Platform For Preclinical Testing Research Articles

Three-dimensional cardiac models: a pre-clinical testing platform.

Major advancements in human pluripotent stem cell (hPSC) technology over recent years have yielded valuable tools for cardiovascular research. Multi-cell type 3-dimensional (3D) cardiac models in particular, are providing complementary approaches to animal studies that are better representatives than simple 2-dimensional (2D) cultures of differentiated hPSCs. These human 3D cardiac models can be broadly divided into two categories; namely those generated through aggregating pre-differentiated cells and those that form self-organizing structures during their in vitro differentiation from hPSCs. These models can either replicate aspects of cardiac development or enable the examination of interactions among constituent cell types, with some of these models showing increased maturity compared with 2D systems. Both groups have already emerged as physiologically relevant pre-clinical platforms for studying heart disease mechanisms, exhibiting key functional attributes of the human heart. In this review, we describe the different cardiac organoid models derived from hPSCs, their generation methods, applications in cardiovascular disease research and use in drug screening. We also address their current limitations and challenges as pre-clinical testing platforms and propose potential improvements to enhance their efficacy in cardiac drug discovery.

Abstract 237: Establishment of an ex vivo tissue culture model as a potential preclinical drug testing platform for hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, and there is a critical need for more effective treatments. The development of new therapies, especially those aiming to modulate the tumor microenvironment (TME), is limited by the lack of in vitro and in vivo models that recapitulate the complexity of the TME and spatial architecture of HCC. Here, we aimed to establish an HCC model to assess the treatment response and hepatotoxicity of selected drugs by ex vivo culturing fresh human liver tumor tissue and adjacent normal tissue, respectively. Materials and Methods: Fresh human liver tissues, including 5 malignant and 20 adjacent-normal, were obtained from surgical resections. PDX tumors were generated from patient-derived organoids. Tissue resections were cut into ultra-thin slices (from 150µm to 350µm) using a vibratome. Related culture conditions such as high oxygen partial pressure, air-liquid interface (ALI) culture, artificial extracellular matrix (AEM), and delay duration were evaluated to optimize the protocol. As a readout, cell viability was quantified by an expert pathologist using Hematoxylin/Eosin staining. The preservation and complexity of the TME were evaluated by immunohistochemistry or flow cytometry using specific antibodies for each cell population (T cells, anti-CD3; stroma cells, a-SMA; endothelial cells, CD34; Kupffer cells, CD68). Liver cell function was tested by measuring ALT/AST activity, albumin release, and CYP340 activity. Results: The highest tissue viability was observed in normal and tumor tissue with a thickness of 150µm and 250µm, respectively. Compared to standard conditions, the combination of ALI and high-oxygen partial pressure conditions dramatically improved tissue viability, with most of the samples having 70% and 50% cell viability at days 3 and 5 of ex vivo culture, respectively (p<0.05). AEM could prevent the efflux of cellular components (p=0.016). The impact of the time delay between the liver resection and the start of slicing was minimal when the slicing occurred within two hours after hepatectomy (p<0.05). Key cellular components including hepatocytes, T cells, fibroblasts, and Kupffer cells also remained better preserved throughout the 5-day culture period in optimized conditions. Conclusion: Our study has successfully optimized the ex vivo tissue culture model of liver tissue, capable of maintaining the tissue for several days while preserving the unique TME. These results highlight the potential of using the ex vivo tissue culture model in the preclinical setting to further investigate and evaluate TME-related treatments and their hepatotoxicity in HCC tissues. Citation Format: Huiche Feng, Miriam Cieri, Cinzia Esposito, Karolina Guja-Jarosz, Daniela Liberati, Lukas Bubendorf, Savas Deniz Soysal, Mairene Coto-Llerena, Luigi Maria Terracciano, Otto Kollmar, Salvatore Piscuoglio. Establishment of an ex vivo tissue culture model as a potential preclinical drug testing platform for hepatocellular carcinoma [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 237.

