Tumor Organoids Research Articles

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.

Imaging-Based Efficacy Evaluation of Cancer Immunotherapy in Engineered Tumor Platforms and Tumor Organoids.

Cancer immunotherapy is used to treat tumors by modulating the immune system. Although the anticancer efficacy of cancer immunotherapy has been evaluated prior to clinical trials, conventional in vivo animal and endpoint models inadequately replicate the intricate process of tumor elimination and reflect human-specific immune systems. Therefore, more sophisticated models that mimic the complex tumor-immune microenvironment must be employed to assess the effectiveness of immunotherapy. Additionally, using real-time imaging technology, a step-by-step evaluation can be applied, allowing for a more precise assessment of treatment efficacy. Here, an overview of the various imaging-based evaluation platforms recently developed for cancer immunotherapeutic applications is presented. Specifically, a fundamental technique is discussed for stably observing immune cell-based tumor cell killing using direct imaging, a microwell that reproduces a confined space for spatial observation, a droplet assay that facilitates cell-cell interactions, and a 3D microphysiological system that reconstructs the vascular environment. Furthermore, it is suggested that future evaluation platforms pursue more human-like immune systems.

Patient-Derived Conditionally Reprogrammed Cells in Prostate Cancer Research.

Prostate cancer (PCa) remains a leading cause of mortality among American men, with metastatic and recurrent disease posing significant therapeutic challenges due to a limited comprehension of the underlying biological processes governing disease initiation, dormancy, and progression. The conventional use of PCa cell lines has proven inadequate in elucidating the intricate molecular mechanisms driving PCa carcinogenesis, hindering the development of effective treatments. To address this gap, patient-derived primary cell cultures have been developed and play a pivotal role in unraveling the pathophysiological intricacies unique to PCa in each individual, offering valuable insights for translational research. This review explores the applications of the conditional reprogramming (CR) cell culture approach, showcasing its capability to rapidly and effectively cultivate patient-derived normal and tumor cells. The CR strategy facilitates the acquisition of stem cell properties by primary cells, precisely recapitulating the human pathophysiology of PCa. This nuanced understanding enables the identification of novel therapeutics. Specifically, our discussion encompasses the utility of CR cells in elucidating PCa initiation and progression, unraveling the molecular pathogenesis of metastatic PCa, addressing health disparities, and advancing personalized medicine. Coupled with the tumor organoid approach and patient-derived xenografts (PDXs), CR cells present a promising avenue for comprehending cancer biology, exploring new treatment modalities, and advancing precision medicine in the context of PCa. These approaches have been used for two NCI initiatives (PDMR: patient-derived model repositories; HCMI: human cancer models initiatives).

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.

The OVAREX study: Establishment of ex vivo ovarian cancer models to validate innovative therapies and to identify predictive biomarkers

BackgroundOvarian cancer is the first cause of death from gynecological malignancies mainly due to development of chemoresistance. Despite the emergence of PARP inhibitors, which have revolutionized the therapeutic management of some of these ovarian cancers, the 5-year overall survival rate remains around 45%. Therefore, it is crucial to develop new therapeutic strategies, to identify predictive biomarkers and to predict the response to treatments. In this context, functional assays based on patient-derived tumor models could constitute helpful and relevant tools for identifying efficient therapies or to guide clinical decision making.MethodThe OVAREX study is a single-center non-interventional study which aims at investigating the feasibility of establishing in vivo and ex vivo models and testing ex vivo models to predict clinical response of ovarian cancer patients. Patient-Derived Xenografts (PDX) will be established from tumor fragments engrafted subcutaneously into immunocompromised mice. Explants will be generated by slicing tumor tissues and Ascites-Derived Spheroids (ADS) will be isolated following filtration of ascites. Patient-derived tumor organoids (PDTO) will be established after dissociation of tumor tissues or ADS, cell embedding into extracellular matrix and culture in specific medium. Molecular and histological characterizations will be performed to compare tumor of origin and paired models. Response of ex vivo tumor-derived models to conventional chemotherapy and PARP inhibitors will be assessed and compared to results of companion diagnostic test and/or to the patient’s response to evaluate their predictive value.DiscussionThis clinical study aims at generating PDX and ex vivo models (PDTO, ADS, and explants) from tumors or ascites of ovarian cancer patients who will undergo surgical procedure or paracentesis. We aim at demonstrating the predictive value of ex vivo models for their potential use in routine clinical practice as part of precision medicine, as well as establishing a collection of relevant ovarian cancer models that will be useful for the evaluation of future innovative therapies.Trial registrationThe clinical trial has been validated by local research ethic committee on January 25th 2019 and registered at ClinicalTrials.gov with the identifier NCT03831230 on January 28th 2019, last amendment v4 accepted on July 18, 2023.

