Zeb1 mediates EMT/plasticity-associated ferroptosis sensitivity in cancer cells by regulating lipogenic enzyme expression and phospholipid composition.

Therapy resistance and metastasis, the most fatal steps in cancer, are often triggered by a (partial) activation of the epithelial-mesenchymal transition (EMT) programme. A mesenchymal phenotype predisposes to ferroptosis, a cell death pathway exerted by an iron and oxygen-radical-mediated peroxidation of phospholipids containing polyunsaturated fatty acids. We here show that various forms of EMT activation, including TGFß stimulation and acquired therapy resistance, increase ferroptosis susceptibility in cancer cells, which depends on the EMT transcription factor Zeb1. We demonstrate that Zeb1 increases the ratio of phospholipids containing pro-ferroptotic polyunsaturated fatty acids over cyto-protective monounsaturated fatty acids by modulating the differential expression of the underlying crucial enzymes stearoyl-Co-A desaturase 1 (SCD), fatty acid synthase (FASN), fatty acid desaturase 2 (FADS2), elongation of very long-chain fatty acid 5 (ELOVL5) and long-chain acyl-CoA synthetase 4 (ACSL4). Pharmacological inhibition of selected lipogenic enzymes (SCD and FADS2) allows the manipulation of ferroptosis sensitivity preferentially in high-Zeb1-expressing cancer cells. Our data are of potential translational relevance and suggest a combination of ferroptosis activators and SCD inhibitors for the treatment of aggressive cancers expressing high Zeb1.

Patient-derived zebrafish xenografts of uveal melanoma reveal ferroptosis as a drug target.

Uveal melanoma (UM) has a high risk to progress to metastatic disease with a median survival of 3.9 months after metastases detection, as metastatic UM responds poorly to conventional and targeted chemotherapy and is largely refractory to immunotherapy. Here, we present a patient-derived zebrafish UM xenograft model mimicking metastatic UM. Cells isolated from Xmm66 spheroids derived from metastatic UM patient material were injected into 2 days-old zebrafish larvae resulting in micro-metastases in the liver and caudal hematopoietic tissue. Metastasis formation could be reduced by navitoclax and more efficiently by the combinations navitoclax/everolimus and flavopiridol/quisinostat. We obtained spheroid cultures from 14 metastatic and 10 primary UM tissues, which were used for xenografts with a success rate of 100%. Importantly, the ferroptosis-related genes GPX4 and SLC7A11 are negatively correlated with the survival of UM patients (TCGA: n = 80; Leiden University Medical Centre cohort: n = 64), ferroptosis susceptibility is correlated with loss of BAP1, one of the key prognosticators for metastatic UM, and ferroptosis induction greatly reduced metastasis formation in the UM xenograft model. Collectively, we have established a patient-derived animal model for metastatic UM and identified ferroptosis induction as a possible therapeutic strategy for the treatment of UM patients.

Zebrafish Patient-Derived Xenograft Model as a Preclinical Platform for Uveal Melanoma Drug Discovery.

Uveal melanoma (UM) is a rare malignant cancer of the eye, with up to 50% of patients dying from metastasis, for which no effective treatment is available. Due to the rarity of the disease, there is a great need to harness the limited material available from primary tumors and metastases for advanced research and preclinical drug screening. We established a platform to isolate, preserve, and transiently recover viable tissues, followed by the generation of spheroid cultures derived from primary UM. All assessed tumor-derived samples formed spheroids in culture within 24 h and stained positive for melanocyte-specific markers, indicating the retention of their melanocytic origin. These short-lived spheroids were only maintained for the duration of the experiment (7 days) or re-established from frozen tumor tissue acquired from the same patient. Intravenous injection of fluorescently labeled UM cells derived from these spheroids into zebrafish yielded a reproducible metastatic phenotype and recapitulated molecular features of the disseminating UM. This approach allowed for the experimental replications required for reliable drug screening (at least 2 individual biological experiments, with n > 20). Drug treatments with navitoclax and everolimus validated the zebrafish patient-derived model as a versatile preclinical tool for screening anti-UM drugs and as a preclinical platform to predict personalized drug responses.

The EMT transcription factor ZEB1 governs a fitness-promoting but vulnerable DNA replication stress response.

