Drug Screening Platform Research Articles

DESIGN AND DEVELOPMENT OF A CUSTOM PLATFORM TO GROW AND CHARACTERIZE OSTEOSARCOMA SPHEROIDS

Osteosarcoma is a highly malignant primary tumor of bone tissue. The 5-year survival rate of patients with metastasis is below 20% and this scenario is unchanged in the last two decades, despite great efforts in pre-clinical and clinical research. Traditional preclinical models of osteosarcoma do not consider the whole complexity of its microenvironment, leading to poor correlation between in vitro/in vivo results and clinical outcomes. Spheroids are a promising in vitro model to mimic osteosarcoma and perform drug-screening tests, as they (i) reproduce the microarchitecture of the tumor, (ii) are characterized by hypoxic regions and necrotic core as the in vivo tumor, (iii) and recapitulate the chemo-resistance phenomena. However, to date, the spheroid model is scarcely used in osteosarcoma research.Our aim is to develop a customized culture dish to grow and characterize spheroids and to perform advanced drug-screening tests. The resulting platform must be adapted to automated image acquisition systems, to overcome the drawbacks of commercial spheroids platforms.To this purpose, we designed and developed a micro-patterned culture dish by casting agarose on a 3D printed mold from a CAD design. We successfully obtained viable and reproducible homotypic osteosarcoma spheroids, with two different cells lines from osteosarcoma (i.e., 143b and MG-63). Using the platform, we performed viability assays and live fluorescent stainings (e.g., Calcein AM) with low reagent consumption. Moreover, the culture dish was validated as drug screening platform, administrating Doxorubicin at different doses, and evaluating its effect on OS spheroids, in terms of morphology and viability. This platform can be considered an attractive alternative to the highly expensive commercial spheroid platforms to obtain homogeneous and reproducible spheroids in a high-throughput and cost effective mode.

Application of biomaterial-based three-dimensional bioprinting for organ-on-a-chip fabrication

An organ-on-a-chip is a microfluidic device that simulates the microenvironment of organs, facilitating the study of human physiology and disease mechanisms. Through the integration of tissue engineering and micromachining technologies, it effectively manages the cellular microenvironment and implements tissue-specific functions and physiological responses with high fidelity. Several factors must be appropriately considered in the fabrication of an organ-on-a-chip, including the choice of biomaterials to simulate the extracellular matrix (ECM), selection of cells constituting the target organ, incorporation of humanized design to realize the primary function and structure of the organ, and the use of appropriate biofabrication methods to build a tissue-specific environment. Notably, three-dimensional (3D) bioprinting has emerged as a promising method for biofabricating organ-on-a-chip. Three-dimensional bioprinting offers versatility in adapting to various biomaterials with different physical properties, allowing precise control of 3D cell arrays and facilitating cyclic movements of fluidic flow within microfluidic platforms. These capabilities enable the precise fabrication of organ-on-a-chip that reflects tissue-specific functions and microenvironments. Additionally, 3D-bioprinted organ-on-a-chip can serve as a disease-on-a-chip platform, achieved through the implementation of pathophysiological environments and integration with devices such as bioreactors. Their significance in pharmacology research lies in their exceptional resemblance to the 3D microenvironment structure of actual organs, which are conducive for the validation of sequential mechanism of drug action. This review describes recent examples of organ-on-a-chip applications for various organs and state-of-the-art 3D bioprinting techniques employed in organ-on-a-chip fabrication. The discussion extends to the future prospects of this technology, encompassing aspects such as commercialization through mass production and its potential application in personalized medicine or drug-screening platforms. Serving as a relevant guide, this review offers insights for future research and developments in in vitro micromodel fabrication.

