cold ECM solution (ST4) and incubated overnight. On day 1, PDO-derived fragmented organoids or epithelial cells (concentration: 3 × 10 6 cells /ml) were seeded at the upper channel. Once cells adhered to the membrane (~ 4 h), the chips were connected to flow at 60 µl/h. Patient matched regional fibroblasts were seeded in the lower stromal channel of the chip on day 2. Fibroblasts were incubated for 4 h at 37°C before chips were reconnected to flow at 60 µl/h. On day 6 post-seeding of epithelial cells, the expansion media in the epithelial inlets were replaced with differentiation media. Overall chips were maintained up to 12 days. The development of PDOs and microtissues (day 0 to day 12), morphological changes and the population of cells undergoing treatment-induced cell death were also determined by brightfield and fluorescence microscopy respectively (EVOS, Invitrogen). Detailed protocol (Standard operating protocol) has been supplied as Document S1. Histological analysis of primary and chip derived microtissues organ chips Primary tissue samples fixed in 10% formalin were dehydrated with alcohol and then embedded in paraffin. 5 µm thick sections were prepared, mounted on glass slides, stained with H&E and analyzed. On Day 13, Tissue-Tek® OCT compound (Thermo Fisher Scientific) was administered through both channels of 4% PFA fixed microtissues and OCT-blocks were created. 30 µm vertical cross-sections were prepared, stained with H&E and imaged (EVOS M7000). Whole-exome sequencing and analysis PDOs were passaged for at least 5 generations to collect ample amount of genomic DNA and develop EAC-chips. H&E-stained tissue sections were reviewed by an expert pathologist before isolation of the DNA. A primary tissue sample with tumor content ≥ 50% was submitted for WES. Genomic DNA was extracted (Qiagen: Cat. No.80204) from snap frozen primary tissue, matched organoids, and chip derived cells from upper epithelial channel. DNA extracted from blood (buffy coat) was used as a germline reference. To compare the genomic similarities between the primary tumor biopsy, organoid and organ-on-chip samples, we aligned the WES reads to the human reference genome (GRCh38) using the Burrows-Wheeler Aligner (BWA)[ 34 ]. Duplicate reads were marked and base quality scores were recalibrated using the genome analysis toolkit (GATK)[ 35 ]. Somatic mutations were called for the primary tumour, organoid and organ-on-chip against a patient matched normal sample using muTect [ 36 ]. Somatic mutations were annotated with ANNOVAR[ 37 ]. Acquired copy number alterations (CNA), including regions of loss of heterozygosity (LOH), were determined using FACETS[ 38 ]. The mutation allele frequencies were determined with bam-readcount [ 39 ]. Immunofluorescence microscopy studies 12-day old chips were fixed with 4% PFA and cut into 300µm vertical cross sections using a vibratome (Leica VT1200). Immunofluorescence staining was carried out using manufacturer’s suggested dilutions of the primary, secondary antibodies and DAPI nuclear stain (Table S4 ). Stained chip sections were imaged using a Zeiss LSM 780 confocal microscope. Whole-mount 3D-PDO culture immunofluorescence was performed via a 3D tissue clearing kit (5730, Corning) according to the manufacturer’s protocol. Images of organoid were captured using either in EVOS M7000 and ZEISS Lattice SIM 3. Scanning Electron Microscopy (SEM) Both treated and untreated EAC-chips were fixed with 4% PFA solution and washed 3 times with PBS. Concomitantly, the chip was trimmed using a razor blade, and 300µm vertical cross sections were prepared using vibratome. Afterward, the samples were gradually dehydrated in 100% ethanol and placed in a critical point dryer (CPD030, Leica) and 20 × 1-minute CO 2 exchange cycles were performed. Samples were sputter-coated (ACE600, Leica) with an 8 nm layer of platinum. images were acquired using an Environmental Scanning Electron Microscope (Quanta 450, FEI). Assessment of apparent permeability The degree of epithelial barrier integrity was determined following an existing protocol ( https://emulatebio.com/wp-content/uploads/2021/06/EP187_v1.0_Barrier_Function_Analysis_Protocol.pdf ). Data were obtained from 10 chips derived from 3 source patients (2–4 chips/donor). Assessment of intrinsic response of intact PDOs to neoadjuvant therapy under static conditions PDOs were enzymatically dissociated and 10,000 cells per 30 ul of Matrigel/well were distributed in a 96-well plate. 200 ul of OEM was dispensed into each well. In vitro drug treatments were conducted in triplicate at concentrations ranging from 0–1 µM of F:C:T docetaxel with ratio of 10:1:1 respectively. Triplicate chemotherapeutics at relevant doses (described in Table S2 ) were added together for 72 h. Solvent percentage was limited to 0.1%. As Matrigel is soft and does not adhere tightly to the surface, replacing the media with or without triplet chemotherapeutics in a 96-well format was technically unfeasible. The percent viability of intact PDOs was measured after 72 h of treatment using the CellTiter-Glo 3D reagent (Promega, G9681) according to the manufacturer’s instructions. Luminescence was measured via Varioskan LUX Multimode Microplate Reader (Thermo Scientific). The experimental readings were normalized to respective solvent controls. Viability curves and IC 50 values were generated using GraphPad Prism 8 software. Clinically relevant drug dosing for static and dynamic culture conditions The actual FLOT regimen and clinically relevant dosing reconstituted for the microfluidic culture is described in Table S2 . Leucovorin was omitted, since it is not a chemotherapeutic agent. Fragmented and dispersed PDOs were seeded in ECM solution coated 96 well plates. Cells were directly exposed to one cycle of FOT on day 8 (Table S2 ). Cellular viability was determined on day 12 as described above. The data are representative of biological and technical triplicates. EAC-Chips achieved human relevant epithelial tissue integrity within 5 days under dynamic conditions and treatment began on day 8. FOT chemotherapy was administered through the stromal channel. At first, a flush cycle (< Cmax, FOT: 52:1.7:1] of 1–2 mins with a flow rate of 1000 µl/h in the bottom channel and 0 µl/h top channel was performed to ensure proper distribution of the drugs only in the stromal microfluidic channel networks. To mimic bolus IV-injection, during the first hour of cycle a mixture of all three chemotherapeutics was administered at a flow rate of 100 µl/h, followed by only a mixture of oxaliplatin (O) and 5FU (F) at the rate 60 µl/h for another hour. Only 5FU (F) was subsequently perfused through the stromal channel at a flow rate of 60 µl/h for a total of 24 h. Measurement of LDH activity On day 9, effluents from epithelial channels were collected and samples were immediately assayed for LDH activity using the Promega™ LDH Cytotoxicity Assay Kit (Promega #G1781). All absorbance-related measurements were performed in a Varioskan LUX Multimode Microplate Reader (Thermo Scientific). The resulting data are shown as percentage of cell cytotoxicity. Two-way ANOVA was used to assess differences in the cell-free LDH activity in the effluents from treated and untreated groups. Propidium Iodide (PI) assay On day 12, media was aspirated from the inlet reservoir of the epithelial channel and replaced with fresh culture medium supplemented with PI (Sigma #P4170) for 5 minutes. The samples were subsequently washed with PBS. Images of 5–6 random regions of each chip were captured using a EVOS microscope. The non-parametric Mann-Whitney U-Wilcoxon test was employed to compare number of dead cells (defined as having an average cell area ≥ 100µm²) in both treated as well as untreated SENS and RES samples. Measurement of tumor-derived extracellular soluble fragments of CK19 On day 11, effluents were collected from outlet reservoirs of epithelial channels of both treatment naïve and FLOT treated chips. Chemo-naïve sera were collected (n = 10) at the time of diagnostic endoscopy. Soluble CK19 fragment concentration in serum and effluents were measured using ELISA (Elabscience®, E-EL-H2077) as per the manufacturer’s instructions. The data analyzed are displayed as a percentage of reduction in the soluble CK19 fragments in treated effluents compared with untreated chips. Statistical analysis GraphPad Prism 8 software ( www.graphpad.com ) was used for statistical analysis. p value < 0.05 was considered as significant for each analysis. *<0.05; **<0.01, ***<0.005 ***<0.001; ns: not significant Results Schematic representation of the strategy adapted to develop personalized human EAC-chips and clinicopathological characteristics of the study cohort. Our comprehensive strategy for modeling patient-derived esophageal adenocarcinoma chips, utilizing both PDOs and associated fibroblasts from the same patient source, for functional precision oncology were demonstrated as a graphical abstract. Esophageal PDOs and matched fibroblasts were generated from endoscopic biopsies of 8 newly diagnosed, treatment-naïve patients with locally advanced EAC (Table 1). Subsequent pathological (tumor regression grade/TRG) and objective radiographic (RECIST v1.1) responses to docetaxel-based triplet neoadjuvant chemotherapy were extracted from the medical records. Patients were sub-grouped into either chemosensitive/SENS (complete [TRG0] or near-complete [TRG1a-b] pathological response) or chemoresistant/RES (moderate [TRG2] or poor [TRG3] pathological response). Representative clinical timelines and longitudinal endoscopic/ PET imaging are depicted in Fig. 1 . Static PDO monocultures enable high throughput at the cost of reduced fidelity. Analysis of selected PDOs (MGE #1181; #0985, #1033, #0099, #1159, #1023) from the EAC patient study cohort revealed that these organoids histologically resemble their primary tissues of origin and maintain cellular complexity, e.g. cohesive morphology (Fig. 2Ai-ii). Tumor tissue-derived 3D PDOs express various epithelial markers, including Pan-cytokeratin (PanCK), a marker of differentiating epithelial tumors, and Ki67, a prognostic and predictive tumor marker. Patient-matched cancer-associated fibroblasts (CAFs) express standard pan-CAF markers such as lumican (LUM) and PDGFRβ. Myofibroblast-like CAFs (myCAFs) are expressing αSMA, while inflammatory CAFs (iCAFs) are expressing PDGFRα (Fig. 2Aiii). Collectively, these data validate the lineage-specific identities of the primary cell types. Static monocultures of treatment naïve 3D-organoids were directly exposed to a defined triplet chemotherapy for 72 h. This approach was designed to achieve patient specific chemosensitivity and minimize additional nonspecific toxicity of chemotherapeutic drugs in vitro . When PDOs are used as an in vitro platform, the area under the drug response curve (AUC), which integrates both drug potency and efficacy, is a more robust and accurate parameter for predicting patient response to