Gibco Cell Culture Heroes Webinar Library

Stem cell’s unique properties confer them a multitude of potential applications in the fields of cellular therapy, disease modelling and drug screening fields. In particular, the ability to differentiate neural progenitors (NP) from human induced pluripotent stem cells (hiPSCs) using chemically-defined conditions provides an opportunity to create a simple and straightforward culture platform for application in these fields. Here, we demonstrated that hiPSCs can undergo neural commitment inside microwells, forming characteristic neural structures resembling neural rosettes and further give rise to glial and neuronal cells. Furthermore, this platform can be applied towards the study of the effect of neurotoxic molecules that impair normal embryonic development. As a proof of concept, the neural teratogenic potential of the antiepileptic drug valproic acid (VPA) was analyzed. It was verified that exposure to VPA, close to typical dosage values (0.3 to 0.75 mM), led to a prevalence of NP structures over neuronal differentiation.

Since prenatal exposure to cannabis has been associated with an altered rate of mental development and significant changes in nervous system functioning, we also investigated the effect of continuous exposure to cannabinoids – cannabidiol, Δ9-THC, and two synthetic cannabinoids (SC) - on developing human neurons, mimicking the prenatal exposure by drug-consuming mothers. In sum, all studied substances have a profound impact on the developing neurons, highlighting the importance of thorough research on the impact of prenatal exposure to natural and SCs.

The methodology proposed herein for the generation and neural differentiation of hiPSC aggregates can potentially be used for modelling the neurodevelopmental toxicity associated with prenatal exposure of different drugs and natural compounds.

The liver is responsible for many metabolic, endocrine and exocrine functions. Approximately 2 million deaths per year are associated with liver failure. Modern 3D bioprinting technologies allied with autologous induced pluripotent stem cells (iPS)-derived grafts could represent a relevant tissue engineering approach to treat end stage liver disease patients. In this presentation, we will show how to differentiate human iPS towards relevant liver cellular phenotypes (hepatocytes, endothelial cells and mesenchymal cells) for 3D bioprinting. Also, we will evaluate the impacts of using single cell dispersion (i.e. obtained from conventional bidimensional differentiation) of iPS-derived parenchymal (i.e. hepatocyte-like cells) versus using iPS-derived hepatocyte-like cells spheroids (i.e. three-dimensional cell culture), both in combination with non-parenchymal cells, into final liver tissue functionality. These results will contribute to improve current liver bioprinting technology towards future regenerative medicine applications and liver physiology and disease modeling.

Knowledge of the three-dimensional structure of therapeutically relevant targets has become an essential step in the pipeline of drug discovery. The X-ray crystallography has been the primary biophysical method for elucidating the 3D- structure of macromolecules and structural information has paved the way for novel pharmaceutical strategies (such as structure-based drug design (SBDD) method). Moreover, protein structures can be exploited by modeling methods to screen for novel ligands or to rationally design ligands. In this webinar, therapeutically relevant proteins will be presented. First, adhesion G protein- coupled receptors (aGPCRs), known for their exceptionally long ectodomains, which extend several hundreds to thousands of amino acids, and they contain important cell adhesion-related domains, including the GPCR autoproteolysis- inducing (GAIN) domain. this domain is conserved among the aGPCR family and is characterized by playing crucial roles in the receptor activation by undergoing an autoproteolysis process. However, the molecular mechanism of this process and the downstream signal transduction mediated by aGPCRs are poorly understood at the structural level. Many researchers have highlighted the critical role of these receptors in promoting or preventing cancer development. Thus, structural and functional studies would trigger future characterization of aGPCRs to explore their potential as therapeutic targets in cancer.

Despite the success of immunotherapy against several malignancies including melanoma and lung adenocarcinoma, glioblastoma remains the exception. CD8+ T cells are the main drivers of the response to immunotherapy, but most of them differentiate into a dysfunctional state inside the tumour. The signals underpinning CD8+ T cell dysfunction in glioblastoma remain uncharacterized. Using a transplantable mouse model of glioblastoma, we have found a high infiltration of CD4 and CD8 T cells expressing several inhibitory receptors such as PD-1, TIM-3 and LAG-3, suggesting the acquisition of a dysfunctional state. We hypothesize that chronic antigen stimulation through the T cell receptor is driving differentiation and the acquisition of a dysfunctional state on T cells, facilitating tumour progression. The main learning objectives of this talk will be to highlight the relevance of studying brain tumour immunology and to make a brief description of the approach we are using in order to identify and validate potential targets to treat this devastating disease.

Osteoporosis causes a decrease in bone density, along with deterioration of the bone’s microarchitecture at a faster rate than normal. This leads to increased risk of fractures, referred to as osteoporotic fractures which often occur in the spine. With age, the risk of fractures increases, due to reduction in bone mineral density as well as the increased rate of falls among the elderly. Bones weakened from metastatic cancer can also break or fracture. The fracture might happen with a fall or injury, but a weak bone can also break during everyday activities. Sudden pain in the middle of the back, for instance, is a common symptom of a bone in the spine breaking and collapsing from cancer.

One of the current treatments for osteoporotic fractures is vertebroplasty. Vertebroplasty is a procedure in which bone cement is injected into the fractured vertebrae for repair and stabilization.