Exploring tumor organoids for cancer treatment

As a life-threatening chronic disease, cancer is characterized by tumor heterogeneity. This heterogeneity is associated with factors that lead to treatment failure and poor prognosis, including drug resistance, relapse, and metastasis. Therefore, precision medicine urgently needs personalized tumor models that accurately reflect the tumor heterogeneity. Currently, tumor organoid technologies are used to generate in vitro 3D tissues, which have been shown to precisely recapitulate structure, tumor microenvironment, expression profiles, functions, molecular signatures, and genomic alterations in primary tumors. Tumor organoid models are important for identifying potential therapeutic targets, characterizing the effects of anticancer drugs, and exploring novel diagnostic and therapeutic options. In this review, we describe how tumor organoids can be cultured and summarize how researchers can use them as an excellent tool for exploring cancer therapies. In addition, we discuss tumor organoids that have been applied in cancer therapy research and highlight the potential of tumor organoids to guide preclinical research.

Optimization of Fluorescence In Situ Hybridization Protocols in the Era of Precision Medicine.

Fluorescence in situ hybridization (FISH) is a cytogenetic assay that is widely used in both clinical and research settings to validate genetic aberrations. Simple in principle, it is based on denaturation and hybridization of a DNA probe and its complementary sequence; however, it is subject to continuous optimization. Here we share how in-house FISH can be optimized using different control tissues to visualize and ultimately validate common and novel genetic abnormalities unearthed by next-generation sequencing (NGS). Seven specific FISH probes were designed and labeled, and conditions for eight tissue types and one patient-derived tumor organoid were optimized. Formalin-fixed paraffin-embedded (FFPE) tissue slides were used for each experiment. Slides were first deparaffinized, then placed in a pretreatment solution followed by a digestion step. In-house FISH probes were then added to the tissue to be denatured and hybridized, and then washed twice. To obtain optimal results, probe concentration, pepsin incubation time, denaturation, and the two post-hybridization washes were optimized for each sample. By modifying the above conditions, all FISH experiments were optimized in separate tissue types to investigate specific genomic alterations in tumors arising in those tissues. Signals were clear and distinct, allowing for visualization of the selected probes. Following this protocol, our lab has quickly optimized 11 directly labeled in-house FISH probes to support genetic aberrations nominated by NGS, including most recent discoveries through whole-genome sequencing analyses. We describe a robust approach of how to advance in-house labeled FISH probes. By following these guidelines, reliable and reproducible FISH results can be obtained to interrogate FFPE slides from benign, tumor tissues, and patient-derived tumor organoid specimens. This is of most relevance in the era of NGS and precision oncology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Metaphase FISH optimization Support Protocol 1: In-house probe labeling and preparation Support Protocol 2: Metaphase spread preparation Basic Protocol 2: Optimization of FISH on formalin-fixed paraffin-embedded tissue.