Biochemical and transcriptomic evaluation of a 3D lung organoid platform for pre-clinical testing of active substances targeting senescence

Chronic lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis are incurable. Epithelial senescence, a state of dysfunctional cell cycle arrest, contributes to the progression of such diseases. Therefore, lung epithelial cells are a valuable target for therapeutic intervention. Here, we present a 3D airway lung organoid platform for the preclinical testing of active substances with regard to senescence, toxicity, and inflammation under standardized conditions in a 96 well format. Senescence was induced with doxorubicin and measured by activity of senescence associated galactosidase. Pharmaceutical compounds such as quercetin antagonized doxorubicin-induced senescence without compromising organoid integrity. Using single cell sequencing, we identified a subset of cells expressing senescence markers which was decreased by quercetin. Doxorubicin induced the expression of detoxification factors specifically in goblet cells independent of quercetin. In conclusion, our platform enables for the analysis of senescence-related processes and will allow the pre-selection of a wide range of compounds (e.g. natural products) in preclinical studies, thus reducing the need for animal testing.

Ex Vivo Mathematical Myeloma Advisor (EMMA) - a Clinical, Molecular, and Phenotypic Platform to Tailor Personalized Therapeutic Strategies for Multiple Myeloma

Multiple myeloma (MM) is a genetically heterogeneous disease, lacking molecular markers for patient stratification and prediction of clinical response. We have created a unique platform for clinical-informed decisions, research advancement, and preclinical compound testing called the “Ex Vivo Mathematical Myeloma Advisor” (EMMA). In this pipeline, MM patients who undergo bone marrow (BM) biopsy and consent to participate in the Total Cancer Care protocol have their clinical data (demographics, therapies received, lab results, clinical history, etc.) abstracted, along with the molecular characterization (FISH/cytogenetics, RNA-Seq, and WES) of their disease. In parallel, fresh BM-derived MM blasts are combined with stromal cells in a collagen-based matrix containing patient plasma and used in an ex vivo drug screening assay. The cells are seeded into 384 or 1536-well plates and exposed to serial dilutions of up to 127 single agents and combinations. The entire setup is imaged in bright field every 30 minutes for a duration of 6 days. Image analysis is used to convert membrane integrity into survival curves, and AUC and LD50 values are estimated. The pattern of these curves facilitates the identification of resistant subclones. This data is further combined with augmented synergy models that are parameterized with PK/PD data for each drug. This integration permits the simulation of “in silico clinical trials”, which are compiled into reports that assist clinicians in selecting the best treatment for their patients. In addition, the immune population of the BM aspirate is characterized by flow cytometry, and soluble factors in the patient's BM plasma are quantified by ELISA. The integration of clinical, molecular, and phenotypic data with image analysis and machine learning tools presents a unique opportunity for finding molecular signatures of drug response (Fig. 1). For instance, our pipeline can be utilized to identify clinical factors (such as ethnicity, age, etc.) and molecular features (mutations, cytogenetic abnormalities, enriched transcription factors, differentially expressed biological pathways, etc.) associated with drug resistance/sensitivity. Additionally, it enables the determination of the mechanisms of action of new compounds and the prediction of potential synergistic/antagonistic drug combinations. The longitudinal acquisition of data in our non-destructive ex vivo drug screen assay facilitates the exploration of drug interaction dynamics over time, enabling the pinpointing of the moment when synergism occurs at maximum intensity. This, in turn, allows us to propose optimized temporal-based therapeutic regimens for clinical use. Up to this point, our pipeline has molecularly characterized nearly 1,000 MM samples, and EMMA has been applied to over 600 samples, forming one of the world's most extensive and comprehensively characterized MM cohorts. While EMMA has proven effective in quantifying the activity of monoclonal antibodies, ongoing efforts are focusing on incorporating individual immune cell components to assess ex vivo activity in the context of evolving cellular immunotherapies. As a proof of principle, we demonstrate the system's capability to measure the effect of daratumumab (CD38+ antibody) on autologous macrophage-mediated MM cell death, thereby distinguishing it from apoptosis caused by direct antibody binding (Fig. 2). Additionally, we evaluate the influence of teclistamab (T-cell engager CD3/BCMA bispecific antibody) on CD8+ T cell activity against autologous MM cells, as well as the efficacy of CAR-T cells in killing MM cells. Through the testing of standard-of-care drugs alongside immune agents, our pipeline has identified the combination of bortezomib + daratumumab as a synergistic approach with significant translational potential, warranting further investigation. In summary, our state-of-the-art platform facilitate drug discovery and unravel the intricate biological phenomena underlying drug interactions in MM. The advantages offered by our system are threefold: i) it enables pharmaceutical companies to test new compounds in a bona fide, patient-derived model; ii) it assists clinicians in selecting the best therapy for their patients, and iii) it helps prevent cancer patients from receiving non-suitable therapies. This approach can also be promptly applied to other liquid cancers, such as leukemia and lymphoma.