combination therapy compared to the half-maximal inhibitory concentration (IC 50 ) [ 12 , 40 ]. We observed enhanced resolution in the survival curves when comparing the responses of chemosensitive and chemoresistant PDOs after 72 h (Fig. 2 B), in contrast to 24 or 48 h (data not shown). We successfully determined subcytotoxic doses (here, IC 50 values) for only 5 of 6 PDOs with no significant differences in the mean IC 50 and AUC values between the groups. Mean percentage of survival at some specified doses frequently overlaps, making it difficult to predict patient response. Additionally, one chemosensitive PDO failed to recapitulate in vivo responses (Fig. 2 B.i). The data indicate additional higher-fidelity follow-up steps are necessary before accurately predicting patient responses. To address the issue of technically difficult media changes with 3D-PDOs in Matrigel, we tested dispersed fragmented 2.5D-PDOs cultured on ECM coated 96-well plates. This High throughput or HTP approach allows us to screen sensitivity of tumor cells against FOT regimen using the precise clinical drug exposure conditions (Table S1 - S2 ; 1 cycle FOT). Here, despite multiple rounds of media replacement, the untreated cells continued to adhere tightly to the ECM-coated surfaces of the 96-well plates, differing from the behavior of cells in 3D Matrigel. However, despite this ability to replicate inpatient dosing, the survival of chemosensitive organoids was not found to be significantly different from resistant organoids (Fig. 2 B.ii), highlighting the need for a more accurate disease modelling platform. Establishment and characterization of esophageal-organ-on-a-chip system We leveraged the microfluidic organ-on-chip platform (Emulate Inc.) to create a more relevant, patient specific, stroma-inclusive, high-fidelity preclinical EAC avatar. The timeline for model development and assessment of treatment response is depicted in Figure S1 A. Esophageal PDO derived epithelial cells were grown in the upper “luminal/epithelial” channel, separated by a porous membrane from the lower “vascular/stromal” channel that was lined with patient-matched fibroblasts. When subjected to a physiologically relevant nutrient flow (60µl/h), the EAC-derived cells proliferate and regenerate at the tumor-stromal interface. Horizontal- and vertical-views of 4% paraformaldehyde (PFA) fixed chips (Fig. 3 A) and live microtissues (Fig. 3 B) revealed the presence of a characteristic tightly anchored and single-layered squamous epithelium in normal esophagus-chips versus the disorganized and multilayered epithelial cells with glandular protrusions in EAC-chips. A low magnification, cross-sectional image of the chip shows the spatial organization of proliferating epithelial/tumor cells (Ki67⁺) and CAFs (vimentin⁺) across the PDMS membrane (Figure S1 B). Figure S2 showcases bright-field images of epithelial/tumor side of all 8 chips developed for this study. Each tumor chip exhibits an unpolarized and distinct disorganized arrangement of epithelial/tumor cells, with no evidence of normal, stratified, polarized squamous-like epithelium, further supporting patient specific, heterogeneous tumorigenesis within the chip. SEM analysis of the EAC-chips reveals stacked and closely packed pleiomorphic tumor cells with microvilli (Fig. 3 C). The EAC-Chip also displayed a decrease in apparent permeability, indicating the formation of a physiologically relevant tissue barrier, within 5 days (Figure S4 A). Interestingly, functional separation between the epithelial and stromal channels of the microfluidic device enables delivery of therapies through the interstitial space ( via the lower channel) to mimic how cancers are exposed to chemotherapy in vivo . EAC chips emulate the histology and genetic background of the source patient tissue Histological analysis of a representative EAC-chip revealed faithful recapitulation of the morphological features of the tumor of origin (Fig. 4 A-B and S3). According to findings from TCGA [ 41 ], and other research teams [ 42 , 43 ], copy number alteration (CNA) and loss of heterozygosity (LOH), such as deletions and amplifications, are more common in EAC patients than point mutations. Whole exome sequencing (WES) unraveled a set of shared subclones with mutations in tumor suppressor genes or other oncogenes across source patient tissue, matching the PDO and EAC-chip. Several noteworthy oncogenic driver mutations in tumor suppressor genes such as TP53, SMAD4, KDM6A, and mutations in oncogenes such as LAMA1, ZNF521, CDH2/7, are found to be shared by matched EAC primary tissue, PDO and microtissue on the chip (as shown in Fig. 4 B and S5). There are mutations within oncogenes that are exclusively shared by primary tissues and chips e.g. APC, and GATA6. Evaluation of clinical mimicry in EAC chips To assess the efficacy of this model for the prediction of treatment efficacy, triplet chemotherapeutic agents were administered via the stromal/fibroblast channels of 3 chemosensitive (#1181, #0985, #1033) and 3 chemoresistant (#0099, #1159, #1023) EAC-chips on the 8th day. Figure S1 B displays the fibroblast channel through which chemotherapy was administered. Each chip was subjected to one cycle of combination chemotherapy (FOT; >24 h, days 8–9) followed by media flow without chemotherapeutics for 72 h (days 9–12). Confocal microscopic and SEM imaging revealed that the epithelial cells in chemosensitive chips were almost absent from the membrane surface after one cycle of FOT; the few remaining cells displayed an involuted morphology with membrane blebbing and tissue discontinuity (Fig. 5Ai-ii). On the other hand, the chemoresistant cancer-derived epithelial layer remained almost intact, and the cells retained their morphology. Notably, fibroblast layers in the lower channel were maintained after treatment in both cohorts, indicating that the chemotherapy perfused directly through the stroma-lined channel at inpatient- equivalent doses selectively kills chemosensitive epithelial cancer cells. In addition, propidium iodide (PI)-based live-dead cell imaging further demonstrated increased cell death following treatment in chemosensitive EAC chips, with greater nuclear incorporation of PI after treatment than in chemoresistant samples (Fig. 5 B and S6). LDH release, a measure of plasma membrane damage and cancer cell death, was significantly higher in the effluents of the epithelial channel of chemosensitive EAC-chips than in those of chemoresistant EAC-chips collected immediately following FOT administration. (Fig. 5 C). Representative images of the disease-free (normal) esophagus-on-a-chip show intact epithelial layer with insignificant number of dead cells following administration of the same doses of FOT, perfused through the stromal channel (Figure S4 C). Interestingly, when the same chemotherapeutic agents were perfused directly into a chemoresistant cancer cells via the top channel, significant cytotoxicity was observed and chemoresistant EAC-Chip failed to accurately replicate the inpatient response (Figure S4 B), confirming that physiologically relevant delivery of these chemotherapeutic agents through the interstitial space (or stromal channel) is a key requirement for mimicking features of the TME and creating a patient-relevant model. Correlation between clinical response (RECIST v1.1) and effectiveness of NACT reconstituted on EAC-chips. Proliferating tumor cells shed fragments of C-terminus of CK19 (40 KDa) or CYFRA 21 − 1 into the serum, and NACT can effectively lower the levels of this circulating tumor marker [ 44 – 49 ]. We measured soluble CK19 fragments in serum of chemo-naïve EAC patients (n = 10) (Table S3 and Figure S7) and chip derived effluents. According to the RECIST 1.1 criteria (objective responses), selected SENS patients (n = 3) experienced a reduction in tumor burden of more than 50% from baseline CT scans (Fig. 6 ). The decrease in CK19 fragment concentration in each treated chip effluent correlates with inpatient (source patient of the chip) chemotherapy-induced decrease in local tumor burden. SENS and RES chips both demonstrated a strong correlation with the corresponding objective responses to the regimen implemented for the chips (Fig. 6 ). Discussion Emerging evidence has indicated that the high degree of chemoresistance in locally advanced EAC is driven by both tumor and stromal heterogeneity. Developing accurate experimental models for EAC remains critically important due to the high morbidity and mortality linked to this aggressive esophageal cancer subtype. Currently, there are no spontaneously developing animal models for EAC. The most commonly used mouse model for EAC was developed by overexpressing interleukin-1β (IL-1β) in the esophagus and forestomach, which induces chronic inflammation, Barrett’s esophagus-like metaplasia, and eventually EAC [ 50 ]. However, this process requires over 3–4 months, yields inconsistent results, as not all mice develop the full spectrum of disease, particularly EAC. While these models replicate certain features of human disease, the keratinized nature of the murine esophagus limits their utility for accurately assessing drug efficacy and toxicity. Patient-derived orthotopic xenograft (PDOX) models are occasionally employed to study EAC. Therefore, the novel model presented in this manuscript holds significant promise for advancing research in the EAC field. Recently, significant advances have been made using patient-derived 3D tumor organoid-based culture systems, as this model faithfully preserves the heterogeneity of primary tumors and replicates patient responses to therapy. While PDOs offer more complex cellular structures than traditional cell cultures do, control over the microenvironment is not as precise as in organ-on-a-chip systems. [ 32 , 51 – 54 ]. Here, we highlighted potential shortcomings in the clinical relevance of static EAC-PDO monocultures when exposed to a combination of chemotherapeutics (only SOC available for locally advanced EAC patients). Standard organoid culture methods, in which PDOs are embedded in fragile Matrigel domes, do not allow for the precise recapitulation of the chemotherapy regimen the patient tumors experience in terms of dosing and timing of delivery. Consequently, chemotherapeutic agents continuously remain in the media for 24h -72h, often leading to bystander cytotoxicity, inconclusive IC 50 or AUC values for determining chemoresistance. Notably, a few PDOs failed to respond consistently to clinically relevant triplet chemotherapeutic dose regimen, under static conditions. Standard organoid cultures allow for the assessment of resistance to monotherapies like oxaliplatin or 5-FU; however, they fall short in reliably replicating resistance to docetaxel, whether used alone or in combination with other drugs. Docetaxel is pharmacokinetically stable and highly toxic, which is why patients typically receive it for only an hour. No human trials have been conducted in which patients received docetaxel for a duration longer than this. Therefore, our findings have aligned with previous clinical studies and indicated that static co-culture methods are not always suitable for such complex investigations. To overcome this issue, we reconstituted the clinical regimen of triplet chemotherapy ex vivo using this novel model. Furthermore, we also illustrated the advantages of integrating both PDO and microfluidic organ chip methodologies to generate a novel patient-specific preclinical EAC platform. Patient specificity, fidelity, quality, functionality, and clinical significance comprise key parameters that we have addressed through the development of this platform. Although very few efforts have been made to create EAC-on-a-chip or 3D-EAC-bioprinted models [ 25 ], no patient-centered, physiologically, and clinically relevant EAC-chip system capable of emulating patients' innate response to chemotherapy is currently available. This platform is an excellent EAC organoid-based readout for assessing disease activity and treatment response. For the first time, we have directly compared the in vitro response to a clinically relevant combinations of chemotherapeutics in 2.5D, 3D tumor PDOs and organ chips. Our results demonstrate the superiority of the EAC-Chip in accurately replicating patient response. In accordance with our protocol (Fig. 1 ), PDOs and corresponding EAC chips can be developed within 5–6 weeks of a diagnostic biopsy, allowing for chemotherapy response prediction within the NACT timeline (approx. 