John Justin Gooding’s research while affiliated with State Library of New South Wales and other places

Precision medicine for paediatric and adult cancers that includes drug sensitivity profiling, can identify effective therapies for individual patients. However, obtaining adequate biopsy samples for high-throughput (HTP) screening remains challenging, with tumours needing to be expanded in culture or patient-derived xenografts - this is time-consuming and often unsuccessful. Herein, we have developed paediatric patient-derived tumour models using an engineered extracellular matrix (ECM) tissue mimic hydrogel system and HTP 3D bioprinting. Gene expression analysis from neuroblastoma and sarcoma patients identified key components of the ECM in these tumour types. Engineered hydrogels with ECM-mimic peptides were used to create patient-specific tumour organoids, modelling tumour growth conditions. Expanded tumour organoids recapitulated the genetic and phenotypic characteristics of the original tumours and retained tumourgenicity. Screening of these models identified individualised drug sensitivities. Our approach offers a timely and clinically relevant technology platform for precision medicine in paediatric cancers, potentially transforming preclinical testing across cancer types.

Childhood cancer patients with high-risk disease have poor prognosis. Therapies given to treat these patients are often highly toxic and determined empirically, exposing children to damaging and ineffective therapies. Precision medicine is a promising strategy for these patients. However, tumor biopsies often lack sufficient sample for phenotypic drug screening, requiring cell expansion via primary cell culture or development of patient-derived xenograft models - a slow process with variable success rates. There is an unmet need for sensitive, predictive, and timely drug testing models. Dynamic interactions between cells and the extracellular matrix (ECM) impact cellular signaling and response to cancer therapy. The development of patient-derived tumor models that are reflective of the patient sample and achieved in a clinically relevant timeframe will be game changing. In-silico analysis of 265 ECM gene expression from a high-risk neuroblastoma and sarcoma patient cohort (n=145) identified collagens and fibronectin to be the most abundantly expressed ECM genes in the tumor samples. To reflect this environment, we developed tuneable tissue ECM-like bioinks where cells are embedded within the bioink that mimics growth constraints within a tumor [1]. Specifically, we functionalized the bioinks with peptides of collagen I, fibronectin, and laminin to mimic the ECM environment and combined this with HTP 3D bioprinting technology to create patient-derived tumoroids. In this proof-of-concept study, we identified conditions that enable the growth and expansion of the neuroblastoma and sarcoma cells in a 3D ECM-like environment. The tumor samples proliferate in the bioinks and HTP drug screening was used to determine drug sensitivity. Importantly, the bioprinted cells reflect the genetic and phenotypic characteristics of the original patient tumors and retained their tumorigenic capacity in vivo. Collectively, we have successfully generated preclinical models that reflect patient tumors and are directly compatible with preclinical drug testing. Importantly, our 3D bioprinting platform has the potential to advance cancer precision medicine in a clinically relevant timeframe. These findings have broader applications for a range of cancer types for preclinical testing, drug discovery and cancer biology.1. Utama, RH, et al. Macromolecular Bioscience, 2021. 21(9):e2100125 Citation Format: Valentina Poltavets, MoonSun Jung, Joanna Skhinas, Kathleen Kimpton, Alvin Kamili, Gabe Tax, Jie Mao, Louise Cui, Marie Wong, Mark J. Cowley, Loretta Lau, Emmy ME Dolman, John J. Gooding, Maria Kavallaris. Development of high-throughput 3D bioprinted pediatric models for precision medicine [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 5475.

This study presents the combination of electrochemical cell based biosensor and fluorescence microscopy on a single platform to track biomolecular processes contributing to the morphology changes of the cells. Initial experiments demonstrate that using interdigitated electrodes where the gold microfingers were comparable in width and spacing to a single cell have the optimal sensitivity in the final electrochemical cell based‐sensing device. This was determined by measuring the cell index, based on the impedance analysis of bare and cell‐covered microelectrodes. The fabrication of the electrodes on a glass substrate enabled the capture of high‐resolution fluorescence microscopy images of single cells and related intracellular calcium release inside the HeLa cells via a window incorporated into the gold microelectrode design. As an illustration of the enhanced capability of the combined approach over traditional impedance cellular assay, the opto‐electric assay was utilized as a functional readout for G protein couple receptor activation. The simultaneous examinations of cells stimulated with histamine demonstrated an association between time courses of changes in cytosolic calcium concentration and reductions in cell‐cell adhesions.

Herein the development of cellular impedance biosensors, electrochemical impedance spectroscopy, and the general principles and terms associated with the cell-electrode interface is reviewed. This family of techniques provides quantitative and sensitive information into cell responses to stimuli in real-time with high temporal resolution. The applications of cell-based impedance biosensors as a readout in cell biology is illustrated with a diverse range of examples. The current state of the field, its limitations, the possible available solutions, and the potential benefits of developing biosensors are discussed.

Stochastic optical reconstruction microscopy (STORM) allows widefield imaging with single molecule resolution through calculating the coordinates of individual fluorophores from the separation of the fluorophore emission in both time and space. Such separation is achieved by photoswitching the fluorophores between a long lived OFF state and an emissive ON state. Despite STORM has revolutionizing cellular imaging, molecular counting in complexes remains challenging due to undercounting errors from photobleached or not-recovered dyes and overcounting artifacts from the repetitive and random blinking of the dyes. Herein we show how an electrochemical approach switching fluorophores for STORM (EC-STORM) has greater control over the switching kinetics, emitter density, and recovery yield than possible photochemically. Using EC-STORM, we demonstrate the capability for molecular counting by applying a programmable electrochemical potential to interrupt the photophysics of dyes. That is, the random blinking of dyes is suppressed by a negative potential but the switching ON event can be activated by a short pulsed positive potential, such that the frequency of ON events scales linearly with the number of underlying dyes. This advance will enable EC-STORM being the widely applicable super resolution imaging technique.

