Tumor Organoid Cultures Research Articles

Assessing the Efficacy and Feasibility of Conditioned Medium as a Culture Reagent for Tumor Organoid Culture: A Comprehensive Evaluation

Tumor organoids are three-dimensional models of tumor cells grown in vitro which derived from primary tumors of patients. Tumor organoids highly mimic the characteristics of the primary tumors, retain tumor heterogeneity, and can be used for functional testing in vitro, including high-throughput drug screening and personalized therapeutic strategies. However, the establishment of tumor organoids facing such dilemma as low success rate and growth rate. The use of appropriate and effective culture reagent is pivotal to the success of tumor organoids culture. Nowadays, the most commonly and long-lasting used media in cell culture is conditioned medium, while its application in organoids culture has been controversial due to undefined components. In response, our study conducts a comprehensive analysis, offering compelling evidence supporting the efficacy of conditioned medium in promoting organoids growth and expansion. This research aims to address uncertainties surrounding its use and contribute valuable insights to enhance tumor organoids culture practices.

A Convexity-Preserving Level-Set Method for the Segmentation of Tumor Organoids.

Tumor organoid cultures play a crucial role in clinical practice, particularly in guiding medication by accurately determining the morphology and size of the organoids. However, segmenting individual tumor organoids is challenging due to their inhomogeneous internal intensity and overlapping structures. This paper proposes a convexity-preserving level-set segmentation 4 model based on the characteristics of tumor organoid images to segment individual tumor organoids precisely. Considering the predominant spherical shape exhibited by organoid growth, we propose a level-set model that includes a data-driven term, a curvature term, and a regularization term. The data-driven term pulls the contour to the vicinity of the boundary; the curvature term ensures the maintenance of convexity in the targeted segmentation, and the regularization term controls the smoothness and propagation of the contour. The proposed model aids in overcoming interference from factors such as overlap and noise, enabling the evolving curve to converge to the actual boundary of the target accurately. Furthermore, we propose a selectable and targeted initialization method that guarantees precise segmentation of specific regions of interest. Experiments on 51 pancreatic ductal adenocarcinoma organoid images show that our model achieved excellent segmentation results. The average Dice value and computation time are 98.81±0.48% and 20.67 s. Compared with the C-V and CPLSE models, it is more accurate and takes less time.

Patient-derived parotid gland pleomorphic adenoma organoid model and its preliminary histological and functional characterization

Objective: To establish patient-derived organoid models of pleomorphic adenomas (PA) of the parotid gland and preliminarily characterize their histology, related biomarkers and functions. Methods: Fresh tumor tissue specimens were collected from surgical procedures of Oral and Maxillofacial Department. The harvested tissues were processed and cultured in a head and neck tumor organoid culture system to establish organoid models from parotid gland pleomorphic adenomas. The in vitro growth of PA organoids was recorded by light microscopy. The successfully established organoids were passaged and cryopreserved, and the cryopreserved PA organoids were revived and re-cultured to observe their viability and organoid regeneration ability. Histological characterization, as well as characterization and detection of related markers and functional proteins, were performed on the organoids, comparing them with the patient-derived tissues. Results: The constructed organoid model of pleomorphic adenoma exhibited a dense and compact three-dimensional spherical structure. Hematoxylin and eosin staining indicated morphological similarities between the organoid and its tissue of origin. Immunohistochemistry showed positive cytoplasmic staining for Calponin, cytokeratin 7, and epithelial membrane antigen in both the organoid and the source tumor tissue, suggesting consistent histopathological characteristics between the organoid and its tissue of origin. Periodic acid-Schiff staining of the organoid showed positive staining for glycogen, with positive staining located in the interior and periphery of the organoid, indicating that the organoid possessed secretory functions like the salivary gland. Conclusions: This study successfully constructed organoids of pleomorphic adenoma derived from patient samples. This model faithfully replicates the tissue morphology and biomarkers of the source tissue and exhibits biological functions associated with mucus secretion. It serves as a valuable in vitro model for studying the development and progression of salivary gland tumors.

