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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.

High-grade serous ovarian cancer (HG-SOC)—often referred to as a “silent killer”—is the most lethal gynecological malignancy. The fallopian tube (murine oviduct) and ovarian surface epithelium (OSE) are considered the main candidate tissues of origin of this cancer. However, the relative contribution of each tissue to HG-SOC is not yet clear. Here, we establish organoid-based tumor progression models of HG-SOC from murine oviductal and OSE tissues. We use CRISPR-Cas9 genome editing to introduce mutations into genes commonly found mutated in HG-SOC, such as Trp53, Brca1, Nf1 and Pten. Our results support the dual origin hypothesis of HG-SOC, as we demonstrate that both epithelia can give rise to ovarian tumors with high-grade pathology. However, the mutated oviductal organoids expand much faster in vitro and more readily form malignant tumors upon transplantation. Furthermore, in vitro drug testing reveals distinct lineage-dependent sensitivities to the common drugs used to treat HG-SOC in patients., The relative contribution of fallopian tube (FT) or ovarian surface epithelium (OSE) to high-grade serous ovarian cancer (HG-SOC) development is unclear. Here, the authors establish organoid models from murine oviductal and OSE tissues that allow cancer modeling via CRISPR-Cas9 genome editing, and report a dual origin of murine HG-SOC.

SummaryCellular immunotherapies are rapidly gaining clinical importance, yet predictive platforms for modeling their mode of action are lacking. Here, we developed a dynamic immuno-organoid 3D imaging-transcriptomics platform; BEHAV3D, to unravel the behavioral and underlying molecular mechanisms of solid tumor targeting. Applied to an emerging cancer metabolome-sensing immunotherapy: TEGs, we first demonstrate targeting of multiple breast cancer subtypes. Live-tracking of over 120,000 TEGs revealed a diverse behavioral landscape and identified a ‘super engager’ cluster with serial killing capability. Inference of single-cell behavior with transcriptomics identified the gene signature of ‘super engager’ killer TEGs, which contained 27 genes with no previously described T cell function. Furthermore, guided by a dynamic type 1 interferon (IFN-I) signaling module induced by high TEG-sensitive organoids, we show that IFN-I can prime resistant organoids for TEG-mediated killing. Thus, BEHAV3D characterizes behavioral-phenotypic heterogeneity of cellular immunotherapies and holds promise for improving solid tumor-targeting in a patient-specific manner.

Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various organs, such methods are lacking for breast tissue. Here we provide an optimized, highly versatile protocol for long-term culture of organoids derived from either normal human breast tissues or breast cancer (BC) tissues, as well as culturing conditions for a panel of 45 biobanked samples, including BC organoids covering all major disease subtypes (triple-negative, estrogen receptor-positive/progesterone receptor-positive and human epidermal growth receptor 2-positive). Additionally, we provide methods for genetic manipulation by Lipofectamine 2000, electroporation or lentivirus and subsequent organoid selection and clonal culture. Finally, we introduce an optimized method for orthotopic organoid transplantation in mice, which includes injection of organoids and estrogen pellets without the need for surgery. Organoid derivation from tissue fragments until the first split takes 7-21 d; generation of genetically manipulated clonal organoid cultures takes 14-21 d; and organoid expansion for xenotransplantation takes >4 weeks.

BRCA1 germ-line mutations are a major cause of hereditary breast cancer and BRCA1-deficient breast cancer shares many characteristics as sporadic basal-like breast cancer (BLBC). Effective therapeutic targets for BRCA1-deficient BLBC remain lacking. By utilizing a BRCA1-deficient BLBC mouse model based on intraductal injection of Krt8-Cre adenovirus to inactivate Brca1 and Trp53 in luminal mammary epithelial cells, here we report that the Wnt receptor Frizzled 7 (FZD7) serves as a biomarker and therapeutic target in the resulting mammary tumor cells and is particularly enriched in cancer stem cells / tumor-initiating cells (CSCs/TICs). Inhibiting FZD7-mediated Wnt signaling using a nontoxic FZD-binding fragment of C. difficile toxin B (TcdBFBD) attenuates growth of BRCA1-deficient tumor organoids and xenografted tumors, without damaging Wnt-sensitive tissues such as bones in vivo. Finally, FZD1/2/7-positive cells are enriched in chemotherapy-resistant cells in both BLBC and luminal breast tumors treated with cisplatin, and TcdBFBD synergizes strongly with cisplatin in inhibiting both tumor types. These findings demonstrate the therapeutic value for targeting FZD1/2/7 in treating breast cancers and establish TcdBFBD as a potential therapeutic agent targeting TICs and chemotherapy-resistant cancer cells.