2–3 months). These rapid results have the potential to offer alternative or salvage therapy options for chemoresistant patients at adjuvant time frames. This EAC-chip enables drug diffusion through the tumor-stroma interface, aiding in the study of differential cellular responses and the effects on tissue integrity and drug PK/PD. Since the current organ-chip system remains a low-throughput model, a static, 3D-PDO model is still required for HTP drug screening. Drugs demonstrating efficacy in the HTP screening can subsequently be evaluated using this high-fidelity, microfluidic organ-chip platform for more accurate prediction. This platform is also perfectly aligned with the guiding principles for ethical use of animals in research or 3Rs (Replacement, Reduction and Refinement). Conclusions This patient-specific EAC-Chip model can serve as a basis for the development of a functional precision oncology platform for this aggressive malignancy. However, this concept of implementing patient-specific tumors-on-a-chip is not limited to EAC; it can also be extended to various other cancer types. Declarations Ethics approval and consent to participate Patient data were used in this study, with informed consent obtained from each patient prior to the diagnostic biopsy. The study was approved by the Research Ethics Board of the McGill University Health Centre (protocols 2007-856 and 2021-7681). Consent for publication The study includes data from individuals who provided consent for their information without identity to be used exclusively for research purposes. Acknowledgements LF and DI contributed equally to the study. We would like to thank the study participants who kindly permitted us to collect and use their biological material and clinical data. Schematic diagrams were prepared using BioRender. We also acknowledge the RI-MUHC BioBank platform for their role in obtaining patient consent and sample collection, the MUHC Pathology department for determining tumor content in primary tissue (Dr. Pierre-Olivier Fiset), Genome Quebec for their role in acquiring the whole exome sequencing data. Additionally, we would like to extend our acknowledgements for their technical assistance to the Histopathology and Molecular Imaging platforms at the Research Institute of the McGill University Health Centre, as well as to the Histology platform of the Goodman Cancer Institute at McGill University. Authors’ contributions Conceptualization: SP, LF, and DI Methodology: SP, ES, VS. Experiments: SP, MK, SFT, SDB, JB, KT. Visualization: SP Funding acquisition: LF, VS Project administration: NB Supervision: LF, VS Writing – original draft: SP Writing – review & editing: SP, LF, DI, NB Research Funding Montreal General Hospital Foundation (LF) Impact Grant award from the Department of Defense-Congressionally Directed Medical Research Programs, Award # CA200572 (LF, VS) STrOmal ReprograMing (STORMing Cancer); Cancer Research UK Grand Challenge. Conflicts of Interest Dr. Donald E. Ingber is a founder, board member, and chairs the SAB of Emulate Inc., and holds equity. The remaining authors disclose no conflicts. Data Availability The primary data supporting the findings in this study are available within the manuscript and its Supplementary Information. The raw source and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request. Authors’ details 1 Cancer Research Program, Research Institute of the McGill University Health Centre; Montreal, Quebec, Canada. 2 Wyss Institute for Biologically Inspired Engineering, Harvard University; Boston, USA. 3 Department of Bioengineering, McGill University; Montreal, Canada. 4 Department of Surgical and Interventional Sciences, McGill University; Montreal, Canada. Supplementary Information Supplementary materials are available as online version. References Morgan E, Soerjomataram I, Rumgay H, Coleman HG, Thrift AP, Vignat J, Laversanne M, Ferlay J, Arnold M. The Global Landscape of Esophageal Squamous Cell Carcinoma and Esophageal Adenocarcinoma Incidence and Mortality in 2020 and Projections to 2040: New Estimates From GLOBOCAN 2020. Gastroenterology. 2022;163(3):649–58. e642.https://doi.org/10.1053/j.gastro.2022.05.054 . Ajani JA, D'Amico TA, Bentrem DJ, Cooke D, Corvera C, Das P, Enzinger PC, Enzler T, Farjah F, Gerdes H, et al. 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Clinicopathological characteristics of the study cohort The study cohort was subdivided into chemosensitive and chemoresistant cohorts based on pathological and clinical response to NACT. Patients were HER2-negative and microsatellite stable. All patients except the underlined patients received FLOT. Those patients received DCF. AC: adenocarcinoma; EGJ: esophageal-gastric junction (Siewert II); F: 5-fluorouracil; L: leucovorin; O: oxaliplatin; T or D: docetaxel; C: cisplatin; Objective Response: RECIST: Response Evaluation Criteria in Solid Tumors; PET-SUV response - CR: complete response; PR: partial response; SD: stable disease. GX: Undetermined grade. MGE: MUHC-Gastroesophageal. Organoids and corresponding CAFs were collected from the group of patients with locally advanced, resectable EAC before undergoing NACT and were utilized to create EAC chips. Patient specimens, except for the underlined ones, were used to evaluate chemotherapy response on EAC chips. Cite Share Download PDF Status: Published Journal Publication published 23 May, 2025 Read the published version in Journal of Translational Medicine → Version 1 posted Editorial decision: Accept 08 May, 2025 Reviewers agreed at journal 13 Apr, 2025 Reviewers invited by journal 13 Apr, 2025 Editor assigned by journal 10 Apr, 2025 First submitted to journal 05 Apr, 2025 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Ingber","email":"","orcid":"","institution":"RI-MUHC: Research Institute of the McGill University Health Centre","correspondingAuthor":false,"prefix":"","firstName":"Donald","middleName":"E.","lastName":"Ingber","suffix":""},{"id":442356674,"identity":"a2c36fa9-216c-44e6-b26e-088dbe846d2b","order_by":10,"name":"Lorenzo Ferri","email":"","orcid":"","institution":"RI-MUHC: Research Institute of the McGill University Health Centre","correspondingAuthor":false,"prefix":"","firstName":"Lorenzo","middleName":"","lastName":"Ferri","suffix":""}],"badges":[],"createdAt":"2025-02-12 18:18:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6017396/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6017396/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12967-025-06593-1","type":"published","date":"2025-05-23T15:57:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80800921,"identity":"3f64b359-775a-4420-82a6-4add1433e61d","added_by":"auto","created_at":"2025-04-17 08:35:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1212271,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePatient treatment timeline.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTimelines of diagnosis, treatment, response to treatment, and PDO generation in representative \u003cstrong\u003e(A) \u003c/strong\u003echemosensitive (SEN) and \u003cstrong\u003e(B)\u003c/strong\u003e chemoresistant (RES) patients.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6017396/v1/109d8c62b23f698b492c2565.png"},{"id":80800378,"identity":"1539895a-408c-47f7-b80f-3f5079903a09","added_by":"auto","created_at":"2025-04-17 08:27:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2834929,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStatic PDO cultures maintained without associated fibroblasts display higher throughput but lower fidelity. (A)\u003c/strong\u003e \u003cstrong\u003ei)\u003c/strong\u003e H\u0026amp;E-stained sections of representative chemonaïve primary tissues and corresponding PDOs. \u003cstrong\u003eii) \u003c/strong\u003eRepresentative EVOS images of mature EAC tissue-derived PDOs stained with DAPI (cyan) and Ki67 (magenta). 20X objective. Scale bars, 50 mm.\u003cstrong\u003e iii) \u003c/strong\u003ePDOs and CAFs were characterized using relevant epithelial and fibroblasts lineage-specific biomarkers.\u003cstrong\u003e (B)\u003c/strong\u003e Response to treatment measured via the CellTiter-Glo® assay. \u003cstrong\u003ei)\u003c/strong\u003e 3D-PDOs were treated with triplet chemotherapy for 72 h, and dose‒response curves are displayed. Differences in subcytotoxic IC\u003csub\u003e50\u003c/sub\u003e doses (determined \u003cem\u003evia\u003c/em\u003e GraphPad) were not significant between chemosensitive (green; n=3) and chemoresistant (red n=3) PDOs. \u003cstrong\u003eii)\u003c/strong\u003e Fragmented PDOs seeded on ECM-coated wells and exposed to either Cmax or subcytotoxic dose (\u0026lt;Cmax) concentrations of FOT (ST2) on day 8. Viability was determined on day 12. The representative bar graphs (means ± SEMs) show no significant differences in response between the groups.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6017396/v1/c8ecd2e7589de78f7ca117b5.png"},{"id":80800368,"identity":"e87d867e-4aed-4705-964c-cf49d70a0bdf","added_by":"auto","created_at":"2025-04-17 08:27:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2162456,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstablishment and characterization of esophageal organ-on-chipsystems. \u003c/strong\u003ePDO-derived normal or EAC epithelial cells were cultured in the upper “epithelial” channel, along with patient- and tissue-matched fibroblasts in the lower “stromal” channel, for 12 days under continuous flow.