Therapeutic Screening Research Articles

Single cell phototransfection of mRNAs encoding SARS-CoV2 spike and nucleocapsid into human astrocytes results in RNA dependent translation interference

Multi-RNA co-transfection is starting to be employed to stimulate immune responses to SARS-CoV-2 viral infection. While there are good reasons to utilize such an approach, there is little background on whether there are synergistic RNA-dependent cellular effects. To address this issue, we use transcriptome-induced phenotype remodeling (TIPeR) via phototransfection to assess whether mRNAs encoding the Spike and Nucleocapsid proteins of SARS-CoV-2 virus into single human astrocytes (an endogenous human cell host for the virus) and mouse 3T3 cells (often used in high-throughput therapeutic screens) synergistically impact host cell biologies. An RNA concentration-dependent expression was observed where an increase of RNA by less than 2-fold results in reduced expression of each individual RNAs. Further, a dominant inhibitory effect of Nucleocapsid RNA upon Spike RNA translation was detected that is distinct from codon-mediated epistasis. Knowledge of the cellular consequences of multi-RNA transfection will aid in selecting RNA concentrations that will maximize antigen presentation on host cell surface with the goal of eliciting a robust immune response. Further, application of this single cell stoichiometrically tunable RNA functional genomics approach to the study of SARS-CoV-2 biology promises to provide details of the cellular sequalae that arise upon infection in anticipation of providing novel targets for inhibition of viral replication and propagation for therapeutic intervention.

Abstract PR-009: Reconstructing the metastatic tumor microenvironment of high grade serous ovarian cancer using human multicellular in vitro models

Abstract Effective treatment of high grade serous ovarian cancer (HGSOC) still represents a clinical challenge. This is partially due to poor understanding of the complexity of this disease and the highly immunosuppressive tumor microenvironment (TME) that makes HGSOC resistant to most of the newly discovered therapies. The lack of relevant and physiological human in vitro models represents a major issue as the development of new treatments are mostly based on mouse models that do not fully recapitulate the human disease. In this study, we developed 3-dimensional (3D) human multicellular in vitro models using as a template and validation, data collected in a previous study that ‘deconstructs’ the metastatic TME of HGSOC. Our human 3D in vitro pentaculture (five cell types) model is composed of patient-derived primary adipocytes, fibroblasts and mesothelial cells isolated from macroscopic normal omentum – the primary site of metastasis in HGSOC – of women undergoing a gynaeco-oncology surgery, combined with different high grade serous malignant cell lines and monocytes isolated from healthy individuals. We found that the peripheral blood monocytes were able to differentiate and polarize to macrophages in our model in a similar way that observed in human HGSOC omental metastasis. Interestingly, our pentaculture models self-rearrange at the cellular level in a different way depending on the malignant cell line used in the culture. RNAseq analysis of the pentacultures showed that the different cultures clustered according to the malignant cell line used suggesting that malignant cells have a major influence on the transcriptome of the TME. Moreover, we demonstrated that our pentacultures partially replicated human biopsies: some of the macrophage and fibroblast clusters identified in a previous scRNAseq experiment on human HGSOC biopsies were also found in the pentacultures. We then confirmed the presence of some these macrophages clusters at the protein level. Finally, these pentacultures reproduce some of the actions of macrophage modifying drugs that have previously been shown in vivo. In conclusion, the study of the immunosuppressive TME of HGSOC in these human 3D in vitro models allows us to identify the contribution of different cell types to tumor growth and spread and provides us a semi-high throughput platform for therapeutic screening. Citation Format: Beatrice Malacrida, Samar Elorbany, Eleni Maniati, Rachel Bryan-Ravenscroft, Sophie L.P. Skingsley, Florian Laforets, Ranjit Manchanda, Frances R. Balkwill. Reconstructing the metastatic tumor microenvironment of high grade serous ovarian cancer using human multicellular in vitro models [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr PR-009.

Patient-Derived Organoids as Therapy Screening Platforms in Cancer Patients.