3D bioprinting platform development for high-throughput cancer organoid models construction and drug evaluation

The evaluation of anti-tumor drugs is critical for their development and clinical guidance. Tumor organoid models are gaining increased attention due to their ability to better mimic real tumor tissues, as well as lower time and economic costs, which makes up for the shortcomings of cell lines and xenograft models. However, current tumor organoid cultures based on the Matrigel have limitations in matching with high-throughput engineering methods due to slow gelation and low mechanical strength. Here, we present a novel composite bioink for culturing colorectal cancer organoids that provides an environment close to real tissue growth conditions and exhibits excellent photocrosslinking properties for rapid gel formation. Most importantly, the tumor organoids viability in the composite bioink after printing was as high as 97%, which also kept multicellular polar structures consistent with traditional culture methods in the Matrigel. Using 3D bioprinting with this composite bioink loaded with organoids, we demonstrated the feasibility of this drug evaluation model by validating it with clinically used colorectal cancer treatment drugs. Our results suggested that the composite bioink could effectively cultivate tumor organoids using 3D bioprinting, which had the potential to replace less reliable manual operations in promoting the application of tumor organoids in drug development and clinical guidance.

Abstract Wrk2-01: Organoid Models of Cancer and the Tumor Microenvironment

Abstract Both cell-intrinsic and microenvironmental influences vitally govern tumor progression. Mutational, chromosomal and epigenetic alterations represent the former, while the latter is influenced by diverse stromal populations including fibroblasts, vasculature and immune cells. In vitro organoid culture provides an experimentally tractable platform for cancer research that is applicable to cell-autonomous and tumor microenvironmental studies. The first part of this lecture will describe our work using organoid culture to functionally validate tumor cell-intrinsic genomic alterations. Notably, cancer research has traditionally lacked in vitro methods where tumors can be holistically cultured in a non-reconstitutive manner that co-preserves neoplastic cells together with endogenous stromal components. Thus, the second part of this lecture will describe advances in 3D tumor organoid culture that allow growth of cancer cells in a manner that preserves their native interaction with diverse tumor-infiltrating immune cells and allows analysis of immune checkpoint inhibition. Citation Format: C. Kuo. Organoid Models of Cancer and the Tumor Microenvironment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr Wrk2-01.

Extracellular vesicles secreted by 3D tumor organoids are enriched for immune regulatory signaling biomolecules compared to conventional 2D glioblastoma cell systems.

Newer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterize extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems. Primary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterization was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analyzed by high-resolution mass spectrometry. We observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR-323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer-associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response. Our results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.

Abstract 4249: Establishment and analysis of patient-derived high-grade serous ovarian cancer organoids for disease modeling and drug testing

Abstract Purpose: Ovarian cancer is the most lethal gynecologic cancer. Majority of ovarian cancers belong to the subgroup of high-grade serous ovarian cancer (HGSC). Its standard treatment is surgery and chemotherapy with platinum and taxane-based compounds, for which almost all patients become resistant. We have established a panel of patient derived 3-dimensional (3D) HGSC long-term tumor organoid cultures for personalized disease modeling, drug screening and evaluation of the efficiency of potential new treatments to be used in preclinical research. Methods: Tumor tissues or ascites from > 30 HGSC tumors were set up as long-term 3D organoid cultures (PDOs). RNA and whole genome sequencing was performed from the original tumors and from PDOs. Pluripotency and proliferation were assessed via high-throughput immunofluorescent detection of PAX8 and Ki67. Resistance towards standard treatment and response to additional selected drugs and drug combinations were evaluated in cell death and proliferation assays. Imaging and image-based quantifications were performed under 3D setup. PDOs were set up for in vivo drug testing in immunodeficient NOD/Shi-scid/IL-2Rgnull (NOG) mice. Summary: A set of HGSC PDOs of different longitudinal, prospective, and multiregional samples has been established representing the three evolutionary states of HGSC: evolving, maintaining and adaptive state. They are ready to be used as rational and effective models for studying drug interventions. Supportively, results of preclinical studies with PI3K inhibitor Alpelisib matched the published patient responses. Tumor evolutionary analysis based on data of longitudinal, prospective, and multiregional collected samples suggest further targetable pathways that can mediate chemotherapy resistance and pinpoint potential targets for drug intervention. Conclusion: Inhibition of these pathways with simultaneous targeting of other cellular functions, such as lysosomal-mediated cell death, may be highly effective alternative treatment option. 3D imaging and efficient image analysis are necessary tools to understand tumor organoid biology and drug responses to assist development and pre-clinical evaluation of new treatments. This study is a part of the DECIDER project (https://www.deciderproject.eu/, NCT04846933). Citation Format: Tuula Kallunki, Anna R. Lauridsen, Aikaterini Skorda, Alexandra Lahtinen, Marie L. Bay, Benita Rasmussen, Kaisa Huhtinen, Taru Muranen, Jaana Oikkonen, Johanna Hynninen, Sampsa Hautaniemi. Establishment and analysis of patient-derived high-grade serous ovarian cancer organoids for disease modeling and drug testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4249.