\u003cstrong\u003e (A)\u003c/strong\u003e \u0026nbsp;Brightfield (left) and immunofluorescence (right) images of vertical sections show cell patterns as well as interfaces between the epithelial layer and fibroblasts. The gray dashed lines indicate the boundaries of the porous PDMS membrane. \u003cstrong\u003e(B)\u003c/strong\u003e Horizontal brightfield images of chip microtissues showing a characteristic squamous epithelium layer (normal esophagus-on-chip) or irregular adenocarcinoma-like glandular formations (EAC chip). \u003cstrong\u003eC.\u003c/strong\u003e SEM images of representative vertical cross-sections of epithelial surfaces of the EAC chip.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6017396/v1/28e58a2869d83d5d7c974c6b.png"},{"id":80800923,"identity":"e440063c-e211-480b-826a-b8eddbddd5f2","added_by":"auto","created_at":"2025-04-17 08:35:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2146814,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEAC chips recapitulate the histology and genetic features of the source patient tissue. (A)\u003c/strong\u003e Representative brightfield image of a horizontal section of the EAC-Chip derived from patient \u003cstrong\u003eMGE-1023\u003c/strong\u003e. \u003cstrong\u003e(B)\u003c/strong\u003e Representative H\u0026amp;E-stained image of an OCT®-embedded cryosection (30 μm) of on-chip EAC epithelium of \u003cstrong\u003eMGE-1023\u003c/strong\u003e(right) demonstrating histological similarity to a matched primary tumor section (5 μm, left). \u003cstrong\u003e(C) \u003c/strong\u003eGenetic materials from EAC patient (\u003cstrong\u003eMGE-1023) \u003c/strong\u003eon-a-chip recapitulates the genetic landscapes of primary tissues despite undergoing \u003cem\u003ein vitro\u003c/em\u003e culture for several generations.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6017396/v1/2d8b68809b37c35de24b6fc2.png"},{"id":80800922,"identity":"7103edcc-18e2-432a-a2f6-c5acf5e2d448","added_by":"auto","created_at":"2025-04-17 08:35:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2132916,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of clinical mimicry in EAC chips.\u003c/strong\u003e EAC chips were subjected to 1 cycle (24 h) of FOT (Supplementary Table 2) at clinically relevant concentrations on day 8 \u003cem\u003evia\u003c/em\u003e the stromal channel. The chips were maintained for another 72 h without chemotherapy. \u003cstrong\u003e(A) i. \u003c/strong\u003eRepresentative confocal image showing near-complete eradication of epithelial cells (CK7\u003csup\u003e+\u003c/sup\u003e) after 1 cycle of chemotherapy in the chemosensitive sample, whereas the epithelial layer remained intact in the chemoresistant sample. \u003cstrong\u003eii. \u003c/strong\u003eRepresentative SEM images demonstrating the disrupted surface of epithelial cell membranes in treated chemosensitive but not chemoresistant EAC-Chips. \u003cstrong\u003e(B) i. \u003c/strong\u003eRepresentative live brightfield and fluorescence micrographs at 72 h posttreatment depict the different sensitivities of chemosensitive and chemoresistant samples. The black arrows indicate intercellular spaces affected by chemotherapy. \u003cstrong\u003eii. \u003c/strong\u003eThe sizes of the dead (PI-positive; average cell area ≥ 100 μm2) regions in 6–8 random fields of intact chips were measured, and the sizes (mean ± SEM) are displayed as a bar graph, which shows larger affected area and additional dead cells in chemosensitive versus chemoresistant samples. \u003cem\u003ep \u003c/em\u003evalue **\u0026lt; 0.01, ns: not significant; Mann‒Whitney U‒Wilcoxon test. \u003cstrong\u003e(C) \u003c/strong\u003eEAC-chip effluents were collected on day 9 from the epithelial channel to determine LDH levels. The data revealed significantly greater LDH release in the chemosensitive samples than in the chemoresistant samples, indicating greater chemotherapy-mediated cytotoxicity. The data are presented as the mean percentage of cytotoxicity ± SEM. \u003cem\u003ep\u003c/em\u003e values \u0026lt;0.05; 2-way ANOVA.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6017396/v1/998f7c572b7010502ad68041.png"},{"id":80800925,"identity":"165baa12-c2a8-474b-91f1-d14487459382","added_by":"auto","created_at":"2025-04-17 08:35:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":75803,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe FOT regimen as reconstituted on EAC chips is highly effective with respect to the clinical response (RECIST v1.1). \u003c/strong\u003eThe merged comparative bar graph depicts matched inpatient objective responses to NACT (4 cycles) and in-chip NACT responses (1 cycle). All SENS patients had a \u0026gt;50% reduction in tumor burden from baseline CT according to RECIST 1.1. Chip effluents were collected on day 11 (48 h after chemotherapy) for the CK19 fragment assay. The alterations in soluble CK19 fragment concentrations within EAC chip effluents before and after triplet chemotherapy are expressed as the mean percentage reduction in CK19 concentration ± SEM. This reduction is calculated separately by comparing the treated EAC-chips to their untreated counterparts.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6017396/v1/e5a3f602cbb817e868f06ec2.png"},{"id":83460070,"identity":"889eed4d-2264-411b-8d59-50c5b36b97dc","added_by":"auto","created_at":"2025-05-26 16:09:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11892087,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6017396/v1/53d0d58d-0baa-4918-ba88-96a8692cc864.pdf"},{"id":80800375,"identity":"4b451586-b1a7-447f-a7c4-867bbd1f828a","added_by":"auto","created_at":"2025-04-17 08:27:27","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9377363,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6017396/v1/fea4459d6c42d14f04478033.pdf"},{"id":80800371,"identity":"257de7d7-f5dc-4982-b776-7d02dc70272f","added_by":"auto","created_at":"2025-04-17 08:27:27","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":315587,"visible":true,"origin":"","legend":"","description":"","filename":"DocumentS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6017396/v1/46119461035d144192ec63fe.pdf"},{"id":80800373,"identity":"f75cba8f-68fa-44a9-af8b-a75725ca1777","added_by":"auto","created_at":"2025-04-17 08:27:27","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":113374,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6017396/v1/18bf58c4c91fb726eb9deb3b.pdf"},{"id":80800380,"identity":"6bef6207-7866-40dd-ab27-66c038cbf068","added_by":"auto","created_at":"2025-04-17 08:27:27","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":682000,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6017396/v1/0822c8a1badec9ef7b360be5.jpeg"},{"id":80800934,"identity":"c7ca7e5c-db98-4006-94ad-ce2af08c7f3d","added_by":"auto","created_at":"2025-04-17 08:35:28","extension":"tiff","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":5763750,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1. Clinicopathological characteristics of the study cohort\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study cohort was subdivided into chemosensitive and chemoresistant cohorts based on pathological and clinical response to NACT. Patients were HER2-negative and microsatellite stable. All patients except the underlined patients received FLOT. Those patients received DCF. AC: adenocarcinoma; EGJ: esophageal-gastric junction (Siewert II); F: 5-fluorouracil; L: leucovorin; O: oxaliplatin; T or D: docetaxel; C: cisplatin; Objective Response: RECIST: Response Evaluation Criteria in Solid Tumors; PET-SUV response - CR: complete response; PR: partial response; SD: stable disease. GX: Undetermined grade. MGE: MUHC-Gastroesophageal.\u003c/p\u003e\n\u003cp\u003eOrganoids and corresponding CAFs were collected from the group of patients with locally advanced, resectable EAC before undergoing NACT and were utilized to create EAC chips. Patient specimens, except for the underlined ones, were used to evaluate chemotherapy response on EAC chips.\u003c/p\u003e","description":"","filename":"Table1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-6017396/v1/70888787b5b1bea1383c0d43.tiff"}],"financialInterests":"","formattedTitle":"Patient-derived esophageal adenocarcinoma organ chip: a physiologically relevant platform for functional precision oncology","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEsophageal adenocarcinoma (EAC) is the fastest-rising malignancy in North America [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The only standard-of-care (SOC) treatment for locally advanced, resectable EAC is perioperative docetaxel-based triplet chemotherapy (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Most EAC tumors exhibit \u003cem\u003eab initio\u003c/em\u003e chemotherapy resistance. Meanwhile, most responders to neoadjuvant chemotherapy (NACT) exhibit high rates of progression and metastasis during the adjuvant period. Given the lack of alternative approaches, each non-responder to NACT continues to receive the SOC as adjuvant therapy. The development of accurate \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e preclinical models, prognostic markers for response to therapy, and alternative effective treatments for chemoresistant EAC patients all remain critical unmet clinical needs.\u003c/p\u003e \u003cp\u003ePatient-derived organoid (PDO) culture recapitulates the heterogeneity of epithelial cell lineages more faithfully than traditional 2-dimentional (2D) cell lines do[\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These cancer organoids have been effectively used for high-throughput (HTP) screening of monotherapies and for predicting patient outcomes [\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. There are also reports highlighting the ineffectiveness of PDOs in predicting the outcome of treatment with 5-fluorouracil (5-FU) combined with oxaliplatin [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTraditionally, organoids grow in complex 3-dimentional (3D)-Matrigel domes, and cultures are maintained under static conditions. Matrigel, a heterogeneous mixture of murine sarcoma-derived ECM proteins, exhibits lot-to-lot varying concentration, stiffness and influences drug diffusion [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Thus, PDO culture format often suffers from poor reproducibility [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. To address this critical issue, several groups have used well-defined micro-engineered hydrogels or alginates as alternatives to Matrigel, especially for incorporating user-defined microenvironmental signals to investigate human epithelial tumors [\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Thus the \u003cem\u003ein vitro\u003c/em\u003e response of PDOs may diverge from the observed response in patients, especially for combination therapy where each drug may have a distinct pharmacokinetic profile and toxicity (e.g. docetaxel).