529P Development and validation of a high-throughput drug screen (HTDS) from patient-derived tumor organoids (PDTO) from gastrointestinal malignancies: Clinical and genomic correlations

529P Development and validation of a high-throughput drug screen (HTDS) from patient-derived tumor organoids (PDTO) from gastrointestinal malignancies: Clinical and genomic correlations

Recapitulating the tumor microenvironment in a dish, one cell type at a time

The tumor microenvironment harbors a variety of different cell types that differentially impact tumor biology. In this issue of Cell Reports Methods, Raffo-Romero etal. standardized and optimized 3D tumor organoids to model the interactions between tumor-associated macrophages and tumor cells invitro.

Sensitivity of colorectal cancer organoids to hyperthermic intraperitoneal chemotherapy with lobaplatin

Objective: To investigate the sensitivity of tumor organoids derived from samples of colorectal cancer to lobaplatin and oxaliplatin hyperthermic perfusion in vitro and to assist clinical development of hyperthermic intraperitoneal chemotherapy. Method: Tumor samples and relevant clinical data were collected from patients with pathologically confirmed colorectal cancer in the Sixth Affiliated Hospital of Sun Yat-sen University from July 2021 to December 2022. Organoids were cultured and tumor tissue were passaged. In vitro hyperthermic perfusion experiments were performed on organoids with good viability. Firstly, 10 organoids were treated with oxaliplatin and lobaplatin at the following six concentrations: 1 000, 250, 62.5, 15.6, 3.9, and 0.98 μmol/L. The organoids were exposed to oxaliplatin at 42℃ for 30 minutes and to lobaplatin at 42℃ for 60 minutes. Dose-response curves of responses to in vitro hyperthermic perfusion with these two drugs were constructed and evaluated. Clinical doses of oxaliplatin and lobaplatin were further tested on 30 organoids. This testing revealed oxaliplatin was effective at 579 µmol/L at a hyperthermic perfusion temperature of 42℃ for 30 min and lobaplatin was effective at 240 µmol/L at a hyperthermic perfusion temperature of 42℃ for 60 minutes. Result: Thirty-two tumor organoids were cultured from samples of colorectal cancer. The median concentration required for oxaliplatin to eliminate 50% of tumor cells (IC50) was 577.45 µmol/L (IQR: 1846.09 µmol/L). The median IC50 for lobaplatin was 85.04 µmol/L (IQR: 305.01 µmol/L).The difference between the two groups was not statistically significant (Z=1.784, P=0.084). In seven of 10 organoids, lobaplatin showed a greater IC50 after in vitro hyperthermic perfusion than did oxaliplatin. Testing of 30 organoids with clinical doses of oxaliplatin and lobaplatin revealed that oxaliplatin achieved an average inhibition rate of 39.6% (95%CI: 32.1%‒47.0%), whereas the average rate of inhibition for lobaplatin was 89.7% (95%CI: 87.0%‒92.3%): this difference is statistically significant (t=‒15.282, P<0.001). Conclusion: The rate of inhibition achieved by hyperthermic perfusion of lobaplatin in vitro is better than that achieved by hyperthermic perfusion with oxaliplatin. Lobaplatin is more effective than oxaliplatin when administered by hyperthermic intraperitoneal perfusion and therefore has the potential to replace oxaliplatin in this setting.

Pyrotinib as a salvage treatment for patients with HER-2 positive advanced lung adenocarcinoma after the progression of afatinib treatment.