The DNA damage response (DDR) and epithelial-to-mesenchymal transition (EMT) are two crucial cellular programs in cancer biology. While the DDR orchestrates cell-cycle progression, DNA repair, and cell death, EMT promotes invasiveness, cellular plasticity, and intratumor heterogeneity. Therapeutic targeting of EMT transcription factors, such as ZEB1, remains challenging, but tumor-promoting DDR alterations elicit specific vulnerabilities. Using multi-omics, inhibitors, and high-content microscopy, we discover a chemoresistant ZEB1-high-expressing sub-population (ZEB1hi) with co-rewired cell-cycle progression and proficient DDR across tumor entities. ZEB1 stimulates accelerated S-phase entry via CDK6, inflicting endogenous DNA replication stress. However, DDR buildups involving constitutive MRE11-dependent fork resection allow homeostatic cycling and enrichment of ZEB1hi cells during transforming growth factor ß (TGF-ß)-induced EMT and chemotherapy. Thus, ZEB1 promotes G1/S transition to launch a progressive DDR benefitting stress tolerance, which concurrently manifests a targetable vulnerability in chemoresistant ZEB1hi cells. Our study thus highlights the translationally relevant intercept of the DDR and EMT.

Novel Treatments of Uveal Melanoma Identified with a Synthetic Lethal CRISPR/Cas9 Screen.

Currently, no systemic treatment is approved as the standard of care for metastatic uveal melanoma (UM). mTOR has been evaluated as a drug target in UM. However, one of the main limitations is dose reduction due to adverse effects. The combination of everolimus with another targeted agent would allow the reduction of the dose of a single drug, thus widening the therapeutic window. In our study, we aimed to identify a synergistic combination with everolimus in order to develop a novel treatment option for metastatic UM. We exploited CRISPR-Cas9 synthetic lethality screening technology to search for an efficient combination. IGF1R and PRKDC and several other genes were identified as hits in the screen. We investigated the effect of the combination of everolimus with the inhibitors targeting IGF1R and DNA-PKcs on the survival of UM cell lines. These combinations synergistically slowed down cell growth but did not induce apoptosis in UM cell lines. These combinations were tested on PDX UM in an in vivo model, but we could not detect tumor regression. However, we could find significant activity of the dual DNA-PKcs/mTOR inhibitor CC-115 on PDX UM in the in vivo model.

Establishment of Embryonic Zebrafish Xenograft Assays to Investigate TGF-ß Family Signaling in Human Breast Cancer Progression.

Transforming growth factor-ß (TGF-ß) family members have pivotal functions in controlling breast cancer progression, acting not only on cancer cells but also on other cells within the tumor microenvironment. Here we describe embryonic zebrafish xenograft assays to investigate how TGF-ß family signaling controls breast cancer cell intravasation, extravasation and regulates tumor angiogenesis. Fluorescently mCherry-labeled breast cancer cells are injected in the perivitelline space or Duct of Cuvier of Tg (fli:EGFP) transgenic Casper zebrafish embryos, in which the zebrafish express enhanced green fluorescent protein in the entire vasculature. The dynamic responses of migratory and invasive human cancer cells, and the induction of new blood vessel formation by the cancer cells in zebrafish host, are visualized using a fluorescent microscope. These assays provide efficient, reliable, low-cost models to investigate the effect of (epi)genetic modulators and pharmacological compounds that perturb the activity of TGF-ß family signaling components on breast cancer cell metastasis and angiogenesis.

Ortho- and Ectopic Zebrafish Xeno-Engraftment of Ocular Melanoma to Recapitulate Primary Tumor and Experimental Metastasis Development.

There are currently no animal models for metastatic ocular melanoma. The lack of metastatic disease models has greatly hampered the research and development of novel strategies for the treatment of metastatic ocular melanoma. In this protocol we delineate a quick and efficient way to generate embryonic zebrafish models for both the primary and disseminated stage of ocular melanoma, using retro-orbital orthotopic and intravascular ectopic cell engraftment, respectively. Combining these two different engraftment strategies we can recapitulate the etiology of cancer in its totality, progressing from primary, localized tumor growth under the eye to a peri-vascular metastasis formation in the tail. These models allow us to quickly and easily modify the cancer cells prior to implantation with specific labeling, genetic or chemical interference; and to treat the engrafted hosts with (small molecular) inhibitors to attenuate tumor development. Here, we describe the generation and quantification of both orthotopic and ectopic engraftment of ocular melanomas (conjunctival and uveal melanoma) using fluorescently labelled stable cell lines. This protocol is also applicable for engraftment of primary cells derived from patient biopsy and patient/PDX derived material (manuscript in preparation). Within hours post engraftment cell migration and proliferation can be visualized and quantified. Both tumor foci are readily available for imaging with both epifluorescence microscopy and confocal microscopy. Using these models, we can confirm or refute the activity of either chemical or genetic inhibition strategies within as little as 8 days after the onset of the experiment, allowing not only highly efficient screening on stable cell lines, but also enables patient directed screening for precision medicine approaches.