PurposeThere remains an unmet need for preclinical models to enable personalized therapy for ovarian cancer (OC) patients. Recently, patient-derived organoid (PDO) cultures of patients with OC have been established that faithfully represent the histopathological features and genomic landscape of the patient tumor. In this study, we evaluate the capacity of OC PDOs to predict clinical drug response and functional consequences of tumor heterogeneity.Experimental design36 genomically characterized PDOs from 23 patients with known clinical histories were exposed to chemotherapeutics and targeted drugs.ResultsOC PDOs maintained genomic features of the original tumor lesion and recapitulated patient response to neoadjuvant carboplatin and paclitaxel combination treatment, according to distinct clinical outcomes (histopathological, biochemical and radiological). PDOs displayed inter-as well as intrapatient drug response heterogeneity, which could in part be explained by genetic aberrations. All PDOs were resistant to PARP-inhibitors, in accordance with homologous recombination pathway fidelity and genome-wide mutation context. KRAS, BRAF and NRAS mutation status predicted response to BRAF-inhibitor vemurafenib and pan-HER-inhibitor afatinib, and explained differential response among four PDOs derived from distinct tumor locations of an individual patient. Importantly, PDO drug screening identified sensitivity to at least one drug for the majority of patients (88%).ConclusionsOC PDOs are a valuable preclinical model system that can provide insights in drug response for individual patients with OC, complementary to genetic testing. Generating PDOs of multiple tumor locations can improve clinical decision making and increase our knowledge on genetic and drug response heterogeneity.

Introduction/Background Epithelial ovarian cancer is characterized by intratumour genetic heterogeneity, chemotherapy resistance and poor survival. The advent of ovarian cancer organoids, that recapitulate histological and genetic features of the original lesions, has providedopportunities for personalized assessment of drug response. Here, we assessed if the ovarian cancer organoid system can predict patients9 clinical response to chemotherapeutics. In addition, we evaluated the link between intratumour genetic heterogeneity and drug response using multiple organoids derived from individual patients. Methodology In total, we included 34 organoid lines derived from 22 patients with ovarian cancer at primary surgery or interval debulking. Organoid drug responses to chemotherapeutics as well as targeted drugs were determined. The IC50 and area under the curve values were computed from the drug response curves. Patients9 recurrence-free and overall survival were compared to the organoid drug response. For patients who received carboplatin and paclitaxel chemotherapy prior to interval debulking (N=7), we additionally compared organoid drug response to biochemical (CA125), pathological, radiological outcomes. The organoids and corresponding tumour tissues were subjected to whole genome sequencing for genomic analysis. Results Organoid drug response correlated to clinical response as defined by histopathological tumour assessment (chemotherapy response score (CRS)). Organoids of patients with no or minimal pathological response (CRS=1) were less sensitive to carboplatin and paclitaxelcompared to organoids of the patients with appreciable pathological response (CRS=2). The presumed additive effect of paclitaxel on carboplatin was only observed in a subset of organoids. Organoid exposure to eight additional drugs revealed that multiple tumour locations of the same patients exhibited varying responses to specific drugs. Genomic analyses are being performed to identify underlying mechanisms of differential drug responses. Conclusion Ovarian cancer organoids provide a valuable preclinical system with the potential to guide treatment choice through prediction of drug response, avoid unnecessary overtreatment and provide insights in response mechanisms. Disclosure No competing interests. Applicable funding sources: EIF | Stand Up To Cancer (SU2C) [Clevers, Kopper] KWF Kankerbestrijding (Dutch Cancer Society) - UU2015-7743 [de Witte, Witteveen, Zweemer, Kloosterman] Gieskes Strijbis Foundation (1816199) [Clevers, Witteveen, Kloosterman, Zweemer, Lohmussaar, Espejo Valle-Inclan, Hami, Snippert]