Stag2-Cohesin Loss Attenuates Flt3 ITD Myeloid Blast Expansion Yet Preserves Mutant HSC

FLT3 is mutated through an internal tandem duplication (ITD) in 20-25% of acute myeloid leukemia (AML), driving aberrant STAT5/AKT signaling and leukemogenesis. Murine modelling of ITD showed marked myeloid progenitor expansion and hematopoietic stem cell (HSC) exhaustion in various co-mutant settings. Here we present a Stag2 Δ Flt3 ITD model with stark reversal of these two aforementioned features. STAG2, a cohesin complex member, maintains the integrity of the 3D genome partitioning structure known as topologically structural domains. Loss of Stag2 impairs the access and engagement of key hematopoietic transcription factors such as PU.1 to their target genes. sAML is diagnosed when patients present leukemia with known history of hematological malignancies or chemotherapy/radiotherapy treatment. Comparing to de novo AML, sAML often arises in older patients and harbors a poor prognosis with a 5-year overall survival rate of <30%. Advances in genomic studies found various epigenetic mutations, such as STAG2, are often associated with sAML and AML-myelodysplastic related changes (MRC) subtype. STAG2 mutations are found in 14-20% of sAML cases and is suggested to reside within a dominant clone during the pre-leukemia phase, MDS to sAML transformation, such as with FLT3 ITD acquisition and persists during remission. To understand the mechanistic contribution of STAG2-cohesin loss with FLT3 ITD, we generated sequential mutagenesis murine models where the order of Stag2 and Flt3 ITD mutation is set as either ITD 1st Stag2 2nd ( de novo like) or Stag2 1stITD 2nd (sAML like) using tamoxifen-inducible Cre/Flpo recombinase or pIpC-inducible Mx1Cre. In the de novo like model, ITD is constitutively active then Stag2 is deleted when mice reach 6-8 weeks of age. Surprisingly, loss of Stag2 attenuates LSK to MP transition at 4 weeks post deletion, while MPP3 remains elevated, suggesting aberrant remodeling of myeloid differentiation. In the sAML like model, Stag2 is deleted via Mx1Cre and waited for 4 months to mimic the MDS phase, which is then followed by activation of ITD mutation via Flpo, which represents the MDS to sAML transformation. After activating both mutations, mice were followed for another 4 months before analyzing the hematopoietic stem and progenitor compartment. In contrast to the de novo like model, sequential Stag2 1stITD 2nd preserves the HSC population defined by either immunophenotyping or transcriptome via scRNAseq ( Figure 1). The mutant HSC is more quiescent but retains the capacity to reconstitute lethally irradiated recipients in the short term. Similar to the de novo like model, sequential Stag2 1stITD 2nd mice also exhibits a blocked myeloid differentiation. Comparing to ITD mutant, Stag2 1stITD 2nd LSK cells have decreased expression of Socs2 and Cish. While functionally determining the role of mutant HSC, we are performing RNA-seq during at early timepoints post ITD activation to determine how preceding Stag2 mutation could have altered the stem cell fate decision. Targeted therapy with inhibitors of FLT3 have had an overall survival benefit in FLT3-mutant AML, though the magnitude of effect has been modest. STAG2 mutations are more likely to be identified in poor responders to FLT3 inhibition as both reported by us with Pexidartinib treatment, as well as in the setting of Crenolanib treatment where expansion of the STAG2-mutant clone was observed during treatment. Thus, remodeling of the chromatin landscape though altered CTCF binding or cohesin function might impact leukemia identity in FLT3-mutant AML. Our data highlights an important regulatory role of Stag2-cohesin in Flt3 ITD mediated leukemogenesis, while generating a model that mimics the genetic evolution of sAML. This model will not only shed light on the sAML pathogenesis but also with creates a pre-clinical testing platform with potential therapeutic relevance.

