Cancers, Vol. 14, Pages 5836: The Chick Embryo Xenograft Model for Malignant Pleural Mesothelioma: A Cost and Time Efficient 3Rs Model for Drug Target Evaluation

1. IntroductionMalignant pleural mesothelioma (MPM) is an aggressive cancer of the lung lining, mainly caused by environmental exposure to asbestos. Despite strict asbestos regulation in most high-income countries, the global incidence of MPM has not yet peaked, and it is predicted to remain a significant cause of morbidity and mortality for decades [1]. MPM is commonly categorised as three main histopathological types, epithelioid, sarcomatoid or biphasic, which may represent a continuous spectrum of disease [2]. Invasion into the surrounding stroma is a diagnostic criterion [3]. MPM has extensive interactions with immune and stromal cells in the tumour microenvironment, in particular fibroblasts and endothelial cells. Asbestos causes fibrotic disease, with a desmoplastic reaction often evident in MPM that suggests involvement of cancer-associated fibroblasts (CAFs) [4]. Indeed, MPM recruits and activates CAFs to promote tumour progression through growth factor and cytokine networks in murine models [5]. Tumour angiogenesis also plays an important role in the pathogenesis of MPM, which commonly express vascular endothelial growth factor (VEGF) [6].MPM is almost invariably incurable, even in early presentation, and chemotherapy benefits are very modest. Combination cisplatin and pemetrexed was the mainstay of treatment for over 20 years [7], with potential additional benefit from the VEGF antibody bevacizumab [8], until the recent licencing of immunotherapy. Combined nivolumab and ipilimumab treatment provides real clinical benefit [9], however not all patients are eligible, and most who are treated progress within a year. Therefore, an urgent need for further treatment options remains. Other targeted therapies have been slow to emerge for MPM, which lacks common oncogenic drivers, and instead is characterised by loss-of-function mutations in tumour suppressors, most commonly BAP1, P16 and NF2 [10,11]. Nevertheless, genetic loss of tumour suppressors or other epigenetic alterations may sensitise MPM to targeted therapies, with arginine deprivation and CDK4/6, PARP or EZH2 inhibitors now showing promise in early phase trials [12,13,14,15]. However, benefits vary widely with histological and genetic features in this heterogeneous disease and may be restricted to a small percentage of patients, whilst robust predictive biomarkers remain elusive. As more complete understanding of MPM biology emerges this offers hope for new therapeutic strategies and suitable patient stratification. However, to realise this potential, effective preclinical models are required to translate discovery science into clinical application.Preclinical models that are currently used or in development for MPM were recently reviewed [16,17]. More than 100 cell lines derived from human MPM are available for in vitro culture to investigate cell biology or drug sensitivity [16]. Their inherent drawbacks, such as adaptation during two-dimensional (2D) culture, and a lack of heterogeneity, 3D architecture or microenvironment interactions, may be partially mitigated by using a sufficient number and diversity of low passage cell lines, growth as spheroid cultures [18], or co-culture with immune or stromal cells [19,20]. Organoid or explant models that better recapitulate tumour heterogeneity, 3D structure, and microenvironment in vitro require access to fresh MPM patient tissue. Explant cultures have been used successfully in MPM [21,22] although explants typically retain tumour architecture for just a few days and are prone to necrosis [23]. In contrast, proposed organoid cultures for MPM [16] could be sustained in longer term culture but lack contextual architecture. Currently, a vascularised 3D architecture and microenvironment can only be achieved using in vivo models in protected animals. Established human MPM cell lines or patient-derived tissues may be used for sub-cutaneous or orthotopic xenografts in immune compromised mice, although this typically precludes study of tumour/immune cell interactions [24,25,26]. Alternative immunocompetent in vivo MPM models rely on induction by asbestos or long carbon nanotubes in rodents, which can accelerate MPM development in genetically engineered mice with mutation of relevant tumour suppressor genes [27,28,29,30,31,32]. However, in addition to being expensive and slow to establish, these models often rely on procedures of high severity, and their utility may be limited by variable histopathology and species-specific gene associations [16]. Thus, a spectrum of MPM preclinical models is required for prioritising new therapeutic candidates and modelling different aspects of the human disease, to deliver meaningful preclinical data whilst supporting the replacement, reduction, and refinement of the use of animals (3Rs). Notably, a 3Rs compliant in vivo model that recapitulates aspects of 3D tumour architecture and microenvironment is currently lacking for MPM.Fertilised hen’s eggs provide a non-protected in vivo model that does not require an animal licence until a specified stage of embryonic development, as defined by national regulations; in the UK this is two thirds of the gestation period. The chick embryo chorioallantoic membrane (CAM) is a rich source of oxygen and growth factors [33,34] and was first used to grow chicken sarcoma cells over a century ago [35]. A wide range of human cancer cells including melanoma, neuroblastoma, breast, colorectal and pancreatic cancer have subsequently been xenografted on the CAM [33]. However, despite one study providing proof of principle for use of patient-derived mesothelioma tissue [36], this has not been adopted by the field, and no CAM models have yet been developed with MPM cell lines. For other cancers, 3D vascularised tumours form within days, providing a rapid and cost-effective alternative to rodent xenografts. The chick embryo model allows monitoring of the major hallmarks of cancer including proliferation, angiogenesis, invasion, and metastasis, whilst recent studies demonstrate the feasibility of drug administration and utility of preclinical imaging [33,34,37,38,39,40]. We propose that incorporating such methodology into new MPM-CAM models will enhance the drug testing pipeline, for example by providing initial in vivo evaluation to reduce the use of protected animal models. We set out to develop robust standard operating protocols (SOPs) for establishing MPM-CAM xenograft models and to characterise the biology of the tumour nodules, which could in future enable a stratified approach to drug assessment.