Toward reproducible tumor organoid culture: focusing on primary liver cancer.

Organoids present substantial potential for pushing forward preclinical research and personalized medicine by accurately recapitulating tissue and tumor heterogeneity in vitro. However, the lack of standardized protocols for cancer organoid culture has hindered reproducibility. This paper comprehensively reviews the current challenges associated with cancer organoid culture and highlights recent multidisciplinary advancements in the field with a specific focus on standardizing liver cancer organoid culture. We discuss the non-standardized aspects, including tissue sources, processing techniques, medium formulations, and matrix materials, that contribute to technical variability. Furthermore, we emphasize the need to establish reproducible platforms that accurately preserve the genetic, proteomic, morphological, and pharmacotypic features of the parent tumor. At the end of each section, our focus shifts to organoid culture standardization in primary liver cancer. By addressing these challenges, we can enhance the reproducibility and clinical translation of cancer organoid systems, enabling their potential applications in precision medicine, drug screening, and preclinical research.

The role of decellularized cell derived extracellular matrix in the establishment and culture of in vitro breast cancer tumor model

Decades of research have shown that two-dimensional cell culture studies are insufficient for preclinical cancer diagnosis and treatment, and that cancer cells in three-dimensional (3D) culture systems have better cell–cell and cell–matrix interactions, gene expression, heterogeneity, and structural complexity that more closely resemble in vivo tumors. Researchers are still optimizing 3D culturing settings for different cancers. Despite promising tumor spheroid research, tumor cell-only aggregates lack the tumor microenvironment and cannot model tumors. Here, MCF-7 breast cancer cell derived decellularized extracellular matrix (CD-dECMs) were obtained and converted into autologous, biologically active, biocompatible, and non-immunogenic hydrogels to be used as micro-environment in both organoid formation and culture. For the production of organoids, CD-dECM doping concentrations ranging from 0.1 mg ml−1 to 1.5 mg ml−1 were evaluated, and the lowest concentration was found to be the most effective. For organoid culture, 8 mg ml−1 CD-dECM, 4 mg ml−1 rat tendon collagen type I (Col I) (4 mg ml−1) and a 1:1 (v/v) mixture of these two were used and the most viable and the biggest organoids were discovered in CD-dECM/Col I (1:1) group. The results show that autologous CD-dECM can replace hydrogels in tumor organoid generation and culture at low and high concentrations, respectively.

Pancreatic Tumor Organoid-Derived Factors from Cachectic Patients Disrupt Contractile Smooth Muscle Cells

Patients with pancreatic cancer often suffer from cachexia and experience gastrointestinal symptoms that may be related to intestinal smooth muscle cell (SMC) dysfunction. We hypothesized that pancreatic tumor organoids from cachectic patients release factors that perturb the SMC’s contractile characteristics. Human visceral SMCs were exposed to conditioned medium (CM) from the pancreatic tumor organoid cultures of cachectic (n = 2) and non-cachectic (n = 2) patients. Contractile proteins and markers of inflammation, muscle atrophy, and proliferation were evaluated by qPCR and Western blot. SMC proliferation and migration were monitored by live cell imaging. The Ki-67-positive cell fraction was determined in the intestinal smooth musculature of pancreatic cancer patients. CM from the pancreatic tumor organoids of cachectic patients did not affect IL-1β, IL-6, IL-8, MCP-1, or Atrogin-1 expression. However, CM reduced the α-SMA, γ-SMA, and SM22-α levels, which was accompanied by a reduced SMC doubling time and increased expression of S100A4, a Ca2+-binding protein associated with the synthetic SMC phenotype. In line with this, Ki-67-positive nuclei were increased in the intestinal smooth musculature of patients with a low versus high L3-SMI. In conclusion, patient-derived pancreatic tumor organoids release factors that compromise the contractile SMC phenotype and increase SMC proliferation. This may contribute to the frequently observed gastrointestinal motility problems in these patients.

