Tissue Organoid Cultures Metabolize Dietary Carcinogens Proficiently and Are Effective Models for DNA Adduct Formation.

Human tissue three-dimensional (3D) organoid cultures have the potential to reproduce in vitro the physiological properties and cellular architecture of the organs from which they are derived. The ability of organoid cultures derived from human stomach, liver, kidney, and colon to metabolically activate three dietary carcinogens, aflatoxin B1 (AFB1), aristolochic acid I (AAI), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was investigated. In each case, the response of a target tissue (liver for AFB1; kidney for AAI; colon for PhIP) was compared with that of a nontarget tissue (gastric). After treatment cell viabilities were measured, DNA damage response (DDR) was determined by Western blotting for p-p53, p21, p-CHK2, and γ-H2AX, and DNA adduct formation was quantified by mass spectrometry. Induction of the key xenobiotic-metabolizing enzymes (XMEs) CYP1A1, CYP1A2, CYP3A4, and NQO1 was assessed by qRT-PCR. We found that organoids from different tissues can activate AAI, AFB1, and PhIP. In some cases, this metabolic potential varied between tissues and between different cultures of the same tissue. Similarly, variations in the levels of expression of XMEs were observed. At comparable levels of cytotoxicity, organoids derived from tissues that are considered targets for these carcinogens had higher levels of adduct formation than a nontarget tissue.

Haplotype-specific assembly of shattered chromosomes in esophageal adenocarcinomas.

The epigenetic landscape of cancer is regulated by many factors, but primarily it derives from the underlying genome sequence. Chromothripsis is a catastrophic localized genome shattering event that drives, and often initiates, cancer evolution. We characterized five esophageal adenocarcinoma organoids with chromothripsis using long-read sequencing and transcriptome and epigenome profiling. Complex structural variation and subclonal variants meant that haplotype-aware de novo methods were required to generate contiguous cancer genome assemblies. Chromosomes were assembled separately and scaffolded using haplotype-resolved Hi-C reads, producing accurate assemblies even with up to 900 structural rearrangements. There were widespread differences between the chromothriptic and wild-type copies of chromosomes in topologically associated domains, chromatin accessibility, histone modifications, and gene expression. Differential epigenome peaks were most enriched within 10 kb of chromothriptic structural variants. Alterations in transcriptome and higher-order chromosome organization frequently occurred near differential epigenetic marks. Overall, chromothripsis reshapes gene regulation, causing coordinated changes in epigenetic landscape, transcription, and chromosome conformation.

A suspension technique for efficient large-scale cancer organoid culturing and perturbation screens.

Organoid cell culture methodologies are enabling the generation of cell models from healthy and diseased tissue. Patient-derived cancer organoids that recapitulate the genetic and histopathological diversity of patient tumours are being systematically generated, providing an opportunity to investigate new cancer biology and therapeutic approaches. The use of organoid cultures for many applications, including genetic and chemical perturbation screens, is limited due to the technical demands and cost associated with their handling and propagation. Here we report and benchmark a suspension culture technique for cancer organoids which allows for the expansion of models to tens of millions of cells with increased efficiency in comparison to standard organoid culturing protocols. Using whole-genome DNA and RNA sequencing analyses, as well as medium-throughput drug sensitivity testing and genome-wide CRISPR-Cas9 screening, we demonstrate that cancer organoids grown as a suspension culture are genetically and phenotypically similar to their counterparts grown in standard conditions. This culture technique simplifies organoid cell culture and extends the range of organoid applications, including for routine use in large-scale perturbation screens.

CELLector: Genomics-Guided Selection of Cancer In Vitro Models.

Selecting appropriate cancer models is a key prerequisite for maximizing translational potential and clinical relevance of in vitro oncology studies. We developed CELLector: an R package and R Shiny application allowing researchers to select the most relevant cancer cell lines in a patient-genomic-guided fashion. CELLector leverages tumor genomics to identify recurrent subtypes with associated genomic signatures. It then evaluates these signatures in cancer cell lines to prioritize their selection. This enables users to choose appropriate in vitro models for inclusion or exclusion in retrospective analyses and future studies. Moreover, this allows bridging outcomes from cancer cell line screens to precisely defined sub-cohorts of primary tumors. Here, we demonstrate the usefulness and applicability of CELLector, showing how it can aid prioritization of in vitro models for future development and unveil patient-derived multivariate prognostic and therapeutic markers. CELLector is freely available at https://ot-cellector.shinyapps.io/CELLector_App/ (code at https://github.com/francescojm/CELLector and https://github.com/francescojm/CELLector_App).

Genomics-guided pre-clinical development of cancer therapies.