TLD1433 Photosensitizer Inhibits Conjunctival Melanoma Cells in Zebrafish Ectopic and Orthotopic Tumour Models.

The ruthenium-based photosensitizer (PS) TLD1433 has completed a phase I clinical trial for photodynamic therapy (PDT) treatment of bladder cancer. Here, we investigated a possible repurposing of this drug for treatment of conjunctival melanoma (CM). CM is a rare but often deadly ocular cancer. The efficacy of TLD1433 was tested on several cell lines from CM (CRMM1, CRMM2 and CM2005), uveal melanoma (OMM1, OMM2.5, MEL270), epidermoid carcinoma (A431) and cutaneous melanoma (A375). Using 15 min green light irradiation (21 mW/cm2, 19 J.cm-2, 520 nm), the highest phototherapeutic index (PI) was reached in CM cells, with cell death occurring via apoptosis and necrosis. The therapeutic potential of TLD1433 was hence further validated in zebrafish ectopic and newly-developed orthotopic CM models. Fluorescent CRMM1 and CRMM2 cells were injected into the circulation of zebrafish (ectopic model) or behind the eye (orthotopic model) and 24 h later, the engrafted embryos were treated with the maximally-tolerated dose of TLD1433. The drug was administrated in three ways, either by (i) incubating the fish in drug-containing water (WA), or (ii) injecting the drug intravenously into the fish (IV), or (iii) injecting the drug retro-orbitally (RO) into the fish. Optimally, four consecutive PDT treatments were performed on engrafted embryos using 60 min drug-to-light intervals and 90 min green light irradiation (21 mW/cm2, 114 J.cm-2, 520 nm). This PDT protocol was not toxic to the fish. In the ectopic tumour model, both systemic administration by IV injection and RO injection of TLD1433 significantly inhibited growth of engrafted CRMM1 and CRMM2 cells. However, in the orthotopic model, tumour growth was only attenuated by localized RO injection of TLD1433. These data unequivocally prove that the zebrafish provides a fast vertebrate cancer model that can be used to test the administration regimen, host toxicity and anti-cancer efficacy of PDT drugs against CM. Based on our results, we suggest repurposing of TLD1433 for treatment of incurable CM and further testing in alternative pre-clinical models.

A NF-ĸB-Activin A signaling axis enhances prostate cancer metastasis.

Metastasis is a main cause of death in prostate cancer (PCa). To dissect the molecular cues from cancer cell-microenvironment interaction that drive metastatic cascade, bone metastatic PCa cells were intravenously implanted into zebrafish embryos and mice tibia forming metastatic lesions. Transcriptomic analysis showed an elevated expression of stemness genes, pro-inflammatory cytokines and TGF-ß family member Activin A in the cancer cells at metastatic onset in both animal models. Consistently, analysis of clinical datasets revealed that the expression of Activin A is specifically elevated in metastases and correlates with poor prognosis in stratified high-risk PCa patients. It is further unveiled that the microenvironment induced Activin A expression by NF-κB activation. The elevated level of Activin A enhanced the invasive ALDHhi CSC-like phenotypes and PCa proliferation by activation of Smad and ERK1/2 signaling driving metastasis. Suppression of Activin A or Activin receptor significantly reduced the CSC-like subpopulation, invasion, metastatic growth, and bone lesion formation in zebrafish and mice xenografts, suggesting a functional role of NF-κB-dependent Activin A in PCa metastasis. Overall, our study demonstrates that human PCa cells can display a comparable response with the microenvironment in zebrafish and mice xenografts. Combining both animal models, we uncovered the microenvironment-dependent activin signaling as an essential driver in PCa metastasis with therapeutic potential.

Human melanoma brain metastases cell line MUG-Mel1, isolated clones and their detailed characterization.