The efficacy of afatinib or pyrotinib has been demonstrated in HER2-positive advanced non-small cell lung cancer (NSCLC) patients; however, the efficacy of pyrotinib after afatinib progression has yet to be determined. Patients with HER2 mutated advanced lung adenocarcinoma administeredafatinib or pyrotinib monotherapy were enrolled. Those who received pyrotinib after afatinib were further analyzed to determine the efficacy and safety of pyrotinib after progression on afatinib. Survival curves were plotted with the Kaplan-Meier method. A swimming plot was used to describe the specific treatments. Additionally, patient-derived tumor organoids (PDTOs) were established from HER2-amplified NSCLC patient samples to investigate the antitumor activity of pyrotinib in HER2-amplified tumor cells in vitro. A total of 99 patients were enrolled, 13 of whom were administered pyrotinib after progression on afatinib. No statistical difference in PFS of pyrotinib was observed between patients whether be treated after afatinib progression or not (6.7months vs. 4.4months, P = 0.817), thus indicating that progression on afatinib did not affect the efficacy of pyrotinib. Further analysis was conducted on the former patients, which comprising eight patients administered interval chemotherapy after progression on afatinib. Two patients achieved PR after pyrotinib treatment. No independent factors were found to influence the PFS of pyrotinib. PDTOs confirmed the anti-tumor activity of pyrotinib in NSCLC tumor cells with HER2 amplification. Progression after prior afatinib treatment does not influence the efficacy of pyrotinib treatment. Pyrotinib may be a salvage option for patients with HER2 mutation who have experienced progression on afatinib.

Ehf controls mammary alveolar lineage differentiation and is a putative suppressor of breast tumorigenesis

The transcription factor EHF is highly expressed in the lactating mammary gland, but its role in mammary development and tumorigenesis is not fully understood. Utilizing a mouse model of Ehf deletion, herein, we demonstrate that loss of Ehf impairs mammary lobuloalveolar differentiation at late pregnancy, indicated by significantly reduced levels of milk genes and milk lipids, fewer differentiated alveolar cells, and an accumulation of alveolar progenitor cells. Further, deletion of Ehf increased proliferative capacity and attenuated prolactin-induced alveolar differentiation in mammary organoids. Ehf deletion also increased tumor incidence in the MMTV-PyMT mammary tumor model and increased the proliferative capacity of mammary tumor organoids, while low EHF expression was associated with higher tumor grade and poorer outcome in luminal A and basal human breast cancers. Collectively, these findings establish EHF as a non-redundant regulator of mammary alveolar differentiation and a putative suppressor of mammary tumorigenesis.

Abstract B025: Luminal-to-basal phenotypic plasticity promotes invasive phenotypes in a live-imaging assay using patient-derived bladder tumor organoids

Abstract Lineage plasticity in cancer is characterized by changes in cell state that are often associated with tumor progression and treatment resistance. While nearly all non-muscle invasive bladder cancer (NMIBC) tumors have a luminal epithelial phenotype, many muscle invasive bladder cancers (MIBC) display a basal identity. NMIBC to MIBC progression may therefore be related to a phenotypic switch from luminal-to-basal subtypes, which in turn may be associated with poorer clinical outcomes. However, current understanding of the molecular processes linking cell plasticity and tumor invasion is limited, due in part to a lack of physiologically relevant in vitro models and assays. To address these questions, we are utilizing a living biobank of 82 patient-derived bladder tumor organoid lines that our group has established. These organoids represent a 3-dimensional, heterogeneous model system for studying bladder cancer, as they can be genetically manipulated and recapitulate the molecular and histopathological features of the parental tumor. Notably, in previous work, we described luminal-to-basal phenotypic plasticity in a subset of organoid lines derived from luminal tumors, which display a shift to basal/squamous identity in culture that is reversible by orthotopic xenografting. In current studies, we are investigating cell migration and invasiveness using a live-imaging assay that we have developed to examine bladder tumor organoid outgrowth in vitro. Importantly, this assay allows longitudinal visualization as well as quantitation of invasiveness over a four-day time span. Analysis of over 25 organoid lines with plastic, stable luminal, and stable basal properties has revealed a broad spectrum of organoid outgrowth phenotypes that do not correlate with cell proliferation or clinical parameters. Instead, the degree of outgrowth correlates precisely with luminal-to-basal phenotypic plasticity, as assessed by transcriptome analyses, immunofluorescence microscopy, and in vivo xenografting. Organoid lines with minimal outgrowth have stable luminal phenotypes, whereas lines that produce their own extracellular matrix to create a leading edge of migrating cells have plastic phenotypes. Furthermore, movies of the invading organoids reveal distinct cell migratory patterns, as several organoid lines display collective migration that generates an invasive front at the leading edge. This collective migration phenotype is distinct from phenotypic plasticity, is at least partially independent from organoid outgrowth, and is undergoing further analysis. Thus, our organoid outgrowth assay identifies two different parameters of invasive potential, one of which correlates with phenotypic plasticity. Our findings characterize bladder tumor invasion patterns for the first time and suggest that lineage plasticity in NMIBC represents a key mechanism that promotes tumor invasion in MIBC. Citation Format: Clementine Le Coz, John R. Christin, Talal Syed, Zejian Wang, Caroline J. Laplaca, Andrew T. Lenis, Michael M. Shen. Luminal-to-basal phenotypic plasticity promotes invasive phenotypes in a live-imaging assay using patient-derived bladder tumor organoids [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B025.