Patient-derived organoids (PDOs) developed ex vivo and in vitro are increasingly used for therapeutic screening. They provide a more physiologically relevant model for drug discovery and development compared to traditional cell lines. However, several challenges remain to be addressed to fully realize the potential of PDOs in therapeutic screening. This paper summarizes recent advancements in PDO development and the enhancement of PDO culture models. This is achieved by leveraging materials engineering and microfabrication technologies, including organs-on-a-chip and droplet microfluidics. Additionally, this work discusses the application of PDOs in therapy screening to meet diverse requirements and overcome bottlenecks in cancer treatment. Furthermore, this work introduces tools for data processing and analysis of organoids, along with their microenvironment. These tools aim to achieve enhanced readouts. Finally, this work explores the challenges and future perspectives of using PDOs in drug development and personalized screening for cancer patients.

Key considerations to improve the normalization, interpretation and reproducibility of morbidity data in mammalian models of viral disease.

Viral pathogenesis and therapeutic screening studies that utilize small mammalian models rely on the accurate quantification and interpretation of morbidity measurements, such as weight and body temperature, which can vary depending on the model, agent and/or experimental design used. As a result, morbidity-related data are frequently normalized within and across screening studies to aid with their interpretation. However, such data normalization can be performed in a variety of ways, leading to differences in conclusions drawn and making comparisons between studies challenging. Here, we discuss variability in the normalization, interpretation, and presentation of morbidity measurements for four model species frequently used to study a diverse range of human viral pathogens - mice, hamsters, guinea pigs and ferrets. We also analyze findings aggregated from influenza A virus-infected ferrets to contextualize this discussion. We focus on serially collected weight and temperature data to illustrate how the conclusions drawn from this information can vary depending on how raw data are collected, normalized and measured. Taken together, this work supports continued efforts in understanding how normalization affects the interpretation of morbidity data and highlights best practices to improve the interpretation and utility of these findings for extrapolation to public health contexts.

Experience of caregivers on the continuum of care and prevention of malnutrition among children with cholera in Ethiopia: a phenomenology study

IntroductionMalnutrition is a public health problem in low- and middle-income countries among children. Although illnesses such as diarrhea are common immediate drivers of childhood malnutrition, their consequences could be averted through optimal sick child feeding and care to ensure the continuum of care. This study aimed to explore the lived experiences of mothers/caregivers on continuum of care to prevent malnutrition among children with cholera in Ethiopia.MethodsA phenomenology study design was applied to explore experiences of mothers/caregivers in the Bale and Guji zones of the Oromia region, southeast Ethiopia, from November to December 2022 using an unstructured interview guide. The saturation of ideas was used to stop the in-depth interview. Translated data were cleaned and imported into ATLAS.ti7 software for analysis. Using an open coding system, the data were coded into a meaningful context. Deeper immersion into data with repeated reading, creating themes, subthemes, and family/category were carried out. In coding and categorization, multiple coders were involved. The finding was presented using well-spoken verbatim/quotes as illustrations and in narratives.ResultsIn this qualitative study, ten participants were taken to explore their lived experience on the continuum of care for children with acute malnutrition and cholera. The study found that poverty, expensive cost of living, and poor utilization of diversified food were challenges. Moreover, health facilities did not provide any services to mothers whose child was admitted for malnutrition treatment. Children five years and above were excluded from both therapeutic food and screening for malnutrition program. Interruptions of supplies, low attention given to child feeding, inadequate knowledge, and lack of time to prepare diversified food were the main findings.ConclusionPoverty, poor feeding habits, supplies interruption and non-inclusion of malnourished children five and above in screening for malnutrition and in the therapeutic feeding program is missed opportunities that lead to decreased early detection and treatment of malnutrition among children with cholera. Moreover, mothers/caregivers did not receive any service from health facilities when their child was admitted for treatment of malnutrition. This situation forces them to stop treatment before their child recovers from malnutrition, which has a negative impact on the continuum of care and prevention of malnutrition. Therefore, we strongly recommend strengthening emergency nutrition within the country’s health system and revising the food and nutrition policy to incorporate emergency nutrition, with a particular focus on children under the age of fifteen. Additionally, it is important that the study’s recommendations underscore the significance of a multi-sectoral approach that involves collaboration among the health sector, government agencies, and non-governmental organizations. Moreover, adaptive agricultural products be made easily accessible to the community which is crucial in effective preventing and reducing malnutrition in children in the study and similar settings.