Utilizing a human TLR selective ligand in a humanized immune system mouse model to investigate human TLR4 signaling.

Mouse models with humanized immune systems are becoming increasingly prevalent in pharmaceutical research as a platform for preclinical testing with potential for greater translatability to clinical applications. However, the presence of both mouse and human cells that respond to TLR ligands poses a challenge for investigating therapeutic modalities targeting TLR signaling. AZ617 is a human TLR4 agonist, which has been shown in vitro to preferentially induce human cytokines via the TLR4 signaling pathway. We sought to examine the ability of AZ617 to preferentially induce human cytokines in CD34+ stem cell-engrafted NOG-EXL mice (huNOG-EXL), to determine its suitability as an in vivo human functional readout. AZ617 elicited a strong human TNFα and IL-6 response in vivo that demonstrated a 10- and 5-fold preference, respectively, over the mouse TNFα and IL-6. To assess efficacy of inhibiting a key protein in the TLR4 signaling pathway, PF-06650833, a small molecule inhibitor of IRAK4, was used as a tool molecule. PF-0660833 was found to effectively inhibit AZ617-induced human TNFα release in vitro. Likewise, PF-06650833 reduced AZ617-induced human TNFα in the huNOG-EXL mouse model, with a weaker effect on human IL-6. A longitudinal study tracking functionality of monocytes revealed that the ability of monocytes to respond to ex vivo stimuli was increased by 21 weeks after engraftment. Taken together, our data suggests that human selective TLR ligands could preferentially drive cytokine production from human cells in huNOG-EXL mice. This model will allow for investigation of pharmacological inhibition of human TLR signaling pathways in an in vivo model system.

Patient-derived precision cut tissue slices from primary liver cancer as a potential platform for preclinical drug testing

The exploitation of anti-tumour immunity, harnessed through immunomodulatory therapies, has fundamentally changed the treatment of primary liver cancer (PLC). However, this has posed significant challenges in preclinical research. Novel immunologically relevant models for PLC are urgently required to improve the translation from bench to bedside and back, explore and predict effective combinatorial therapies, aid novel drug discovery and develop personalised treatment modalities. We used human precision-cut tissue slices (PCTS) derived from resected tumours to create a patient-specific immunocompetent disease model that captures the multifaceted and intricate heterogeneity of the tumour and the tumour microenvironment. Tissue architecture, tumour viability and treatment response to single agent and combination therapies were assessed longitudinally over 8 days of exvivo culture by histological analysis, detection of proliferation/cell death markers, ATP content via HPLC. Immune cell infiltrate was assessed using PCR and immunofluorescence. Checkpoint receptor expression was quantified via Quantigene RNA assay. After optimising the culture conditions, PCTS maintained the original tissue architecture, including tumour morphology, stroma and tumour-infiltrated leukocytes. Moreover, PCTS retained the tumour-specific immunophenotype over time, suggesting the utility of PCTS to investigate immunotherapeutic drug efficacy and identify non-responsiveness. Here we have characterised the PCTS model and demonstrated its effectiveness as a robust preclinical tool that will significantly support the development of successful (immuno)therapeutic strategies for PLC. Foundation for Liver Research, London.