Polymethyl methacrylate (PMMA) is at present the most commonly used bone cement. However, there are several problems with using PMMA such as the mismatch of mechanical properties with that of surrounding bone. As well as the release of cytotoxic monomers and the high heat polymerization of the material, resulting in reduced integration with the fractured bone. Numerous efforts have been made to develop bone composites with antibacterial properties and remineralising potential, however none of the experimental formulations to date have been ideal. Therefore, it is a necessity to improve bioactive, remineralising bone composites with anti-cancer properties.

Our bone composite consists of Monocalcium phosphate (MCPM) and polylysine (PLS). MCPM encourage remineralisation to allow for interaction with bone and PLS can inhibit bacteriophage development, due to its cationic nature.

Recent advances in in vitro 3D cellular culture technologies, such as organoids, rapidly developed and established novel, more physiologically relevant models for basic biology and clinical applications. Traditionally, high-throughput microfluidics and other similar technologies have relied on two-dimensional (2D) cell models for the study of various human conditions and diseases, yet they are not compatible with 3D culture. I will discuss the development of an automatic and dynamic microfluidic ex vivo organoid culture modeling system to facilitate and accelerate preclinical research and the development of personalized treatment strategies. This integrated platform combines microfluidics, automatic dynamic and temporal stimulations (i.e., combinatorial drug screenings), combined with in silico data analysis and prediction tools. The platform can be used to advance the capabilities of research on cancer organoids models, screen and mirror real life patient chemotherapy treatments and ultimately facilitate treatment decisions for the development of personalized therapies.

Purpose
Liver x receptors (LXRs) are hypothesized to serve as a link between lipid metabolism and inflammation by promoting cholesterol efflux as well as exhibiting anti-inflammatory properties. NAD-dependent deacetylase SIRT1 is known to promote insulin secretion, reduce glucose tolerance and to play a critical role in regulating inflammation. SIRT1 has also been shown to interact with LXR to promote LXR activation. Additionally, previous literature has shown that starvation increases SIRT1 levels in mice. The purpose of this study was to investigate the role of SIRT1-LXR activation in control of inflammation and subsequent metabolic changes in retinal endothelial cells.

Methods
Bovine retinal endothelial cells (BRECs) were treated with diabetic relevant stimulus TNFα (10ng/ml); LXR activator, DMHCA (1uM); or SIRT1 activator, SRT1720 (1uM). In order to model calorie restriction in vitro BRECs were serum starved (0% FBS) for 24hrs. SIRT1, IL1β, ABCA1 and ABCG1 were analyzed by qRT-PCR. Sirt1 activity was measured via histone deacetylase activity (HDAC) assay. LXR acetylation was measured via western blot analysis. Results: Treatment with pro-inflammatory cytokine, TNFα (10ng/ml) for 24hrs significantly increased cholesterol levels (p=0.0233, n=6), IL1β expression, IL6 expression and resulted in decreased levels of HDAC activity in BRECs. Activation of LXR or SIRT1 prevented TNFα-induced inflammation. Serum starvation resulted in a significant increase in HDAC activity (p=0.0005, n=6) and SIRT1 expression levels. Lastly, serum starvation caused a decrease in LXR acetylated levels in BRECs.

Conclusion
The results of this study demonstrate that serum starvation promotes activation of the SIRT1-LXR pathway metabolism in retinal endothelial cells. Therefore, this study suggests that therapeutic fasting may serve to activate the SIRT1-LXR pathway providing the dual benefits of decreasing inflammation and promoting cholesterol metabolism in the retina

Multiple myeloma is a disease of terminally differentiated plasma cells with the massive production of monoclonal immunoglobulins in the bone marrow.

Today there are many treatment modalities ongoing targeting the myeloma microenvironment, but especially immunotherapy seems to be a promising approach.

Immune checkpoints are critical components in the regulation of immune cell responses. If immune cells are activated due to the contact to an antigen, they also need to be controlled to prevent an overactive immune response. These control checkpoints are often over-expressed in cancer cells and therefore protect them from the tumour-specific immune response enabling tumour immune escape.

We investigate the role of the PD-1/PD-L1 immune checkpoint during myeloma disease progression and determine combinatorial approaches of immunotherapeutic drugs to increase myeloma cell killing efficiency.

A major limitation for the development of 3D engineered tissues is the absence of viable and perfusable vasculature. As a precursor to vascularized adipose tissue, cylindrical channels were formed in a cast gelatin methacrylate (GelMA) construct by printing sacrificial networks of Pluronic F127. Human umbilical vein endothelial cells (HUVECs) were seeded and cultured within the 3D printed channels, while adipose-derived stem cells (ADSCs) were cultured in the GelMA prior to casting the 3D printed channels.

The hydrogel was characterized by NMR, surface tension, contact angle and DMA. Pluronic filaments were printed onto glass slides using a robotic printer I&J 7300-LF (Fishnar, UK). HUVECs (PromoCell, UK) were cultured on GelMA substrate, whilst ADSCs (Thermo Fisher Scientific) were embedded within the GelMA. LIVE/DEAD and alamarBlue assays were used to assess the cells’ viability and proliferation respectively. Phalloidin staining was used to assess actin cytoskeleton organization.