Accordingly, we report here validated protocols that allow the efficient generation of xenografts on the CAM using a panel of well-characterised low passage MPM cell lines, which represent the spectrum of histopathology and tumour suppressor inactivation seen in the human disease. Importantly, the engrafted MPM nodules exhibit interactions with chick fibroblasts and vasculature, mimicking key facets of clinical MPM that are absent from in vitro cultures and which may influence therapeutic outcomes. We also show that bioluminescence imaging (BLI) can readily be used to evaluate MPM tumour burden over time, reducing inter-egg variability in tumour nodule measurements to minimise the number of embryos required to power studies. The protocols we describe for this new MPM-CAM preclinical model make it amenable to rapid, medium throughput evaluation of MPM cell biology in relation to genetics or therapeutics. Readouts, including multimodal imaging, transcriptional and histopathological analysis can be combined to monitor tumour size, survival, vascularisation, invasion, stromal composition, and proliferative capacity, and may in future be expanded to include markers of drug sensitivity or target engagement.

2. Materials and Methods 2.1. Cell Lines and Cell CultureThe MSTO-211H mesothelioma cell line [41] was obtained from ATCC. The other mesothelioma cell lines: MESO-7T, MESO-8T, MESO-12T, MESO-23T and MESO-29T [42] or MPM#2, MPM#26, MPM#26 and MPM#34 [43] were supplied by MesobanK [44] (MesobanK, Cambridge, UK). All cell lines were grown in RPMI-1640 Glutamax (Gibco, Waltham, MA, USA) with 10% foetal bovine serum (Gibco, Waltham, MA, USA) in a 5% CO2 humidified incubator at 37 °C. Growth media for cell lines designated MESO was supplemented with 20 ng/mL EGF (Peprotech, London, UK), 1 mg/mL hydrocortisone (Sigma, St. Louis, MO, USA) and 2 mg/mL heparin (Sigma, St. Louis, MO, USA). Cell lines were split every 3–4 days at approximately 80% confluency and grown for a maximum of 20 passages. All cell lines were authenticated by STR profiling and routinely confirmed as mycoplasma free. 2.2. Dual Labelling of Cell LinesMesothelioma cell lines were transduced with lentiviral particles carrying pHIV-Luc-ZsGreen (a gift from Bryan Welm; RRID: Addgene_39196). Lentiviral particle generation and cell line transduction were performed as previously described [45]. Transduction efficiency was assessed via fluorescence using a Nikon Eclipse Ti and CFI Plan-Fluor 10X (N.A.0.3) objective (Nikon, Tokyo, Japan). 2.3. In Vitro Bioluminescence Measurement

Dual labelled cells were seeded in 96 well plates (black walled, clear bottom; Corning, Glendale, AZ, USA; cat#3603) using a 1:2 serial dilution to give a range of 500 to 500,000 cells/well. The luciferase assay was performed after 4 h once cells had adhered to the plate. Media was replaced with 100 µL media containing 150 µg/mL luciferin (Promega, Madison, WI, USA; E1605). After 10 min incubation at room temperature, the bioluminescence signal was acquired using an IVIS Spectrum In Vivo Imaging System (Perkin Elmer, Waltham, MA, USA; Open filter, FOV = C, Imaging subject: in vivo). All bioluminescent signal was quantified using the Living Image Software (IVIS Imaging Systems, Perkin Elmer, Waltham, MA, USA).