Mapping and modeling human colorectal carcinoma interactions with the tumor microenvironment

The initiation and progression of cancer are intricately linked to the tumor microenvironment (TME). Understanding the function of specific cancer-TME interactions poses a major challenge due in part to the complexity of the in vivo microenvironment. Here we predict cancer-TME interactions from single cell transcriptomic maps of both human colorectal cancers (CRCs) and mouse CRC models, ask how these interactions are altered in human tumor organoid (tumoroid) cultures, and functionally recapitulate human myeloid-carcinoma interactions in vitro. Tumoroid cultures suppress gene expression programs involved in inflammation and immune cell migration, providing a reductive platform for re-establishing carcinoma-immune cell interactions in vitro. Introduction of human monocyte-derived macrophages into tumoroid cultures instructs macrophages to acquire immunosuppressive and pro-tumorigenic gene expression programs similar to those observed in vivo. This includes hallmark induction of SPP1, encoding Osteopontin, an extracellular CD44 ligand with established oncogenic effects. Taken together, these findings offer a framework for understanding CRC-TME interactions and provide a reductionist tool for modeling specific aspects of these interactions.

Exploring Tumor-Immune Interactions in Co-Culture Models of T Cells and Tumor Organoids Derived from Patients.

The use of patient-derived tumor tissues and cells has led to significant advances in personalized cancer therapy and precision medicine. The advent of genomic sequencing technologies has enabled the comprehensive analysis of tumor characteristics. The three-dimensional tumor organoids derived from self-organizing cancer stem cells are valuable ex vivo models that faithfully replicate the structure, unique features, and genetic characteristics of tumors. These tumor organoids have emerged as innovative tools that are extensively employed in drug testing, genome editing, and transplantation to guide personalized therapy in clinical settings. However, a major limitation of this emerging technology is the absence of a tumor microenvironment that includes immune and stromal cells. The therapeutic efficacy of immune checkpoint inhibitors has underscored the importance of immune cells, particularly cytotoxic T cells that infiltrate the vicinity of tumors, in patient prognosis. To address this limitation, co-culture techniques combining tumor organoids and T cells have been developed, offering diverse avenues for studying individualized drug responsiveness. By integrating cellular components of the tumor microenvironment, including T cells, into tumor organoid cultures, immuno-oncology has embraced this technology, which is rapidly advancing. Recent progress in co-culture models of tumor organoids has allowed for a better understanding of the advantages and limitations of this novel model, thereby exploring its full potential. This review focuses on the current applications of organoid-T cell co-culture models in cancer research and highlights the remaining challenges that need to be addressed for its broader implementation in anti-cancer therapy.

Abstract 453: Targeting FOXL2C402Gvulnerabilities in adult type ovarian granulosa cell tumors

Abstract Background: Nearly all adult-type granulosa cell tumors of the ovary (aGCTs) carry the same oncogenic mutation affecting the second winged helix domain of the Forkhead family transcription factor FOXL2 (c.C402G; p.C134W). There are no currently available therapies that exploit the high prevalence of FOXL2 mutations in this disease. Objectives: Exploit FOXL2-C402G oncogenic addiction in order to identify FDA-approved compounds with differential potency in KGN-FOXL2WT/C402G cells and isogenic KGN-FOXL2—/— cells. Methods: CRISPR/Cas9 was used to generate isogenic derivatives of KGN, a cell line derived from a recurrent aGCT. Parental cells (KGN-FOXL2WT/C402G) or isogenic cells lacking both FOXL2 alleles (KGN-FOXL2—/—) were functionally characterized and loss of Foxl2 protein was confirmed by immunoblot. Two drug libraries comprising a total of 2,416 compounds (majority FDA approved) were screened for effectiveness in KGN-FOXL2WT/C402G cells and KGN-FOXL2—/— cells. Our assay included staining with DAPI, followed by automated image analysis of each well to enumerate nuclei/cell numbers. Based on Fraction Affected (FA) statistics, compounds were ranked according to effectiveness in KGN-FOXL2WT/C402G cells. FA-area under the curve (FA-AUC) cutoff >0.1 yielded 152 drug candidates, categorized into 13 drug classes. 40 drug candidates, including the top 1-4 hits in each drug class were chosen for further validation. Validation rate in the confirmatory screen was at 95%. A novel primary tumor organoid culture system for aGCT was developed and validated using whole exome sequencing and immunophenotype. Selected candidate compounds were further investigated in aGCT organoid cultures from two relapsed tumors. Results: We identified classes of drugs such as the inhibitors of Bromodomain family proteins and Hydroxy-Camptothecin analogs, with enhanced activity in KGN-FOXL2WT/C402G cells relative to KGN-FOXL2—/— cells. Unexpectedly glucocorticoids as a class were found to stimulate proliferation of KGN-FOXL2WT/C402G cells whereas they lacked this activity in KGN-FOXL2—/— cells. For 36 glucocorticoids present in the screen, FA-AUC ranged from -0.3191 to -0.1342 (highest proliferation index among all test compounds) in KGN-FOXL2WT/C402G cells compared to -0.0353 to +0.1667 in KGN-FOXL2—/— cells. In recurrent aGCT organoids, the high-potency glucocorticoid dexamethasone induced cell proliferation and co-treatment with glucocorticoids increased the IC50 of paclitaxel in KGN-FOXL2WT/C402G cells from 1.43 nM to 4.04 nM. Conclusion: Mutant FOXL2 mediates a proliferative response to glucocorticoids in cultured cells and primary aGCT organoids. Paclitaxel is a standard of care chemotherapy in the treatment of aGCTs and is often administered with glucocorticoid pre-medication. The adverse effect of glucocorticoids on paclitaxel efficiency shown here should thus be taken into consideration in the clinic. Citation Format: Thomas Welte, Veena Vuttaradhi, Eleonora Khlebus, Barrett Lawson, Robert T. Hillman, Nghi Nguyen, Mary Sobieski, Reid T. Powell, Clifford Stephan. Targeting FOXL2C402Gvulnerabilities in adult type ovarian granulosa cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 453.