RESULTS: Once methacrylation has occurred NMR peaks are seen at 6ppm and 2ppm corresponding to lysine and methacrylated grafts of hydroxyl groups. Viability assays confirmed that HUVECs and ADSCs were viable after 48 hours. alamarBlue data indicated an increase in cell metabolism over a 7-day period. Phalloidin staining demonstrated good organization of the actin cytoskeleton of HUVECs on GelMA. Data on HUVECs injected within the printed 2D networks and 3D culture of ADSCs within the GelMA matrix will also be presented.

DISCUSSION & CONCLUSIONS: Collectively, our data illustrate that HUVECs could potentially grow and fully line the printed networks.

The choroid plexus, which makes up the blood-cerebrospinal fluid barrier in the central nervous system (CNS), lines the ventricles, produces cerebrospinal fluid, and protects the brain via a physical barrier. Perivascular macrophages line the stromal capillaries through it, and tight junctions between the apical sides of the epithelial cells regulate the microenvironment. The choroid plexus is also believed to be an immune interface between peripheral and CNS immune systems and plays a major role in the resolution of neuroinflammation by recruiting monocytes and leukocytes into the CNS. It has also been proposed to be a target of viral infection, such as a human immunodeficiency virus (HIV) reservoir, and a site of damage in cerebral hemorrhage, stroke, and hypoxia. Since the choroid plexus can allow transmigration of leukocytes, recruit myeloid cells, control diffusion of small molecules and water, and control drug permeability into the CSF, it is important to have an ex vivo culture model.

We designed a rhesus macaque 2D choroid plexus epithelial cell culture, as well as a modified 3D cell culture model, in order to study activation, diffusion, and migration through the choroid plexus. Our hope is to understand the response of the blood-CSF barrier to peripheral HIV infection, as the rhesus macaque is an ideal animal model of infection. This model can also be used to study pro- and anti-inflammatory challenges, as well as barrier properties.

At present cancer research focuses on three major areas: cancer diagnostics, drugs development, and next-generation therapies. About 90% of the in vitro research relies on traditional two-dimensional (2D) monolayer cell culture systems. 2D cell culture systems fail to accurately recapitulate the structure, function, physiology of living tissues, and this means that the results from studies involving efficacy of new drugs, gene expression, metabolic pathways, and cell proliferation do not correlate to actual in vivo scenarios. In contrast, a 3D cell culture system promotes many biological relevant functions that are not observed in 2D. The primary reason for this is attributed to two reasons: 1)In vivo cancer cells experience limited diffusion of oxygen, nutrients and signaling molecules in a dynamic way, which the 2D fails to mimic. 2)Cellular interaction, function, growth and signaling all occurs in a highly complex 3D architecture with the influence of extracellular matrix and other regulatory factors, which cannot be recapitulated in 2D systems.

To achieve this dynamic coordination, microfluidic cell culture systems can be employed that can provide a continuous flow of nutrients, exchange of gases and other regulatory factors in a well-controlled manner. Such a system is ideal to mimic the in vivo environment of cells. Coupling microfluidics with 3D cell culture system will allow study of cellular functions such as proliferation in dynamic systems, cell-cell interaction and cellular response to the external environment in a much more realistic environment. Further, microfluidic systems give an opportunity to study cellular interaction by fabricating microstructures and artificial scaffolds to study cellular movements and the underlying mechanobiology. Using the microfluidic approach better drugs and therapies can be developed which can be easily translated to in vivo systems and hence can bridge the gap between in vitro and in vivo systems.

Although mesenchymal stem/stromal cells (MSCs) chondrogenic differentiation has been thoroughly investigated, the rudiments for enhancing chondrogenesis have remained largely dependent on external cues. Since aggregation of MSCs, a prerequisite for chondrogenesis, generates tension within the cell agglomerate, we theorized that the initial number of the cells within the aggregate could function as an intrinsic activator of a mechanobiology paradigm and alter the outcomes.

We discovered that reducing aggregate cell number (ACN) from 500k to 70k leads to activation and acceleration of the chondrogenic differentiation, independent of soluble chondro-inductive factors, via β-catenin dependent TCF/LEF transcriptional activity and expression of anti-apoptotic protein survivin. Our state-of-the-art mechanical testing revealed a correlation between progression of chondrogenesis and emergence of stiffer cell phenotype. In-depth Affymetrix gene array analysis proposed that the down-regulation of genes associated with lipid synthesis and regulation could account for observed outcomes. Furthermore, we illustrate that implanting aggregates within collagenous matrix not only decreases the necessity for high quantity of cells but also leads to drastic improvement in quality of the deposited tissue.

In summary, our study presents a simple and donor-independent strategy to enhance the efficiency of MSCs chondrogenic differentiation and demonstrates a correlation between MSCs chondrogenesis and mechanical properties with potential translational applications.

