Multicellular 3D Models to Study Tumour-Stroma Interactions

Two-dimensional (2D) cell cultures have been the standard for many different applications, ranging from basic research to stem cell and cancer research to regenerative medicine, for most of the past century. Hence, almost all of our knowledge about fundamental biological processes has been provided by primary and established cell lines cultured in 2D monolayer. However, cells in tissues and organs do not exist as single entities, and life in multicellular organisms relies on the coordination of several cellular activities, which depend on cell–cell communication across different cell types and tissues. In addition, cells are embedded within a complex non-cellular structure known as the extracellular matrix (ECM), which anchors them in a three-dimensional (3D) formation. Likewise, tumour cells interact with their surrounding matrix and tissue, and the physical and biochemical properties of this microenvironment regulate cancer differentiation, proliferation, invasion, and metastasis. 2D models are unable to mimic the complex and dynamic interactions of the tumour microenvironment (TME) and ignore spatial cell–ECM and cell–cell interactions. Thus, multicellular 3D models are excellent tools to recapitulate in vitro the spatial dimension, cellular heterogeneity, and molecular networks of the TME. This review summarizes the biological significance of the cell–ECM and cell–cell interactions in the onset and progression of tumours and focuses on the requirement for these interactions to build up representative in vitro models for the study of the pathophysiology of cancer and for the design of more clinically relevant treatments.     

Assessment of Normal Tissue Radiosensitivity by Evaluating DNA Damage and Repair Kinetics in Human Brain Organoids

DNA-double strand break (DSB), detected by immunostaining of key proteins orchestrating repair, like γH2AX and 53BP1, is well established as a surrogate for tissue radiosensitivity. We hypothesized that the generation of normal brain 3D organoids (“mini-brains”) from human induced pluripotent stem cells (hiPSC) combined with detection of DNA damage repair (DDR) may hold the promise towards developing personalized models for the determination of normal tissue radiosensitivity. In this study, cerebral organoids, an in vitro model that stands in its complexity between 2D cellular system and an organ, have been used. To quantify radiation-induced response, immunofluorescent staining with γH2AX and 53BP1 were applied at early (30 min, initial damage), and late time points (18 and 72 h, residual damage), following clinical standard 2 Gy irradiation. Based on our findings, assessment of DDR kinetics as a surrogate for radiosensitivity in hiPSC derived cerebral organoids is feasible. Further development of mini-brains recapitulating mature adult neuronal tissue and implementation of additional signaling and toxicity surrogates may pave the way towards development of next-generation personalized assessment of radiosensitivity in healthy neuronal tissue.     

A Drug Screening Pipeline Using 2D and 3D Patient-Derived In Vitro Models for Pre-Clinical Analysis of Therapy Response in Glioblastoma

Glioblastoma is one of the most common and lethal types of primary brain tumor. Despite aggressive treatment with chemotherapy and radiotherapy, tumor recurrence within 6–9 months is common. To overcome this, more effective therapies targeting cancer cell stemness, invasion, metabolism, cell death resistance and the interactions of tumor cells with their surrounding microenvironment are required. In this study, we performed a systematic review of the molecular mechanisms that drive glioblastoma progression, which led to the identification of 65 drugs/inhibitors that we screened for their efficacy to kill patient-derived glioma stem cells in two dimensional (2D) cultures and patient-derived three dimensional (3D) glioblastoma explant organoids (GBOs). From the screening, we found a group of drugs that presented different selectivity on different patient-derived in vitro models. Moreover, we found that Costunolide, a TERT inhibitor, was effective in reducing the cell viability in vitro of both primary tumor models as well as tumor models pre-treated with chemotherapy and radiotherapy. These results present a novel workflow for screening a relatively large groups of drugs, whose results could lead to the identification of more personalized and effective treatment for recurrent glioblastoma.     

The Rise of Retinal Organoids for Vision Research

Retinal degenerative diseases lead to irreversible blindness. Decades of research into the cellular and molecular mechanisms of retinal diseases, using either animal models or human cell-derived 2D systems, facilitated the development of several therapeutic interventions. Recently, human stem cell-derived 3D retinal organoids have been developed. These self-organizing 3D organ systems have shown to recapitulate the in vivo human retinogenesis resulting in morphological and functionally similar retinal cell types in vitro. In less than a decade, retinal organoids have assisted in modeling several retinal diseases that were rather difficult to mimic in rodent models. Retinal organoids are also considered as a photoreceptor source for cell transplantation therapies to counteract blindness. Here, we highlight the development and field’s improvements of retinal organoids and discuss their application aspects as human disease models, pharmaceutical testbeds, and cell sources for transplantations.     