Droplet-based bioprinting for fabrication of tumor spheroids

Cancer is now one of the leading causes of mortality worldwide, and the cancer treatment development is still slow due to the lack of efficient in vitro tumor models for studying tumorigenesis and facilitating drug development. Multicellular tumor spheroids can recapitulate the critical properties of tumors in vivo, including spatial organization, physiological responses, and metabolism, and are considered powerful platform for disease study and drug screening. Although several spheroid fabrication methods have been developed, most of them result in uncontrolled cell aggregations, yielding spheroids of variable size and function. Droplet-based bioprinting is capable of depositing cells in spatiotemporal manner so as to control the composition and distribution of printed biological constructs, thereby facilitating high-throughput fabrication of complicated and reproducible tumor spheroids. In this review, we introduce the progress of droplet-based bioprinting technology for the fabrication of tumor spheroids. First, different droplet-based bioprinting technologies are compared in terms of their strengths and shortcomings, which should be taken into account while fabricating tumor spheroids. Second, the latest advances in modeling distinct types of cancers and the enabled applications with tumor spheroids are summarized. Finally, we discuss the challenges and potentials revolving around the advances of bioprinting technology, improvement of spheroid quality, and integration of different technologies.

DEVELOPMENT OF A BONE-ON-A-CHIP MICROFLUIDIC DEVICE AS A MODEL OF OSTEOPOROSIS

Osteoporosis is a progressive, chronic disease of bone metabolism, characterized by decreased bone mass and mineral density, predisposing individuals to an increased risk of fractures. The use of animal models, which is the gold standard for the screening of anti-osteoporosis drugs, raises numerous ethical concerns and is highly debated because the composition and structure of animal bones is very different from human bones. In addition, there is currently a poor translation of pre-clinical efficacy in animal models to human trials, meaning that there is a need for an alternative method of screening and evaluating new therapeutics for metabolic bone disorders, in vitro.The aim of this project is to develop a 3D Bone-On-A-Chip that summarizes the spatial orientation and mutual influences of the key cellular components of bone tissue, in a citrate and hydroxyapatite-enriched 3D matrix, acting as a 3D model of osteoporosis. To this purpose, a polydimethylsiloxane microfluidic device was developed by CAD modelling, stereolithography and replica molding. The device is composed by two layers: (i) a bottom layer for a 3D culture of osteocytes embedded in an osteomimetic collagen-enriched matrigel matrix with citrate-doped hydroxyapatite nanocrystals, and (ii) a upper layer for a 2D perfused co-culture of osteoblasts and osteoclasts seeded on a microporous PET membrane.Cell vitality was evaluated via live/dead assay. Bone deposition and bone resorption was analysed respectively with ALP, Alizarin RED and TRACP staining. Osteocytes dendrite expression was evaluated via immunofluorescence. Subsequently, the model was validated as drug screening platform inducing osteocytes apoptosis and administrating standard anti-osteoporotic drugs.This device has the potential to substitute or minimize animal models in pre-clinical studies of osteoporosis, contributing to pave the way for a more precise and punctual personalized treatment.

Gastrointestinal tract organoids as novel tools in drug discovery.

Organoids, characterized by their high physiological attributes, effectively preserve the genetic characteristics, physiological structure, and function of the simulated organs. Since the inception of small intestine organoids, other organoids for organs including the liver, lungs, stomach, and pancreas have subsequently been developed. However, a comprehensive summary and discussion of research findings on gastrointestinal tract (GIT) organoids as disease models and drug screening platforms is currently lacking. Herein, in this review, we address diseases related to GIT organoid simulation and highlight the notable advancements that have been made in drug screening and pharmacokinetics, as well as in disease research and treatment using GIT organoids. Organoids of GIT diseases, including inflammatory bowel disease, irritable bowel syndrome, necrotizing enterocolitis, and Helicobacter pylori infection, have been successfully constructed. These models have facilitated the study of the mechanisms and effects of various drugs, such as metformin, Schisandrin C, and prednisolone, in these diseases. Furthermore, GIT organoids have been used to investigate viruses that elicit GIT reactions, including Norovirus, SARS-CoV-2, and rotavirus. Previous studies by using GIT organoids have shown that dasabuvir, gemcitabine, and imatinib possess the capability to inhibit viral replication. Notably, GIT organoids can mimic GIT responses to therapeutic drugs at the onset of disease. The GIT toxicities of compounds like gefitinib, doxorubicin, and sunset yellow have also been evaluated. Additionally, these organoids are instrumental for the study of immune regulation, post-radiation intestinal epithelial repair, treatment for cystic fibrosis and diabetes, the development of novel drug delivery systems, and research into the GIT microbiome. The recent use of conditioned media as a culture method for replacing recombinant hepatocyte growth factor has significantly reduced the cost associated with human GIT organoid culture. This advancement paves the way for large-scale culture and compound screening of GIT organoids. Despite the ongoing challenges in GIT organoid development (e.g., their inability to exist in pairs, limited cell types, and singular drug exposure mode), these organoids hold considerable potential for drug screening. The use of GIT organoids in this context holds great promises to enhance the precision of medical treatments for patients living with GIT diseases.