2.4. Xenograft Generation

Fertilised Bovan Brown eggs (Henry Stewart Co., Ltd., Fakenham, UK) were incubated at 37 °C and 45% humidity (embryonic day 0; E0) in a specialised poultry incubator (Brinsea OvaEasy 380 Advance EX Series II Automatic Egg Incubator). Eggs were laid horizontally in incubation trays (Brinsea, Weston-super-Mare, UK), and the upward facing side marked to indicate the location for the window to be cut. For the duration of E0–E3, the incubator shelves were set to alternate tilting 45 degrees every 45 min. For in ovo experiments, E3 eggs were windowed by puncturing the wide base of the egg (air cell) with an egg piercer to remove about 5 mL of albumen with a 23 G needle, before sealing the hole with Nev’s Ink tape. Another hole was pierced on the labelled side of the egg, a 3 cm piece of 25 mm 3 M Scotch Magic invisible tape applied, and sharp scissor tips inserted into the pierced hole to carefully cut three sides (2 cm × 1 cm × 2 cm) of a rectangle to create a window in the eggshell. The fenestration area was sealed with approximately 4 cm of 25 mm 3 M Scotch Magic invisible tape, leaving a small tab to enable re-opening of the window, and eggs were placed back into the incubator until E7.

For ex ovo experiments, eggs were gently cracked at E3 and the contents transferred to UV-sterilised black weighing boats (Starlab, Hamburg, Germany). These were placed inside sterile 150 cm2 tissue-culture flasks with re-closable lids (Techno Plastic Products AG, Trasadingen, Switzerland) containing sterile water to maintain humidity and incubated until E7 in a Brinsea OvaEasy 360 incubator.

Prior to implantation on E7, cells were collected by trypsinisation, counted, washed in sterile phosphate-buffered saline (PBS), and pelleted via centrifugation. Experiments were optimised by implanting between 0.5 × 106 and 2 × 106 cells per egg in 10–15 µL; with 2 × 106 cells implanted on a minimum of 12 eggs per cell line for the main experiments. Prior to adding the cells, the CAM was traumatised using a 1 cm wide strip of sterile gauze swab, causing a small bleed. The cells were directly pipetted onto this region of the CAM, without any support structure such as Matrigel or a silicon ring. Eggs were resealed and incubated until E14. Chick survival and tumour progression were monitored during experiments, with bioluminescence imaging (BLI) performed prior to fluorescence imaging and tumour dissection at E14.

2.5. In Vivo Bioluminescence Imaging (BLI)

Prior to dissection on E14, viability of tumours was routinely assessed by BLI with images acquired using an IVIS Spectrum In Vivo Imaging System (Open filter, FOV = C, Imaging subject: Other). For BLI, 250 µL of 15 mg/mL luciferin (Promega, Madison, WI, US; E1605) was injected into the yolk sac using a BD Micro-Fine 0.5 mL insulin syringe with 29 G needle (BD Biosciences, Franklin Lakes, NJ, USA), avoiding blood vessels. To determine the optimal post-injection timepoint for in vivo imaging, a chick with a visible tumour nodule at E14 was selected for each dual-labelled cell line and bioluminescence images acquired every 3 min for 2 h. A steady state maximal signal was reached by 45 min in each case, so this timepoint was used for all image acquisition. Where longitudinal BLI was carried out, the same procedure was followed on the indicated days. Bioluminescent signal was reported as total flux (radiance: p/sec/cm2/sr).