Modelling Fabry disease with kidney organoids derived from human embryonic stem cells using CRISPR-Cas9 mediated gene editing

Using gene editing to generate a nephron organoid model which recapitulates the hallmarks of human kidney disease is an attractive strategy, especially due to the potential for drug discovery in the context of rare genetic diszmfltmxeases. The present study aimed to develop an organoid-based Fabry disease model derived from human embryonic stem cells which have undergone CRISPR/Cas9-mediated α-galactosidase A gene knockout. Fabry disease modelling via kidney organoids holds promise for elucidating disease mechanisms and establishing in vitro screening platform therapeutic drug screening approaches.

Gastric cancer patient-derived organoids model for the therapeutic drug screening

BackgroundGastric cancer (GC) is a highly heterogeneous disease featuring many various histological and molecular subtypes. Therefore, it is imperative to have well-characterized in vitro models for personalized treatment development. Gastric cancer patient-derived organoids (PDOs), re-capitulating in vivo conditions, exhibit high clinical efficacy in predicting drug sensitivity to facilitate the development of cancer precision medicine. MethodsPDOs were established from surgically resected GC tumor tissues. Histological and molecular characterization of PDOs and primary tissues were performed via IHC and sequencing analysis. We also conducted drug sensitivity tests using PDO cultures with five chemotherapeutic drugs and twenty-two targeted drugs. ResultsWe have successfully constructed a PDOs biobank that included EBV+, intestinal/CIN, diffuse/GS, mixed and Her2+ GC subtypes, and these PDOs captured the pathological and genetic characteristics of corresponding tumors and exhibited different sensitivities to the tested agents. In a clinical case study, we performed an additional drug sensitivity test for a patient who reached an advanced progressive stage after surgery. We discovered that the combination of napabucasin and COTI-2 exhibited a stronger synergistic effect than either drug alone. ConclusionPDOs maintained the histological and genetic characteristics of original cancer tissues. PDOs biobank opens up new perspectives for studying cancer cell biology and personalized medicine as a preclinical study platform.

Abstract C058: Identifying cell-intrinsic drivers of transcriptional plasticity in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with few effective therapies. Contributing to the complexity of this deadly cancer, PDAC tumors with similar genomic backgrounds can express divergent transcriptional programs with clinical importance. These transcriptional subtypes, termed “cell states”, have prognostic significance: PDAC tumors in the “classical state” generally have better outcomes and are thought to be more responsive to chemotherapy while those in the “basal state” are more aggressive. Currently, cell state is not used to direct therapy. Cell state is an integrative property shaped by both cell-intrinsic (e.g., mutations, epigenetics) and cell-extrinsic (e.g., growth factors, cytokines) factors. In our prior work, single-cell analysis of clinical PDAC samples demonstrated notable intra-tumoral cell state heterogeneity with evidence for cells that co-expressed both basal and classical markers, suggestive of cell state plasticity. In addition, longitudinal monitoring of patient-derived organoids has shown that cells can shift from classical to coexpressor/basal states as the environment changes, and this shift in cell states can drastically influence drug response. However, it remains unknown which signaling and transcriptional pathways induce basal or classical states, and whether these gene regulatory networks can be targeted therapeutically. Thus, there is a pressing need to devise scalable methods to determine essential drivers for prognostically-relevant cancer cell states. Here, we aim to define transcriptional drivers of basal and classical cell states in pancreatic cancer. We are currently optimizing a flow cytometry-fluorescence in situ hybridization (Flow-FISH) workflow to enable a multi-gene cell state readout. We plan to perform a transcription factor over-expression screen within a cohort of PDAC models using this Flow-FISH readout to identify those factors that can drive cells toward basal (n=3), classical (n=2), or coexpressor (n=3) states. As a proof of concept, we have confirmed that TP63A overexpression induces basal state expression, even in models that initially express a classical phenotype. Through these efforts, we aim to construct high-fidelity isogenic but state-variant PDAC models for use both in therapeutic screening and as a substrate for investigating mechanisms governing cancer cell state plasticity. Citation Format: Yuzhou Evelyn Tong, Walaa E. Katan, Alex Bott, Julia Joung, Alex K. Shalek, Peter S. Winter, Srivatsan Raghavan. Identifying cell-intrinsic drivers of transcriptional plasticity in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C058.