Introduction/Background Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumour heterogeneity of OC are limited and hard to establish. Methodology We present a protocol that enables efficient derivation and long-term expansion of OC organoids. Results Utilizing this protocol, we have established 56 organoid lines from 32 patients, representing all main subtypes of OC. OC organoids recapitulate histological and genomic features of the pertinent lesion from which they were derived, illustrating intra- and interpatient heterogeneity, and can be genetically modified. We show that OC organoids can be used for drug-screening assays and capture different tumour subtype responses to the gold standard platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. Finally, OC organoids can be xenografted, enabling in vivo drug-sensitivity assays. Conclusion Taken together, this demonstrates their potential application for research and personalized medicine. Disclosure No competing interests. Applicable funding sources: EIF | Stand Up To Cancer (SU2C) [Clevers, Kopper, Balgobind, Begthel] KWF Kankerbestrijding (Dutch Cancer Society) - UU2015-7743 [de Witte, van Roosmalen, Witteveen, Zweemer, Kloosterman] Gieskes Strijbis Foundation (1816199) [Clevers, Witteveen, Kloosterman, Zweemer, Lohmussaar, Espejo Valle-Inclan, Hami, Bos, Snippert] Published in Nature Medicine. April 2019. dx.doi.org/10.1038/s41591-019-0422-6.

Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and hard to establish. We present a protocol that enables efficient derivation and long-term expansion of OC organoids. Utilizing this protocol, we have established 56 organoid lines from 32 patients, representing all main subtypes of OC. OC organoids recapitulate histological and genomic features of the pertinent lesion from which they were derived, illustrating intra- and interpatient heterogeneity, and can be genetically modified. We show that OC organoids can be used for drug-screening assays and capture different tumor subtype responses to the gold standard platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. Finally, OC organoids can be xenografted, enabling in vivo drug-sensitivity assays. Taken together, this demonstrates their potential application for research and personalized medicine. A biobank of ovarian cancer organoids recapitulates the histopathological and molecular hallmarks of patient tumors and provides a resource for preclinical research.

Breast cancer (BC) comprises multiple distinct subtypes that differ genetically, pathologically, and clinically. Here, we describe a robust protocol for long-term culturing of human mammary epithelial organoids. Using this protocol, >100 primary and metastatic BC organoid lines were generated, broadly recapitulating the diversity of the disease. BC organoid morphologies typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor. DNA copy number variations as well as sequence changes were consistent within tumor-organoid pairs and largely retained even after extended passaging. BC organoids furthermore populated all major gene-expression-based classification groups and allowed in vitro drug screens that were consistent with in vivo xeno-transplantations and patient response. This study describes a representative collection of well-characterized BC organoids available for cancer research and drug development, as well as a strategy to assess in vitro drug response in a personalized fashion. The heterogeneity of breast cancer subtypes can be captured using organoid cultures that can facilitate drug screens that corroborate with patient responses.

Over the last few years several studies led to a significant change in the field of ovarian cancer and it is currently believed that the fallopian tube and not the ovary surface epithelium (OSE) is the main origin of high-grade serous ovarian cancer (HG-SOC). Nevertheless, due to the lack of unique markers and adequate model systems the relative contribution of each tissue is not yet clear and the notion that OSE has a role in HG-SOC development was not cast aside altogether. In this work we have established novel organoid systems derived from both mouse OSE and oviduct (Ovi, the equivalent of human fallopian tube). These systems recapitulate their tissue of origin and demonstrate differences in medium requirements as well as gene expression. To establish comparable tumor progression models for both OSE and Ovi we used CRISPR-Cas9 technique and targeted commonly mutated genes in ovarian cancer (Trp53, Brca1, Nf1 and Pten). Thus, we were able to establish clones with different combinations of mutations. Histological, metaphase spread and gene expression analysis of the mutated organoid clones from both OSE and Ovi demonstrated different degrees of deviation from their wild type counterpart. This deviation became more evident as the amount of introduced mutations increased. Preliminary transplantation experiments showed that only triple mutants gave rise to tumors (Trp53, Brca1 and Pten or Trp53, Brca1 and Nf1). Moreover, Ovi mutant clones were more prone to grow into tumors in comparison to OSE as no tumors have been detected in mice transplanted with the OSE equivalent clones so far. Taken together, in this study we present the first comparable Ovi/OSE research platform that enables addressing questions related to origin and early stages of HG-SOC development. Citation Format: Kadi Lõhmussaar, Oded Kopper, Hans C. Clevers. BUILDING AN ORGANOID-BASED MODEL FOR OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-039.