Development of a Three-Dimensional Multicellular Model of Human Neuroblastoma Using Matrigel as an Extracellular Matrix Analogue.

Neuroblastoma, the most prevalent extracranial solid tumor in children, poses therapeutic challenges due to its variable clinical course and propensity for metastasis. Despite advances in treatment strategies like chemotherapy, drug resistance remains a significant concern, highlighting the need for improved models to study tumor behavior and drug responses. This chapter proposes the development of a three-dimensional multicellular model of human neuroblastoma using Matrigel as an ECM analogue. Such models aim to replicate the complexity of the tumor microenvironment, providing valuable insights into tumor progression and drug resistance mechanisms. By recapitulating key features of neuroblastoma within a physiologically relevant context, these models offer a platform for preclinical drug screening and therapeutic development.

Engineering vascularised organoid-on-a-chip: strategies, advances and future perspectives.

In recent years, advances in microfabrication technology and tissue engineering have propelled the development of a novel drug screening and disease modelling platform known as organoid-on-a-chip. This platform integrates organoids and organ-on-a-chip technologies, emerging as a promising approach for in vitro modelling of human organ physiology. Organoid-on-a-chip devices leverage microfluidic systems to simulate the physiological microenvironment of specific organs, offering a more dynamic and flexible setting that can mimic a more comprehensive human biological context. However, the lack of functional vasculature has remained a significant challenge in this technology. Vascularisation is crucial for the long-term culture and in vitro modelling of organoids, holding important implications for drug development and personalised medical approaches. This review provides an overview of research progress in developing vascularised organoid-on-a-chip models, addressing methods for in vitro vascularisation and advancements in vascularised organoids. The aim is to serve as a reference for future endeavors in constructing fully functional vascularised organoid-on-a-chip platforms.

Establishment of an in Vitro Three-Dimensional Vascularized Micro-Tumor Model and Screening of Chemotherapeutic Drugs.

Breast cancer is the most common malignancy in women worldwide, and major challenges in its treatment include drug resistance and metastasis. Three-dimensional cell culture systems have received widespread attention in drug discovery studies but existing models have limitations, that warrant the development of a simple and repeatable three-dimensional culture model for high-throughput screening. In this study, we designed a simple, reproducible, and highly efficient microencapsulated device to co-culture MCF-7 cells and HUVECs in microcapsules to establish an in vitro vascularized micro-tumor model for chemotherapeutic drug screening. First, to construct a three-dimensional micro-tumor model, cell encapsulation devices were created using three different sizes of flat-mouthed needles. Immunohistochemistry and immunofluorescence assays were conducted to determine vascular lumen formation. Cell proliferation was detected using the Cell Counting Kit-8 assay. Finally, to observe the drug reactions between the models, anticancer drugs (doxorubicin or paclitaxel) were added 12 h after the two-dimensional cultured cells were plated or 7 days after cell growth in the core-shell microcapsules. Vascularized micro-tumors were obtained after 14 days of three-dimensional culture. The proliferation rate in the three-dimensional cultured cells was slower than that of two-dimensional cultured cells. Three-dimensional cultured cells were more resistant to anticancer drugs than two-dimensional cultured cells. This novel sample encapsulation device formed core-shell microcapsules and can be used to successfully construct 3D vascularized micro-tumors in vitro. The three-dimensional culture model may provide a platform for drug screening and is valuable for studying tumor development and angiogenesis.