\u003c/p\u003e \u003cp\u003eSince organoids lack various cellular (stromal and immune) and physiological factors (oncotic pressure and interstitial fluid movement) of the tumor microenvironment (TME), they often fail to fully capture the intricacies of drug efficacy and distribution observed in actual tumors [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. There is thus significant room to improve on cancer organoid technology by combining organoids and stromal cells to develop a higher-fidelity platform that more effectively recapitulates human-relevant esophageal organ structure, cellular/tissue heterogeneity, physiology, pathogenesis, and drug distribution and is more likely to accurately predict in patient response to therapy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough still a low-throughput system, microfluidic organ-chip technology offers multiple advanced features that are lacking in static organoid culture technology, including recapitulation of the organ-level TME [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Organ Chips provide dynamic fluid flow and thus provide a unique opportunity to mimic human relevant pharmacokinetic and pharmacodynamic (PK/PD) profiles of drugs \u003cem\u003ein vitro\u003c/em\u003e to recapitulate treatment regimen-specific drug efficacy and toxicity [\u003cspan additionalcitationids=\"CR28 CR29 CR30 CR31\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Additionally, organ chips also demonstrated some translatable endpoints similar to those observed for human pathophysiology [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, we highlight a key limitation of 3D PDO culture technology and propose an improved approach by integrating PDO and cancer-associated fibroblast (CAF) co-cultures with microfluidic organ-on-a-chip technology. We describe a first-generation, syngeneic human EAC-Chip platform containing PDO-derived EAC cells separated by a porous membrane from patient-matched CAFs), co-cultured under continuous flows of nutrient media. Furthermore, we also demonstrate that the EAC-chip platform achieves clinical mimicry when a triplet chemotherapy regimen is perfused through the bottom (stromal) channel lined with CAFs.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eStudy cohort\u003c/h2\u003e\n \u003cp\u003eConsented patients with locally advanced resectable EAC (n\u0026thinsp;=\u0026thinsp;8) who received triplet NACT were selected for the current study (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Patients were divided into two groups, \u003cstrong\u003e(i) Chemosensitive (SENS)\u003c/strong\u003e (n\u0026thinsp;=\u0026thinsp;4) patients who demonstrated either complete or partial objective response (OR) to NACT. \u003cstrong\u003e(ii) Chemoresistant (RES)\u003c/strong\u003e (n\u0026thinsp;=\u0026thinsp;4) patients were either clinically assessed for stable disease (SD) or progressive disease (PD) following NACT.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eIsolation and propagation of a syngeneic patient-derived epithelial/tumor niche and associated fibroblasts\u003c/h3\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTissue specimens were collected from the adenocarcinoma and adjacent normal regions at the time of diagnostic endoscopic biopsy. Organoids and regional fibroblasts were established from the specimens and propagated using the protocol below.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eBriefly, the tissue was minced and enzymatically digested with tissue digestion buffer in a GentleMACSTM Octo Dissociator with heaters (Milltenyi Biotec.). The cell pellets were obtained and trypsinized to obtain single cells. The resulting cells were resuspended and were allowed to stand undisturbed for 10min. Later a fraction was transferred to a new tube for fibroblast culture and centrifuged.\u003c/p\u003e\n\u003cp\u003e\u003cspan\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003ea. For fibroblast propagation\u003c/span\u003e: Cell pellets were resuspended in 1ml fibroblast propagation medium and seeded in Type I collagen precoated plates.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan\u003eb.\u0026nbsp;\u003c/span\u003e\u003cspan\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eFor organoid propagation\u003c/span\u003e: Cell pellet was resuspended in ice cold Matrigel and plated. Organoids in Matrigel domes were maintained in\u0026nbsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eexpansion medium\u003c/span\u003e. Mature PDOs were passaged after 10\u0026ndash;12 days. Single cells were obtained through enzymatic digestion and trypsinization.\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eBoth PDOs and associated fibroblasts were cultured under hypoxic conditions, i.e., 3% O\u003csub\u003e2\u003c/sub\u003e. All consumables used for each method are listed in Table \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eFor analysis of organoid histology, Histogel blocks were prepared following an existing protocol (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ccr.cancer.gov/sites/default/files/2022-11/Histogel_Protocol_0.pdf\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eCulture of stroma-exclusive, fragmented and dispersed PDOs under static conditions\u003c/h3\u003e\n\u003cp\u003eFragmentated PDOs were obtained with TrypLE\u0026trade; (Invitrogen 12605010) at 37\u0026deg;C for 1 minute. 5000 fragmented PDOs were seeded on ECM precoated 96 well plates and maintained in organoid expansion medium. On the sixth day post-seeding, the \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eexpansion medium\u003c/span\u003e (EM) was replaced with \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003edifferentiation medium (DM)\u003c/span\u003e which was maintained throughout the experiment. Cells were maintained for 12 days and harvested for measuring cell viability and microscopy.\u003c/p\u003e\n\u003ch3\u003eEstablishment of syngeneic esophageal tissue-on-a-chip\u003c/h3\u003e\n\u003cp\u003eThe complete organ-on-a-chip setup (Chip-S1\u0026trade; stretchable organ chips, POD\u0026trade; portable modules, Zoe-CM-1\u0026trade; culture module and Orb-HM1\u0026trade; Hub Module) was procured from Emulate Inc. The chip design comprises two parallel microfluidic channels, separated by an optically transparent 50 \u0026micro;m PDMS porous membrane (7 \u0026micro;m diameter). On day 0, chip surfaces were activated with S1 chip activation solution and high-power UV light. The activated membrane was coated with freshly prepared ice-cold ECM solution (ST4) and incubated overnight. On day 1, PDO-derived fragmented organoids or epithelial cells (concentration: 3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells /ml) were seeded at the upper channel. Once cells adhered to the membrane (~\u0026thinsp;4 h), the chips were connected to flow at 60 \u0026micro;l/h. Patient matched regional fibroblasts were seeded in the lower stromal channel of the chip on day 2. Fibroblasts were incubated for 4 h at 37\u0026deg;C before chips were reconnected to flow at 60 \u0026micro;l/h. On day 6 post-seeding of epithelial cells, the expansion media in the epithelial inlets were replaced with differentiation media. Overall chips were maintained up to 12 days. The development of PDOs and microtissues (day 0 to day 12), morphological changes and the population of cells undergoing treatment-induced cell death were also determined by brightfield and fluorescence microscopy respectively (EVOS, Invitrogen).\u003c/p\u003e\n\u003cp\u003eDetailed protocol (Standard operating protocol) has been supplied as Document S1.\u003c/p\u003e\n\u003ch3\u003eHistological analysis of primary and chip derived microtissues organ chips\u003c/h3\u003e\n\u003cp\u003ePrimary tissue samples fixed in 10% formalin were dehydrated with alcohol and then embedded in paraffin. 5 \u0026micro;m thick sections were prepared, mounted on glass slides, stained with H\u0026amp;E and analyzed. On Day 13, Tissue-Tek\u0026reg; OCT compound (Thermo Fisher Scientific) was administered through both channels of 4% PFA fixed microtissues and OCT-blocks were created. 30 \u0026micro;m vertical cross-sections were prepared, stained with H\u0026amp;E and imaged (EVOS M7000).\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eWhole-exome sequencing and analysis\u003c/h2\u003e\n \u003cp\u003ePDOs were passaged for at least 5 generations to collect ample amount of genomic DNA and develop EAC-chips. H\u0026amp;E-stained tissue sections were reviewed by an expert pathologist before isolation of the DNA. A primary tissue sample with tumor content\u0026thinsp;\u0026ge;\u0026thinsp;50% was submitted for WES. Genomic DNA was extracted (Qiagen: Cat. No.80204) from snap frozen primary tissue, matched organoids, and chip derived cells from upper epithelial channel. DNA extracted from blood (buffy coat) was used as a germline reference.\u003c/p\u003e\n \u003cp\u003eTo compare the genomic similarities between the primary tumor biopsy, organoid and organ-on-chip samples, we aligned the WES reads to the human reference genome (GRCh38) using the Burrows-Wheeler Aligner (BWA)[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. Duplicate reads were marked and base quality scores were recalibrated using the genome analysis toolkit (GATK)[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. Somatic mutations were called for the primary tumour, organoid and organ-on-chip against a patient matched normal sample using muTect [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. Somatic mutations were annotated with ANNOVAR[\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. Acquired copy number alterations (CNA), including regions of loss of heterozygosity (LOH), were determined using FACETS[\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. The mutation allele frequencies were determined with bam-readcount [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eImmunofluorescence microscopy studies\u003c/h3\u003e\n\u003cp\u003e12-day old chips were fixed with 4% PFA and cut into 300\u0026micro;m vertical cross sections using a vibratome (Leica VT1200). Immunofluorescence staining was carried out using manufacturer\u0026rsquo;s suggested dilutions of the primary, secondary antibodies and DAPI nuclear stain (Table \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e). Stained chip sections were imaged using a Zeiss LSM 780 confocal microscope. Whole-mount 3D-PDO culture immunofluorescence was performed \u003cem\u003evia\u003c/em\u003e a 3D tissue clearing kit (5730, Corning) according to the manufacturer\u0026rsquo;s protocol. Images of organoid were captured using either in EVOS M7000 and ZEISS Lattice SIM 3.