Nanoparticle-based magnetic contrast agents have opened the potential for magnetic resonance imaging (MRI) to be used for early non-invasive diagnosis of Alzheimer’s disease (AD). Accumulation of amyloid pathology in the brain has shown association with cognitive decline and tauopathy; hence, it is an effective biomarker for the early detection of AD. The aim of this study was to develop a biocompatible magnetic nanoparticle targeted to amyloid beta (Aβ) plaques to increase the sensitivity of T2-weighted MRI for imaging of amyloid pathology in AD. We presented novel iron core-iron oxide nanoparticles stabilized with a dimercaptosuccinic acid coating and functionalized with an anti-Aβ antibody. Nanoparticle biocompatibility and cellular internalization were evaluated in vitro in U-251 glioblastoma cells using cellular assays, proteomics, and transmission electron microscopy. Iron nanoparticles demonstrated no significant in vitro cytotoxicity, and electron microscopy results showed their movement through the endocytic cycle within the cell over a 24 h period. In addition, immunostaining and bio-layer interferometry confirmed the targeted nanoparticle’s binding affinity to amyloid species. The iron nanoparticles demonstrated favourable MRI contrast enhancement; however, the addition of the antibody resulted in a reduction in the relaxivity of the particles. The present work shows promising preliminary results in the development of a targeted non-invasive method of early AD diagnosis using contrast-enhanced MRI.

Herein is explored a dual optical and electrical cell based biosensor that can provide insights into cellular events. The fabrication steps along with the electrical characterization of the microelectrodes is described. Initial experiments show that the ability of indium tin oxide (ITO) to detect biological cells at the electrode‐cell layer interface mainly depends on the size of sensing area. Following this, the impact of conductivity was also explored to assess the real time impedance signal during the adhesion of a non‐confluent layer of the cells on different substrates. Electrodes with a higher conductivity than ITO gave much higher senstivity of impedance changes which allowed the dynamics of subtle cellular morphology changes to be monitored at densities far lower than a confluent layer of cells. Finally, the capability of ITO and gold microelectrode as a functional readout for G protein couple receptor activation were determined. These set of analyses highlighted the challenges and opportunities of ITO substrate as a dual cell‐based assay for quantitative analysis of subtle changes in cell morphology.

Although immunotherapy has revolutionized oncotherapy, only ∼15% of head and neck squamous cell carcinoma (HNSCC) patients benefit from the current therapies. An immunosuppressive tumor microenvironment (TME) and dysregulation of the polycomb ring finger oncogene BMI1 are potential reasons for the failure. Herein, to promote immunotherapeutic efficacy against HNSCC, we developed an injectable nanocomposite hydrogel with a polymer framework (PLGA‐PEG‐PLGA) that is loaded with both imiquimod encapsulated CaCO3 nanoparticles (RC) and cancer cell membrane (CCM) coated mesoporous silica nanoparticles containing a peptide‐based proteolysis‐targeting chimeras (PROTAC) for BMI1 and paclitaxel (PepM@PacC). Upon injection, this nanocomposite hydrogel undergoes in situ gelation, after which it degrades in the TME over time, releasing RC and PepM@PacC nanoparticles to respectively perform immunotherapy and chemotherapy. Specifically, the RC particles selectively manipulate tumor‐associated macrophages and dendritic cells to activate a T‐cell immune response, while CCM‐mediated homologous targeting and endocytosis delivers the PepM@PacC particles into cancer cells, where endogenous glutathione promotes disulfide bond cleavage to release the PROTAC peptide for BMI1 degradation and frees the paclitaxel from the particle pores to elicit apoptosis meanwhile enhance immunotherapy. Thus, our nanocomposite hydrogel, which was designed to exploit multiple known vulnerabilities of HNSCC, succeeds in suppressing both growth and metastasis of HNSCC. This article is protected by copyright. All rights reserved

Gold coated magnetic nanoparticles (Au@MNPs), modified with DNA sequences give dispersible electrodes that can detect ultralow amounts of microRNAs and other nucleic acids but, as with most other sensors, they require calibration. Herein we show how to adapt a calibration free approach for electrochemical aptamer-based sensors on bulk electrodes to microRNA (miR-21) detection with methylene blue terminated DNA modified Au@MNPs. The electrochemical square wave voltammetry signal from the DNA-Au@MNPs when collected at a bulk electrode under magnetic control, decreases upon capture of miR-21. We show that the square wave voltammogram has concentration dependent and independent frequencies that can be used to give a calibration free signal.

Single molecule experiments have recently attracted enormous interest. Many of these studies involve the encapsulation of a single molecule into nanoscale containers (such as vesicles, droplets and nanowells). In such cases, the single molecule encapsulation efficiency is a key parameter to consider in order to get a statistically significant quantitative information. It has been shown that such encapsulation typically follows a Poisson distribution and such theory of encapsulation has only been applied to the encapsulation of single molecules into perfectly sized monodispersed containers. However, experimentally nanocontainers are usually characterized by a size distribution, and often just a single binding pair (rather than a single molecule) is required to be encapsulated. Here the use of Poisson distribution is extended to predict the encapsulation efficiency of two different molecules in an association equilibrium. The Poisson distribution is coupled with a log-normal distribution in order to consider the effect of the container size distribution, and the effect of adsorption to the container is also considered. This theory will allow experimentalists to determine what single molecule encapsulation efficiency can be expected as a function of the experimental conditions. Two case studies, based on experimental data, are given to support the theoretical predictions.