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Background Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient–derived plasma samples. Methods Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity. Results Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane–based methods, samples extracted with magnetic beads–based kits revealed an overall lower total ccfDNA yield (−29%; P Conclusions In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane–based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies.

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Previous studies have described that tumor organoids can capture the diversity of defined human carcinoma types. Here, we describe conditions for long-term culture of human mucosal organoids. Using this protocol, a panel of 31 head and neck squamous cell carcinoma (HNSCC)–derived organoid lines was established. This panel recapitulates genetic and molecular characteristics previously described for HNSCC. Organoids retain their tumorigenic potential upon xenotransplantation. We observe differential responses to a panel of drugs including cisplatin, carboplatin, cetuximab, and radiotherapy in vitro. Additionally, drug screens reveal selective sensitivity to targeted drugs that are not normally used in the treatment of patients with HNSCC. These observations may inspire a personalized approach to the management of HNSCC and expand the repertoire of HNSCC drugs.Significance:This work describes the culture of organoids derived from HNSCC and corresponding normal epithelium. These tumoroids recapitulate the disease genetically, histologically, and functionally. In vitro drug screening of tumoroids reveals responses to therapies both currently used in the treatment of HNSCC and those not (yet) used in clinical practice.See related commentary by Hill and D'Andrea, p. 828.This article is highlighted in the In This Issue feature, p. 813

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Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a ‘super engager’ behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies.

Background: Organoids are in vitro three-dimensional structures that can be grown from patient tissue. Head and neck cancer (HNC) is a collective term used for multiple tumor types including squamous cell carcinomas and salivary gland adenocarcinomas.Methods: Organoids were established from HNC patient tumor tissue and characterized using immunohistochemistry and DNA sequencing. Organoids were exposed to chemo- and radiotherapy and a panel of targeted agents. Organoid response was correlated with patient clinical response. CRISPR-Cas9-based gene editing of organoids was applied for biomarker validation.Findings: A HNC biobank consisting of 110 models, including 65 tumor models, was generated. Organoids retained DNA alterations found in HNC. Comparison of organoid and patient response to radiotherapy (primary [n = 6] and adjuvant [n = 15]) indicated potential for guiding treatment options in the adjuvant setting. In organoids, the radio-sensitizing potential of cisplatin and carboplatin could be validated. However, cetuximab conveyed radioprotection in most models. HNC-targeted treatments were tested on 31 models, indicating possible novel treatment options with the potential for treatment stratification in the future. Activating PIK3CA mutations did not predict alpelisib response in organoids. Protein arginine methyltransferase 5 (PRMT5) inhibitors were identified as a potential treatment option for cyclin-dependent kinase inhibitor 2A (CDKN2A) null HNC.Conclusions: Organoids hold potential as a diagnostic tool in personalized medicine for HNC. In vitro organoid response to radiotherapy (RT) showed a trend that mimics clinical response, indicating the predictive potential of patient-derived organoids. Moreover, organoids could be used for biomarker discovery and validation.

Adult stem cell-based organoid technology is a versatile tool for the generation and long-term maintenance of near-native 3D epithelial tissues in vitro. The generation of cancer organoids from primary patient material enables a range of therapeutic agents to be tested in the resulting organoid cultures. Patient-derived cancer organoids therefore hold great promise for personalized medicine. Here, we provide an overview of the protocols used by different groups to establish organoids from various epithelial tissues and cancers, plus the different protocols subsequently used to test the in vitro therapy sensitivity of these patient-derived organoids. We also provide an in-depth protocol for the generation of head and neck squamous cell carcinoma organoids and their subsequent use in semi-automated therapy screens. Establishment of organoids and subsequent screening can be performed within 3 months, although this timeline is highly dependent on a.o. starting material and the number of therapies tested. The protocol provided may serve as a reference to successfully establish organoids from other cancer types and perform drug screenings thereof.