Embedded bioprinted multicellular spheroids modeling pancreatic cancer bioarchitecture towards advanced drug therapy.

The desmoplastic bioarchitecture and microenvironment caused by fibroblasts have been confirmed to be closely related to the drug response behavior of pancreatic ductal adenocarcinoma (PDAC). Despite the extensive progress in developing PDAC models as in vitro drug screening platforms, developing efficient and controllable approaches for the construction of physiologically relevant models remains challenging. In the current study, multicellular spheroid models that emulate pancreatic cancer bioarchitecture and the desmoplastic microenvironment are bioengineered. An extrusion-based embedded dot bioprinting strategy was established to fabricate PDAC spheroids in a one-step process. Cell-laden hydrogel beads were directly deposited into a methacrylated gelatin (GelMA) suspension bath to generate spherical multicellular aggregates (SMAs), which further progressed into dense spheroids through in situ self assembly. By modulating the printing parameters, SMAs, even from multiple cell components, could be manipulated with tunable size and flexible location, achieving tunable spheroid patterns within the hydrogel bath with reproducible morphological features. To demonstrate the feasibility of this printing strategy, we fabricated desmoplastic PDAC spheroids by printing SMAs consisting of tumor cells and fibroblasts within the GelMA matrix bath. The produced hybrid spheroids were further exposed to different concentrations of the drug gemcitabine to verify their potential for use in cell therapy. Beyond providing a robust and facile bioprinting system that enables desmoplastic PDAC bioarchitecture bioengineering, this work introduces an approach for the scalable, flexible and rapid fabrication of cell spheroids or multi-cell-type spheroid patterns as platforms for advanced drug therapy or disease mechanism exploration.

PINK1‐mitophagy activator (PA‐01) overcomes mitochondrial hyperfusion conferring retinal neuroprotection in type‐1 diabetes

Aims/Purpose: We hypothesize that exacerbated mitochondrial fusion abrogates mitophagy in diabetic retinopathy [DR]. Our aim was to rescue this process pharmacologically to prevent vision loss in a mouse model of type‐1 diabetes.Methods: We generated a drug screening platform mimicking mitochondrial hyperfusion in DR. To this end, retinal Müller cells (mouse primary and MIO‐M1 cell line) were antagonized for mitochondrial fission using a Drp1 inhibitor peptide under elevated glucose (30.5 mM) conditions. Pharmacological ‘hits’ rescuing mitophagy in conditions of diabetes‐induced hyperfusion were assessed for their capability to improve mitochondrial health (membrane potential [ΔΨm]) and bioenergetics (Seahorse). The bio‐activity of lead compounds were corroborated in vivo using diabetic mitophagy reporter mice (Mito‐QC Ins2Akita), while effectiveness to alleviate retinal degeneration was evaluated via electroretinography (ERG), retinal thickness (SD‐OCT) and morphometric analysis of retinal neurons.Results: Among all drugs screened, we identified PA‐01 (a PINK1‐mitophagy activator) as capable of safely rescuing mitophagy under conditions of diabetes‐induced mitochondrial hyperfusion. Accordingly, PA‐01 rescued mitochondrial health (ΔΨm) and bioenergetics in Müller glia cultures, by optimizing metabolic flux (basal‐ and ATP‐linked respiration). The bio‐activity of PA‐01 was demonstrated in vivo, where long‐term oral administration rescued mitochondrial hyperfusion and mitophagy in the diabetic retina, while further preventing neurodegeneration. This was reflected in i) improved scotopic ERG responses (increased a‐wave and b‐wave amplitudes), ii) improved SD‐OCT retinal thickness and iii) neuroprotection to photoreceptors, synaptic terminals and amacrine cells.Conclusions: Exacerbated mitochondrial fusion contributes to DR pathology by inhibiting mitophagy in Müller glia. Rescuing this process pharmacologically holds promising therapeutic potential to alleviate vision loss in DR.