A Promising New Model: Establishment of Patient-Derived Organoid Models Covering HPV-Related Cervical Pre-Cancerous Lesions and Their Cancers.

The lack of human-derived in vitro models that recapitulate cervical pre-cancerous lesions has been the bottleneck in researching human papillomavirus (HPV) infection-associated pre-cancerous lesions and cancers for a long time. Here, a long-term 3D organoid culture protocol for high-grade squamous intraepithelial lesions and cervical squamous cell carcinoma that stably recapitulates the two tissues of origin is described. Originating from human-derived samples, a small biobank of cervical pre-tumoroids and tumoroids that faithfully retains genomic and transcriptomic characteristics as well as the causative HPV genome is established. Cervical pre-tumoroids and tumoroids show differential responses to common chemotherapeutic agents and grow differently as xenografts in mice. By coculture organoid models with peripheral blood immune cells (PBMCs) stimulated by HPV antigenic peptides, it is illustrated that both organoid models respond differently to immunized PBMCs, supporting organoids as reliable and powerful tools for studying virus-specific T-cell responses and screening therapeutic HPV vaccines. In this study, a model of cervical pre-cancerous lesions containing HPV is established for the first time, overcoming the bottleneck of the current model of human cervical pre-cancerous lesions. This study establishes an experimental platform and biobanks for in vitro mechanistic research, therapeutic vaccine screening, and personalized treatment for HPV-related cervical diseases.

Hydrophobic surface induced pro-metastatic cancer cells for in vitro extravasation models

In vitro vascularized cancer models utilizing microfluidics have emerged as a promising tool for mechanism study and drug screening. However, the lack of consideration and preparation methods for cancer cellular sources that are capable of adequately replicating the metastatic features of circulating tumor cells contributed to low relevancy with in vivo experimental results. Here, we show that the properties of cancer cellular sources have a considerable impact on the validity of the in vitro metastasis model. Notably, with a hydrophobic surface, we can create highly metastatic spheroids equipped with aggressive invasion, endothelium adhesion capabilities, and activated metabolic features. Combining these metastatic spheroids with the well-constructed microfluidic-based extravasation model, we validate that these metastatic spheroids exhibited a distinct extravasation response to epidermal growth factor (EGF) and normal human lung fibroblasts compared to the 2D cultured cancer cells, which is consistent with the previously reported results of in vivo experiments. Furthermore, the applicability of the developed model as a therapeutic screening platform for cancer extravasation is validated through profiling and inhibition of cytokines. We believe this model incorporating hydrophobic surface-cultured 3D cancer cells provides reliable experimental data in a clear and concise manner, bridging the gap between the conventional in vitro models and in vivo experiments.

A light-activatable theranostic combination for ratiometric hypoxia imaging and oxygen-deprived drug activity enhancement

While performing oxygen-related tumour treatments such as chemotherapy and photodynamic therapy, real-time monitoring hypoxia of tumour is of great value and significance. Here, we design a theranostic combination for light-activated ratiometric hypoxia imaging, hypoxia modulating and prodrug activation. This combination consisted of an oxygen-sensitive near-infrared-emitting ratiometric phosphorescence probe and a hypoxia-activated prodrug-loaded covalent organic framework. In this combination, the probe plays two roles, including quantitative monitoring of oxygen concentration by ratiometric imaging and consuming the oxygen of tumour under light excitation by photodynamic therapy. Meanwhile, the enhanced hypoxia microenvironment of tumour can raise the cytotoxicity of prodrug loaded in covalent organic framework, resulting in boosting antitumour therapeutic effects in vivo. This theranostic combination can precisely provide therapeutic regime and screen hypoxia-activated prodrugs based on real-time tumour hypoxia level, offering a strategy to develop hypoxia mediated tumour theranostics with hypoxia targeted prodrugs.