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a near-native state. When derived from resected or biopsied tumor tissue, organoids closely recapitulate in vivo tumor morphology and can be used to study therapy response in vitro. Biobanks of tumor organoids reflect the vast variety of clinical tumors and patients and therefore hold great promise for preclinical and clinical applications. This paper presents a method for medium-throughput drug screening using head and neck squamous cell carcinoma and colorectal adenocarcinoma organoids. This approach can easily be adopted for use with any tissue-derived organoid model, both normal and diseased. Methods are described for in vitro exposure of organoids to chemo- and radiotherapy (either as single-treatment modality or in combination). Cell survival after 5 days of drug exposure is assessed by measuring adenosine triphosphate (ATP) levels. Drug sensitivity is measured by the half-maximal inhibitory concentration (IC50), area under the curve (AUC), and growth rate (GR) metrics. These parameters can provide insight into whether an organoid culture is deemed sensitive or resistant to a particular treatment.

Loss of histone 3 lysine 27 trimethylation (H3K27me3) has been described as a diagnostic marker for malignant peripheral nerve sheath tumor (MPNST), also discriminating MPNST with rhabdomyoblastic differentiation (malignant Triton tumor) from rhabdomyosarcoma (RMS). We studied the immunohistochemical expression of H3K27me3 in embryonal RMSs (ERMSs), performed methylation profiling in order to support the diagnosis and RNA-sequencing for comparison of the transcriptome of H3K27me3-positive and -negative cases. Of the 25 ERMS patients, 17 were males and 8 were females with an age range from 1 to 67 years (median, 6 years). None were known with neurofibromatosis type 1. One patient had Li-Fraumeni syndrome. Tumor localization included paratesticular (n = 9), genitourinary (n = 6), head/neck (n = 5), retroperitoneal (n = 4) and lower arm (n = 1). Five MPNSTs served as reference group. All ERMS had classical features including a variable spindle cell component. Immunohistochemical loss (partial or complete) of H3K27me3 was detected in 18/25 cases (72%). Based on methylation profiling, 22/22 cases were classified as ERMS. Using RNA sequencing, the ERMS group (n = 14) had a distinct gene expression profile in contrast to MPNSTs, confirming that the H3K27me3 negative ERMS cases do not represent malignant Triton tumors. When comparing H3K27me3-negative and -positive ERMSs, gene set enrichment analysis revealed differential expression of genes related to histone acetylation and normal muscle function with H3K27me3 negative ERMSs being associated with acetylation. Conclusion: Loss of H3K27me3 frequently occurs in ERMSs and correlates with H3K27 acetylation. H3K27me3 is not a suitable marker to differentiate ERMS (with spindle cell features) from malignant Triton tumor.

Summary Establishment of patient-derived adult stem cell-based pancreatic (tumor) organoids was first described in 2015. Since then, multiple laboratories have demonstrated the robustness of this method. We recently described the generation of a pancreatic cancer biobank containing 30 well-characterized tumor organoid models. Here, we describe the applied methods in detail. Use of tumor-selective media prevents contamination with normal cells. Generated organoids can be cryopreserved and can serve as a living biobank of pancreatic cancer for biomarker identification and drug screening. For complete information on the generation and use of this protocol, please refer to Driehuis et al. (2019)., Graphical Abstract, Highlights • Protocol to establish pancreatic patient-derived organoids from resections and biopsies • Detailed procedure to generate and expand both normal and tumor organoid models • Methods for freezing and thawing organoids to successfully build living biobanks, Establishment of patient-derived adult stem cell-based pancreatic (tumor) organoids was first described in 2015. Since then, multiple laboratories have demonstrated the robustness of this method. We recently described the generation of a pancreatic cancer biobank containing 30 well-characterized tumor organoid models. Here, we describe the applied methods in detail. Use of tumor-selective media prevents contamination with normal cells. Generated organoids can be cryopreserved and can serve as a living biobank of pancreatic cancer for biomarker identification and drug screening.