Human embryonic stem cells have the potential to differentiate into all human cell types and therefore hold a great therapeutic promise. Differentiation into the embryonic endoderm and its derivatives is of special interest since it can provide a cure for severe widespread clinical conditions such as diabetes and hepatic failure. In this work we established a unique experimental outline that enables the study of early human endoderm development and can help improve and create new differentiation protocols. To this end we started with mesendoderm cells and separated them into early endoderm and mesoderm progenitor cells using CXCR4 and PDGFRA cell surface markers. We molecularly characterized the different lineages, and demonstrated the importance of the TGFβ pathway in definitive endoderm initiation. The endoderm progenitor cells were then purified creating an endodermal differentiation niche that is not affected by other cell populations. We followed the differentiation of these cells at different time points, and demonstrated an up regulation of genes indicative to differentiation into both foregut and hindgut. Surprisingly, upon continued culture, there was significant down regulation of the hepatic gene signature. This down regulation could be rescued with FGF2 treatment demonstrating its importance in hepatic cell maintenance. In conclusion, we suggest that isolating endoderm progenitor cells is crucial for the analysis of their fate, and enables the identification of factors involved in their differentiation and maintenance.

New sources of beta cells are needed in order to develop cell therapies for patients with diabetes. An alternative to forced expansion of post-mitotic beta cells is the induction of differentiation of stem-cell derived progenitor cells that have a natural self-expansion capacity into insulin-producing cells. In order to learn more about these progenitor cells at different stages along the differentiation process in which they become progressively more committed to the final beta cell fate, we took the approach of identifying, isolating and characterizing stage specific progenitor cells. We generated human embryonic stem cell (HESC) clones harboring BAC GFP reporter constructs of SOX17, a definitive endoderm marker, and PDX1, a pancreatic marker, and identified subpopulations of GFP expressing cells. Using this approach, we isolated a highly enriched population of pancreatic progenitor cells from hESCs and examined their gene expression with an emphasis on the expression of stage-specific cell surface markers. We were able to identify novel molecules that are involved in the pancreatic differentiation process, as well as stage-specific cell markers that may serve to define (alone or in combination with other markers) a specific pancreatic progenitor cell. These findings may help in optimizing conditions for ultimately generating and isolating beta cells for transplantation therapy.

Human embryoid bodies (HEBs) are cell aggregates that are produced during the course of embryonic stem cell differentiation in suspension. Mature HEBs have been shown to contain derivatives of the three embryonic germ layers. In this study, using a combination of laser capture microscopy followed by DNA microarray analysis and cell sorting, we demonstrate that early HEBs are composed of three major cell populations. These cell populations can be defined by the expression of specific cell markers, namely: (i) OCT4+, REX1−; (ii) NCAD+, OCT4−; and (iii) EPOR+, OCT4−. By analyzing gene expression in embryonic tissues, these cell populations could respectively be assigned to the embryonic ectoderm, mesendoderm, and extraembryonic endoderm lineages. We show that the extraembryonic endoderm, which selectively expresses platelet-derived growth factor B (PDGF-B), negatively affects the mesendoderm lineage, which selectively expresses the receptor PDGFRA. Our analysis suggests that early HEBs are spatially patterned and that cell differentiation is governed by interactions between the different cell types.

Human embryonic stem cells (HESCs) have a tremendous clinical and scientific importance since they may serve as a cell source for transplantation and as a system for the study of human development and disease. The genetic engineering of HESCs has become instrumental in achieving these goals. Here we discuss various methodologies to genetically manipulate HESCs and propose a variety of applications of the modified cells in basic and applied research.

The boundaries of embryonic stem cell (ESC) research have extended considerably in recent years in several important ways. Alongside a deeper understanding of the pluripotent state, ESCs have been successfully integrated into various fields, such as genomics, epigenetics, and disease modeling. Significant progress in cell fate control has pushed directed differentiation and tissue engineering further than ever before and promoted clinical trials. The geographical distribution of research activity has also expanded, especially for human ESCs. This review outlines these developments and future challenges that remain.