Heart-on-a-chip systems: disease modeling and drug screening applications.

Cardiovascular disease (CVD) is the leading cause of death worldwide, casting a substantial economic footprint and burdening the global healthcare system. Historically, pre-clinical CVD modeling and therapeutic screening have been performed using animal models. Unfortunately, animal models oftentimes fail to adequately mimic human physiology, leading to a poor translation of therapeutics from pre-clinical trials to consumers. Even those that make it to market can be removed due to unforeseen side effects. As such, there exists a clinical, technological, and economical need for systems that faithfully capture human (patho)physiology for modeling CVD, assessing cardiotoxicity, and evaluating drug efficacy. Heart-on-a-chip (HoC) systems are a part of the broader organ-on-a-chip paradigm that leverages microfluidics, tissue engineering, microfabrication, electronics, and gene editing to create human-relevant models for studying disease, drug-induced side effects, and therapeutic efficacy. These compact systems can be capable of real-time measurements and on-demand characterization of tissue behavior and could revolutionize the drug development process. In this review, we highlight the key components that comprise a HoC system followed by a review of contemporary reports of their use in disease modeling, drug toxicity and efficacy assessment, and as part of multi-organ-on-a-chip platforms. We also discuss future perspectives and challenges facing the field, including a discussion on the role that standardization is expected to play in accelerating the widespread adoption of these platforms.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) for modeling cardiac arrhythmias: strengths, challenges and potential solutions.

Ion channels and cytoskeletal proteins in the cardiac dyad play a critical role in maintaining excitation-contraction (E-C) coupling and provide cardiac homeostasis. Functional changes in these dyad proteins, whether induced by genetic, epigenetic, metabolic, therapeutic, or environmental factors, can disrupt normal cardiac electrophysiology, leading to abnormal E-C coupling and arrhythmias. Animal models and heterologous cell cultures provide platforms to elucidate the pathogenesis of arrhythmias for basic cardiac research; however, these traditional systems do not truly reflect human cardiac electro-pathophysiology. Notably, patients with the same genetic variants of inherited channelopathies (ICC) often exhibit incomplete penetrance and variable expressivity which underscores the need to establish patient-specific disease models to comprehend the mechanistic pathways of arrhythmias and determine personalized therapies. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) inherit the genetic background of the patient and reflect the electrophysiological characteristics of the native cardiomyocytes. Thus, iPSC-CMs provide an innovative and translational pivotal platform in cardiac disease modeling and therapeutic screening. In this review, we will examine how patient-specific iPSC-CMs historically evolved to model arrhythmia syndromes in a dish, and their utility in understanding the role of specific ion channels and their functional characteristics in causing arrhythmias. We will also examine how CRISPR/Cas9 have enabled the establishment of patient-independent and variant-induced iPSC-CMs-based arrhythmia models. Next, we will examine the limitations of using human iPSC-CMs with respect to in vitro arrhythmia modeling that stems from variations in iPSCs or toxicity due to gene editing on iPSC or iPSC-CMs and explore how such hurdles are being addressed. Importantly, we will also discuss how novel 3D iPSC-CM models can better capture in vitro characteristics and how all-optical platforms provide non-invasive and high- throughput electrophysiological data that is useful for stratification of emerging arrhythmogenic variants and drug discovery. Finally, we will examine strategies to improve iPSC-CM maturity, including powerful gene editing and optogenetic tools that can introduce/modify specific ion channels in iPSC-CMs and tailor cellular and functional characteristics. We anticipate that an elegant synergy of iPSCs, novel gene editing, 3D- culture models, and all-optical platforms will offer a high-throughput template to faithfully recapitulate in vitro arrhythmogenic events necessary for personalized arrhythmia monitoring and drug screening process.

Engineered Heart Tissues for Standard 96-Well Tissue Culture Plates.