198. MODELING CANCER CACHEXIA USING PATIENT TUMOR ORGANOID-DERIVED XENOGRAFTS IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract Background Cancer cachexia is a metabolic and functional multifactorial body wasting syndrome and is one of the major causes of death in late-stage patients. Esophageal squamous cell carcinoma (ESCC) presents one of the highest morbidity and mortality rates due to cancer cachexia. However, there is limited effective and approved targeted treatment. Cancer cachexia animal models are highly limited, with few cancer types available as a pre-clinical drug testing platform and with none related to esophageal carcinoma. Methods We have developed a panel of ESCC patient-derived organoid xenograft (PDOX) mouse models based on our latest patient-derived organoid cultures from Hong Kong patients. Results Our PDOX mouse model displays cachexic phenotypes that traditional cell line-derived xenografts lack, including body wasting and functional signs. The slow-proliferating characteristic of the ESCC PDOX on mice allows development of cachexic conditions before the humane endpoint. Our comprehensive panel of PDOX lines allowed us to identify full spectrum and differential cachexia severities across mice bearing different PDOXs, indicating a heterogenous nature and allowing faithful mimicking of various patient conditions. Molecular analysis showed that several classic cachexia-related gene expressions were up-regulated in the isolated liver and inguinal adipose tissues of cachexic mice, along with elevated circulating Il-6 levels. Conclusion Our ESCC PDOX model potentially serves as a useful pre-clinical cachexia-targeted drug testing platform, providing more treatment options for ESCC patients for better disease management. Acknowledgments We acknowledge the Research Grants Council (Theme-based Research Scheme T12–701/17-R to MLL) and the Food and Health Bureau (Health and Medical Research Fund 06171566 to VZY) of the Hong Kong Special Administrative Region for funding support. We acknowledge DSMZ (German Collection of Microorganisms and Cell Culture) for the KYSE cell lines. We acknowledge the University of Hong Kong LKS Faculty of Medicine Centre for PanorOmic Sciences for providing imaging facilities.

Immune-infiltrated kidney organoid-on-chip model for assessing T cell bispecific antibodies

T cell bispecific antibodies (TCBs) are the focus of intense development for cancer immunotherapy. Recently, peptide-MHC (major histocompatibility complex)-targeted TCBs have emerged as a new class of biotherapeutics with improved specificity. These TCBs simultaneously bind to target peptides presented by the polymorphic, species-specific MHC encoded by the human leukocyte antigen (HLA) allele present on target cells and to the CD3 coreceptor expressed by human T lymphocytes. Unfortunately, traditional models for assessing their effects on human tissues often lack predictive capability, particularly for "on-target, off-tumor" interactions. Here, we report an immune-infiltrated, kidney organoid-on-chip model in which peripheral blood mononuclear cells (PBMCs) along with nontargeting (control) or targeting TCB-based tool compounds are circulated under flow. The target consists of the RMF peptide derived from the intracellular tumor antigen Wilms' tumor 1 (WT1) presented on HLA-A2 via a bivalent T cell receptor-like binding domain. Using our model, we measured TCB-mediated CD8+ T cell activation and killing of RMF-HLA-A2-presenting cells in the presence of PBMCs and multiple tool compounds. DP47, a non-pMHC-targeting TCB that only binds to CD3 (negative control), does not promote T cell activation and killing. Conversely, the nonspecific ESK1-like TCB (positive control) promotes CD8+ T cell expansion accompanied by dose-dependent T cell-mediated killing of multiple cell types, while WT1-TCB* recognizing the RMF-HLA-A2 complex with high specificity, leads solely to selective killing of WT1-expressing cells within kidney organoids under flow. Our 3D kidney organoid model offers a platform for preclinical testing of cancer immunotherapies and investigating tissue-immune system interactions.

Autologous humanized PDX modeling for immuno-oncology recapitulates features of the human tumor microenvironment

BackgroundInteractions between immune and tumor cells are critical to determining cancer progression and response. In addition, preclinical prediction of immune-related drug efficacy is limited by interspecies differences between human and...

EPS3.03 Patient-derived organoids as a platform for preclinical drug testing, identification of responsive mutations and personlized treatment diagnosis in cystic fibrosis

Objective: A novel technology to generate patient-specific organoid cultures derived from adult stem cells, or HUB Organoids™ has gained widespread interest for its application in the development of new therapies and as a predictive diagnostic tool. In this study we aim to utilize CF patient-derived HUB Organoids to identify the combinatory treatment effect of read-through agent, correctors and potentiator on rare mutations.

Comparative Evaluation of the Cytotoxicity of Doxorubicin in BT-20 Triple-Negative Breast Carcinoma Monolayer and Spheroid Cultures.