2.6. Fluorescence Imaging and Tumour Dissection

Following BLI at E14, tumours were imaged under a Leica M165FC fluorescence stereomicroscope with 16.5:1 zoom optics, fitted with a Leica DFC425 C camera (Leica Biosystems, Wetzlar, Germany). Tumours were imaged in ovo on the CAM and then dissected. Briefly, extra-fine straight-tip tweezers were utilised to manipulate the CAM to allow a sizeable circumference to be cut around the tumour nodules with spring bow micro scissors. Excised tumour nodules were placed in sterile PBS for ex ovo imaging. After removing any excess CAM, dissected tumours were weighed on a fine balance, and processed for immunohistochemical or transcriptional analyses. Following removal of the tumour, embryos were terminated in accordance with the UK Animals Scientific Procedures Act 1986 (amended 2012), under which the chick embryo is classified as non-protected until two thirds of gestation is reached at E14. No home office approval is required, and procedures were reviewed by the Liverpool Animal Welfare and Ethical Review Body.

2.7. Immunohistochemistry

Immediately after dissection tumour samples were placed in 1 mL 10% neutral buffered formalin (Sigma, St. Louis, MO, USA) for 16 h and then transferred to 70% ethanol. Following automated tissue processing, samples were embedded in paraffin blocks and sectioned at 4 µm onto SuperFrost Plus slides. Immunohistochemical staining was performed on the fully automated Leica BOND RXM. Primary antibodies: mouse anti-Pan cytokeratin (MNF116; Invitrogen, Walthan, MA, USA; MA1-26237 [human specific]; or AE1/AE3; Santa Cruz, Dallas, TX, USA; sc-81714 [human and chicken reactivity]) used at a dilution of 1:80 and pH9; rabbit anti-αSMA (Abcam ab5694 [human and chicken reactivity]) 1:200 and pH9; and mouse anti-Ki-67 (Novocastra, Newcastle-upon-Tyne, UK; NCL-L-Ki67-MM1) 1:200 and pH9. Antibody binding was visualised with diaminobenzidine (DAB), samples were counterstained with haematoxylin to assist in distinguishing between human tumour and chick cell nuclei, and sections were mounted with DPX mountant (Sigma, St. Louis, MO, USA). Images of the slides were acquired using digital slide scanners (Leica Aperio CS2 digital slide scanner or Zeiss Axioscan Z1).

2.8. Digital Image Analysis of Ki-67Digitalised whole slide images of tumour nodules stained for Ki-67 were scored using open-source, digital pathology software QuPath version 0.2.0-m7 [46]. Briefly, 10 DAB-positive and 10 DAB-negative cells were manually selected as a training cohort to identify Ki-67 positive and negative cells, respectively. The tumour nodule was delineated manually using the wand tool prior to watershed nucleus detection (settings: requested pixel size 0.5 μm; background radius 8.0 μm; sigma 1.5 μm; min/max area 10/400 µm; threshold 0.1; maximum background intensity 2.0; and cell expansion 5 μm), which was optimised visually. The detection threshold value for Ki-67 positive cells (nucleus DAB OD mean) was manually set to 0.1 for all slides, following visual assessment. All measurements were exported into Excel to calculate the percentage of Ki-67 positive cells. 2.9. RNA Extraction and qRT-PCRTumours harvested from the CAM were rinsed in ice-cold PBS and transferred to RNAlater (Ambion, Life Technologies, Carlsbad, CA, USA). Total RNA was extracted as previously described [45] using a NucleoSpin RNA Tissue Kit (Macherey-Nagel, Dueren, Germany). For cell lines cultured in vitro, total RNA was extracted as previously described [47] using RNeasy columns (Qiagen, Hilden, Germany). Complementary DNA was reverse transcribed from 1μg RNA with RevertAid H-minus M-MuLV reverse transcriptase (Fermentas, Waltham, MA, USA) using an oligo-dT primer (Promega, Madison, WI, USA). qRT-PCR was performed using a CFX real-time PCR detection system (Bio-Rad, Hercules, CA, USA) and SYBR Green. Primer sequences were: GAPDH (for: 5′-CAATGACCCCTTCATTGACC-3′, rev: 5′-GACAAGCTTCCCGTTCTCAG-3′), ACTB (for: 5′-CACCTTCTACAATGAGCTGCGTGTG-3′, rev: 5′-ATAGCACAGCCTGGATAGCAACGTAC-3′), MMP9 (for: 5′-TTCTGCCCGGACCAAGGATA-3′, rev: 5′-ATGCCATTCACGTCGTCCTT-3′) and VEGF (for: 5-CTCCACCATGCCAAGTGGTC-3′, rev: 5′-GCAGTAGCTGCGCTGATAGA-3′). Relative transcript expression is reported for genes of interest normalised to the mean values for ACTB and GAPDH (2ΔCq). 2.10. Analysis of CAM Vasculature

Microscopy RGB images of tumour nodules on top of the CAM were cropped to an area of approximately 1000 µm around the tumour nodule. A region of interest excluding the tumour was selected for analysis. The IKOSA CAM Assay (KML Vision, Graz, Austria) was used to determine the mean vessel thickness, as well as the total vessel area, total vessel length, and number of branching points normalised to the area analysed.