\u003c/p\u003e\n\u003ch3\u003eScanning Electron Microscopy (SEM)\u003c/h3\u003e\n\u003cp\u003eBoth treated and untreated EAC-chips were fixed with 4% PFA solution and washed 3 times with PBS. Concomitantly, the chip was trimmed using a razor blade, and 300\u0026micro;m vertical cross sections were prepared using vibratome. Afterward, the samples were gradually dehydrated in 100% ethanol and placed in a critical point dryer (CPD030, Leica) and 20 \u0026times; 1-minute CO\u003csub\u003e2\u003c/sub\u003e exchange cycles were performed. Samples were sputter-coated (ACE600, Leica) with an 8 nm layer of platinum. images were acquired using an Environmental Scanning Electron Microscope (Quanta 450, FEI).\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eAssessment of apparent permeability\u003c/h2\u003e\n \u003cp\u003eThe degree of epithelial barrier integrity was determined following an existing protocol (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://emulatebio.com/wp-content/uploads/2021/06/EP187_v1.0_Barrier_Function_Analysis_Protocol.pdf\u003c/span\u003e\u003c/span\u003e). Data were obtained from 10 chips derived from 3 source patients (2\u0026ndash;4 chips/donor).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eAssessment of intrinsic response of intact PDOs to neoadjuvant therapy under static conditions\u003c/h2\u003e\n \u003cp\u003ePDOs were enzymatically dissociated and 10,000 cells per 30 ul of Matrigel/well were distributed in a 96-well plate. 200 ul of OEM was dispensed into each well. \u003cem\u003eIn vitro\u003c/em\u003e drug treatments were conducted in triplicate at concentrations ranging from 0\u0026ndash;1 \u0026micro;M of F:C:T docetaxel with ratio of 10:1:1 respectively. Triplicate chemotherapeutics at relevant doses (described in Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e) were added together for 72 h. Solvent percentage was limited to 0.1%. As Matrigel is soft and does not adhere tightly to the surface, replacing the media with or without triplet chemotherapeutics in a 96-well format was technically unfeasible. The percent viability of intact PDOs was measured after 72 h of treatment using the CellTiter-Glo 3D reagent (Promega, G9681) according to the manufacturer\u0026rsquo;s instructions. Luminescence was measured via Varioskan LUX Multimode Microplate Reader (Thermo Scientific). The experimental readings were normalized to respective solvent controls. Viability curves and IC\u003csub\u003e50\u003c/sub\u003e values were generated using GraphPad Prism 8 software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eClinically relevant drug dosing for static and dynamic culture conditions\u003c/h2\u003e\n \u003cp\u003eThe actual FLOT regimen and clinically relevant dosing reconstituted for the microfluidic culture is described in Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e. Leucovorin was omitted, since it is not a chemotherapeutic agent. Fragmented and dispersed PDOs were seeded in ECM solution coated 96 well plates. Cells were directly exposed to one cycle of FOT on day 8 (Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). Cellular viability was determined on day 12 as described above. The data are representative of biological and technical triplicates.\u003c/p\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eEAC-Chips achieved human relevant epithelial tissue integrity within 5 days under dynamic conditions and treatment began on day 8. FOT chemotherapy was administered through the stromal channel. At first, a flush cycle (\u0026lt;\u0026thinsp;Cmax, FOT: 52:1.7:1] of 1\u0026ndash;2 mins with a flow rate of 1000 \u0026micro;l/h in the bottom channel and 0 \u0026micro;l/h top channel was performed to ensure proper distribution of the drugs only in the stromal microfluidic channel networks. To mimic bolus IV-injection, during the first hour of cycle a mixture of all three chemotherapeutics was administered at a flow rate of 100 \u0026micro;l/h, followed by only a mixture of oxaliplatin (O) and 5FU (F) at the rate 60 \u0026micro;l/h for another hour. Only 5FU (F) was subsequently perfused through the stromal channel at a flow rate of 60 \u0026micro;l/h for a total of 24 h.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eMeasurement of LDH activity\u003c/h2\u003e\n \u003cp\u003eOn day 9, effluents from epithelial channels were collected and samples were immediately assayed for LDH activity using the Promega\u0026trade; LDH Cytotoxicity Assay Kit (Promega #G1781). All absorbance-related measurements were performed in a Varioskan LUX Multimode Microplate Reader (Thermo Scientific). The resulting data are shown as percentage of cell cytotoxicity. Two-way ANOVA was used to assess differences in the cell-free LDH activity in the effluents from treated and untreated groups.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003ePropidium Iodide (PI) assay\u003c/h2\u003e\n \u003cp\u003eOn day 12, media was aspirated from the inlet reservoir of the epithelial channel and replaced with fresh culture medium supplemented with PI (Sigma #P4170) for 5 minutes. The samples were subsequently washed with PBS. Images of 5\u0026ndash;6 random regions of each chip were captured using a EVOS microscope. The non-parametric Mann-Whitney U-Wilcoxon test was employed to compare number of dead cells (defined as having an average cell area\u0026thinsp;\u0026ge;\u0026thinsp;100\u0026micro;m\u0026sup2;) in both treated as well as untreated SENS and RES samples.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eMeasurement of tumor-derived extracellular soluble fragments of CK19\u003c/h2\u003e\n \u003cp\u003eOn day 11, effluents were collected from outlet reservoirs of epithelial channels of both treatment na\u0026iuml;ve and FLOT treated chips. Chemo-na\u0026iuml;ve sera were collected (n\u0026thinsp;=\u0026thinsp;10) at the time of diagnostic endoscopy. Soluble CK19 fragment concentration in serum and effluents were measured using ELISA (Elabscience\u0026reg;, E-EL-H2077) as per the manufacturer\u0026rsquo;s instructions. The data analyzed are displayed as a percentage of reduction in the soluble CK19 fragments in treated effluents compared with untreated chips.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eGraphPad Prism 8 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.graphpad.com\u003c/span\u003e\u003c/span\u003e) was used for statistical analysis. \u003cem\u003ep value\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as significant for each analysis. *\u0026lt;0.05; **\u0026lt;0.01, ***\u0026lt;0.005 ***\u0026lt;0.001; ns: not significant\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eSchematic representation of the strategy adapted to develop personalized human EAC-chips and clinicopathological characteristics of the study cohort.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur comprehensive strategy for modeling patient-derived esophageal adenocarcinoma chips, utilizing both PDOs and associated fibroblasts from the same patient source, for functional precision oncology were demonstrated as a graphical abstract. Esophageal PDOs and matched fibroblasts were generated from endoscopic biopsies of 8 newly diagnosed, treatment-na\u0026iuml;ve patients with locally advanced EAC (Table\u0026nbsp;1). Subsequent pathological (tumor regression grade/TRG) and objective radiographic (RECIST v1.1) responses to docetaxel-based triplet neoadjuvant chemotherapy were extracted from the medical records. Patients were sub-grouped into either \u003cb\u003echemosensitive/SENS\u003c/b\u003e (complete [TRG0] or near-complete [TRG1a-b] pathological response) or \u003cb\u003echemoresistant/RES\u003c/b\u003e (moderate [TRG2] or poor [TRG3] pathological response). Representative clinical timelines and longitudinal endoscopic/ PET imaging are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatic PDO monocultures enable high throughput at the cost of reduced fidelity.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAnalysis of selected PDOs (MGE #1181; #0985, #1033, #0099, #1159, #1023) from the EAC patient study cohort revealed that these organoids histologically resemble their primary tissues of origin and maintain cellular complexity, e.g. cohesive morphology (Fig.\u0026nbsp;2Ai-ii). Tumor tissue-derived 3D PDOs express various epithelial markers, including Pan-cytokeratin (PanCK), a marker of differentiating epithelial tumors, and Ki67, a prognostic and predictive tumor marker. Patient-matched cancer-associated fibroblasts (CAFs) express standard pan-CAF markers such as lumican (LUM) and PDGFRβ. Myofibroblast-like CAFs (myCAFs) are expressing αSMA, while inflammatory CAFs (iCAFs) are expressing PDGFRα (Fig.\u0026nbsp;2Aiii). Collectively, these data validate the lineage-specific identities of the primary cell types. Static monocultures of treatment na\u0026iuml;ve 3D-organoids were directly exposed to a defined triplet chemotherapy for 72 h. This approach was designed to achieve patient specific chemosensitivity and minimize additional nonspecific toxicity of chemotherapeutic drugs \u003cem\u003ein vitro\u003c/em\u003e. When PDOs are used as an \u003cem\u003ein vitro\u003c/em\u003e platform, the area under the drug response curve (AUC), which integrates both drug potency and efficacy, is a more robust and accurate parameter for predicting patient response to combination therapy compared to the half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. We observed enhanced resolution in the survival curves when comparing the responses of chemosensitive and chemoresistant PDOs after 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), in contrast to 24 or 48 h (data not shown). We successfully determined subcytotoxic doses (here, IC\u003csub\u003e50\u003c/sub\u003e values) for only 5 of 6 PDOs with no significant differences in the mean IC\u003csub\u003e50\u003c/sub\u003e and AUC values between the groups. Mean percentage of survival at some specified doses frequently overlaps, making it difficult to predict patient response. Additionally, one chemosensitive PDO failed to recapitulate \u003cem\u003ein vivo\u003c/em\u003e responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB.i). The data indicate additional higher-fidelity follow-up steps are necessary before accurately predicting patient responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo address the issue of technically difficult media changes with 3D-PDOs in Matrigel, we tested dispersed fragmented 2.5D-PDOs cultured on ECM coated 96-well plates. This High throughput or HTP approach allows us to screen sensitivity of tumor cells against FOT regimen using the precise clinical drug exposure conditions (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-\u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e; 1 cycle FOT). Here, despite multiple rounds of media replacement, the untreated cells continued to adhere tightly to the ECM-coated surfaces of the 96-well plates, differing from the behavior of cells in 3D Matrigel. However, despite this ability to replicate \u003cem\u003einpatient\u003c/em\u003e dosing, the survival of chemosensitive organoids was not found to be significantly different from resistant organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB.ii), highlighting the need for a more accurate