Three-dimensional cell culture models are increasingly adopted as preferred pre-clinical drug testing platforms, as they circumvent limitations associated with traditional monolayer cell cultures. However, many of these models are not fully characterized. This study aimed to characterize a BT-20 triple-negative breast carcinoma spheroid model and assess its susceptibility to doxorubicin in comparison to a monolayer model. Spheroids were developed using the liquid overlay method. Phenotypic attributes were analyzed by characterizing changes in size, gross morphology, protein content, metabolic activity, hypoxic status, and cell-cell junctions. The cytotoxic range of doxorubicin in monolayers was determined using the sulforhodamine B assay, and the comparative effect of toxic and sub-toxic concentrations was assessed in both spheroids and monolayers. Similar to the in vivo microenvironment, spheroids had a heterogeneous spatial cytoarchitecture, inherent hypoxia and strong adherens junctions. Doxorubicin induced dose-dependent cytotoxicity in monolayers (IC25: 130 nM, IC50: 320 nM and IC75: 1580 nM); however, these concentrations did not alter the spheroid size or acid phosphatase activity. Only concentrations ≥6 µM had any effect on spheroid integrity. In comparison to monolayers, the BT-20 spheroid model has decreased sensitivity to doxorubicin and could serve as a better model for susceptibility testing in triple-negative breast cancer.

Abstract 363: Modelling cancer cachexia using a comprehensive panel of heterogeneous esophageal squamous cell carcinoma xenografts for therapeutic application

Abstract Background: Esophageal squamous cell carcinoma (ESCC) is one of deadliest esophageal malignancies in Asia. Cancer cachexia is a multifactorial body wasting syndrome and is one of the major causes of death in late-stage patients (1). It is characterized by skeletal muscle loss and atrophy, adipose and body wasting, and metabolic dysregulations and stress (1). ESCC is one of the cancer types with the highest morbidity and mortality rates due to cancer cachexia (2). However, there are few effective and approved treatments targeting cancer cachexia. Currently, cancer cachexia animal models are highly limited with few cancer types available as a pre-clinical drug testing platform, with none related to esophageal carcinoma (3). Results: Here we have developed a panel of ESCC patient-derived organoid xenograft (PDOX) mouse model based on our latest patient-derived organoid cultures originating from Hong Kong patients. Our immunodeficient mouse-based PDOX model resembles different cachexic phenotypes that cell line-derived xenograft lacks, including body (skeletal muscle and adipose tissue) wasting and functional signs (e.g. lack of body movement, reduced muscle strength, and loss of appetite). The slow-proliferating characteristic of the ESCC PDOX allows development of a cachexic condition before humane endpoint. Furthermore, our full panel of PDOX lines allowed us to identify the full spectrum, differential cachexia severity across mice bearing different PDOXs, indicating a heterogenous nature. We have comprehensively characterized the cachexia phenotypes and performed proof-of-concept preclinical drug tests using established anti-cachexic drugs; treated PDOX-bearing mice have improved body condition and function compared to the control, which were further verified by histological examinations of tissue samples. Molecular analysis showed that several classic cachexia related gene expressions were down-regulated in isolated liver (Il1b) and inguinal adipose tissues (adipose triglyceride lipase). Conclusions: Our ESCC PDOX model potentially serves as a great pre-clinical cachexia-targeted drug testing platform, providing more treatment options for ESCC patients for better disease management. Acknowledgement: We acknowledge the Research Grants Council (TRS T12-701/17-R to MLL) and the Food and Health Bureau (HMRF 06171566 to VZY) of Hong Kong Special Administrative Region for funding supports. We acknowledge DSMZ for the KYSE cell lines. We acknowledge the HKUMed-CPOS for providing imaging facilities.