2.11. Magnetic Resonance Imaging (MRI)

MRI was performed using a horizontal bore 9.4 T Bruker Biospec 94/20 USR system (Bruker Scientific Instruments, Billerica, MA, USA). Following 10 min incubation at 4 °C, the egg was placed in a custom-built cradle, and an actively decoupled 3 cm diameter surface coil was affixed on the eggshell above the site of the tumour. An 86 mm volume coil was used for signal transmission, while the surface coil was used for signal detection. Initially scout images were acquired using a three-plane gradient echo sequence to localise the tumour. High resolution 3D images were subsequently acquired using a 3D TurboRARE (spin-echo, T2-weighted) pulse sequence with the following parameters: field of view 40 mm × 40 mm, matrix size 400 × 400 × 20, TR/TE = 1800/6.8 ms, effective TE: 80.74 ms, echo train length 8, slab thickness = 2 mm, averages 1, flip angle 90, scan time of approximately 8 min.

2.12. Statistical Analysis

Statistical analyses were performed in GraphPad Prism version 9 for Mac (GraphPad Software, San Diego, CA, USA). Distribution of data was assessed by Shapiro–Wilk test and data were analysed by parametric or non-parametric tests as appropriate. The number of independent samples, definition of error bars, and the statistical test employed are described in relevant figure legends. p values less than 0.05 were considered significant.

4. Discussion

MPM remains an area of critical unmet clinical need, despite a growing understanding of the processes driving its development and spread. Capitalising on this is unfortunately slow and costly, with drug development often taking 10 years or more from concept to clinical application, and with an estimated cost of around a billion dollars in research costs for each drug entering practice. Research performed using cultured cell lines, or rodent models, has historically corresponded poorly to outcome in eventual clinical trials. Thus, a continuum of preclinical models is required to embody different aspects of a human disease to screen and validate drug responses. Here, we describe protocols and analysis for a novel 3Rs-compliant CAM model for MPM that is rapid, economical, scalable, and adaptable, and which covers the spectrum of MPM histopathological types and common genetic alterations. It provides a wide range of in vivo readouts of tumour biology and viability within days rather than months lending itself to development as a useful preclinical model.

The CAM proved to be a conducive environment for MPM, as all 10 cell lines that we tested engrafted very effectively, particularly the epithelioid MPM where, on average, viable nodules formed in 80% of cases. As the CAM model is very economical compared to in vivo approaches in rodents, this facilitates use of a sufficient number and diversity of low passage cell lines to capture some of the heterogeneity of MPM and encompass the common genetic changes. For future evaluation of therapeutics, this could facilitate identification of relatively small subgroups that are likely to respond and may prevent discard of viable compounds. The diverse readouts available for CAM xenografts can be tuned depending on the experimental question, enabling specific hallmarks of cancer to be monitored, for example when assessing anti-proliferative, anti-invasive or anti-angiogenic therapeutic compounds.