Three-dimensional in vitro model of bone metastases of neuroblastoma as a tool for pharmacological evaluations.

In vitro metastatic models are foreseen to introduce a breakthrough in the field of preclinical screening of more functional small-molecule pharmaceuticals and biologics. To achieve this goal, the complexity of current in vitro systems requests an appropriate upgrade to approach the three-dimensional (3D) in vivo metastatic disease. Here, we explored the potential of our 3D β-tricalcium phosphate (β-TCP) model of neuroblastoma bone metastasis for drug toxicity assessment. Tailor-made scaffolds with interconnected channels were produced by combining 3D printing and slip casting method. The organization of neuroblastoma cells into a mesenchymal stromal cell (MSC) network, cultured under bioactive conditions provided by β-TCP, was monitored by two-photon microscopy. Deposition of extracellular matrix protein Collagen I by MSCs and persistent growth of tumor cells confirmed the cell-supportive performance of our 3D model. When different neuroblastoma cells were treated with conventional chemotherapeutics, the β-TCP model provided the necessary reproducibility and accuracy of experimental readouts. Drug efficacy evaluation was done for 3D and 2D cell cultures, highlighting the need for a higher dose of chemotherapeutics under 3D conditions to achieve the expected cytotoxicity in tumor cells. Our results confirm the importance of 3D geometry in driving native connectivity between nonmalignant and tumor cells and sustain β-TCP scaffolds as a reliable and affordable drug screening platform for use in the early stages of drug discovery.

Multifunctional cardiac microphysiological system based on transparent ITO electrodes for simultaneous optical measurement and electrical signal monitoring.

Drug-induced cardiotoxicity is a significant contributor to drug recalls, primarily attributed to limitations in existing drug screening platforms. Traditional heart-on-a-chip platforms often employ metallic electrodes to record cardiomyocyte electrical signals. However, this approach hinders direct cardiomyocyte morphology observation and typically yields limited functionality. Consequently, this limitation may lead to an incomplete understanding of cardiomyocyte characteristics. To address these challenges, we introduce a multifunctional cardiac microphysiological system featuring transparent indium tin oxide electrodes. This innovative design aims to overcome the limitations of conventional heart-on-a-chip systems where metal electrodes interfere with the observation of cells and increase the difficulty of subsequent image processing of cell images. In addition to facilitating optical measurement combined with image processing capabilities, this system integrates a range of electrodes with diverse functionalities. These electrodes can realize cellular electrical stimulation, field potential monitoring, and impedance change tracking, enabling a comprehensive investigation of various cardiomyocyte traits. To demonstrate its versatility, we investigate the effects of four cardiac drugs with distinct pharmacological profiles on cardiomyocytes using this system. This platform provides a means for quantitatively and predictively assessing cardiac toxicity, which could be applied to conduct a comprehensive evaluation during the drug discovery process.

Humanized mouse models: A valuable platform for preclinical evaluation of human cancer.

Animal models are routinely employed to assess the treatments for human cancer. However, due to significant differences in genetic backgrounds, traditional animal models are unable to meet bioresearch needs. To overcome this restriction, researchers have generated and optimized immunodeficient mice, and then engrafted human genes, cells, tissues, or organs in mice so that the responses in the model mice could provide a more reliable reference for treatments. As a bridge connecting clinical application and basic research, humanized mice are increasingly used in the preclinical evaluation of cancer treatments, particularly after gene interleukin 2 receptor gamma mutant mice were generated. Human cancer models established in humanized mice support exploration of the mechanism of cancer occurrence and provide an efficient platform for drug screening. However, it is undeniable that the further application of humanized mice still faces multiple challenges. This review summarizes the construction approaches for humanized mice and their existing limitations. We also report the latest applications of humanized mice in preclinical evaluation for the treatment of cancer and point out directions for future optimization of these models.