Cataract, a clouding of the ocular lens, is the leading cause of blindness worldwide. Currently the only means of treatment is through surgical intervention. Given the sheer prevalence of cataract worldwide, surgical intervention places a significant financial burden on the health-care system. Hence, there is a need to develop pharmacological treatments to maintain the transparency of the lens. This webinar will explore the molecular and cellular basis of how cataract forms with a particular focus on the role of transforming growth factor-beta (TGFβ) and its downstream signaling pathways. The Lens Research Laboratory at the University of Sydney led by Professor Frank Lovicu seeks to unravel the complex interplay of growth factor signaling pathways involved in the formation of cataract and in doing so, find novel drug targets to combat cataract. The lens epithelial explant culture system was developed in the Lens Research Laboratory in the 1980s and has enabled the discovery of many now well-accepted phenomena about lens epithelial cell behavior. Using this model, observations can be made while primary lens epithelial cells are adherent to their native basement membrane, known as the lens capsule, thus enabling a closer representation of the in vivo situation compared to. This webinar will explain how lens epithelial explants are generated and utilized for experiments.

Prostate cancer is the second most common form of cancer in males, affecting one in eight men by the time they reach the age of 75. The disease continues to be a major cause of morbidity and mortality in men, but a method for accurate prognosis in these patients is yet to be developed necessitating a search for new molecular markers and continued investigation of prostate cancer cell biology.

Cell lines have been used in the search for biomarkers that are suitable for prostate cancer diagnosis. Many studies have only involved single cell lines, partially characterised cell lines or were performed without non-malignant controls, potentially undermining effective biomarker discovery.

By utilising a larger panel of prostate cell lines, we discovered that cell lines transfected with HPV-18 display an aberrant pattern of protein and gene expression compared to SV40 or non-immortalised cancer cells. Through this discovery process we found that the critical process of endosomal biogenesis might be altered in prostate cancer. Microarray analysis of clinical cohorts confirmed these changes and were further delineated in fresh-frozen prostate cancer tissue by qRT-PCR.

The discrimination of prostate cancer cell lines can be further achieved using a various spectroscopic methods; utilising quantitative mass spectrometry (LC-ESI-MS/MS), non-invasive FTIR analysis and fluorescent detection of lipids in live cells by imaging with confocal microscopy we identified specific phospholipids and cholesteryl ester species that could distinguish between aggressive and non-aggressive cellular phenotypes and that could act as potential biomarkers for prostate cancer. It is possible to discriminate between cancer and non-malignant cells using a luminescent metal complex ReZolve-L1 that interacts with polar lipids. These technologies can be employed to find new candidates that have significant biomarker potential.

Osteosarcoma is the most common type of primary bone cancer affecting adolescents and children attributed to rapid bone growth and turnover with a peak incidence at 18 years old. It is a rare, incurable, and often fatal disease. The current osteosarcoma treatments include standard chemotherapy, limb salvage surgery or amputation. The 5-year relative survival rate is 70% for localized forms, however, metastasis is present in about one fifth to one quarter of patients at diagnosis, which then contributes to a 5-year survival rate of around 20%. The survival statistics have remained constant with no advances in treatment options for decades highlighting the need for new therapeutic options.

In healthy tissue, a reservoir of stem cells gives rise to non-stem cells while simultaneously maintaining their own population. This enables potentially endless growth and recovery from damage. Cancer stem cells (CSC) sustain cancer growth in the same way as normal stem cells do in healthy tissues. They give rise to both CSC and non-CSC tumour cells, the latter which lack stemness but make up the bulk of a tumour. DNA damaging agents (radiation and chemotherapy) can stop tumour growth by killing non-CSC tumour cells but CSC are resistant to these treatments. Surviving CSC could cause cancer relapse and progression, resulting in higher rates of patient mortality. There is growing interest in developing treatments which are specifically effective against CSC.

We are investigating how CSC could be treated by T cell immunotherapy. T cells can kill specific cancer cells by recognizing antigens, protein fragments on the cells’ surface. Another advantage of T cell immunotherapy is the potential for (dormant) T cells to persist in the body which can actively recognise the re-emergence of cancer. Our lab focuses on prostate cancer, the second most common cancer in men worldwide. Early-stage treatment is typically successful but relapse (biochemical) occurs in 15-40% of patients. Additionally, the survival rate of patients that present with or progress to metastatic disease is only 30%. We are characterizing prostate CSC and identifying their antigens to target them with T cells. This could prevent disease relapse and progression and improve long term patient outcomes.

In this talk I will discuss characterisation of CSC in prostate and other cancers. I will describe how we experimentally identified CSC antigens and discuss the utility of antigen datasets in developing immunotherapy treatments. Our current research is focused on isolating and testing the killing efficacy of T cells which recognise the CSC antigens, and how they will lead to future anti-cancer treatments.

Breast cancers are classified into three main subtypes according to their receptor status: estrogen receptor-positive (ER+), human epidermal growth factor receptor 2-positive (HER2+), and triple negative. These classifications provide information about the biology of the disease and help clinicians determine which treatments to use for patients. Targeted therapy for HER2+ breast cancer, including trastuzumab and pertuzumab, and for ER+ breast cancer, including Tamoxifen and aromatase inhibitors, have drastically improved survival outcomes for patients with these disease subtypes. However, when tumors are both HER2+ and ER+, treatment decisions and outcomes are less clear. HER2+/ER+ breast cancers treated with HER2 targeted therapies do not respond as well as HER2+/ER-negative tumors. Our lab is interested in understanding the biology behind these differences in response as well as better defining treatment options for HER2+/ER+ breast cancer. We have established long-term estrogen deprived (LTED) cell models of HER2+/ER+ breast cancer utilizing phenol-red free media as a surrogate for aromatase inhibitor treatment and Fulvestrant (a selective estrogen receptor degrader) resistant cell lines using increasing doses of Fulvestrant over time. We are testing combination therapy over different time courses then measure proliferation and targets of downstream signaling including protein phosphorylation by western blot and gene transcription by qRT-PCR. Ultimately, these results will help inform clinicians about the most effective treatment for patients with HER2+/ER+ breast cancer.