Clinical Application Perspectives of Lung Cancers 3D Tumor Microenvironment Models for In Vitro Cultures

Despite the enormous progress and development of modern therapies, lung cancer remains one of the most common causes of death among men and women. The key element in the development of new anti-cancer drugs is proper planning of the preclinical research phase. The most adequate basic research exemplary for cancer study are 3D tumor microenvironment in vitro models, which allow us to avoid the use of animal models and ensure replicable culture condition. However, the question tormenting the scientist is how to choose the best tool for tumor microenvironment research, especially for extremely heterogenous lung cancer cases. In the presented review we are focused to explain the key factors of lung cancer biology, its microenvironment, and clinical gaps related to different therapies. The review summarized the most important strategies for in vitro culture models mimicking the tumor–tumor microenvironmental interaction, as well as all advantages and disadvantages were depicted. This knowledge could facilitate the right decision to designate proper pre-clinical in vitro study, based on available analytical tools and technical capabilities, to obtain more reliable and personalized results for faster introduction them into the future clinical trials.     

Modulatory Effects of Biosynthesized Gold Nanoparticles Conjugated with Curcumin and Paclitaxel on Tumorigenesis and Metastatic Pathways—In Vitro and In Vivo Studies

Background: Breast cancer is the most common cancer in women globally, and diagnosing it early and finding potential drug candidates against multi-drug resistant metastatic breast cancers provide the possibilities of better treatment and extending life. Methods: The current study aimed to evaluate the synergistic anti-metastatic activity of Curcumin (Cur) and Paclitaxel (Pacli) individually, the combination of Curcumin–Paclitaxel (CP), and also in conjugation with gold nanoparticles (AuNP–Curcumin (Au-C), AuNP–Paclitaxel (Au-P), and AuNP–Curcumin–Paclitaxel (Au-CP)) in various in vitro and in vivo models. Results: The results from combination treatments of CP and Au-CP demonstrated excellent synergistic cytotoxic effects in triple-negative breast cancer cell lines (MDA MB 231 and 4T1) in in vitro and in vivo mouse models. Detailed mechanistic studies were performed that reveal that the anti-cancer effects were associated with the downregulation of the expression of VEGF, CYCLIN-D1, and STAT-3 genes and upregulation of the apoptotic Caspase-9 gene. The group of mice that received CP combination therapy (with and without gold nanoparticles) showed a significant reduction in the size of tumor when compared to the Pacli alone treatment and control groups. Conclusions: Together, the results suggest that the delivery of gold conjugated Au-CP formulations may help in modulating the outcomes of chemotherapy. The present study is well supported with observations from cell-based assays, molecular and histopathological analyses.     

Three-Dimensional Cell Culture Models to Investigate Oral Carcinogenesis: A Scoping Review

Three-dimensional (3-D) cell culture models, such as spheroids, organoids, and organotypic cultures, are more physiologically representative of the human tumor microenvironment (TME) than traditional two-dimensional (2-D) cell culture models. They have been used as in vitro models to investigate various aspects of oral cancer but, to date, have not be widely used in investigations of the process of oral carcinogenesis. The aim of this scoping review was to evaluate the use of 3-D cell cultures in oral squamous cell carcinoma (OSCC) research, with a particular emphasis on oral carcinogenesis studies. Databases (PubMed, Scopus, and Web of Science) were systematically searched to identify research applying 3-D cell culture techniques to cells from normal, dysplastic, and malignant oral mucosae. A total of 119 studies were included for qualitative analysis including 53 studies utilizing spheroids, 62 utilizing organotypic cultures, and 4 using organoids. We found that 3-D oral carcinogenesis studies had been limited to just two organotypic culture models and that to date, spheroids and organoids had not been utilized for this purpose. Spheroid culture was most frequently used as a tumorosphere forming assay and the organoids cultured from human OSCCs most often used in drug sensitivity testing. These results indicate that there are significant opportunities to utilize 3-D cell culture to explore the development of oral cancer, particularly as the physiological relevance of these models continues to improve.     

