Abstract
An Organoid-based Screening Platform
Brain metastases occur in many cancers, and so the increased availability of physiologically relevant models would contribute to improved knowledge and therapeutic options. A recent article reported the development of an organoid-based platform that can be used to test drugs against brain metastases in a cost- and time-effective manner.1 The approach, referred to as METPlatform, employed organotypic cultures derived from established brain metastases (from lung and breast cancer) and used bioluminescence imaging and immunofluorescence as the viability readouts. The team assessed the anti-tumoral activity of a library of 114 compounds and compared their results with those obtained with a traditional cell-based assay. Interestingly, the METPlatform selected hits that would not have been considered with other traditional approaches (7 out of 17 hits). The platform permits the assessment of not only the established metastases, but also the earlier stages of the disease, as initial organ colonisation can be mimicked ex vivo by plating cancer cells on top of tumour-free organotypic brain cultures. In addition, by using specific markers for different brain cell types, it would be possible to evaluate potential unselective toxicity toward non-cancer cell types.
The paper illustrates how METPlatform was used to study the vulnerabilities of macrometastases, and how it identified heat shock protein 90 (HSP90) as a potential target. METPlatform does not consider blood–brain barrier permeability, but the team selected and tested a brain-permeable second-generation HSP90 inhibitor (DEBIO-0932), which showed high potency against mouse and human metastases. Furthermore, they used in situ proteomics to probe the downstream effects of HSP90 inhibition, identifying novel mediators of brain metastasis, biomarkers of the disease, and combination strategies to overcome resistance.
An advantage of this platform is its potential to bridge the gap between animal and human studies, as it can be adapted to include patient-derived organotypic cultures by using fresh surgically resected human tissue. The authors pointed out that: “Given the limited efforts to test drugs currently available or under clinical trials in patients with brain metastasis, METPlatform provides an additional strategy to generate initial data on this potential application.”1
Reference
1. Zhu L, Retana D, García-Gómez P, et al. A clinically compatible drug-screening platform based on organotypic cultures identifies vulnerabilities to prevent and treat brain metastasis. EMBO Mol Med 2022; 14: e14552. DOI: 10.15252/emmm.202114552.
An In Vitro Lung Exposure System
Exposure systems that mimic cell exposure to airborne substances have the potential to replace animals, but they must reproduce the in vivo conditions in the lungs (i.e. 37°C and 100% relative humidity) — this represents an important technical challenge. In air–liquid interface (ALI)-based exposure approaches, the test samples are delivered directly to the apical side of cells on inserts inside an exposure chamber. Thus, the conditions inside the exposure chamber (e.g. temperature, relative humidity, airflow) should promote optimal cell health and be well-defined for a given system — however, this information is often lacking or incomplete in many publications. Furthermore, while many ALI exposure approaches exist, in order to gain regulatory acceptance, standardisation and harmonisation are crucial.
A study by Guénette et al.1 aimed to address some of these challenges by developing a reliable and reproducible system to expose cells to gases at the ALI. They set out to establish a way of verifying and optimising ALI exposure conditions — more specifically, the temperature, relative humidity, airflow parameters and dose. The senior author, Errol Thomson, told ATLA: “Air–liquid interface exposures offer the potential to conduct toxicity testing of airborne pollutants using human cell and tissue models rather than using animal models. Unlike in conventional submerged cell culture exposures, lung cells cultured at the air–liquid interface can be directly exposed to air pollutants, meaning that we can conduct exposures that better model conditions in the lungs. As air–liquid interface exposures are technically challenging, establishing conditions that maintain cell viability and enable reproducible delivery of airborne pollutants to the cells is an important first step to ensuring that effects can be attributed to the pollutant and not be obscured by technical variability. Better modelling of human exposures should ultimately increase the utility of the data for regulatory purposes.”
The team focused on a horizontal-flow ALI exposure system, which is a specific type of exposure whereby airflow is directed horizontally above the cells. To verify and optimise testing conditions, particularly those affecting the viability of lung epithelial cells (A549), they determined temperature and humidity/condensation throughout the system, while exposure stability, dosimetry and toxicity were tested with the air pollutant, ozone. They found that, for optimal cell viability during the 2-hour exposure period to clean air, it was crucial that the defined temperature and relative humidity of the environment within the exposure chamber was reached and maintained.
As the authors rightly pointed out, understanding the exposure conditions and optimising cell viability are crucial, because test substance-exposed cells are usually compared to control cells. Thus, any effects resulting from poorly optimised exposures could influence the conclusions made regarding the actual effects of the test substances.
Reference
1. Guénette J, Breznan D and Thomson EM. Establishing an air–liquid interface exposure system for exposure of lung cells to gases. Inhal Toxicol 2022; 34: 80–89.