We have recently established a protocol to grow wildtype human oral mucosa organoids. These three-dimensional structures can be maintained in culture long-term, do not require immortalization, and recapitulate the multilayered composition of the epithelial lining of the oral mucosa. Here, we validate the use of this model to study the effect of Leucovorin (LV) on Methotrexate (MTX)-induced toxicity. MTX is a chemotherapeutic agent used in the treatment of pediatric acute lymphoblastic leukemia. Although effective, the use of MTX often results in severe side-effects, including oral mucositis, which is characterized by epithelial cell death. Here, we show that organoids are sensitive to MTX, and that the addition of LV reduces MTX toxicity, in both a concentration- and timing-dependent manner. Additionally, we show that a 24 hour 'pretreatment' with LV reduces MTX-induced cell death, suggesting that such a pretreatment could decrease mucositis in patients. Taken together, we provide the first in vitro model to study the effect of MTX on wildtype oral mucosa cells. Our findings underscore the relevance of the clinically applied LV regimen and highlight the potential of this model to further optimize modifications in dosing and timing of Leucovorin on oral mucosa cells.

WNT signalling is an essential signalling pathway for all multicellular animals. Although first described more than 30 years ago, new components and regulators of the pathway are still being discovered. Considering its importance in both embryonic development and adult homeostasis, it is not surprising that this pathway is often deregulated in human diseases such as cancer. Recently, it became clear that in addition to cytoplasmic components such as β-catenin, other, membrane-bound or extracellular, components of the WNT pathway are also altered in cancer. This review gives an overview of the recent discoveries on WNT signalling events near the cell membrane. Furthermore, membrane-associated components of the WNT pathway, which are more accessible for therapeutic intervention, as well therapeutic approaches that already target those components will be discussed. In this way, we hope to stimulate the development of effective anti-cancer therapies that target this fascinating pathway. Linked Articles This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc

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.

This protocol describes a strategy for the generation of 3D prostate organoid cultures from healthy mouse and human prostate cells (either bulk or FACS-sorted single luminal and basal cells), metastatic prostate cancer lesions and circulating tumor cells. Organoids derived from healthy material contain the differentiated luminal and basal cell types, whereas organoids derived from prostate cancer tissue mimic the histology of the tumor. We explain how to establish these cultures in the fully defined serum-free conditioned medium that is required to sustain organoid growth. Starting with the plating of digested tissue material, full-grown organoids can usually be obtained in ∼2 weeks. The culture protocol we describe here is currently the only one that allows the growth of both the luminal and basal prostatic epithelial lineages, as well as the growth of advanced prostate cancers. Organoids established using this protocol can be used to study many different aspects of prostate biology, including homeostasis, tumorigenesis and drug discovery.