A pancreatic cancer organoid platform identifies an inhibitor specific to mutant KRAS

KRAS mutations, mainly G12D and G12V, are found in more than 90% of pancreatic ductal adenocarcinoma (PDAC) cases. The success of drugs targeting KRASG12C suggests the potential for drugs specifically targeting these alternative PDAC-associated KRAS mutations. Here, we report a high-throughput drug-screening platform using a series of isogenic murine pancreatic organoids that are wild type (WT) or contain common PDAC driver mutations, representing both classical and basal PDAC phenotypes. We screened over 6,000 compounds and identified perhexiline maleate, which can inhibit the growth and induce cell death of pancreatic organoids carrying the KrasG12D mutation both invitro and invivo and primary human PDAC organoids. scRNA-seq analysis suggests that the cholesterol synthesis pathway is upregulated specifically in the KRAS mutant organoids, including the key cholesterol synthesis regulator SREBP2. Perhexiline maleate decreases SREBP2 expression levels and reverses the KRAS mutant-induced upregulation of the cholesterol synthesis pathway.

Combining fibril-induced alpha-synuclein aggregation and 6-hydroxydopamine in a mouse model of Parkinson's disease and the effect of cerebral dopamine neurotrophic factor on the induced neurodegeneration.

The existent pre-clinical models of Parkinson's disease do not simultaneously recapitulate severe degeneration of dopamine neurons and the occurrence of alpha-synuclein (aSyn) aggregation in one study system. In this study, we injected aSyn pre-formed fibrils (PFF) and 6-hydroxydopamine (6-OHDA) unilaterally into the striatum of C57BL/6 wild-type male mice at an interval of 2 weeks to induce aggregation of aSyn protein and trigger the loss of dopamine neurons simultaneously in one model and studied the behavioural effects of the combination in these mice. 6-OHDA was tested at three different doses, and 2μg of 6-OHDA combined with PFF-induced aSyn aggregation was found to produce the most optimal disease phenotype. At 14 weeks timepoint, mice injected with a combination of PFF and 6-OHDA sustained significant damage to the nigrostriatal pathway and exhibited aSyn-positive aggregation. Our data suggest that the neurons that formed large aSyn aggregates were particularly vulnerable to 6-OHDA-induced degeneration. We also demonstrate the manifestation of a relatively aggressive pathology in 2- to 4-month-old mice, as compared to younger 7- to 9-week-old ones. Furthermore, cerebral dopamine neurotrophic factor (CDNF) administered intrastriatally rescued dopamine neurons and motor behaviour of the animals to some extent from 6-OHDA toxicity. However, no such effect could be seen in the novel 6-OHDA + PFFs combination model. For the first time, we demonstrate the combined effect of PFF and 6-OHDA simultaneously in one model. We further discuss the scope for further optimizing this combination model to develop it as a promising pre-clinical platform for drug screening and development.

Abstract PR016: Largescale organoid panel drug screening to short-track clinically-relevant output