Synthesis of Multifunctional Plasmonic Nanodarts through One‐End Deposition on Gold Nanobipyramids for Tumor Organoid Ablation and Antimicrobial Applications

AbstractPlasmonic nanomaterials with multiple components and designed structures show diverse properties and functions owing to the synergistic effects of the different components. Engineering the compositional and structural complexity of plasmonic nanomaterials is a powerful technique for expanding their range of applications. A facile approach to an unprecedented dart‐like type of nanostructure with AgPd bimetal selectively deposited at one end of Au nanobipyramids, forming AgPd nanodarts, is reported here. The synthetic process involves both galvanic replacement and Ostwald ripening. The morphological evolution of the AgPd nanodarts is thoroughly investigated and the growth mechanism is put forward. The as‐obtained AgPd nanodarts exhibit an outstanding photothermal conversion efficiency and peroxidase activity, which make them powerful for photothermally–catalytically combined tumor organoid ablation. In addition, two types of AgPd nanodart‐derived single‐end nanostructures, AgPd@ZIF‐8, and AgPd@TiO2 nanodarts, are prepared for the first time. The ZIF‐8 and TiO2 shell are preferentially coated at the AgPd end of the AgPd nanodarts, respectively. Both AgPd@ZIF‐8 and AgPd@TiO2 nanodarts show superior antimicrobial activities. The protocol and concept of modulating the structure of the nanodarts in this work can be extended to the development of a new class of plasmonic nanostructures with multifunctionalities.

Biomimetic hydrogels with mesoscale collagen architecture for patient-derived tumor organoids culture

Patient-derived tumor organoids (PDTOs) shows great potential as a preclinical model. However, the current methods for establishing PDTOs primarily focus on modulating local properties, such as sub-micrometer topographies. Nevertheless, they neglect to capture the global millimeter or intermediate mesoscale architecture that have been demonstrated to influence tumor response to therapeutic treatment and tumor progression. In this study, we present a rapid technique for generating collagen bundles with an average length of 90 ± 27 μm and a mean diameter of 5 ± 1.5 μm from tumor tissue debris that underwent mechanical agitation following enzymatic digestion. The collagen bundles were subsequently utilized for the fabrication of biomimetic hydrogels, incorporating microbial transglutaminase (mTG) crosslinked gelatin. These biomimetic hydrogels, referred to as MC-gel, were specifically designed for patient-derived tumor organoids. The lung cancer organoids cultured in MC-gel exhibited larger diameters and higher cell viability compared to those cultured in gels lacking the mesoscale collagen bundle; moreover, their irregular morphology more closely resembled that observed in vivo. The MC-gel-based lung cancer organoids effectively replicated the histology and mutational landscapes observed in the original donor patient’s tumor tissue. Additionally, these lung cancer organoids showed a remarkable similarity in their gene expression and drug response across different matrices. This recently developed model holds great potential for investigating the occurrence, progression, metastasis, and management of tumors, thereby offering opportunities for personalized medicine and customized treatment options.