Melanoma is a leading cause of high mortality that frequently spreads to the brain and is associated with deterioration in quality and quantity of life. Treatment opportunities have been restricted until now and new therapy options are urgently required. Our focus was to reveal the potential heterogeneity of melanoma brain metastasis. We succeeded to establish a brain melanoma metastasis cell line, namely MUG-Mel1 and two resulting clones D5 and C8 by morphological variety, differences in lipidome, growth behavior, surface, and stem cell markers. Mutation analysis by next-generation sequencing, copy number profiling, and cytogenetics demonstrated the different genetic profile of MUG-Mel1 and clones. Tumorigenicity was unsuccessfully tested in various mouse systems and finally established in a zebra fish model. As innovative treatment option, with high potential to pass the blood-brain barrier a peptide isolated from lactoferricin was studied in potential toxicity. Brain metastases are a major clinical challenge, therefore the development of relevant in vitro and in vivo models derived from brain melanoma metastases provides valuable information about tumor biology and offers great potential to screen for new innovative therapies.

Animal Models of Breast Cancer Bone Metastasis.

This chapter is designed to provide a comprehensive overview outlining the different in vivo models available for research into breast cancer bone metastasis. The main focus is to guide the researcher through the methodological processes required to establish and utilize these models within their own laboratory. These detailed methods are designed to enable the acquisition of accurate and meaningful results that can be used for publication and future translation into clinical benefit for women with breast cancer-induced bone metastasis.

Mesenchymal stromal cells prevent progression of liver fibrosis in a novel zebrafish embryo model.

Chronic liver damage leads to the onset of fibrogenesis. Rodent models for liver fibrosis have been widely used, but are less suitable for screening purposes. Therefore the aim of our study was to design a novel model for liver fibrosis in zebrafish embryos, suitable for high throughput screening. Furthermore, we evaluated the efficacy of mesenchymal stromal cells (MSCs) to inhibit the fibrotic process and thereby the applicability of this model to evaluate therapeutic responses. Zebrafish embryos were exposed to TAA or CCL4 and mRNA levels of fibrosis-related genes (Collagen-1α1, Hand-2, and Acta-2) and tissue damage-related genes (TGF-ß and SDF-1a, SDF-1b) were determined, while Sirius-red staining was used to estimate collagen deposition. Three days after start of TAA exposure, MSCs were injected after which the fibrotic response was determined. In contrast to CCL4, TAA resulted in an upregulation of the fibrosis-related genes, increased extracellular matrix deposition and decreased liver sizes suggesting the onset of fibrosis. The applicability of this model to evaluate therapeutic responses was shown by local treatment with MSCs which resulted in decreased expression of the fibrosis-related RNA markers. In conclusion, TAA induces liver fibrosis in zebrafish embryos, thereby providing a promising model for future mechanistic and therapeutic studies.

Endoglin Expression on Cancer-Associated Fibroblasts Regulates Invasion and Stimulates Colorectal Cancer Metastasis.

PURPOSE: Cancer-associated fibroblasts (CAF) are a major component of the colorectal cancer tumor microenvironment. CAFs play an important role in tumor progression and metastasis, partly through TGF-ß signaling pathway. We investigated whether the TGF-ß family coreceptor endoglin is involved in CAF-mediated invasion and metastasis. EXPERIMENTAL DESIGN: CAF-specific endoglin expression was studied in colorectal cancer resection specimens using IHC and related to metastases-free survival. Endoglin-mediated invasion was assessed in vitro by transwell invasion, using primary colorectal cancer-derived CAFs. Effects of CAF-specific endoglin expression on tumor cell invasion were investigated in a colorectal cancer zebrafish model, whereas liver metastases were assessed in a mouse model. RESULTS: CAFs specifically at invasive borders of colorectal cancer express endoglin and increased expression intensity correlated with increased disease stage. Endoglin-expressing CAFs were also detected in lymph node and liver metastases, suggesting a role in colorectal cancer metastasis formation. In stage II colorectal cancer, CAF-specific endoglin expression at invasive borders correlated with poor metastasis-free survival. In vitro experiments revealed that endoglin is indispensable for bone morphogenetic protein (BMP)-9-induced signaling and CAF survival. Targeting endoglin using the neutralizing antibody TRC105 inhibited CAF invasion in vitro. In zebrafish, endoglin-expressing fibroblasts enhanced colorectal tumor cell infiltration into the liver and decreased survival. Finally, CAF-specific endoglin targeting with TRC105 decreased metastatic spread of colorectal cancer cells to the mouse liver. CONCLUSIONS: Endoglin-expressing CAFs contribute to colorectal cancer progression and metastasis. TRC105 treatment inhibits CAF invasion and tumor metastasis, indicating an additional target beyond the angiogenic endothelium, possibly contributing to beneficial effects reported during clinical evaluations.See related commentary by Becker and LeBleu, p. 6110.