disease modelling platform. \u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment and characterization of esophageal-organ-on-a-chip system\u003c/h2\u003e \u003cp\u003eWe leveraged the microfluidic organ-on-chip platform (Emulate Inc.) to create a more relevant, patient specific, stroma-inclusive, high-fidelity preclinical EAC avatar. The timeline for model development and assessment of treatment response is depicted in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA. Esophageal PDO derived epithelial cells were grown in the upper \u0026ldquo;luminal/epithelial\u0026rdquo; channel, separated by a porous membrane from the lower \u0026ldquo;vascular/stromal\u0026rdquo; channel that was lined with patient-matched fibroblasts. When subjected to a physiologically relevant nutrient flow (60\u0026micro;l/h), the EAC-derived cells proliferate and regenerate at the tumor-stromal interface. Horizontal- and vertical-views of 4% paraformaldehyde (PFA) fixed chips (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and live microtissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) revealed the presence of a characteristic tightly anchored and single-layered squamous epithelium in normal esophagus-chips versus the disorganized and multilayered epithelial cells with glandular protrusions in EAC-chips. A low magnification, cross-sectional image of the chip shows the spatial organization of proliferating epithelial/tumor cells (Ki67⁺) and CAFs (vimentin⁺) across the PDMS membrane (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e showcases bright-field images of epithelial/tumor side of all 8 chips developed for this study. Each tumor chip exhibits an unpolarized and distinct disorganized arrangement of epithelial/tumor cells, with no evidence of normal, stratified, polarized squamous-like epithelium, further supporting patient specific, heterogeneous tumorigenesis within the chip. SEM analysis of the EAC-chips reveals stacked and closely packed pleiomorphic tumor cells with microvilli (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The EAC-Chip also displayed a decrease in apparent permeability, indicating the formation of a physiologically relevant tissue barrier, within 5 days (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA). Interestingly, functional separation between the epithelial and stromal channels of the microfluidic device enables delivery of therapies through the interstitial space (\u003cem\u003evia\u003c/em\u003e the lower channel) to mimic how cancers are exposed to chemotherapy \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEAC chips emulate the histology and genetic background of the source patient tissue\u003c/h2\u003e \u003cp\u003eHistological analysis of a representative EAC-chip revealed faithful recapitulation of the morphological features of the tumor of origin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B and S3). According to findings from TCGA [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], and other research teams [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], copy number alteration (CNA) and loss of heterozygosity (LOH), such as deletions and amplifications, are more common in EAC patients than point mutations. Whole exome sequencing (WES) unraveled a set of shared subclones with mutations in tumor suppressor genes or other oncogenes across source patient tissue, matching the PDO and EAC-chip. Several noteworthy oncogenic driver mutations in tumor suppressor genes such as TP53, SMAD4, KDM6A, and mutations in oncogenes such as LAMA1, ZNF521, CDH2/7, are found to be shared by matched EAC primary tissue, PDO and microtissue on the chip (as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and S5). There are mutations within oncogenes that are exclusively shared by primary tissues and chips e.g. APC, and GATA6.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of clinical mimicry in EAC chips\u003c/h2\u003e \u003cp\u003eTo assess the efficacy of this model for the prediction of treatment efficacy, triplet chemotherapeutic agents were administered \u003cem\u003evia\u003c/em\u003e the stromal/fibroblast channels of 3 chemosensitive (#1181, #0985, #1033) and 3 chemoresistant (#0099, #1159, #1023) EAC-chips on the 8th day. Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB displays the fibroblast channel through which chemotherapy was administered. Each chip was subjected to one cycle of combination chemotherapy (FOT; \u0026gt;24 h, days 8\u0026ndash;9) followed by media flow without chemotherapeutics for 72 h (days 9\u0026ndash;12). Confocal microscopic and SEM imaging revealed that the epithelial cells in chemosensitive chips were almost absent from the membrane surface after one cycle of FOT; the few remaining cells displayed an involuted morphology with membrane blebbing and tissue discontinuity (Fig.\u0026nbsp;5Ai-ii). On the other hand, the chemoresistant cancer-derived epithelial layer remained almost intact, and the cells retained their morphology. Notably, fibroblast layers in the lower channel were maintained after treatment in both cohorts, indicating that the chemotherapy perfused directly through the stroma-lined channel at \u003cem\u003einpatient-\u003c/em\u003eequivalent doses selectively kills chemosensitive epithelial cancer cells. In addition, propidium iodide (PI)-based live-dead cell imaging further demonstrated increased cell death following treatment in chemosensitive EAC chips, with greater nuclear incorporation of PI after treatment than in chemoresistant samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and S6). LDH release, a measure of plasma membrane damage and cancer cell death, was significantly higher in the effluents of the epithelial channel of chemosensitive EAC-chips than in those of chemoresistant EAC-chips collected immediately following FOT administration. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRepresentative images of the disease-free (normal) esophagus-on-a-chip show intact epithelial layer with insignificant number of dead cells following administration of the same doses of FOT, perfused through the stromal channel (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eC). Interestingly, when the same chemotherapeutic agents were perfused directly into a chemoresistant cancer cells \u003cem\u003evia\u003c/em\u003e the top channel, significant cytotoxicity was observed and chemoresistant EAC-Chip failed to accurately replicate the \u003cem\u003einpatient\u003c/em\u003e response (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB), confirming that physiologically relevant delivery of these chemotherapeutic agents through the interstitial space (or stromal channel) is a key requirement for mimicking features of the TME and creating a patient-relevant model.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCorrelation between clinical response (RECIST v1.1) and effectiveness of NACT reconstituted on EAC-chips.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eProliferating tumor cells shed fragments of C-terminus of CK19 (40 KDa) or CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1 into the serum, and NACT can effectively lower the levels of this circulating tumor marker [\u003cspan additionalcitationids=\"CR45 CR46 CR47 CR48\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. We measured soluble CK19 fragments in serum of chemo-na\u0026iuml;ve EAC patients (n\u0026thinsp;=\u0026thinsp;10) (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e and Figure S7) and chip derived effluents. According to the RECIST 1.1 criteria (objective responses), selected SENS patients (n\u0026thinsp;=\u0026thinsp;3) experienced a reduction in tumor burden of more than 50% from baseline CT scans (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The decrease in CK19 fragment concentration in each treated chip effluent correlates with inpatient (source patient of the chip) chemotherapy-induced decrease in local tumor burden. SENS and RES chips both demonstrated a strong correlation with the corresponding objective responses to the regimen implemented for the chips (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eEmerging evidence has indicated that the high degree of chemoresistance in locally advanced EAC is driven by both tumor and stromal heterogeneity. Developing accurate experimental models for EAC remains critically important due to the high morbidity and mortality linked to this aggressive esophageal cancer subtype. Currently, there are no spontaneously developing animal models for EAC. The most commonly used mouse model for EAC was developed by overexpressing interleukin-1β (IL-1β) in the esophagus and forestomach, which induces chronic inflammation, Barrett\u0026rsquo;s esophagus-like metaplasia, and eventually EAC [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. However, this process requires over 3\u0026ndash;4 months, yields inconsistent results, as not all mice develop the full spectrum of disease, particularly EAC. While these models replicate certain features of human disease, the keratinized nature of the murine esophagus limits their utility for accurately assessing drug efficacy and toxicity. Patient-derived orthotopic xenograft (PDOX) models are occasionally employed to study EAC. Therefore, the novel model presented in this manuscript holds significant promise for advancing research in the EAC field.\u003c/p\u003e \u003cp\u003eRecently, significant advances have been made using patient-derived 3D tumor organoid-based culture systems, as this model faithfully preserves the heterogeneity of primary tumors and replicates patient responses to therapy. While PDOs offer more complex cellular structures than traditional cell cultures do, control over the microenvironment is not as precise as in organ-on-a-chip systems. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan additionalcitationids=\"CR52 CR53\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Here, we highlighted potential shortcomings in the clinical relevance of static EAC-PDO monocultures when exposed to a combination of chemotherapeutics (only SOC available for locally advanced EAC patients). Standard organoid culture methods, in which PDOs are embedded in fragile Matrigel domes, do not allow for the precise recapitulation of the chemotherapy regimen the patient tumors experience in terms of dosing and timing of delivery. Consequently, chemotherapeutic agents continuously remain in the media for 24h -72h, often leading to bystander cytotoxicity, inconclusive IC\u003csub\u003e50\u003c/sub\u003e or AUC values for determining chemoresistance. Notably, a few PDOs failed to respond consistently to clinically relevant triplet chemotherapeutic dose regimen, under static conditions. Standard organoid cultures allow for the assessment of resistance to monotherapies like oxaliplatin or 5-FU; however, they fall short in reliably replicating resistance to docetaxel, whether used alone or in combination with other drugs. Docetaxel is pharmacokinetically stable and highly toxic, which is why patients typically receive it for only an hour. No human trials have been conducted in which patients received docetaxel for a duration longer than this. Therefore, our findings have aligned with previous clinical studies and indicated that static co-culture methods are not always suitable for such complex investigations. To overcome this issue, we reconstituted the clinical regimen of triplet chemotherapy \u003cem\u003eex vivo\u003c/em\u003e using this novel model. Furthermore, we also illustrated the advantages of integrating both PDO and microfluidic organ chip methodologies to generate a novel patient-specific preclinical EAC platform.