Patient-Derived Tumor Organoids Can Predict the Progression-Free Survival of Patients With Stage IV Colorectal Cancer After Surgery.

Recent studies have shown patient-derived tumor organoids can predict the drug response of patients with cancer. However, the prognostic value of patient-derived tumor organoid-based drug tests in predicting the progression-free survival of patients with stage IV colorectal cancer after surgery remains unknown. This study aimed to explore the prognostic value of patient-derived tumor organoid-based drug tests in patients with stage IV colorectal cancer after surgery. Retrospective cohort study. Surgical samples were obtained from patients with stage IV colorectal cancer at the Nanfang Hospital. A total of 108 patients who underwent surgery with successful patient-derived tumor organoid culture and drug testing were recruited between June 2018 and June 2019. Patient-derived tumor organoid culture and chemotherapeutic drug testing. Progression-free survival. According to the patient-derived tumor organoid-based drug test, 38 patients were drug sensitive and 76 patients were drug resistant. The median progression-free survival was 16.0 months in the drug-sensitive group and 9.0 months in the drug resistant group ( p < 0.001). Multivariate analyses showed that drug resistance (HR, 3.38; 95% CI, 1.84-6.21; p < 0.001), right-sided colon (HR, 3.50; 95% CI, 1.71-7.15; p < 0.001), mucinous adenocarcinoma (HR, 2.47; 95% CI, 1.34-4.55; p = 0.004), and non-R0 resection (HR, 2.70; 95% CI, 1.61-4.54; p < 0.001) were independent predictors of progression-free survival. The new patient-derived tumor organoid-based drug test model, which includes the patient-derived tumor organoid-based drug test, primary tumor location, histological type, and R0 resection, was more accurate than the traditional clinicopathological model in predicting progression-free survival ( p = 0.001). A single-center cohort study. Patient-derived tumor organoids can predict progression-free survival in patients with stage IV colorectal cancer after surgery. Patient-derived tumor organoid drug resistance is associated with shorter progression-free survival, and the addition of patient-derived tumor organoid drug tests to existing clinicopathological models improves the ability to predict progression-free survival.

Abstract PD5-02: PD5-02 An Organoid Model System to Study Resistance Mechanisms, Predictive Biomarkers, and New Strategies to Overcome Therapeutic Resistance in Early-Stage Triple-Negative Breast Cancer