Breast cancer (BC) is the most common malignancy with 1.7 million new diagnoses/year and is responsible for more than 450,000 yearly deaths worldwide. Two thirds of BC express the estrogen receptor (ER) and/or progesterone receptor and are referred to as hormone receptor-positive (HR+) BC. Endocrine therapy (ET) is usually active in these tumors, although drug resistance and side effects limit its benefit. Growth factor signaling through the PI3K/AKT/mammalian target of rapamycin (mTOR) and MAP kinase axes enhances ER activity and is a key mechanism underlying endocrine resistance. Water-only fasting (fasting) or plant-based, low-calorie, carbohydrate- and protein-restricted fasting-mimicking diets (FMDs) reduce circulating growth factors, such as insulin and IGF1 Therefore, we hypothesized that these dietary interventions could be used to enhance the activity of ET and delay the occurrence of resistance. For our in vitro experiments we used the HR+ BC cell lines, MCF7, T47D, and ZR-75-1, as well as metastases-derived organoids from patients with HR+ BC. Our in vivo experiments in mouse xenografts of human BC cell lines, were conducted in six-to-eight-week old female NOD SCID or athymic Nude-FoxN1 mice treated with ET w/ or w/o 48-72 hours of fasting/FMD. We monitored tumor growth and mouse survival and collected tumor masses and blood to detect circulating levels of several growth factors, adipokines and cytokines. In vivo add back experiments with fasting-reduced factors were done with IGF1, insulin and leptin. Circulating growth factors and adipo-cytokines were also detected in blood samples from 36 patients with HR+ BC, who were enrolled in either one of two clinical trials (NCT03595540 and NCT03340935) assessing safety and feasibility of periodic FMD in cancer patients. Patient nutritional status and response to treatment were also monitored in our clinical trials.We found that in HR+ BC models, periodic fasting or FMD enhanced tamoxifen and fulvestrant activity by lowering circulating IGF1, insulin, and leptin levels and by blocking AKT-mTOR signaling via EGR1 and PTEN upregulation. When fulvestrant was combined with palbociclib (a cyclin-dependent kinase 4/6 inhibitor), adding periodic FMD cycles promoted long-lasting tumour regressions and reverted acquired resistance to this regime. Moreover, both fasting and FMD prevented tamoxifen-induced endometrial hyperplasia. In HR+ BC patients receiving ET, FMD cycles caused metabolic changes analogous to those observed in mice, including reduced leptin and IGF1 levels, which were found to remain low for extended periods. In mice, these long-lasting effects were associated with carryover anticancer activity. Overall, our results provide the rationale for conducting further clinical studies of fasting-based dietary strategies as an adjuvant to ET w/ or w/o CDK4/6 inhibitors in patients with HR+ BC. Citation Format: Irene Caffa, Vanessa Spagnolo, Pamela Becherini, Francesca Valdemarin, Claudio Vernieri, Min Wei, Sebastian Brandhorst, Chiara Zucal, Else Driehuis, Lorenzo Ferrando, Luca Mastracci, Michele Cilli, Francesco Piacente, Anna Laura Cremonini, Mario Passalacqua, Valerio Vellone, Gabriele Zoppoli, Michele Cea, Giulia Salvadori, Salvatore Cortellino, Hans Clevers, Filippo De Braud, Alessandro Provenzani, Valter D. Longo, Alessio Nencioni. Fasting-mimicking diet and hormone therapy modulates metabolic factors to promote breast cancer regression and reduce side effects [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT075.

Head and neck squamous cell carcinoma (HNSCC) cancer is a broad category of tumor types arising from the inner lining of various anatomic structures including the oral cavity, pharynx, larynx and salivary glands. Treatment often involves an intensive combination of surgery, radiotherapy and chemotherapy. Despite this, tumor recurrence rates remain high and survival rates are relatively poor. Here, we describe a novel high throughput drug screening platform combining the OrganoPlate®, a microfluidic based 3D-culture system, and HN cancer-derived organoids. MIMETAS develops Organ-on-a-Chip-based models for evaluation of new medicines. Our unique microfluidic technology enables testing of compounds on miniaturized 3D organ models in high-throughput. Hereby we show the establishment of HNSCC cancer-derived organoids in 2-lane OrganoPlate®, and its usefulness for phenotypic drug screenings. The aim of the study is to capture differential sensitivity of HN organoid lines to either a common platinum-based chemotherapy or to a targeted drug still not used in the clinic. Organoid lines (T1, T3 and T5) were embedded in the 2-lane OrganoPlate® as single cells in an extracellular matrix gel. At day 2, cultures were treated with cisplatin or PARP-inhibitor niraparib for 120 hours. Additionally, cultures exposed to high doses of cisplatin were evaluated after one week of recovery in order to determine treatment failure and recapitulate potential patient relapse. Drug response was evaluated by assessment of morphology (phase contrast), cell viability (total nuclei count, alamar blue assay), proliferation (EdU incorporation) and DNA damage (phosphorylated histone H2AX). Organoid cultures grow well under medium perfusion in the 2-lane OrganoPlate® and selective sensitivity to cisplatin and niraparib is revealed by the used readouts. The high-throughput, microfluidic 2-lane OrganoPlate® platform offers an attractive method for growing HNSCC cancer-derived organoids, supporting development of individualized tumour models for phenotypic drug screenings. Citation Format: Silvia Bonilla, Else Driehuis, Henriëtte Lanz, Hans Clevers, Jos Joore, Karla Queiroz, Paul Vulto. A novel HNSCC organoid-on-a-chip platform for preclinical investigation and drug screening [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2794.