\u003c/p\u003e \u003cp\u003ePatient specificity, fidelity, quality, functionality, and clinical significance comprise key parameters that we have addressed through the development of this platform. Although very few efforts have been made to create EAC-on-a-chip or 3D-EAC-bioprinted models [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], no patient-centered, physiologically, and clinically relevant EAC-chip system capable of emulating patients' innate response to chemotherapy is currently available. This platform is an excellent EAC organoid-based readout for assessing disease activity and treatment response.\u003c/p\u003e \u003cp\u003eFor the first time, we have directly compared the \u003cem\u003ein vitro\u003c/em\u003e response to a clinically relevant combinations of chemotherapeutics in 2.5D, 3D tumor PDOs and organ chips. Our results demonstrate the superiority of the EAC-Chip in accurately replicating patient response. In accordance with our protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), PDOs and corresponding EAC chips can be developed within 5\u0026ndash;6 weeks of a diagnostic biopsy, allowing for chemotherapy response prediction within the NACT timeline (approx. 2\u0026ndash;3 months). These rapid results have the potential to offer alternative or salvage therapy options for chemoresistant patients at adjuvant time frames. This EAC-chip enables drug diffusion through the tumor-stroma interface, aiding in the study of differential cellular responses and the effects on tissue integrity and drug PK/PD. Since the current organ-chip system remains a low-throughput model, a static, 3D-PDO model is still required for HTP drug screening. Drugs demonstrating efficacy in the HTP screening can subsequently be evaluated using this high-fidelity, microfluidic organ-chip platform for more accurate prediction. This platform is also perfectly aligned with the guiding principles for ethical use of animals in research or 3Rs (Replacement, Reduction and Refinement).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis patient-specific EAC-Chip model can serve as a basis for the development of a functional precision oncology platform for this aggressive malignancy. However, this concept of implementing patient-specific tumors-on-a-chip is not limited to EAC; it can also be extended to various other cancer types.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatient data were used in this study, with informed consent obtained from each patient prior to the diagnostic biopsy. \u0026nbsp;The study was approved by the Research Ethics Board of the McGill University Health Centre (protocols 2007-856 and 2021-7681).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study includes data from individuals who provided consent for their information without identity to be used exclusively for research purposes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLF and DI contributed equally to the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe would like to thank the study participants who kindly permitted us to collect and use their biological material and clinical data. Schematic diagrams were prepared using BioRender. We also acknowledge the RI-MUHC BioBank platform for their role in obtaining patient consent and sample collection, the MUHC Pathology department for determining tumor content in primary tissue (Dr.\u0026nbsp;Pierre-Olivier Fiset), Genome Quebec for their role in acquiring the whole exome sequencing data. Additionally, we would like to extend our acknowledgements for their technical assistance to the Histopathology and Molecular Imaging platforms at the Research Institute of the McGill University Health Centre, as well as to the Histology platform of the Goodman Cancer Institute at McGill University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: SP, LF, and DI\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMethodology: SP, ES, VS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExperiments: SP, MK, SFT, SDB, JB,\u0026nbsp;KT.\u003c/p\u003e\n\u003cp\u003eVisualization: SP\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding acquisition: LF, VS\u003c/p\u003e\n\u003cp\u003eProject administration: NB\u003c/p\u003e\n\u003cp\u003eSupervision: LF, VS\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draft: SP\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; review \u0026amp; editing: SP, LF, DI, NB\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMontreal General Hospital Foundation (LF)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImpact Grant award from the Department of Defense-Congressionally Directed Medical Research Programs, Award # CA200572 (LF, VS)\u003c/p\u003e\n\u003cp\u003eSTrOmal ReprograMing (STORMing Cancer); Cancer Research UK Grand Challenge.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. Donald E. Ingber is a founder, board member, and chairs the SAB of Emulate Inc., and holds equity. The remaining authors disclose no conflicts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe primary data supporting the findings in this study are available within the manuscript and its Supplementary Information. The raw source and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eCancer Research Program, Research Institute of the McGill University Health Centre; Montreal, Quebec, Canada.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eWyss Institute for Biologically Inspired Engineering, Harvard University; Boston, USA.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eDepartment of Bioengineering, McGill University; Montreal, Canada.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003eDepartment of Surgical and Interventional Sciences, McGill University; Montreal, Canada.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary materials are available as online version.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMorgan E, Soerjomataram I, Rumgay H, Coleman HG, Thrift AP, Vignat J, Laversanne M, Ferlay J, Arnold M. 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Cell Mol Gastroenterol Hepatol. 2021;12(5):1719\u0026ndash;41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jcmgh.2021.07.004\u003c/span\u003e\u003cspan address=\"10.1016/j.jcmgh.2021.07.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is 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":"[email protected]","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 adenocarcinoma, Organ-on-a-chip, Organoids, Cancer-associated fibroblasts","lastPublishedDoi":"10.21203/rs.3.rs-6017396/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6017396/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEsophageal adenocarcinoma (EAC) is the sixth most deadly cancer worldwide, with increasing incidence in North America. As no targeted therapy or immunotherapy has revolutionized the management of EAC, chemotherapy is the only standard of care. Most patients with EAC experience poor outcomes because of the inherent or acquired resistance to chemotherapy.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eAdapting a patient-centered approach, we leveraged a microfluidic cell culture technology platform (Emulate), organoids derived from treatment-naive patient tumors or adjacent normal tissues, and patient-matched cancer-associated or normal fibroblasts respectively, to develop a novel, physiologically relevant, high-fidelity preclinical esophagus-on-a-chip model. H\u0026amp;E, immunofluorescence staining, live/dead assay, LDH assay, and ELISA-based detection of tumor biomarkers were used to assess treatment responses.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eEach patient-specific stroma-inclusive microfluidic esophageal adenocarcinoma on-a-chip (EAC chip) faithfully recreates the tumor-stroma interface while preserving the full diversity of two cell types (epithelia and fibroblasts), genetic landscapes and histological architecture of the source tumors. EAC chips also accurately predict the response to neoadjuvant chemotherapy (NACT) within a clinically useful timeframe (approx. 12 days). A docetaxel-based triplet chemotherapy regimen matched with the treatment of the source patient was successfully perfused through the interstitial space within this model. Therefore, EAC chips more accurately recapitulate inpatient pathological and objective responses than the corresponding static 3D-organoid-only cultures.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOverall, this model is an effective tool for predicting patients\u0026rsquo; responses to chemotherapy and testing tumor- or stroma-targeted alternative therapies. Moreover, these high-fidelity, low-throughput EAC chips effectively complement high-throughput PDO culture-based drug testing and provide improved insights into drug efficacy before human studies.\u003c/p\u003e","manuscriptTitle":"Patient-derived esophageal adenocarcinoma organ chip: a physiologically relevant platform for functional precision oncology","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 08:27:22","doi":"10.21203/rs.3.rs-6017396/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2025-05-08T10:41:44+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-04-13T21:10:09+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-13T20:51:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-10T11:13:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2025-04-05T19:03:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","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":"12ff7c81-6452-43d5-9b9c-2b776a37d041","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-26T16:02:50+00:00","versionOfRecord":{"articleIdentity":"rs-6017396","link":"https://doi.org/10.1186/s12967-025-06593-1","journal":{"identity":"journal-of-translational-medicine","isVorOnly":false,"title":"Journal of Translational Medicine"},"publishedOn":"2025-05-23 15:57:09","publishedOnDateReadable":"May 23rd, 2025"},"versionCreatedAt":"2025-04-17 08:27:22","video":"","vorDoi":"10.1186/s12967-025-06593-1","vorDoiUrl":"https://doi.org/10.1186/s12967-025-06593-1","workflowStages":[]},"version":"v1","identity":"rs-6017396","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6017396","identity":"rs-6017396","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}