β-hydroxybutyrate restrains colitis-associated tumorigenesis by inhibiting HIF-1α-mediated angiogenesis

Decreased levels of β-hydroxybutyrate (BHB), a lipid metabolic intermediate known to slow the progression of colorectal cancer (CRC), have been observed in the colon mucosa of patients with inflammatory bowel diseases (IBD). In particular, patients with recurrent IBD present an increased risk of developing colitis-associated colorectal cancer (CAC). The role and molecular mechanism of BHB in the inflammatory and carcinogenic process of CAC remains unclear. Here, the anti-tumor effect of BHB was investigated in the Azoxymethane (AOM)/Dextran Sulfate Sodium (DSS)-induced CAC model and tumor organoids derivatives. The underlying mechanisms were studied using transcriptome and non-target metabolomic assay and further validated in colon tumor cell lineage CT26 in vitro. The tumor tissues and the nearby non-malignant tissues from colon cancer patients were collected to measure the expression levels of ketogenic enzymes. The exogenous BHB supplement lightened tumor burden and angiogenesis in the CAC model. Notably, transcriptome analysis revealed that BHB effectively decreased the expression of VEGFA in the CAC tumor mucosa. In vitro, BHB directly reduced VEGFA expression in hypoxic-treated CT26 cells by targeting transcriptional factor HIF-1α. Conversely, the deletion of HIF-1α largely reversed the inhibitory effect of BHB on CAC tumorigenesis. Additionally, decreased expression of ketogenesis-related enzymes in tumor tissues were associated with poor survival outcomes in patients with colon cancer. In summary, BHB carries out anti-angiogenic activity in CAC by regulating HIF-1α/VEGFA signaling. These findings emphasize the role of BHB in CAC and may provide novel perspectives for the prevention and treatment of colonic tumors.

A promising breakthrough in pancreatic cancer research: The potential of spheroids as 3D models

Pancreatic ductal adenocarcinoma (PDAC) stands as the fourth leading cause of cancer-related deaths, primarily attributable to its resistance to chemotherapy, resulting in a nearly universal fatality rate. Despite the promise exhibited by numerous drugs in preclinical studies, their subsequent failure in clinical trials underscores the inherent limitations of conventional two-dimensional cell culture models commonly employed in early drug screening endeavors. The inadequacies of two-dimensional (2D) models prompted the exploration of three-dimensional (3D) culture systems, which more faithfully recapitulate the native tumor microenvironment. These 3D systems have distinct advantages over 2D models in morphology, proliferation, drug response, and protein expression. Among these 3D platforms, tumor organoids and spheroids, generated through different methodologies, have emerged as next-generation models that closely mirror aspects of pancreatic tumor biology. This comprehensive review scrutinizes pancreatic cancer spheroids' techniques, tissue sources, and applications, offering a nuanced analysis of their advantages and limitations. By comparing these distinct 3D culture systems, researchers gain valuable insights to inform the selection of optimal model designs aligned with their specific experimental objectives. The utilization of these advanced models holds significant promise for enhancing the clinical relevance of both in vitro and in vivo cancer research, thereby contributing to the development of improved therapeutics against pancreatic cancer.

Enhancing the Efficacy of HIPEC Through Bromelain: A Preclinical Investigation in Appendiceal Cancer