Application of Drug Testing Platforms in Circulating Tumor Cells and Validation of a Patient-Derived Xenograft Mouse Model in Patient with Primary Intracranial Ependymomas with Extraneural Metastases

Primary intracranial ependymoma is a challenging tumor to treat despite the availability of multidisciplinary therapeutic modalities, including surgical resection, radiotherapy, and adjuvant chemotherapy. After the completion of initial treatment, when resistant tumor cells recur, salvage therapy needs to be carried out with a more precise strategy. Circulating tumor cells (CTCs) have specifically been detected and validated for patients with primary or recurrent diffused glioma. The CTC drug screening platform can be used to perform a mini-invasive liquid biopsy for potential drug selection. The validation of potential drugs in a patient-derived xenograft (PDX) mouse model based on the same patient can serve as a preclinical testing platform. Here, we present the application of a drug testing model in a six-year-old girl with primary ependymoma on the posterior fossa, type A (EPN-PFA). She suffered from tumor recurrence with intracranial and spinal seeding at 2 years after her first operation and extraneural metastases in the pleura, lung, mediastinum, and distant femoral bone at 4 years after initial treatment. The CTC screening platform results showed that everolimus and entrectinib could be used to decrease CTC viability. The therapeutic efficacy of these two therapeutic agents has also been validated in a PDX mouse model from the same patient, and the results showed that these two therapeutic agents significantly decreased tumor growth. After precise drug screening and the combination of focal radiation on the femoral bone with everolimus chemotherapy, the whole-body bone scan showed significant shrinkage of the metastatic tumor on the right femoral bone. This novel approach can combine liquid biopsy, CTC drug testing platforms, and PDX model validation to achieve precision medicine in rare and challenging tumors with extraneural metastases.

Advances in respiratory physiology in mouse models of experimental asthma.

Recent advances in mouse models of experimental asthma coupled with vast improvements in systems that assess respiratory physiology have considerably increased the accuracy and human relevance of the outputs from these studies. In fact, these models have become important pre-clinical testing platforms with proven value and their capacity to be rapidly adapted to interrogate emerging clinical concepts, including the recent discovery of different asthma phenotypes and endotypes, has accelerated the discovery of disease-causing mechanisms and increased our understanding of asthma pathogenesis and the associated effects on lung physiology. In this review, we discuss key distinctions in respiratory physiology between asthma and severe asthma, including the magnitude of airway hyperresponsiveness and recently discovered disease drivers that underpin this phenomenon such as structural changes, airway remodeling, airway smooth muscle hypertrophy, altered airway smooth muscle calcium signaling, and inflammation. We also explore state-of-the-art mouse lung function measurement techniques that accurately recapitulate the human scenario as well as recent advances in precision cut lung slices and cell culture systems. Furthermore, we consider how these techniques have been applied to recently developed mouse models of asthma, severe asthma, and asthma-chronic obstructive pulmonary disease overlap, to examine the effects of clinically relevant exposures (including ovalbumin, house dust mite antigen in the absence or presence of cigarette smoke, cockroach allergen, pollen, and respiratory microbes) and to increase our understanding of lung physiology in these diseases and identify new therapeutic targets. Lastly, we focus on recent studies that examine the effects of diet on asthma outcomes, including high fat diet and asthma, low iron diet during pregnancy and predisposition to asthma development in offspring, and environmental exposures on asthma outcomes. We conclude our review with a discussion of new clinical concepts in asthma and severe asthma that warrant investigation and how we could utilize mouse models and advanced lung physiology measurement systems to identify factors and mechanisms with potential for therapeutic targeting.

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.

Predictors of success in establishing orthotopic patient-derived xenograft models of triple negative breast cancer

Patient-derived xenograft (PDX) models of breast cancer are an effective discovery platform and tool for preclinical pharmacologic testing and biomarker identification. We established orthotopic PDX models of triple negative breast cancer (TNBC) from the primary breast tumors of patients prior to and following neoadjuvant chemotherapy (NACT) while they were enrolled in the ARTEMIS trial (NCT02276443). Serial biopsies were obtained from patients prior to treatment (pre-NACT), from poorly responsive disease after four cycles of Adriamycin and cyclophosphamide (AC, mid-NACT), and in cases of AC-resistance, after a 3-month course of different experimental therapies and/or additional chemotherapy (post-NACT). Our study cohort includes a total of 269 fine needle aspirates (FNAs) from 217 women, generating a total of 62 PDX models (overall success-rate = 23%). Success of PDX engraftment was generally higher from those cancers that proved to be treatment-resistant, whether poorly responsive to AC as determined by ultrasound measurements mid-NACT (p = 0.063), RCB II/III status after NACT (p = 0.046), or metastatic relapse within 2 years of surgery (p = 0.008). TNBC molecular subtype determined from gene expression microarrays of pre-NACT tumors revealed no significant association with PDX engraftment rate (p = 0.877). Finally, we developed a statistical model predictive of PDX engraftment using percent Ki67 positive cells in the patient’s diagnostic biopsy, positive lymph node status at diagnosis, and low volumetric reduction of the patient’s tumor following AC treatment. This novel bank of 62 PDX models of TNBC provides a valuable resource for biomarker discovery and preclinical therapeutic trials aimed at improving neoadjuvant response rates for patients with TNBC.