Evaluating drug efficacy and toxicology in three dimensions: using synthetic extracellular matrices in drug discovery

The acceptance of the new paradigm of 3-D cell culture is currently constrained by the lack of a biocompatible material in the marketplace that offers ease of use, experimental flexibility, and a seamless transition from in vitro to in vivo applications. I describe the development of a covalently cross-linked mimic of the extracellular matrix (sECM), now commercially available, for 3-D culture of cells in vitro and for translational use in vivo. These bio-inspired, biomimetic materials can be used "as is" in drug discovery, toxicology, cell banking, and, ultimately, medicine. For cell therapy and the development of clinical combination products, the sECM biomaterials must be highly reproducible, manufacturable, approvable, and affordable. To obtain integrated, functional, multicellular systems that recapitulate tissues and organs, the needs of the true end users, physicians and patients, must dictate the key design criteria. In chemical terms, the sECM consists of chemically-modified hyaluronan (HA), other glycosaminoglycans (GAGs), and ECM polypeptides containing thiol residues that are cross-linked using biocompatible polyvalent electrophiles. For example, co-cross-linking the semisynthetic thiol-modified HA-like GAG with thiol-modified gelatin produces Extracel as a hydrogel. This hydrogel may be formed in situ in the presence of cells or tissues to provide an injectable cell-delivery vehicle. Alternately, an Extracel hyrogel can be lyophilized to create a macroporous scaffold, which can then be employed for 3-D cell culture. In this Account, we describe four applications of sECMs that are relevant to the evaluation of drug efficacy and drug toxicity. First, the uses of sECMs to promote both in vitro and in vivo growth of healthy cellularized 3-D tissues are summarized. Primary or cell-line-derived cells, including fibroblasts, chondrocytes, hepatocytes, adult and embryonic stem cells, and endothelial and epithelial cells have been used. Second, primary hepatocytes retain their biochemical phenotypes and achieve greater longevity in 3-D culture in Extracel. This constitutes a new 3-D method for rapid evaluation of hepatotoxicity in vitro. Third, cancer cell lines are readily grown in 3-D culture in Extracel, offering a method for rapid evaluation of new anticancer agents in a more physiological ex vivo tumor model. This system has been used to evaluate signal transduction modifiers obtained from our research on lipid signaling. Fourth, a new "tumor engineering" xenograft model uses orthotopic injection of Extracel-containing tumor cells in nude mice. This approach allows production of patient-specific mice using primary human tumor samples and offers a superior metastatic cancer model. Future applications of the injectable cell delivery and 3-D cell culture methods include chemoattractant and angiogenesis assays, high-content automated screening of chemical libraries, pharmacogenomic and toxicogenomic studies with cultured organoids, and personalized treatment models. In summary, the sECM technology offers a versatile "translational bridge" from in vitro to in vivo to facilitate drug discovery in both academic and pharmaceutical laboratories.     

Tumour Microenvironment: Roles of the Aryl Hydrocarbon Receptor,O-GlcNAcylation,Acetyl-CoA and Melatonergic Pathway in Regulating Dynamic Metabolic Interactions across Cell Types—Tumour Microenvironment and Metabolism

This article reviews the dynamic interactions of the tumour microenvironment, highlighting the roles of acetyl-CoA and melatonergic pathway regulation in determining the interactions between oxidative phosphorylation (OXPHOS) and glycolysis across the array of cells forming the tumour microenvironment. Many of the factors associated with tumour progression and immune resistance, such as yin yang (YY)1 and glycogen synthase kinase (GSK)3β, regulate acetyl-CoA and the melatonergic pathway, thereby having significant impacts on the dynamic interactions of the different types of cells present in the tumour microenvironment. The association of the aryl hydrocarbon receptor (AhR) with immune suppression in the tumour microenvironment may be mediated by the AhR-induced cytochrome P450 (CYP)1b1-driven ‘backward’ conversion of melatonin to its immediate precursor N-acetylserotonin (NAS). NAS within tumours and released from tumour microenvironment cells activates the brain-derived neurotrophic factor (BDNF) receptor, TrkB, thereby increasing the survival and proliferation of cancer stem-like cells. Acetyl-CoA is a crucial co-substrate for initiation of the melatonergic pathway, as well as co-ordinating the interactions of OXPHOS and glycolysis in all cells of the tumour microenvironment. This provides a model of the tumour microenvironment that emphasises the roles of acetyl-CoA and the melatonergic pathway in shaping the dynamic intercellular metabolic interactions of the various cells within the tumour microenvironment. The potentiation of YY1 and GSK3β by O-GlcNAcylation will drive changes in metabolism in tumours and tumour microenvironment cells in association with their regulation of the melatonergic pathway. The emphasis on metabolic interactions across cell types in the tumour microenvironment provides novel future research and treatment directions.     