TMPRSS2-ERG fusions are common genetic events in prostate cancer. Until now, this genetic alteration was modelled by ERG overexpression. In this short communication, we report the creation of mouse prostate organoids that have undergone gene fusion through a CRISPR/Cas9-based strategy. The genetic fusion of TMPRSS2 and ERG results in ERG overexpression. This effect is androgen receptor-mediated, as expression of the fusion transcript can be restored to wildtype ERG levels by treatment with the androgen receptor antagonist Nilutamide.

Head and neck (HN) cancer is a broad category of tumor types arising from various anatomic structures including the craniofacial bones, soft tissues, salivary glands, skin, and mucosal membranes. Treatment often involves an intensive combination of surgery, radiotherapy and chemotherapy. Despite this, tumour recurrence rates remain high and survival rates are relatively poor. Here, we describe a novel high throughput drug screening platform combining the OrganoPlate, a microfluidic based 3D culture plate, and HN cancer-derived organoids. MIMETAS develops Organ-on-a-Chip-based models for evaluation of new medicines. Our unique microfluidic technology enables testing of compounds on miniaturized 3D organ models in high-throughput. These models are expected to show better predictivity as compared to laboratory animals and conventional 2D cell culture models, without compromising throughput or ease of use. Hereby we show the establishment of HN cancer-derived organoids in 2-lane OrganoPlate, and its usefulness for phenotypic drug screenings. The aim of the study is to evaluate the 2-lane OrganoPlate as a platform for growing HN cancer organoids and drug screening. Organoid lines (T2, T3 and T4) were embedded in the 2-lane OrganoPlate as single cells in an Extracellular Matrix gel. At day 3, cultures were treated with Cisplatin or Carboplatin for 120 hours. Drug response was evaluated by assessment of morphology (phase contrast), Cell Viability (Alamar blue) and proliferation (EdU incorporation). Organoids cultures grow well under perfusion in the 2-lane OrganoPlate and different sensitivity to cisplatin is captured by the used readouts. The high-throughput, microfluidic 2-lane OrganoPlate platform offers an attractive method for growing HN cancer-derived organoids, supporting development of individualized tumour models for phenotypic drug screenings. Citation Format: Karla Queiroz, Else Driehuis, Silvia Bonilla, Henriëtte Lanz, Hans Clevers, Jos Joore, Paul Vulto. A novel high throughput platform for head & neck cancer organoids drug screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1173.

WNT signalling is an essential signalling pathway for all multicellular animals. Although first described more than 30 years ago, new components and regulators of the pathway are still being discovered. Considering its importance in both embryonic development and adult homeostasis, it is not surprising that this pathway is often deregulated in human diseases such as cancer. Recently, it became clear that in addition to cytoplasmic components such as β-catenin, other, membrane-bound or extracellular, components of the WNT pathway are also altered in cancer. This review gives an overview of the recent discoveries on WNT signalling events near the cell membrane. Furthermore, membrane-associated components of the WNT pathway, which are more accessible for therapeutic intervention, as well therapeutic approaches that already target those components will be discussed. In this way, we hope to stimulate the development of effective anti-cancer therapies that target this fascinating pathway.This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc.

Organoids are three-dimensional (3D) structures derived from adult or embryonic stem cells that maintain many structural and functional features of their respective organ. Recently, genome editing based on the bacterial defense mechanism CRISPR/Cas9 has emerged as an easily applicable and reliable laboratory tool. Combining organoids and CRISPR/Cas9 creates exciting new opportunities to study organ development and human disease in vitro. The potential applications of CRISPR in organoids are only beginning to be explored.