Abstract Background: While new treatments and improved subtyping schemas are anticipated to improve treatment response in triple-negative breast cancer (TNBC) patients, therapeutic resistance remains a significant challenge. Moreover, there is an urgent need for additional research model systems to study resistance and residual disease in breast cancer, including aggressive subtypes of breast cancer. Organoid culture is a promising technology used for growing breast cancer cells with high efficiency; however, the extent to which treatment resistance can be modeled using this system is unknown. This research used patient-derived organoid cultures in the context of computational analyses of large molecular and clinical datasets to study resistance mechanisms, biomarkers, and alternative treatment strategies to overcome drug resistance in early-stage TNBC. Methods: Organoid cultures were derived from breast tumor samples (taken from lumpectomy, mastectomy, or core biopsy samples), digested to the organoid level using collagenase, and grown in three dimensional cultures using a basement membrane extract and a fully-defined organoid medium (Dekkers et al. Nat Protoc 2021). An evaluation of all available I-SPY2 biomarker data (Wolf et al. Cancer Cell 2022) was performed focusing on genes, proteins, and pathways associated with resistance. These were then used to study whether resistance biomarkers identified in patient tumors are also present in organoids propagated from breast cancer post-treatment residual disease. To this end, bulk RNA sequencing data of organoids were normalized and merged with the TCGA dataset (Hoadley et al. Cell 2018) to enable analysis in a larger context, and immunofluorescence staining of organoids was performed. A high-throughput 386 anti-cancer drug compound screen and subsequent synergy testing with the most promising compounds were performed to identify and predict alternative treatment strategies. Additional assays to explore kinome activity in this organoid model are in progress. Results: A TNBC organoid biobank was established (n=31), which was enriched for inflammatory breast cancer (IBC; n=15), an aggressive form of breast cancer. Most organoids were derived from residual disease after neoadjuvant therapy. Bulk RNA sequencing analysis performed on 10 TNBC organoids revealed 3 subsets that were characterized predominantly by either normal-like/luminal androgen receptor or basal-like features or expressed distinct gene expression profiles, with IBC cases present in all 3 subsets. Intriguingly, the IBC organoids were characterized by higher expression of a number of immune-related signatures, despite an absence of clear immune cells in culture. A residual disease IBC/TNBC organoid resistant to chemotherapy was used to perform the 386-drug compound screen. The organoid model showed resistance to veliparib-cisplatin, consistent with the expression of gene/protein biomarkers predictive of drug resistance found in I-SPY2 (low PARPi7 levels and high pFOXO1 and pMEK1/2 expression). In addition, the screen identified multiple classes of inhibitors as promising synergistic/additive candidates for overcoming resistance to cisplatin. Conclusion: In this proof-of-principle study, we demonstrated the utility of matching I-SPY2 resistance biomarkers and signatures to residual disease tumor organoid cultures. We show that tumor organoid cultures modeling drug resistance states are a useful complement to existing research models of breast cancer and can be used for compound testing. We have developed a pipeline to propagate residual tumors from patients enrolled in I-SPY2 into organoid cultures to create a broader platform for preclinical drug testing informed by tumor biology with the ultimate goal of enabling faster, more successful translational studies and increased treatment options for resistant disease. Citation Format: Tam Binh V. Bui, Denise M. Wolf, Kaitlin Moore, Isaac S. Harris, Pravin Phadatare, Christina Yau, Lamorna A. Brown Swigart, Laura J. Esserman, Jean-Philippe Coppe, Julia Wulfkuhle, Emanuel F. Petricoin, Michael Campbell, Laura M. Selfors, Deborah A. Dillon, Beth Overmoyer, Filipa Lynce, Laura Van ’t Veer, Jennifer Rosenbluth. PD5-02 An Organoid Model System to Study Resistance Mechanisms, Predictive Biomarkers, and New Strategies to Overcome Therapeutic Resistance in Early-Stage Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-02.

Abstract P3-09-01: Characterization of residual disease after neoadjuvant selective estrogen receptor degrader (SERD) therapy using tumor organoids in the I-SPY Endocrine Optimization Protocol (EOP)