Extracellular vesicles (EVs) are cell-secreted nanoparticles that play a pivotal role in intercellular communication that affects both physiological and pathological processes. Therefore, EVs have great potential for diagnostic and therapeutic applications for cancer. EVs can be isolated from cell culture media and patient-derived biological fluids, and exploited as biomarkers or delivery vehicles. In this project, the uptake of EVs in various cancer cells has been evaluated. Moreover, EV RNA profiling has been used as a biomarker of various liver cancer cell lines. Additionally, EVs isolated from cancer cells have been projected into a 2D supported lipid bilayer as the initial step to develop a first of its kind EV biosensor. Taken together, cancer cell derived EVs have great potential for improving the diagnosis and treatment of this disease.

While the significance of the microbiome is unprecedented, a thorough study to dissect the role of individual populations of the natural gut microbiome in healthy and diseased states is still lacking. Currently available in vitro models of the human intestine have certain limitations, e.g., lack of immune cells or microbiota. Therefore, systems closely mirroring the human intestine are needed to gain a better understanding of intestinal dysfunction, host–pathogen interactions and for the development of more effective therapies for cancer. It is important to establish a patient derived, fast and efficient system which would mimic the in vivo architecture in humans and permit precise studies on interactions of the gut microbes with the epithelium and the immune cells. The in vitro 3D “mini-gut” system developed in the Clevers lab displays many important functions of the normal intestinal epithelium. Along with the developed toolbox for the analysis of these organoids (including FACS-based cell sorting, confocal imaging, RNA Sequencing, mass-spec proteomics and CRISPR-Cas9), it has proven to serve as a powerful system to investigate regulatory and pathological mechanisms of the intestinal epithelium on a molecular level.

To this end, we are developing a triple co-culture system of epithelial cells, immune cells and microbes to better understand the functional role of the gut microbiota, enabling personalized healthcare for the benefit of patients suffering from GI tract diseases. It might be possible in the future to determine the microbiome composition of cancer patients and provide personalized immunotherapy drug treatment.

Harnessing the immune system has emerged as a powerful therapeutic strategy in oncology. However, the limited ability of cytotoxic CD8+ T cells to infiltrate solid tumors presents a major roadblock to develop effective immunotherapy. Cytotoxic CD8+ T cells, in fact, have to infiltrate solid tumors, attack and kill cancer cells in order to provide an effective antitumor response. CD8+ T cell effector functions depend on Ca2+ influx into the T cell, which is controlled by two potassium (K+) channels: the voltage-dependent Kv1.3 and the Ca2+-activated KCa3.1. Our laboratory studies the contribution of these channels to T cell effector functions in patients with head and neck squamous cell carcinoma (HNSCC). We recently reported a decreased Kv1.3 function accompanied by a decrease in Ca2+ influx in tumor infiltrating lymphocytes (TILs) isolated from HNSCC patients. Furthermore, CD8+ TILs expressing high Kv1.3 levels and showing increased cell proliferation and cytotoxicity preferentially accumulated in the stroma. We also reported a role for K+ channels in regulating CD8+ T cell infiltration in tumors. Various intratumoral factors, especially the nucleoside adenosine limit the accumulation of TILs. We analyzed the migration of CD8+ T cells from HNSCC patients using a 3D chemotaxis assay and observed that adenosine inhibited the chemotaxis of CD8+ T cells from HNSCC patients to a greater degree than CD8+ T cells from healthy individuals. This increased sensitivity of HNSCC CD8+ T cells to adenosine correlated with their inability to infiltrate the tumor and was due to a decrease in KCa3.1 activity. Thus, our data indicate that defects in the K+ channels in T cells limit their effector functions and migration into the tumors, thereby contributing to the reduced anti-tumor immune response. Positive modulators of these channels could improve cancer immune surveillance, thus potentially opening new avenues for cancer immunotherapy.