Progress of 3D Organoid Technology for Preclinical Investigations: Towards Human In Vitro Models

Review Progress of 3D Organoid Technology for Preclinical Investigations: Towards Human In Vitro Models Yingjuan Liu *, Honglin Xu, Sabu Abraham, Xin Wang, and Bernard D. Keavney* Division of Cardiovascular Sciences, Faculty of Biology, Medicine and Health, University of Manchester, M13 9PT, UK. * Correspondence: yingjuan.liu@manchester.ac.uk (Yingjuan Liu); bernard.keavney@manchester.ac.uk (Bernard D. Keavney) Received: 1 November 2022 Accepted: 24 November 2022 Published: 21 December 2022 Abstract: Currently, with an increased requirement for new therapeutic strategies, preclinical drug testing or screening platforms have rapidly evolved in recent years. In comparison to traditional 2D cell cultures, 3D organoids or spheroids with or without scaffolds improve the microenvironment of in vitro cultures, advancing the in vitro biological observation and enabling mechanistic studies of drug reactions in the human tissue-like environment. 3D organoids and spheroids are straightforward to produce, and relatively uniform in size and shape. This helps to facilitate high throughput screening requirements. Spheroids and organoids have been applied in anti-cancer drug testing, toxicity evaluations, as well as mechanism studies for variable organ systems, including the intestine, liver, pancreas, brain, and heart. Among 3D cultures of spheroids and organoids, ‘tumour spheroids’ formed by dissociated tumour tissues or cancer cell lines are relatively simple in composition and commonly applied to anticancer drug screening. The ‘healthy organoids’ differentiated from hiPSCs/hESCs are more complex in cell composition, distribution, structure and function with higher similarity to in vivo organs, and have found applications in toxicity tests, personalised medicine, and therapeutic and mechanistic studies. In most cases, the multicellular 3D organoids are more resistant and stable in reaction to stimulations or chemicals in vitro , suggesting more accurate modelling of in vivo responses. Here, we review recent progress in human-origin organoid/spheroid systems and their applications in preclinical studies.

Brain Alterations in Aged OVT73 Sheep Model of Huntington's Disease: An MRI Based Approach.

Huntington's disease (HD) is a fatal neurodegenerative autosomal dominant disorder with prevalence of 1 : 20000 that has no effective treatment to date. Translatability of candidate therapeutics could be enhanced by additional testing in large animal models because of similarities in brain anatomy, size, and immunophysiology. These features enable realistic pre-clinical studies of biodistribution, efficacy, and toxicity. Here we non-invasively characterized alterations in brain white matter microstructure, neurochemistry, neurological status, and mutant Huntingtin protein (mHTT) levels in cerebrospinal fluid (CSF) of aged OVT73 HD sheep. Similar to HD patients, CSF mHTT differentiates HD from normal sheep. Our results are indicative of a decline in neurological status, and alterations in brain white matter diffusion and spectroscopy metric that are more severe in aged female HD sheep. Longitudinal analysis of aged female HD sheep suggests that the decline is detectable over the course of a year. In line with reports of HD human studies, white matter alterations in corpus callosum correlates with a decline in gait of HD sheep. Moreover, alterations in the occipital cortex white matter correlates with a decline in clinical rating score. In addition, the marker of energy metabolism in striatum of aged HD sheep, shows a correlation with decline of clinical rating score and eye coordination. This data suggests that OVT73 HD sheep can serve as a pre-manifest large animal model of HD providing a platform for pre-clinical testing of HD therapeutics and non-invasive tracking of the efficacy of the therapy.

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.