Since the approval of trastuzumab for the treatment of breast cancers more than two decades ago, many clinically effective targeted anti-cancer therapies have been developed. Here we consider the evidence that supports genomics-guided drug development and review the concept of oncogene addiction, including recent findings that inform this therapeutic approach. We consider non-oncogene addiction and how this synthetic-lethal paradigm could expand the range of new therapies, particularly for currently undruggable cancers. We discuss how CRISPR-based genetic screening is enhancing the ability to identify new targets. We conclude by considering opportunities for expanding the scope and refining the use of precision cancer medicines.

Pancreatic cancer organoids recapitulate disease and allow personalized drug screening.

We report the derivation of 30 patient-derived organoid lines (PDOs) from tumors arising in the pancreas and distal bile duct. PDOs recapitulate tumor histology and contain genetic alterations typical of pancreatic cancer. In vitro testing of a panel of 76 therapeutic agents revealed sensitivities currently not exploited in the clinic, and underscores the importance of personalized approaches for effective cancer treatment. The PRMT5 inhibitor EZP015556, shown to target MTAP (a gene commonly lost in pancreatic cancer)-negative tumors, was validated as such, but also appeared to constitute an effective therapy for a subset of MTAP-positive tumors. Taken together, the work presented here provides a platform to identify novel therapeutics to target pancreatic tumor cells using PDOs.

Erratum to: Drug Sensitivity Assays of Human Cancer Organoid Cultures.

The protocol Drug Sensitivity Assays of Human Cancer Organoid Cultures has now been made available open access under a CC BY 4.0 license.

Cell Model Passports-a hub for clinical, genetic and functional datasets of preclinical cancer models.

In vitro cancer cell cultures are facile experimental models used widely for research and drug development. Many cancer cell lines are available and efforts are ongoing to derive new models representing the histopathological and molecular diversity of tumours. Cell models have been generated by multiple laboratories over decades and consequently their annotation is incomplete and inconsistent. Furthermore, the relationships between many patient-matched and derivative cell lines have been lost, and accessing information and datasets is time-consuming and difficult. Here, we describe the Cell Model Passports database; cellmodelpassports.sanger.ac.uk, which provides details of cell model relationships, patient and clinical information, as well as access to associated genetic and functional datasets. The Passports database currently contains curated details and standardized annotation for >1200 cell models, including cancer organoid cultures. The Passports will be updated with newly derived cell models and datasets as they are generated. Users can navigate the database via tissue, cancer-type, genetic feature and data availability to select a model most suitable for specific applications. A flexible REST-API provides programmatic data access and exploration. The Cell Model Passports are a valuable tool enabling access to high-dimensional genomic and phenotypic cancer cell model datasets empowering diverse research applications.

Drug Sensitivity Assays of Human Cancer Organoid Cultures.

Drug sensitivity testing utilizing preclinical disease models such as cancer cell lines is an important and widely used tool for drug development. Importantly, when combined with molecular data such as gene copy number variation or somatic coding mutations, associations between drug sensitivity and molecular data can be used to develop markers to guide patient therapies. The use of organoids as a preclinical cancer model has become possible following recent work demonstrating that organoid cultures can be derived from patient tumors with a high rate of success. A genetic analysis of colon cancer organoids found that these models encompassed the majority of the somatic variants present within the tumor from which it was derived, and capture much of the genetic diversity of colon cancer observed in patients. Importantly, the systematic sensitivity testing of organoid cultures to anticancer drugs identified clinical gene-drug interactions, suggestive of their potential as preclinical models for testing anticancer drug sensitivity. In this chapter, we describe how to perform medium/high-throughput drug sensitivity screens using 3D organoid cell cultures.

Organoid cultures recapitulate esophageal adenocarcinoma heterogeneity providing a model for clonality studies and precision therapeutics.

Esophageal adenocarcinoma (EAC) incidence is increasing while 5-year survival rates remain less than 15%. A lack of experimental models has hampered progress. We have generated clinically annotated EAC organoid cultures that recapitulate the morphology, genomic, and transcriptomic landscape of the primary tumor including point mutations, copy number alterations, and mutational signatures. Karyotyping of organoid cultures has confirmed polyclonality reflecting the clonal architecture of the primary tumor. Furthermore, subclones underwent clonal selection associated with driver gene status. Medium throughput drug sensitivity testing demonstrates the potential of targeting receptor tyrosine kinases and downstream mediators. EAC organoid cultures provide a pre-clinical tool for studies of clonal evolution and precision therapeutics.

Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.

Human liver cancer research currently lacks in vitro models that can faithfully recapitulate the pathophysiology of the original tumor. We recently described a novel, near-physiological organoid culture system, wherein primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumors. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumor, allowing for discrimination between different tumor tissues and subtypes, even after long-term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumorogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug-screening testing and led to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized-medicine approaches for the disease.

What role could organoids play in the personalization of cancer treatment?