Abstract Background: Treatment of estrogen receptor (ER)-positive breast cancer with selective estrogen receptor degraders (SERDs) frequently results in the loss or reduction of ER expression. Whether these changes are due to on-target effects of SERDs degrading ER or arise as a mechanism of tumor resistance with associated changes in cellular phenotypes remains unknown. It is critical to distinguish between these possibilities to accurately assess treatment response and determine the most appropriate subsequent therapy. To this end, we created and conducted molecular analyses on patient-derived organoid cultures from post-treatment tissue in patients receiving neoadjuvant SERD therapy for early-stage ER+ breast cancer in the I-SPY2 Endocrine Optimization Protocol (EOP). Methods: The I-SPY2 EOP study is a prospective, randomized substudy within the I-SPY TRIAL testing the oral SERD amcenestrant alone or in combination with letrozole or abemaciclib in stage 2/3 ER+ Her2-negative breast cancer. Enrollment is ongoing, with patients receiving amcenestrant neoadjuvantly for 6 months until the day before surgery. Tumor tissue is collected at baseline, 3 weeks, and at surgery. Organoids were generated from post-treatment surgical samples. Organoid cultures were optimized based on established methods (Dekkers et al., Nature Protocols, 2021) to assess ER levels and activity. Pre- and post-treatment tissue samples were also assessed for ER, PR, Ki67, and GATA3, a luminal marker and transcription factor that is functionally linked with ER, via immunohistochemistry. Results: In 7 patients with both pre- and post-treatment tissue samples including fresh surgical samples for organoid generation, the ER in baseline tumor tissue was &amp;gt;=90% in all patients, PR ranged from 40-90%, and Ki67 ranged from 5-30%. In post-treatment surgical tissue from these cases, ER ranged from 0-30%, PR from 0-50%, Ki67 from &amp;lt; 1%-10%, and GATA3 was positive in 5 of 5 cases tested to-date. The creation of organoids from residual disease at surgery was attempted for these 7 patients, with organoids successfully propagated in 5 cases thus far. 3 of 5 organoid cultures were ready for analysis and in all cases strong ER and PR expression in organoids was observed after culture for &amp;gt; 1 month in the absence of amcenestrant. Detailed gene expression profiling (including Mammaprint/Blueprint) and gene set enrichment analyses (GSEA) to assess for intrinsic breast cancer subtype and ER activity in each sample and corresponding organoid culture are in progress and will be reported with the full dataset. Conclusion: Patient-derived organoid culturing of residual disease after neoadjuvant endocrine therapy is feasible. Neoadjuvant treatment with a SERD can render ER and PR low or absent at the time of surgical resection, which does not necessarily imply the presence of endocrine therapy resistant disease. The use of organoids and additional IHC markers (GATA3) demonstrate that receptor negativity may be an indicator of the drug hitting its target, suggesting ER signaling is still intact. In general, patient-derived tumor organoid cultures modeling residual disease states can be a useful adjunct to existing methods used to monitor the effects of neoadjuvant endocrine therapy and is being explored in the I-SPY EOP trial. Citation Format: Jennifer Rosenbluth, Christopher J. Schwartz, Tam Binh Bui, Shruti Warhadpande, Pravin Phadatare, Sigal Eini, Michael Bruck, Julissa Molina-Vega, Kami Pullakhandam, Nicole Schindler, Lamorna A. Brown Swigart, Christina Yau, Gillian Hirst, Rita Mukhtar, Karthik V. Giridhar, Olufunmilayo I. Olopade, Kevin Kalinsky, Cheryl A. Ewing, Jasmine M. Wong, Michael D. Alvarado, Laura Van’t Veer, Laura J. Esserman, Jo Chien. Characterization of residual disease after neoadjuvant selective estrogen receptor degrader (SERD) therapy using tumor organoids in the I-SPY Endocrine Optimization Protocol (EOP) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-09-01.

269. PATIENT-DERIVED TUMOR ORGANOIDS AND XENOGRAFTS AS BASIC AND PRECLINICAL TRANSLATIONAL ESOPHAGEAL SQUAMOUS CELL CARCINOMA MODELS

Abstract Patient-derived xenografts have been gold standards for cancer modeling and drug testing. However, low establishment efficiency and high maintenance cost have hindered the development in the field. Recently, patient-derived tumor organoid (PDO) cultures have been developed as an efficient and economic model to mimic and recapitulate the original tumor tissue. Due to the limited availability of current esophageal squamous cell carcinoma (ESCC) models, we aimed to establish PDO cultures for ESCC study. We have established and biobanked a panel of ESCC PDO cultures in vitro from Chinese ESCC treatment-naïve patients undergoing an endoscopic examination or upfront surgery, or patients undergoing neoadjuvant chemoradiotherapy (CRT) followed by surgery in Hong Kong. We also established and maintained a panel of PDO-derived xenograft (PDOX) in immunocompromised mice. Genomic profiling showed consistent mutational landscapes of the established PDOs, as compared to ESCC patient profiles. Transcriptomic profiling suggests PDOs closely resemble patient tumor samples. In vitro chemotherapy treatments followed by prolonged recovery suggests the existence of a subpopulation of persistent cells likely enriched by neoadjuvant CRT and well-preserved by organoid cultivation. Coupled with live-cell bioluminescent-based quantification techniques, PDO cultures can be employed in high-throughput therapeutic screening. Immunohistochemical examination confirmed the squamous origin of the PDOs and PDOXs. Slow-proliferating PDOXs allow adequate interaction between the cancer cells and the host and are useful as a suitable preclinical drug testing platform. ESCC PDOs can be efficiently established from highly limited patient tissue samples and maintained long-term in vitro and can be feasibly integrated with mouse models. ESCC PDOs and PDOXs serve as novel models for ESCC basic and translational studies.