Evaluation of the PSMA-Binding Ligand 212Pb-NG001 in Multicellular Tumour Spheroid and Mouse Models of Prostate Cancer

Radioligand therapy targeting the prostate-specific membrane antigen (PSMA) is rapidly evolving as a promising treatment for metastatic castration-resistant prostate cancer. The PSMA-targeting ligand p-SCN-Bn-TCMC-PSMA (NG001) labelled with ²¹²Pb efficiently targets PSMA-positive cells in vitro and in vivo. The aim of this preclinical study was to evaluate the therapeutic potential of ²¹²Pb-NG001 in multicellular tumour spheroid and mouse models of prostate cancer. The cytotoxic effect of ²¹²Pb-NG001 was tested in human prostate C4-2 spheroids. Biodistribution at various time points and therapeutic effects of different activities of the radioligand were investigated in male athymic nude mice bearing C4-2 tumours, while long-term toxicity was studied in immunocompetent BALB/c mice. The radioligand induced a selective cytotoxic effect in spheroids at activity concentrations of 3–10 kBq/mL. In mice, the radioligand accumulated rapidly in tumours and was retained over 24 h, while it rapidly cleared from nontargeted tissues. Treatment with 0.25, 0.30 or 0.40 MBq of ²¹²Pb-NG001 significantly inhibited tumour growth and improved median survival with therapeutic indexes of 1.5, 2.3 and 2.7, respectively. In BALB/c mice, no signs of long-term radiation toxicity were observed at activities of 0.05 and 0.33 MBq. The obtained results warrant clinical studies to evaluate the biodistribution, therapeutic efficacy and toxicity of ²¹²Pb-NG001.     

Bazedoxifene,a GP130 Inhibitor,Modulates EMT Signaling and Exhibits Antitumor Effects in HPV-Positive Cervical Cancer

Persistent HPV (Human Papillomavirus) infection is the primary cause of cervical cancer. Despite the development of the HPV vaccine to prevent infections, cervical cancer is still a fatal malignant tumor and metastatic disease, and it is often difficult to treat, so a new treatment strategy is needed. The FDA-approved drug Bazedoxifene is a novel inhibitor of protein–protein interactions between IL-6 and GP130. Multiple ligand simultaneous docking and drug repositioning approaches have demonstrated that an IL-6/GP130 inhibitor can act as a selective estrogen modulator. However, the molecular basis for GP130 activation in cervical cancer remains unclear. In this study, we investigated the anticancer properties of Bazedoxifene in HPV-positive cervical cancer cells. In vitro and in vivo experiments showed that Bazedoxifene inhibited cell invasion, migration, colony formation, and tumor growth in cervical cancer cells. We also confirmed that Bazedoxifene inhibits the GP130/STAT3 pathway and suppresses the EMT (Epithelial-mesenchymal transition) sub-signal. Thus, these data not only suggest a molecular mechanism by which the GP130/STAT3 pathway may promote cancer, but also may provide a basis for cervical cancer replacement therapy.     

Organoids: An Emerging Tool to Study Aging Signature across Human Tissues. Modeling Aging with Patient-Derived Organoids

The biology of aging is focused on the identification of novel pathways that regulate the underlying processes of aging to develop interventions aimed at delaying the onset and progression of chronic diseases to extend lifespan. However, the research on the aging field has been conducted mainly in animal models, yeast, Caenorhabditis elegans, and cell cultures. Thus, it is unclear to what extent this knowledge is transferable to humans since they might not reflect the complexity of aging in people. An organoid culture is an in vitro 3D cell-culture technology that reproduces the physiological and cellular composition of the tissues and/or organs. This technology is being used in the cancer field to predict the response of a patient-derived tumor to a certain drug or treatment serving as patient stratification and drug-guidance approaches. Modeling aging with patient-derived organoids has a tremendous potential as a preclinical model tool to discover new biomarkers of aging, to predict adverse outcomes during aging, and to design personalized approaches for the prevention and treatment of aging-related diseases and geriatric syndromes. This could represent a novel approach to study chronological and/or biological aging, paving the way to personalized interventions targeting the biology of aging.     