Abstract Introduction Patient-derived organoids (PDOs) have emerged over the last decade as representative in vitro replicas of tumor biology, bringing increased accuracy in predicting clinical outcomes based on pre-clinical results. Large and deeply characterized organoid biobanks, covering broad varieties in indications and genetic backgrounds, have been generated that allow large scale (combination) drug testing, reliable biomarker predictions, and improved drug repurposing and patient stratification studies. However, relatively long timelines and high expenses hold the organoids back to live up to their promise. To overcome these hurdles, we have optimized processes, and here present a largescale organoid panel drug screening platform consisting of over 50 organoid models that predicts drug responses across at least seven indications in a robust, but also fast and cost-efficient manner. Methods Colorectal, breast, lung, pancreatic, ovarian, cervix and melanoma organoid models were selected for subtype and driver mutation variety, banked in large batches, and preserved in an assay-ready format to reduce lead-in time and organoid expansion phase. Drug responses of the full organoid panel were characterized with standard of care compounds (cisplatin, gemcitabine and paclitaxel) and AMG510 targeted therapy (KRAS G12C inhibitor, sotorasib) by measuring ATP levels after a 5-day exposure using a 9-dose curve. Experiments were executed using automated liquid handling equipment to standardize procedures and increase consistency. Control variability (CV), Z-factors and assay windows were calculated to assess assay performance, and IC50 values and area under the curves (AUC) were calculated to express drug sensitivity. Experiments were repeated in multiple labs to test for assay robustness. Results The largescale panel screening platform exhibits a high average assay performance, with high Z-factors (>0.6), low intra- and interplate variability (CV <15%) and reproducible IC50 outputs. Data report of the full panel was prepared within 6 weeks of project kick-off; shortening timelines compared to regular organoid screens over 6-fold, from months to weeks. Using IC50 values and AUC, drug sensitivity testing distinguished sensitive from partially and insensitive models, and models with specific genetic features (KRAS G12C vs G12X) behaved as expected when a AMG510 therapy was tested. Conclusion and Discussion Assay-ready organoid technology-based largescale panel screening as presented here has resulted in efficient timeline and cost reductions and will therefore further harness the large potential of PDO technology in the drug development pipeline. Largescale organoid panel screening enables identification of drug synergy by testing drug combinations. With the majority of included models also being available as PDX models, it will facilitate a seamless translation into subsequent in vivo studies. Moreover, combining panel screens with advanced biomarker analysis will allow early patient stratification, further aiding clinical development of novel oncology drugs. Citation Format: Liza Wijler, Annelot Staes, Linda van Seeters, Lama Alhaj Hasan, Bram Herpers, Leo Price, Mariusz Madej, Michiel Fokkelman, Xiaoxi Xu, Marrit Putker. Largescale organoid panel drug screening to short-track clinically-relevant output [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR016.

Abstract PR004: PRISM high-throughput screening of antibody-drug conjugates uncovers clinically relevant targets

Abstract Antibody-drug conjugates (ADCs) are a rapidly emerging class of cancer therapeutics composed of an antibody scaffold, a cytotoxic payload, and a joining linker. They exhibit properties of both biologics and small molecule inhibitors, can exert activity through multiple mechanisms, and some are capable of inducing target-independent cell killing via a phenomenon known as the bystander effect. Due to this inherent structural and biological complexity, cell-based functional screening is an essential component of ADC development. However, the majority of preclinical in vitro studies for ADCs are limited to a small number of cell lines and thus provide minimal insights into their activity. High-throughput drug-screening platforms, such as PRISM (Profiling Relative Inhibition Simultaneously in Mixtures), that have been highly effective in characterizing small molecule inhibitors could facilitate characterization of ADCs at an unprecedented scale and provide more comprehensive insights into ADC mechanisms, target specificity, and potency. To test the suitability of the PRISM platform for ADC screening, we investigated several ADCs conjugated to the HER2-targeting antibody trastuzumab that differed in terms of linker type (cleavable and non-cleavable), payload class, and drug-to-antibody ratio. ADCs were screened in parallel with controls including unconjugated antibodies and free payloads across a panel of nearly 500 cell lines consisting of diverse cell lineages. We explored whether ADC selectivity was associated with target antigen expression and found a clear association between cell line sensitivity and ERBB2 (HER2) gene expression, with the strongest effects on viability observed in a subset of cell lines overexpressing ERBB2. Univariate biomarker analysis identified ERBB2 gene expression as a significantly correlated feature of sensitivity to all HER2-targeting ADCs. Notably, this correlation was retained across ADCs with payloads capable of exerting bystander killing of cells with low ERBB2 expression. Upon further comparison of biomarker profiles across different ADCs, established relationships between payload resistance and ABCB1 efflux pumps emerged for a subset of ADC payloads. These results indicate that PRISM pooled cell line screening is able to distinguish ADCs with different payload characteristics while maintaining selectivity of the primary target. Overall, our study highlights how PRISM can be leveraged to analyze ADC activity and uncover clinically relevant targets. This approach can aid in evaluating potential ADC candidates by providing a better understanding of ADC mechanisms of action and how their unique structural components drive cytotoxic activity. Citation Format: Jillian N Eskra, Ellen Nguyen, Aydin Golabi, Shiker Nair, Antonella Masciotti, Anthony Fazio, Mustafa Kocak, Melissa Ronan, Matthew G Rees, Jennifer A Roth. PRISM high-throughput screening of antibody-drug conjugates uncovers clinically relevant targets [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR004.