The three-dimension preclinical models for ferroptosis monitoring.

As a new programmed cell death process, ferroptosis has shown great potential and uniqueness in experimental and treatment-resistant cancer models. Currently, the main tools for drug research targeting ferroptosis are tumor cells cultured in vitro and tumor models established in rodents. In contrast, increasing evidence indicates that reactivity may differ from modifications in mice or humans in the process of drug screening. With the blossoming of 3D culture technology, tumor organoid culture technology has gradually been utilized. Compared with traditional 2D culture and tumor tissue xenotransplantation, tumor organoids have a significantly higher success rate. They can be cultured quickly and at a lower cost, which is convenient for gene modification and large-scale drug screening. Thus, combining 3D cell culture technology, drug monitoring, and ferroptosis analysis is necessary to develop the impact of ferroptosis-related agents in tumor treatment.

Targeting DNA Repair with Combined Inhibition of NHEJ and MMEJ Induces Synthetic Lethality in TP53-Mutant Cancers.

Combined inhibition of NHEJ and MMEJ using two nontoxic, targeted DNA repair inhibitors can effectively induce toxic DNA damage to treat TP53-deficient cancers.

Abstract 192: Ex vivo screening and analysis of novel effective treatments for ovarian cancer

Abstract Purpose: Ovarian cancer is the most lethal gynecological cancer. Histologically, about 90% of the ovarian cancers have epithelial origin and more than 75% are characterized as high-grade serous ovarian cancer (HGSOC). Despite having a good primary response to the standard platinum-taxane based chemotherapy, almost all HGSOC patients experience a relapsed disease. The 5-year survival rate of ovarian cancer is only 38% globally, indicating need for the development of novel efficient treatments to fight the resistance. Here we have established a personalized drug screening and evaluation platform using HGSOC patient samples and medicines approved for treatment of different types of cancers. Methods: Tissues collected from ovarian cancer patients were grown as 3-dimensional (3D) ex vivo long-term tumor organoid cultures. Seven HGSOC cell lines were additionally grown as spheroids or as xenograft tumors to establish them as 3D tumor organoid cultures for drug efficiency evaluation. Cancer stemness and further classification of samples were determined via immunofluorescent staining with relevant antibodies (pax8, Ki67, acetylated tubulin, p53, cytokeratin-8) and consequent high-throughput imaging using the ImageXpress Micro Confocal microscope (Molecular Devices). The images were analyzed and quantified using MetaXpress software. Organoid growth and response to tested treatments was validated via invasive growth and survival assays. Both assays were used to monitor resistance of organoids towards cisplatin and carboplatin treatment and towards suggested potential new treatments. Cancer drugs were selected according to suggestions based on RNA seq, DNA and/or drug screenings done in cancer cell lines. A preclinical mouse model (in immunodeficient NOD/Shi-scid/IL-2Rγnull (NOG) mice) was set up to be used for in vivo validation by monitoring the tumor growth in response to the treatment. Results: Currently, 20 patient tumor samples received from Turku University Hospital, Finland (KH, JH) have been cultured in 4 different culture media to obtain highest possible survival rates for each sample. Organoids from ovarian cancer cell lines are characterized for their resistance to increasing concentrations of platinum treatment. Notably, organoid analysis protocols and screening conditions have been setup for the high-throughput microscopy. These include organoid growth, survival, and invasiveness/aggressiveness. We are currently set up for screening organoids with the first drugs suggested by bioinformatics-based genetic pathway analysis and drug screening studies in cancer cell lines. The results of the screen will be presented. This study is a part of a large EU-funded ovarian cancer project DECIDER (https://www.deciderproject.eu/). Citation Format: Aikaterini Skorda, Kaisa Huhtinen, Wojciech Senkowski, Krister Wennerberg, Sampsa Hautaniemi, Johanna Hynninen, Tuula Kallunki. Ex vivo screening and analysis of novel effective treatments for ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 192.