Engineered heart tissues (EHTs) have been shown to be a valuable platform for disease investigation and therapeutic testing by increasing human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) maturity and better recreating the native cardiac environment. The protocol detailed in this chapter describes the generation of miniaturized EHTs (mEHTs) incorporating hiPSC-CMs and human stromal cells in a fibrin hydrogel. This platform utilizes an array of silicone posts designed to fit in a standard 96-well tissue culture plate. Stromal cells and hiPSC-CMs are cast in a fibrin matrix suspended between two silicone posts, forming an mEHT that produces synchronous muscle contractions. The platform presented here has the potential to be used for high throughput characterization and screening of disease phenotypes and novel therapeutics through measurements of the myocardial function, including contractile force and calcium handling, and its compatibility withimmunostaining.

Animal models of Dry Eye Disease: Application to Drug Discovery

Dry eye disease (DED), a multifactorial disorder of the ocular surface and tear film, affects 5-50% of the global population. Currently, no satisfactory treatments of DED exist. Ongoing efforts to identify novel therapeutic agents are handicapped by the limitations of its preclinical animal models, which to some extent reflect the pathophysiological complexities of DED.. A plethora of DED models employing multiple animal species (mice, rats, cats, rabbits, dogs, and non-human primates) has been reported, each aiming to capture components of DED that appear to determine its pathophysiology and response to novel treatments. Here, we review the main animal models of DED and attempt to place each in the context of drug discovery. We also discuss a nascent method for ex vivo culture of human conjunctival cells that may abbreviate early screening of candidate therapeutics. Despite the remaining challenges, there is justified optimism that with the contribution of these preclinical models, the development of an efficacious and safe treatment of DED will be forthcoming.

Embedded 3D Bioprinting for Engineering Miniaturized In Vitro Tumor Models.

Embedded extrusion 3D bioprinting is a rapidly emerging additive manufacturing methodology that provides a precise spatial deposition of synthetic or natural-origin low-viscosity bioinks during the extrusionprinting process. Such a strategy has to date unlocked the freeform extrusion biofabrication of complex micro-to-macro-scale living architectures for numerous applications, including tissue engineering and in vitro disease modeling. In this chapter, we describe a suspension bioprinting methodology leveraging a continuous viscoelastic biopolymer supporting bath functionalized with divalent calcium cations to enable a rapid processing of user-defined bioinks toward architecturally complex 3D in vitro tumor models. This highly simple and cost-effective viscoelasticsupporting bath enables a full freeform biofabrication of cell-laden 3D tumor-mimetic architectures that exhibit structural stability in culture post-printing. The cytocompatibility of the supporting bath, its ease of removal from biofabricated living constructs, and its adaptability for processing different ECM-mimetic bioinks open avenues for multi-scale fabrication of numerous types of physiomimetic 3D tumor models for preclinical screening of candidate therapeutics.

ROS1 kinase inhibition reimagined: identifying repurposed drug via virtual screening and molecular dynamics simulations for cancer therapeutics.

Precision medicine has revolutionized modern cancer therapeutic management by targeting specific molecular aberrations responsible for the onset and progression of tumorigenesis. ROS proto-oncogene 1 (ROS1) is a receptor tyrosine kinase (RTK) that can induce tumorigenesis through various signaling pathways, such as cell proliferation, survival, migration, and metastasis. It has emerged as a promising therapeutic target in various cancer types. However, there is very limited availability of specific ROS1 inhibitors for therapeutic purposes. Exploring repurposed drugs for rapid and effective treatment is a useful approach. In this study, we utilized an integrated approach of virtual screening and molecular dynamics (MD) simulations of repurposing existing drugs for ROS1 kinase inhibition. Using a curated library of 3648 FDA-approved drugs, virtual screening identified drugs capable of binding to ROS1 kinase domain. The results unveil two hits, Midostaurin and Alectinib with favorable binding profiles and stable interactions with the active site residues of ROS1. These hits were subjected to stability assessment through all-atom MD simulations for 200 ns. MD results showed that Midostaurin and Alectinib were stable with ROS1. Taken together, the study showed a rational framework for the selection of repurposed Midostaurin and Alectinib with ROS1 inhibitory potential for therapeutic management after further validation.