The failure of current chemotherapeutic strategies in the fight against cancer can be largely attributed to the occurrence of drug resistance. Drug resistance is a major concern, especially in aggressive and highly metastatic tumours with a poor prognosis. In vitro cell-based models cultured as traditional two-dimensional (2D) cultures are commonly used for cancer research, including drug resistance studies. However, the inconsistencies between 2D in vitro results and in vivo or clinical findings have raised doubts about the accountability of 2D in vitro models as accurate representatives of in vivo tumours. Seeing as cancer cells cultured as three-dimensional (3D) spheroids have been shown to more closely mimic the complex microenvironment of an in vivo tumour, these models may overcome the aforementioned discrepancies. We aim to develop novel 3D micro-gravity spheroid-based cancer models to investigate drug resistance. This is done with different small cell lung cancer (SCLC) cell lines with varying levels of efflux transporters, which are known to be frequently involved in drug resistance mechanisms. Each of these models is validated through comparison of standard anticancer drug efficacy to published in vivo or clinical findings. Our main application of these models, currently, is investigating the potential of traditional herbal medicines to reduce or overcome efflux transporter-based drug resistance in SCLC.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with a 5 year-survival rate of approximately 6&. Despite recent efforts in developing novel therapies, chemotherapy remains the standard of care for advance pancreatic cancer patients. MicroRNAs (miRNAs) are short non-coding RNAs that act as post-transcriptional negative regulators of gene expression. Our laboratory is interested in investigating how miRNAs dysregulation contribute to PDAC tumourigenesis. One of the major signalling pathways that drives tumourigenesis in PDAC is the TGF-β pathway. This pathway promotes epithelial-to-mesenchymal transition (EMT), metastasis and stemness. In this study we aimed to find out whether miRNAs were involved in the TGF-β response, as this was yet largely unknown. We used miRNA expression profiling and identified miRNAs regulated by TGF-β. We then focused on the top two miRNAs upregulated by TGF-β and characterized their role in PDAC. We used several strategies to inhibit the function of these miRNAs, including the genome editing approach CRISPR. With these systems we showed that silencing of these miRNAs impaired EMT, motility, stemness in vitro and tumourigenesis and metastasis in vivo. Furthermore, we identified globally the targets of these miRNAs by integrating AGO2-RIP sequencing with RNA-sequencing upon overexpression of the miRNA of interest. We found that the candidate miRNA targets significantly overlap and mainly inhibit p53 and cell to cell junctions’ pathways, which are all important in PDAC progression. We also showed that the candidate miRNAs were up-regulated in PDAC patient samples, and their specific tumoral expression strongly correlated with reduced overall survival and disease-free survival. These findings demonstrate a fundamental role of miRNAs within the TGF-β response and represent potential novel targets for therapeutic intervention in PDAC.

There is significant epidemiological evidence to suggest that the consumption of a high-broccoli diet is associated with a reduced risk of cancer and cardiovascular disease. Human intervention studies have shown that a broccoli-rich diet can reduce cholesterol levels and rebalance central cell metabolism. Our studies also show that the effect of diet is also influenced by the allelic status of a gene known as PAPOLG, an RNA poly(A) polymerase not previously associated metabolic homeostasis. Through the use of in vitro cell models, high throughput metabolomics and live cell energy phenotyping, the nutrigenetic relationship between PAPOLG and the broccoli-rich diet can be explored. In this webinar I will expand upon the link between broccoli bioactives and metabolic homeostasis, highlight the potential role of RNA turnover in metabolic control, and offer insights into the potential implications for the dietary prevention of disease.

The current gold standard in in vitro pre-clinical cancer treatment screening remain cell lines, grown on static flat surfaces—generally referred to known as traditional two-dimensional cultures (2D). When considering drug discovery and development to discern possible treatment options, ideally one should to implement an experimental model that best mimics the in vivo environment of man.Organs boast a unique three-dimensional cellular architecture, with cell-cell and cell-matrix interactions, creating a complex communication network through biochemical and mechanical signals. More recently, proof of concept that three-dimensional cell culturing (3D) is revolutionizing the evaluation of lead compounds has been shown. However, important and distinct differences exist between 2D and 3D cell culturing, as well as the in vivo situation. These critical differences culminate in discrepancies in treatment responses between these systems, suggesting that 3D models may be able to provide a more accurate representation of how a specific organ or cancer would react, compared to 2D. Various types of 3D cell culture model systems are currently available and being explored. It is important to note that the choice of system depends on the hypothesis, study design or target organ, and not one system is superior to the other and each offers various advantages and disadvantages. The dynamic micro-gravity spheroid 3D system, exhibits the ability to overcome many of the shortcomings of traditional 2D cell cultures. In implementing this system in our laboratories, we aim to establish specific spheroid models and platforms to answer the pressing and relevant questions currently in cancer research.

Glioblastoma (GBM) and Medulloblastoma (MB) are the most common adult and pediatric brain tumors, both of which can have devastating consequences. Patients diagnosed with GBM have a life expectancy of around 15 months, whereas, for MB, the survival rate is higher. However, commonly used treatments for MB can have a negative impact on a child’s developing brain. Therefore, it is imperative that further research is conducted to understand the complex cell biology of these tumors to enable us to improve current treatment protocols.

Both GBM and MB are defined as hypoxic as their O2 levels are lower than the physiological 5% O2 found in the brain. Tumor hypoxia is known to enhance the ability cancer cells to invade other tissues and form tumors at secondary sites (metastasis), as well as causing resistance to chemotherapy and radiotherapy. Currently, little is known about how the chronic hypoxic tumor environment causes long-lasting cellular adaptations within tumor cells resulting in their resistant phenotype.

To further understand this, our lab investigates how long-term hypoxia exposure impacts DNA repair mechanisms within brain tumor cell lines, and how these changes can affect the response of cells to DNA damaging agents such as chemotherapeutic drugs and X-ray irradiation. We use a variety of complementary techniques including cell culture, gene expression analysis and advanced confocal microscopy.