IntroductionAppendiceal cancer (AC) excessive mucin production is a barrier to heated intraperitoneal chemotherapy (HIPEC) drug delivery. Bromelain is a pineapple stem extract with mucolytic properties. We explored bromelain treatment effects against mucinous AC in a patient-derived tumor organoid (PTO) model and an AC cell line.Patients and MethodsPTOs were fabricated from tumor specimens obtained from patients with AC undergoing cytoreductive surgery with HIPEC. PTOs underwent HIPEC treatment with bromelain, cisplatin, and mitomycin C (MMC) at 37 °C and 42 °C with and without bromelain pretreatment.ResultsFrom October 2020 to May 2023, 16 specimens were collected from 13 patients with low-grade (12/16, 75%) and high-grade AC (4/16, 25%). The mucin-depleting effects of bromelain were most significant in combination with N-acetylcysteine (NAC) compared with bromelain (47% versus 10%, p = 0.0009) or NAC alone (47% versus 12.8%, p = 0.0027). Bromelain demonstrated > 31% organoid viability reduction at 60 min (p < 0.001) and > 66% in 48 h (p < 0.0001). Pretreatment with bromelain increased cytotoxicity of both cisplatin and MMC HIPEC conditions by 31.6% (p = 0.0001) and 35.5% (p = 0.0001), respectively. Ki67, CK20, and MUC2 expression decreased after bromelain treatment; while increased caspase 3/7 activity and decreased Bcl-2 (p = 0.009) and Bcl-xL (p = 0.01) suggest induction of apoptosis pathways. Furthermore, autophagy proteins LC3A/B I (p < 0.03) and II (p < 0.031) were increased; while ATG7 (p < 0.01), ATG 12 (p < 0.04), and Becline 1(p < 0.03), expression decreased in bromelain-treated PTOs.ConclusionsBromelain demonstrates cytotoxicity and mucolytic activity against appendiceal cancer organoids. As a pretreatment agent, it potentiates the cytotoxicity of multiple HIPEC regimens, potentially mediated through programmed cell death and autophagy.

Abstract PO2-14-08: Development and characterization of breast cancer organoids representing epithelial heterogeneity and drug response

Abstract Organoids serve as an important preclinical model in multiple cancers including breast cancer, demonstrating preservation of biological features and feasibility of drug screening, with reasonable cost and high scalability. It has been reported that patient-derived organoids (PDOs) can be robustly derived from both primary and metastatic breast tumors, with high resemblance to source tissues in histological, genomic, and transcriptomic features, as well as consistency in drug response compared to xeno-transplantations or patients (Sachs et al, 2018). Since 2018, the Institute for Precision Medicine (IPM) has developed a panel of breast cancer organoids. The current inventory now consists of 128 PDOs from 94 primary tumors, 12 metastases, 12 rapid-autopsy tumors, and 10 normal breast tissues with a 60% success rate of culture; as well as 33 organoids from animal models including 11 patient-derived xenograft organoids (PDXOs), 15 rat (RDO) and 7 mouse (MDO) tumor organoids with 85% success rate. For PDOs, the protocol includes in-lab processing of fresh, deidentified tissue within 60 minutes of surgical operation, thanks to close collaboration with surgeons, consented patients, and an institutional biospecimen core. BC organoids have been comprehensively characterized in terms of morphology, histology, genomics, transcriptomics, and functional experiments. Key biological features are preserved. PDOs (n=56) exhibit a mutational spectrum consistent with human breast cancers. ER expression is detectable in a subset of cultures with robust estradiol response indicated by GREB1 expression; and PDOs from invasive lobular carcinoma (ILC) mostly showed more discohesive structures than PDOs from no special type (NST), with clear E-cadherin loss under immunofluorescence. We have also successfully constructed transcriptionally and genetic modified BC PDOs with shRNA and CRISPR, which showed replicable performance in proliferation assay, drug response, and signaling by western blot. Of note, longitudinal tracking of PDOs with single cell RNA-sequencing (scRNA-seq) revealed well-preserved heterogeneity compared to primary tumor, while transcriptomic clonality was mildly decreased in later passages from pilot data. Future work will include expansion of current collection from various sources, longitudinal characterization with scRNA-seq and DNA sequencing, as well as more functional studies. We aim to construct a robust resource of well-characterized and reliably-usable model bank for preclinical breast cancer research. Citation Format: Daniel Brown, Kai Ding, Fangyuan Chen, Jian Chen, Steffi Oesterreich, Peter Lucas, Priscilla McAuliffe, Jennifer Atkinson, ADRIAN LEE. Development and characterization of breast cancer organoids representing epithelial heterogeneity and drug response [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 PO2-14-08.

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.