Xenografts are less able to recapitulate the histology of the original tumour when derived from cell lines established in 2D culture, compared to fresh patient derived samples, whatever the host organism. However, the CAM model enables MPM cell lines to adopt a 3D architecture, which is characteristic for each cell line and partly addresses the importance of the tumour microenvironment. We observed pronounced interaction of MPM tumour nodules with chick fibroblasts as well as the chick vasculature. Comparing MSTO-211H xenografts in SCID mice [5] with MSTO-211H CAM xenografts shows the same diffuse infiltration of morphologically similar αSMA-stained fibroblast-like cells (Figure S8). This could therefore provide a more holistic model to assess drug responses and potentially facilitate testing of therapeutics targeting fibroblast and MPM interactions, or tumour angiogenesis, which are less easily modelled in vitro. For example, CAFs in MPM produce CTGF, which promotes MPM growth and correlates with survival outcomes, providing a potential therapeutic target [4,54]. The VEGF inhibitor bevacizumab improves chemotherapy outcomes for some patients [8] and, although other anti-angiogenic therapies have not been successful in trials, there remains an interest in targeting abnormal tumour vasculature in MPM [55].Like immunodeficient mouse models, at early stages of embryonic development the chick lacks a fully functioning immune system which facilitates engraftment of human tumour cells on the CAM. The chick immune system develops during the period when tumour nodules grow, and by E18 fully immunocompetent chick lymphocytes can be detected [56]. However, in the UK non-protected models are terminated at E14 and, although immune cells including macrophages and lymphocytes are observed earlier in development [56], their presence in CAM xenografts at E14 has not been reported. Thus, in common with rodent flank xenografts in non-humanised models, CAM xenografts of cancer cell lines cannot be used to test immune modulators or inhibitors at E14. There is however potential to further increase the complexity of the CAM tumour microenvironment, by co-culture of MPM cells with autologous or heterologous human immune cells to investigate cellular interactions and potentially allow limited assessment of the effects of drugs on this interaction.One challenge in fully realising the potential of CAM models in assessing drug responses is selecting the best methodology to evaluate tumour growth or regression. Tumour dimensions may be estimated, or excised nodules weighed, however these methods have limitations. Using dual-labelled MPM cell lines allowed tumours to be monitored by both fluorescence and bioluminescence, and we found the latter invaluable in distinguishing viable tumour nodules that were fully engrafted and vascularised during the experimental timeline. Although the utility of BLI could potentially be limited by the transduction efficiency, our experience is that many cell types transduce with high efficiency, as was the case with all the MPM cell lines tested. The reliance of BLI on ATP makes it superior to fluorescence imaging as BLI only detects metabolically active tumour cells, whilst the reliance on transduced luciferase ensures that only tumour cells and not chick cells are quantified. The latter is particularly important for MPM CAM xenografts, where we saw substantial infiltration of fibroblast-like chick cells. Importantly, whilst the BLI signal showed moderate correlation with tumour weight, we found a high correlation of BLI with Ki-67 staining as an endpoint measure of viable tumour cells. BLI methods have been published for other CAM xenografted cancer cell types, for example urothelial carcinoma [39] and pancreatic ductal adenocarcinoma [37]. However, our methodology differs in using yolk sac injection of luciferin, rather than topical application, to provide reproducible delivery into viable vascularised tumours and facilitate longitudinal measurement of tumour viability.In other studies, CAM xenografts have been successfully treated with drugs administered by topical application [37], intravenous injection [57], or allantoic/yolk sac injection [40]. The choice of administration route is in part influenced by the nature of the drug and any delivery vehicle. Whilst topical application is simple, it is less amenable to accurate dosing. Intravenous injection provides direct administration of drug via the tumour vasculature, but is technically challenging and may preclude multiple dosing, whilst allantoic/yolk sac injection is an easier route to enable drug delivery via the tumour vasculature that is more amenable to repeat dosing. Here, for the purposes of establishing an experimental timeline, we demonstrate yolk sac administration of both luciferin for BLI monitoring and double injection of a vehicle treatment. Using pre- and post-dosing BLI can substantially reduce the effect of inter-egg variability on estimation of tumour response to drugs, and so reduce the number of chick embryos required to fully power studies in line with 3Rs principles. Further studies will be required to refine drug delivery approaches using agents with greater activity in MPM.

While the MPM-CAM xenograft model cannot fully replace rodent or other higher organismal model systems, it provides a complementary 3Rs compliant model to study tumour biology that we believe will in future allow more efficient screening of targets and help identification of subgroups more likely to benefit from therapy. Our studies suggest that certain MPM cell lines with highly responsive BLI signals may be most amenable for testing anti-proliferative therapies, including MESO-7T, MESO12-T and MSTO-211H. In contrast, for anti-invasive therapies histological analysis of MESO-8T nodules would be preferable, whilst many cell lines appear appropriate for evaluating anti-angiogenic therapies using IKOSA CAM analysis combined with immunohistochemistry. Furthermore, these initial MPM-CAM xenograft models for cultured MPM cell lines open the door to ongoing development that can incorporate further aspects of the MPM tumour microenvironment and architecture, for example by co-engrafting MPM cell lines together with human fibroblasts and/or immune cells, and engrafting patient-derived cells or tissue explants coupled with alternative preclinical imaging methods.

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