We have observed down-regulation of key DNA repair proteins induced by hypoxia, causing the cells not to ‘recognize' certain types of DNA damage. Therefore, the cells are less likely to trigger apoptosis after cancer treatment. Additionally, further changes in DNA repair genes may cause a reduction in the efficiency of DNA repair, influencing the cell response to cancer therapy. It is hoped that gaining a deeper understanding of the effect of hypoxia in GBM and MB will aid in the development of more successful treatment methods.

Glioblastoma (GBM) is the most aggressive primary brain cancer, with nearly universal recurrence after treatment. GBMs are highly heterogeneous at the cellular level, and there is much evidence that recurrence, chemoresistance, and invasion are driven by a rare and specialized population of tumor initiating cells (TICs) within the tumor. These TICs are thought to share some similarities with stem cells in that they can both self-renew and differentiate to produce a range of cell types found in the bulk tumor. Because glioblastoma is above all a disease of tissue invasion and because invasion involves complex mechanical signaling between the microenvironment and the invading cells, we probed how TICs respond to mechanical cues. We found that in contrast to the majority of other cell types, TICs surprisingly showed very little stiffness-dependent change in cell shape and migration. Furthermore, we found that by increasing cellular force generation we could increase mechanosensitivity and extend survival in a mouse xenograft model. We next asked how the mechanosensitivity of these TICs changes as they are exposed to bone morphogenetic protein 4 (BMP4), which has been previously shown to elicit a differentiation-like effect on GBM TICs and extend survival in a xenograft model. We found that TICs treated with BMP4 showed increased stiffness-dependent changes in cell shape and reduced tissue invasion. We next performed RNA sequencing for a systems-level picture of how differentiation impacts mechanical signaling in TICs. We identified several pathways that showed mechanically-regulated changes impacted by differentiation, particularly those governing cell-extracellular matrix adhesions. These findings demonstrate that manipulation of mechanotransductive signaling can be leveraged to control tumor growth and invasion, and provide insight on alterations in mechanical signaling in stem-like and differentiated tumor initiating cells.

Colorectal cancer (CRC) develops during a multi-step process from small lesions of the intestinal epithelium. Genetic mutations in the canonical Wnt signaling pathway are considered to be the initial step of tumor formation. Using 3D organoid technology, tumor organoid cultures from CRC patients can be established. CRC organoids closely recapitulate key properties of the original tumor epithelium, including histological appearance, general gene and protein expression pattern and mutational load. Tumor organoids are amenable to radiation treatment, gene-drug association studies and high-throughput drug screens. Organoid cultures from adjacent healthy colon mucosa that retain the identity of the healthy intestinal epithelium in vitro can also be generated. Sequential introduction of the most commonly mutated CRC genes (APC, P53, KRAS and SMAD4) into healthy colon organoids using CRISPR/Cas9 genome editing technology allows for the modelling of tumorigenesis in vitro. Xenotransplantation of mutated organoids into mice recapitulates critical features of CRC progression and metastasis. In sum, (cancer) organoid technology can be used as experimental tool for basic research as well as diagnostic and therapeutic tool for (personalized) medicine.

Oncolytic virotherapy, the use of viral vectors to treat cancer, holds huge promise. Viruses are natural DNA delivery vehicles evolved to target specific tissues and transform them. Oncolytic virotherapies harness these abilities for therapeutic rather than pathological results. By engineering the virus to target cancerous cells rather than healthy cells we can create virotherapies which self-amplify at the point of need. Whilst historically safety focused, the field has now pivoted to enhanced efficacy following the first approved oncolytic virotherapy, T-VEC, for melanoma.

Our laboratory develops Adenoviruses (Ads) as oncolytics. Ads are versatile platforms offering large transgene capacity, ease of manipulation, and lytic potential with an excellent safety profile. However, current Ad-based therapies are hampered by high levels of pre-existing immunity within the population and off-target effects caused by the promiscuity of Ads’ canonical receptor: CAR.

We address these issues by a “bottom up” engineering approach to enhance the well characterized Ad5 serotype combined with a “top down” investigation of understudied Adenoviruses with advantageous phenotypes. By engineering Ad5 we can ablate natural tropism and facilitate specific infection of cancer cells; demonstrated by both in vitro, and in vivo models of cancer. Concurrently, we can develop rare Adenovirus serotypes devoid of pre-existing immunity, namely neutralizing antibody activity. Integrating proteins from these serotypes into therapeutic vectors enables us to radically improve cancer cell transduction.

Once targeted, the viruses must be capable of efficient cancer cell killing. We have developed Ad vectors with a variety of transgenes to manipulate signaling pathways for therapeutic benefit. Current research focuses on combining the above aptitudes into a single virus with a 3-pronged therapeutic action:

• Inherent viral immunogenicity
• Direct cancer cell lysis
• Stimulation of anti-tumor immunity