Optical Fibre-Enabled Photoswitching for Localised Activation of an Anti-Cancer Therapeutic Drug

Local activation of an anti-cancer drug when and where needed can improve selectivity and reduce undesirable side effects. Photoswitchable drugs can be selectively switched between active and inactive states by illumination with light; however, the clinical development of these drugs has been restricted by the difficulty in delivering light deep into tissue where needed. Optical fibres have great potential for light delivery in vivo, but their use in facilitating photoswitching in anti-cancer compounds has not yet been explored. In this paper, a photoswitchable chemotherapeutic is switched using an optical fibre, and the cytotoxicity of each state is measured against HCT-116 colorectal cancer cells. The performance of optical-fibre-enabled photoswitching is characterised through its dose response. The UV–Vis spectra confirm light delivered by an optical fibre effectively enables photoswitching. The activated drug is shown to be twice as effective as the inactive drug in causing cancer cell death, characterised using an MTT assay and fluorescent microscopy. This is the first study in which a photoswitchable anti-cancer compound is switched using an optical fibre and demonstrates the feasibility of using optical fibres to activate photoswitchable drugs for potential future clinical applications.     

The Progress of Intestinal Epithelial Models from Cell Lines to Gut-On-Chip

Over the past years, several preclinical in vitro and ex vivo models have been developed that helped to understand some of the critical aspects of intestinal functions in health and disease such as inflammatory bowel disease (IBD). However, the translation to the human in vivo situation remains problematic. The main reason for this is that these approaches fail to fully reflect the multifactorial and complex in vivo environment (e.g., including microbiota, nutrition, and immune response) in the gut system. Although conventional models such as cell lines, Ussing chamber, and the everted sac are still used, increasingly more sophisticated intestinal models have been developed over the past years including organoids, InTESTine™ and microfluidic gut-on-chip. In this review, we gathered the most recent insights on the setup, advantages, limitations, and future perspectives of most frequently used in vitro and ex vivo models to study intestinal physiology and functions in health and disease.     

Circulating Cell-Free Tumour DNA in the Management of Cancer

With the development of new sensitive molecular techniques, circulating cell-free tumour DNA containing mutations can be identified in the plasma of cancer patients. The applications of this technology may result in significant changes to the care and management of cancer patients. Whilst, currently, these “liquid biopsies” are used to supplement the histological diagnosis of cancer and metastatic disease, in the future these assays may replace the need for invasive procedures. Applications include the monitoring of tumour burden, the monitoring of minimal residual disease, monitoring of tumour heterogeneity, monitoring of molecular resistance and early diagnosis of tumours and metastatic disease.     

Best Practices and Progress in Precision-Cut Liver Slice Cultures

Thirty-five years ago, precision-cut liver slices (PCLS) were described as a promising tool and were expected to become the standard in vitro model to study liver disease as they tick off all characteristics of a good in vitro model. In contrast to most in vitro models, PCLS retain the complex 3D liver structures found in vivo, including cell–cell and cell–matrix interactions, and therefore should constitute the most reliable tool to model and to investigate pathways underlying chronic liver disease in vitro. Nevertheless, the biggest disadvantage of the model is the initiation of a procedure-induced fibrotic response. In this review, we describe the parameters and potential of PCLS cultures and discuss whether the initially described limitations and pitfalls have been overcome. We summarize the latest advances in PCLS research and critically evaluate PCLS use and progress since its invention in 1985.     

Drosophila Larval Models of Invasive Tumorigenesis for In Vivo Studies on Tumour/Peripheral Host Tissue Interactions during Cancer Cachexia

Cancer cachexia is a common deleterious paraneoplastic syndrome that represents an area of unmet clinical need, partly due to its poorly understood aetiology and complex multifactorial nature. We have interrogated multiple genetically defined larval Drosophila models of tumourigenesis against key features of human cancer cachexia. Our results indicate that cachectic tissue wasting is dependent on the genetic characteristics of the tumour and demonstrate that host malnutrition or tumour burden are not sufficient to drive wasting. We show that JAK/STAT and TNF-α/Egr signalling are elevated in cachectic muscle and promote tissue wasting. Furthermore, we introduce a dual driver system that allows independent genetic manipulation of tumour and host skeletal muscle. Overall, we present a novel Drosophila larval paradigm to study tumour/host tissue crosstalk in vivo, which may contribute to future research in cancer cachexia and impact the design of therapeutic approaches for this pathology.