Cleanroom-Free Microfluidic Device for Natural Induction of Hypoxia in 2D and 3D Tumor Models.

Hypoxia is a common and critical feature of solid tumors that contributes to the plasticity and heterogeneity of the cancer cells. Cancer cell populations take on a region-specific adaptation induced by hypoxia, and each cancer cell population will show different levels of sensitivity and resistance to cancer therapeutics. Therefore, a faithful recapitulation of tumor hypoxia that allows for accurate assessments of hypoxia-induced adaptations, heterogeneity, and response to therapy is needed to develop new therapeutic approaches. The existing hypoxic tumor models rely on complex fabrication methods and external gas sources that make them unfavorable for the early-stage screening of new therapeutics. Here, we demonstrate how to establish a cleanroom-free microfluidic device that supports both 2D and 3D hypoxic tumor modeling through natural cancer cell metabolism and confirm the induction of the hypoxic gradient.

Advancing cancer therapeutics: Integrating scalable 3D cancer models, extracellular vesicles, and omics for enhanced therapy efficacy.

Cancer remains as one of the highest challenges to human health. However, anticancer drugs exhibit one of the highest attrition rates compared to other therapeutic interventions. In part, this can be attributed to a prevalent use of in vitro models with limited recapitulative potential of the in vivo settings. Three dimensional (3D) models, such as tumor spheroids and organoids, offer many research opportunities to address the urgent need in developing models capable to more accurately mimic cancer biology and drug resistance profiles. However, their wide adoption in high-throughput pre-clinical studies is dependent on scalable manufacturing to support large-scale therapeutic drug screenings and multi-omic approaches for their comprehensive cellular and molecular characterization. Extracellular vesicles (EVs), which have been emerging as promising drug delivery systems (DDS), stand to significantly benefit from such screenings conducted in realistic cancer models. Furthermore, the integration of these nanomedicines with 3D cancer models and omics profiling holds the potential to deepen our understanding of EV-mediated anticancer effects. In this chapter, we provide an overview of the existing 3D models used in cancer research, namely spheroids and organoids, the innovations in their scalable production and discuss how omics can facilitate the implementation of these models at different stages of drug testing. We also explore how EVs can advance drug delivery in cancer therapies and how the synergy between 3D cancer models and omics approaches can benefit in this process.

Advances in 3D tissue models for neural engineering: self-assembled versus engineered tissue models.

Neural tissue engineering has emerged as a promising field that aims to create functional neural tissue for therapeutic applications, drug screening, and disease modelling. It is becoming evident in the literature that this goal requires development of three-dimensional (3D) constructs that can mimic the complex microenvironment of native neural tissue, including its biochemical, mechanical, physical, and electrical properties. These 3D models can be broadly classified as self-assembled models, which include spheroids, organoids, and assembloids, and engineered models, such as those based on decellularized or polymeric scaffolds. Self-assembled models offer advantages such as the ability to recapitulate neural development and disease processes in vitro, and the capacity to study the behaviour and interactions of different cell types in a more realistic environment. However, self-assembled constructs have limitations such as lack of standardised protocols, inability to control the cellular microenvironment, difficulty in controlling structural characteristics, reproducibility, scalability, and lengthy developmental timeframes. Integrating biomimetic materials and advanced manufacturing approaches to present cells with relevant biochemical, mechanical, physical, and electrical cues in a controlled tissue architecture requires alternate engineering approaches. Engineered scaffolds, and specifically 3D hydrogel-based constructs, have desirable properties, lower cost, higher reproducibility, long-term stability, and they can be rapidly tailored to mimic the native microenvironment and structure. This review explores 3D models in neural tissue engineering, with a particular focus on analysing the benefits and limitations of self-assembled organoids compared with hydrogel-based engineered 3D models. Moreover, this paper will focus on hydrogel based engineered models and probe their biomaterial components, tuneable properties, and fabrication techniques that allow them to mimic native neural tissue structures and environment. Finally, the current challenges and future research prospects of 3D neural models for both self-assembled and engineered models in neural tissue engineering will be discussed.