PhD program in Basic and Applied Oncology Prof. Marco E. Bianchi Title: Establishing human mesothelioma organoids Project overview:

Malignant Pleural Mesothelioma (MPM) is currently an incurable cancer that develops many decades after inhalation of asbestos dust. Few treatment options exist and most patients die within a year (1,2). Previous clinical trials of cytotoxic drugs, targeted agents and immune checkpoint inhibitors have disappointed. The MPM tumour genome is dominated by tumour suppressor loss, a low mutational burden and few oncogenic drivers, posing major challenges for drug development and a new approach is urgently required. In animal models, longitudinal studies demonstrate epigenomic events that precede evolution of MPM, providing a clear rationale for precision prediction and early treatment. Similar longitudinal studies in humans could effectively direct new drug development, but have so far been impossible due to the non-availability of matched pre-MPM/MPM tissues.

We are thus developing patients-derived mesothelioma organoids, comprising mesothelial cells, but then also reconstituted with the immune cell population, given the dominant role of macrophages in asbestos-related pleural inflammation, and their link to disease evolution via ROS-induced DNA damage (3).

Data from single-cell transcriptomics of donor MPM biopsies (n=5) and first-passage organoids (n=6) demonstrates a large fraction of ‘mesenchymal’ cells, plus clusters of macrophages, B and T cells and neutrophils (in that order of abundance), endothelial cells and rare fibroblasts. However, the immune cells are rapidly depleted; transcriptomic analyses focused on the processes enriched in these models are underway. Therefore, our immediate goal will be to re-supplement organoids with macrophages. We will later investigate the epigenomic changes in macrophages that follow theirincorporation into organoids.

In brief, we will provide a pre-clinical platform for initial drug screen targeted to mesothelioma, and subsequent in vivo validation. This is part of a large Accelerator collaboration to be funded by CRUK-AIRC-AECC. The Ph.D. student will: - learn to establish and culture organoids - learn bioinformatic approaches to single-cell transcriptomics - use organoids to test new drugs, as part of the Accelerator collaboration. REFERENCES 1. Beckett, P. et al. Demographics, management and survival of patients with malignant pleural mesothelioma in the National Lung Cancer Audit in England and Wales. Lung cancer 88, 344–348 (2015). 2. Kidd, A. C. et al. Survival prediction in mesothelioma using a scalable Lasso regression model: instructions for use and initial performance using clinical predictors. BMJ Open Resp Res 5, e000240 (2018). 3. Benedetti, S., Nuvoli, B., Catalani, S. & Galati, R. Reactive oxygen species a double-edged sword for mesothelioma. Oncotarget 6, 16848–16865 (2015).

Tumours from xenografts maintained thesame morphology and IHC pattern as theinitial tumour. Organotypics were obtainedfrom 2 different xenografts, indicating biopsyfragments can be expanded to obtain these

Figure 13. Summary of MPM Organotypic Model Development. This technology will be expanded in WP3.1, by Bianchi, including reconstitution of the macrophage component, and used for initial drug screening validation

Milan Brescia

Histopathological Characterisation, Standard MPM IHC markers

17 MPM Epithelioid MPM organotypics grown in matrigel on days 4-8-12 post-seeding. These form various structures; round and compact, small tubes or 'clusters’. 1st generation organotypics grow to a diameter ~200 µm, after 1st passage they grow slowly.

Expansion into PDX modelsMPM biopsy fragmentsimplanted into NOD-SCIDGamma (NSG) mice (n=23)

4 x 1st generation xenograft1 x 2nd generation organotypics

15x 1st generation organotypic7x 2nd generation organotypic5x 3rd generation organotypic1x 4th generation organotypic

MPM Patient: MOSR3

Multiple Biopsy Fragments:Ø Histology/Standard IHC: Mesothelin/Calret/CK5/WT1+Ø DNA extraction, WES, RNA Seq and scRNA SeqØ Organotypic models up to 4th generationØ PDX mice

Organotypic structure with an internal-external polarity; central collagen matrix; IHC pattern maintained

Fresh Frozen tissue collected from 23 patients with MPM and 4 with benign disease at Surgery

Novara

Adapted from data provided by M Bianchi47