Decoy peptides effectively inhibit the binding of SARS-CoV-2 to ACE2 on oral epithelial cells

The entry of SARS-CoV-2 into host cells involves the interaction between the viral spike protein and the human angiotensin-converting enzyme 2 (ACE2) receptor. Given that the spike protein evolves rapidly to evade host immunity, therapeutics that block ACE2 accessibility, such as spike decoys, could serve as an alternative strategy for attenuating viral infection. Here, we constructed a drug screening platform based on oral epithelial cells to rapidly identify peptides or compounds capable of blocking the spike-ACE2 interaction. We engineered short decoy peptides, 8 to 14 amino acids in length, using the spike protein's receptor-binding motif (RBM) and demonstrated that these peptides can effectively inhibit virus attachment to host cells. Additionally, we discovered that diminazene aceturate (DIZE), an ACE2 activator, similarly inhibited virus binding. Our research thus validates the potential of decoy peptides as a new therapeutic strategy against SARS-CoV-2 infections, opening avenues for further development and study.

Direct contractility measurement in cardiomyocytes via thin PDMS diaphragm integration with a moving coil actuator: Synergistic maturation through combined topographical and mechanical stimulation

Mechanical stimulation within diaphragm-based cell culture platforms presents a promising strategy to enhance cardiomyocyte maturation. However, most of the proposed techniques have met with limited success due to their inability to directly measure the contractile force of cardiomyocytes. This issue primarily stems from the typically thicker diaphragm structure which is necessary to withstand high strain ranges induced by mechanical stimulation. Herein, we propose a moving coil actuator integrated with a thin polydimethylsiloxane (PDMS) diaphragm for precise mechanical stimulation, cell culture, and drug screening applications. This mechanical stimulation system generates desired mechanical deformation to cultured cardiomyocytes without compromising the integrity of the diaphragm structure. The 10 µm thin diaphragm produces significant displacement from the contractility of cultured cardiomyocytes, which can be directly measured using a laser vibrometer system. Cardiomyocytes cultured on nanogroove PDMS diaphragms with and without stimulation and flat diaphragms with stimulation exhibited 4.25-, 2.81-, 2.39-fold enhancements in contraction force and 1.09-, 1.06-, 1.04-, and 1.32-, 1.17-, 1.16-fold enhancements in sarcomere lengths and Connexin 43 (Cx43) compared to counterparts cultured on flat and nanogroove PDMS diaphragms without stimulation. The practicality of the device as a drug screening platform is demonstrated by examining the drug-induced effects on cardiomyocytes using cardiovascular drugs.

Engineered 3D tumor microenvironment recapitulating stiffness of lung tissue to explore drug resistance of lung carcinoma

Despite clinical advances, the development of anticancer drugs faces high failure rates owing to cancer drug resistance and insufficient understanding of the native tumor microenvironment. Polymeric hydrogels are promising engineered 3D cancer models owing to their tunable properties and structural similarities to the native extracellular matrix. However, precisely mimicking the native cancer microenvironment remains challenging. In this study, we present interpenetrating polymer network (IPN) hydrogels with independent stiffness control as an engineered 3D lung carcinoma model for drug screening and basic cancer research. These IPN hydrogels are formed via horseradish peroxidase/hydrogen peroxide (H2O2)-mediated dual-crosslinking reactions, showing independently tunable stiffness at various H2O2 concentrations. Using this ability, we create engineered 3D lung cancer models that simulate normal and cancerous lung tissue stiffness with encapsulation of non-small cell lung cancer cells. We perform drug resistance tests using this system and compare the drug responses in various preclinical cancer models in vitro and in vivo. Finally, we investigate the biological mechanism underlying the acquired resistance of lung carcinoma cells to epidermal growth factor receptor inhibitors. In summary, our engineered 3D cancer model can serve as an advanced preclinical platform for drug screening and cancer biology, including the biological mechanisms of acquired drug resistance.