Zebrafish Model for Anti-TB Compounds
The emergence of drug-resistant strains of Mycobacterium tuberculosis highlights the need for novel antibiotics against tuberculosis (TB). The current approach for the identification of lead compounds for TB treatment (i.e. phenotypic high-throughput screening (HTS)) is biased toward lipophilic and poorly water-soluble molecules — characteristics that are usually associated with unfavourable pharmacokinetic activity and which are more difficult to evaluate in subsequent testing. In an attempt to overcome some of these hurdles, Knudsen Dal et al.1 exploited a zebrafish embryo TB model, which relies on infection with a genetically-close relative of M. tuberculosis (Mycobacterium marinum) that results in a disease that recapitulates many key features of the early stages of M. tuberculosis infection.
The team used the embryo model to evaluate the in vivo toxicity and efficacy of five nitronaphthofuran (nNF) compounds that had been shown to have anti-M. tuberculosis activity by using phenotypic HTS. To improve the solubility and administration of the test molecules, the team formulated them in biocompatible polymeric micelles. They delivered the test drugs directly to the zebrafish embryos by intravenous injection, which avoided issues related to their delivery through bath immersion (e.g. unclear route of administration and drug uptake). This approach permitted the administration of precise and reproducible doses of the lipophilic compounds, and required only minimal amounts of the chemicals, which can be an important factor to consider. Based on the data obtained with the set of in vivo assays, three of the five compounds were shown to be the most promising for further development, due to their low toxicity and their ability to significantly reduce bacterial load and improve embryo survival.
The team pointed out that toxicity data from in vitro assays do not always correlate well with the in vivo situation as one of the compounds showed minimal toxicity in the cell-based assay but was highly toxic to the zebrafish embryos. The authors stated that: “In conclusion, the zebrafish embryo can fill a void in the preclinical evaluation of new TB drug candidates by bridging the gap between in vitro studies in bacterial or mammalian cell cultures and time- and cost-intensive in vivo studies in mammalian TB models.”1
Reference
1. Knudsen Dal NJ, Speth M, Johann K, et al. The zebrafish embryo as an in vivo model for screening nanoparticle-formulated lipophilic anti-tuberculosis compounds. Dis Model Mech 2022; 15: dmm049147.
Formation of Microvasculature in 3-D Tissues
In vitro models based on 3-D cultures are more physiologically relevant than 2-D cultures, but the absence of microvasculature limits their ability to mimic native tissue. While various strategies exist to introduce some level of vasculature in 3-D tissues, issues related to lack of reproducibility, or potential damage to cells and scaffold, remain a challenge. A recent paper proposed a new approach, referred to as cavitation moulding, which is based on laser-induced cavitation and relies on femtosecond infrared laser pulses for non-ablative restructuring of collagen hydrogels. In contrast to laser-based photoablation that degrades the exposed material to create cavities, this approach creates cavities due to the expansion of the cavitation bubbles and the generation of a denser shell around the bubble.
The team generated fibrillar type I collagen hydrogels, and used near-infrared femtosecond laser irradiation to generate channels and cavities inside the hydrogel scaffolds. They successfully populated the scaffolds with a liquid suspension of human umbilical vein endothelial cells (HUVECs), which confirmed that the channels could be used as guiding structures and that their surface permitted cell adhesion, survival and proliferation. The 3-D patterning approach was also tested on hydrogels previously populated with cell spheroids (U87 glioblastoma cells), showing that artificial blood vessels can also be created on cell-containing hydrogels without damaging the cells.
Furthermore, the compatibility of cavitation moulding with different proportions of collagen hydrogel was also assessed. Results showed that the efficiency of cavity formation and the uniformity of channel diameter increased with the concentration of collagen in the hydrogel, while the resulting channel diameter decreased with increasing collagen concentration (possibly because the hydrogel becomes stiffer and limits the expansion of the cavitation bubble). Thus, a minimum fibre density and elastic response is thought to be required to permit cavitation moulding. While only collagen hydrogels were assessed, the team believes that cavitation moulding should work on any material that can support local permanent densification and that the technique should be compatible with most cell lines that thrive in soft hydrogels.
They summarised that: “Thus, our approach for 3D patterning collagen scaffolds by cavitation molding enables design freedom and flexibility for realizing 3D tissue models with unprecedented complexity, which hold great promise for applications in drug development, medical research, and artificial organs, leading to exciting new research.”1
Reference
1. Enrico A, Voulgaris D, Östmans R, et al. 3D Microvascularized tissue models by laser-based cavitation molding of collagen. Adv Mater 2022; 34: e2109823.
Nominations for Lush Prize 2022
Since 2012, the Lush Prize has encouraged and rewarded activity to end animal use in toxicity testing, specifically focusing on the ‘One R’ of absolute replacement rather than all Three Rs. The various prize categories are specifically designed to connect the various strands of work required to introduce non-animal testing methods and get them accepted by regulatory bodies.
The Lush Prize 2022, with a global prize fund of £250,000 to support initiatives to end or replace animal testing, has opened for nominations. The five main categories are Science, Training, Young Researcher, Lobbying and Public Awareness. There is also a non-financial Political Achievement Award (for elected political officials).
Submissions are welcomed from across the whole world and nominations close on Friday 17 June 2022. Further information on each award category can be found at lushprize.org