Successful tumor outgrowth requires the coordination of a variety of cell intrinsic and cell extrinsic signaling events. These events include those establishing a tumor microenvironment (TME) that both enables tumor cell survival and disables anti-tumor immunity. Recent reports demonstrate that these events require molecules produced by resident tumor cells. Tumor-borne signals within the tumor microenvironment propagate tumor cell fitness and immune hijacking. The endoplasmic reticulum (ER) stress is an adaptive response to a variety of TME insults, including hypoxia and nutrient deprivation, raising the possibility that ER stress could serve as a potential source of tumor cell fitness and immune dysregulation. To that end, we induced cancer cells of various origin to undergo ER stress and harvested the resulting conditioned medium (CM) to explore its effects on both recipient cancer cells and immune cells. Cell culture medium became an invaluable tool to our studies as it allowed us to recreate stimuli existent within the tumor microenvironment under controlled conditions. Our results revealed that the CM of cancer cells undergoing ER stress transmits ER stress to recipient cells. On the one hand, myeloid cells (macrophages and dendritic cells) treated with the CM of ER stressed cancer cells acquire a mixed pro-inflammatory and immune suppressive phenotype, which restrained T cell anti-tumor immunity and facilitated tumor growth in vivo. On the other hand, cancer cells treated with the CM of ER stressed cancer cells acquire cellular fitness to a variety of challenges including nutrient deprivation and chemotherapies. When implanted into immune competent hosts, ER stress experienced cancer cells grew at markedly faster rates than inexperienced ones. These findings support the existence of a novel mechanism used by tumor cells to restrain anti-tumor immunity while enhancing cellular fitness with the TME.

The use of gene therapy is well studied due to its potential to treat cancer, the second leading cause of death worldwide. The goal of gene therapy is to introduce functional genetic material into human cells to be transcribed and translated in order to regulate, repair or suppress a molecular mechanism that contributes to a disease state. Compared to traditional cancer therapies such as surgery, chemotherapy or radiation therapy, gene therapy is a more personalized and targeted approach because it is based on understanding the genetic profile of a patient’s tumor. Genes being developed for cancer therapy code for a variety of proteins including tumor suppressors, specific antigens, transcription factors, cell cycle regulators, receptors and cytokines. The cytokine Interleukin-24 (IL-24), is of special interest for gene therapy because of its selective killing effect on numerous cancer cell types while having no effect on corresponding normal cells. Due to this property, IL-24 is being investigated in Phase II clinical trials as a gene therapeutic to treat cancer patients. To understand how IL-24 exerts its specific killing effect, our lab studies the signaling pathways that IL-24 activates to induce programmed cell death also known as apoptosis. We use various cancer cell lines to understand which proteins IL-24 modulates to produce its killing effect. Currently, we are exploring how IL-24 blocks protein synthesis in cancer cells to promote cell death. Our aim is to further develop IL-24 as an anti-cancer therapeutic for gene therapy and to reveal targets for combination therapies that will work synergistically with IL-24 to produce a cancer specific killing effect.

Recent studies show that cancer cells can resist treatment by changing into a different cell type. Many treatments for specific cancers, such as breast, prostate, or lung, target vital pathways active in healthy tissue. The reliance of cancer cells on these pathways suggest that they retain properties of healthy cells. A prominent example of targeted treatment is androgen deprivation therapy for advanced prostate cancer. This therapy limits the production and effectiveness of androgen hormones because prostate cancer cells depend on androgen hormones, just like their healthy counterparts. Prostate cancers that become resistant to multiple rounds of therapy often no longer express the target of therapy. These resistant or ‘reprogrammed’ tumor cells are more likely to express different cell lineage markers. These markers are expressed by neuroendocrine cells, a rare cell type in healthy and untreated cancerous prostate tissue. Once prostate cancer cells are reprogrammed, current therapies are ineffective and patients quickly succumb to their disease. Our laboratory studies reprogramming in prostate cancer cells with the aim of developing new drugs to treat these resistant patients. We use murine models and 3D organoid culture of murine and human tumors to understand how prostate cancer cells acquire the ability to reprogram and become resistant. Organoid culture is a valuable tool in our research because it allows the formation of structures that include multiple cell types. In the future, we will use organoids of aggressive prostate cancer in screens of drug candidates and assess drug effectiveness in weeks, rather than the months or years required for classic in vivo studies.

Radiation therapy is a critical tool for the treatment of brain tumors, however, exposure to high doses of ionizing radiation (IR) causes numerous central nervous system side-effects, including declines in cognitive function, memory, and attention. Brain injury from IR is characterized by numerus inflammatory effects, including white matter damage from the loss of myelin-producing oligodendrocyte cells. While neuro-oncology outcomes are often concerned with survival, strategies to understand and ameliorate radiation-induced damage after IR treatment are needed to preserve and improve patient quality of life. Our lab is interested in studying the differential effects of radiation on oligodendrocyte cells, as they comprise the majority of white matter in the brain, and methods to halt radiation-induced damage. We have established a mass spectrometry-based metabolomics method to study radiation-injury in cells, tissue, and biofluids, and are applying this technique to study radiation effects on the MO3.13 oligodendrocyte cell line.

We are currently investigating the ability of dimethyl fumarate (DMF), an established neuroprotective agent, to amend damage and demyelination to oligodendrocyte cells versus glioma cells, after X-irradiation. Using metabolomics, we noted that oligodendrocyte cells upregulated tricarboxylic acid (TCA) cycle intermediates in response to DMF treatment, with sustained levels after radiation. In addition, measured levels of glutathione were elevated, and markers for generalized oxidative stress were comparably lower with DMF pretreatment. Ultimately, this information could be used to prevent radiation-induced demyelination, promoting patient quality of life.