Cancer treatments are increasingly being targeted to specific patient populations based on the molecular and genetic features of their tumor, so called precision or personalized cancer medicine. Preclinical cancer models are essential tools for cancer research, but unfortunately our current models often fail to effectively represent patient tumors and can be poorly predictive of clinical responses. In this perspective, we discuss the use of new in vitro 3D cell models called 'organoids' as preclinical cancer models in the context of other commonly used models, namely cancer cell lines and patient-derived xenografts. We consider the relative strengths and limitations of each model, and discuss how organoid culture models could facilitate the personalization of cancer medicine.

Prospective derivation of a living organoid biobank of colorectal cancer patients.

In Rspondin-based 3D cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. We report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely recapitulate several properties of the original tumor. The spectrum of genetic changes within the "living biobank" agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell-line- and xenograft-based drug studies, and allow personalized therapy design. PAPERCLIP.

Fulvestrant-induced expression of ErbB3 and ErbB4 receptors sensitizes oestrogen receptor-positive breast cancer cells to heregulin ß1.

INTRODUCTION: We have previously reported that induction of epidermal growth factor receptor and ErbB2 in response to antihormonal agents may provide an early mechanism to allow breast cancer cells to evade the growth-inhibitory action of such therapies and ultimately drive resistant cell growth. More recently, the other two members of the ErbB receptor family, ErbB3 and ErbB4, have been implicated in antihormone resistance in breast cancer. In the present study, we have investigated whether induction of ErbB3 and/or ErbB4 may provide an alternative resistance mechanism to antihormonal action in a panel of four oestrogen receptor (ER)-positive breast cancer cell lines. METHODS: MCF-7, T47D, BT474 and MDAMB361 cell lines were exposed to fulvestrant (100 nM) for seven days, and effects on ErbB3/4 expression and signalling, as well as on cell growth, were assessed. Effects of heregulin ß1 (HRGß1) were also examined in the absence and presence of fulvestrant to determine the impact of ER blockade on the capacity of this ErbB3/4 ligand to promote signalling and cell proliferation. RESULTS: Fulvestrant potently reduced ER expression and transcriptional activity and significantly inhibited growth in MCF-7, T47D, BT474 and MDAMB361 cells. However, alongside this inhibitory activity, fulvestrant also consistently induced protein expression and activity of ErbB3 in MCF-7 and T47D cells and ErbB4 in BT474 and MDAMB361 cell lines. Consequently, fulvestrant treatment sensitised all cell lines to the actions of the ErbB3/4 ligand HRGß1 with enhanced ErbB3/4-driven signalling activity, reexpression of cyclin D1 and significant increases in cell proliferation being observed when compared to untreated cells. Indeed, in T47D and MDAMB361 HRGß1 was converted from a ligand having negligible or suppressive growth activity into one that potently promoted cell proliferation. Consequently, fulvestrant-mediated growth inhibition was completely overridden by HRGß1 in all four cell lines. CONCLUSIONS: These findings suggest that although antihormones such as fulvestrant may have potent acute growth-inhibitory activity in ER-positive breast cancer cells, their ability to induce and sensitise cells to growth factors may serve to reduce and ultimately limit their inhibitory activity.

Antihormone induced compensatory signalling in breast cancer: an adverse event in the development of endocrine resistance.

Using MCF7 breast cancer cells, it has been shown that antihormones promote expression/activity of oestrogen-repressed tyrosine kinases, notably EGFR, HER2 and Src. These inductive events confer responsiveness to targeted inhibitors (e.g., gefitinib, trastuzumab, saracatinib). We observed that these antihormone-induced phenomena are common to ER+HER2- and ER+HER2+ breast cancer models in vitro, where targeting of EGFR, HER2 or Src alongside antihormone improves antitumour response and delays/prevents endocrine resistance. Such targeted inhibitors also subvert acquired endocrine resistant cells which retain increased EGFR, HER2 and Src (e.g., TAMR and FASR models derived after 6-12 months of tamoxifen or Faslodex treatment). Thus, antihormone-induced tyrosine kinases comprise "compensatory signalling" crucial in limiting maximal initial antihormone response and subsequently driving acquired resistance in vitro. However, despite such convincing preclinical findings from our group and others, clinical trials examining equivalent antigrowth factor strategies have proved relatively disappointing. Our new studies deciphering underlying causes reveal that further antihormone-promoted events could be pivotal in vivo. Firstly, Faslodex induces HER3 and HER4 which sensitise ER+ cells to heregulin, a paracrine growth factor that overcomes endocrine response and diminishes antitumour effect of agents targeting EGFR, HER2 or Src alongside antihormone. Secondly, extended antihormone exposure (experienced by ER+ cells prior to adjuvant clinical relapse) can "reprogramme" the compensatory kinase profile in vitro, hindering candidate antigrowth factor targeting of endocrine resistance. Faslodex resistant cells maintained with this antihormone for 3 years in vitro lose EGFR/HER2 dependency, gaining alternative mitogenic/invasion kinases. Deciphering these previously unrecognised antihormone-induced events could provide superior treatments to control endocrine relapse in the clinic.