Overexpression of EZH2 in conjunctival melanoma offers a new therapeutic target.

Malignant melanoma of the conjunctiva (CM) is an uncommon but potentially deadly disorder. Many malignancies show an increased activity of the epigenetic modifier enhancer of zeste homolog 2 (EZH2). We studied whether EZH2 is expressed in CM, and whether it may be a target for therapy in this malignancy. Immunohistochemical analysis showed that EZH2 protein expression was absent in normal conjunctival melanocytes and primary acquired melanosis, while EZH2 was highly expressed in 13 (50%) of 26 primary CM and seven (88%) of eight lymph node metastases. Increased expression was positively associated with tumour thickness (p =0.03). Next, we targeted EZH2 with specific inhibitors (GSK503 and UNC1999) or depleted EZH2 by stable shRNA knockdown in three primary CM cell lines. Both pharmacological and genetic inactivation of EZH2 inhibited cell growth and colony formation and influenced EZH2-mediated gene transcription and cell cycle profile in vitro. The tumour suppressor gene p21/CDKN1A was especially upregulated in CM cells after EZH2 knockdown in CM cells. Additionally, the potency of GSK503 against CM cells was monitored in zebrafish xenografts. GSK503 profoundly attenuated tumour growth in CM xenografts at a well-tolerated concentration. Our results indicate that elevated levels of EZH2 are relevant to CM tumourigenesis and progression, and that EZH2 may become a potential therapeutic target for patients with CM. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Evaluation of (fli:GFP) Casper Zebrafish Embryos as a Model for Human Conjunctival Melanoma.

Purpose: Conjunctival melanoma (CM) is a rare malignant disease that can lead to recurrences and metastases. There is a lack of effective treatments for the metastases, and we set out to develop a new animal model to test potential therapies. Zebrafish are being used as a model for many diseases, and our goal was to test whether this animal could be used to study CM. Methods: Three human CM cell lines (CRMM-1 and CM2005.1, which both harbor a B-RAF mutation, and CRMM-2, which has an N-RAS mutation) were injected into the yolk sac, around the eye, and into the duct of Cuvier of transgenic (fli:GFP) Casper zebrafish embryos. Fluorescent and confocal images were taken to assess the phenotype and the behavior of engrafted cells and to test the effect of Vemurafenib as a treatment against CM. Results: While the cells that had been injected inside the yolk sac died and those injected around the eye sporadically went into the circulation, the cells that had been injected into the duct of Cuvier colonized the zebrafish: cells from all three cell lines proliferated and disseminated to the eyes, where they formed clusters, and to the tail, where we noticed extravasation and micrometastases. Vemurafenib, a potent agent for treatment of B-RAF V600E-positive melanoma, inhibited outgrowth of CRMM-1 and CM2005.1 cells in a mutation-dependent way. Conclusions: The (fli:GFP) Casper zebrafish embryo can be used as an efficient animal model to study metastatic behavior of human CM cells and warrants further testing of drug efficacy to aid care of CM patients.

Innovative approaches to establish and characterize primary cultures: an ex vivo 3D system and the zebrafish model.

Patient-derived specimens are an invaluable resource to investigate tumor biology. However, in vivo studies on primary cultures are often limited by the small amount of material available, while conventional in vitro systems might alter the features and behavior that characterize cancer cells. We present our data obtained on primary dedifferentiated liposarcoma cells cultured in a 3D scaffold-based system and injected into a zebrafish model. Primary cells were characterized in vitro for their morphological features, sensitivity to drugs and biomarker expression, and in vivo for their engraftment and invasiveness abilities. The 3D culture showed a higher enrichment in cancer cells than the standard monolayer culture and a better preservation of liposarcoma-associated markers. We also successfully grafted primary cells into zebrafish, showing their local migratory and invasive abilities. Our work provides proof of concept of the ability of 3D cultures to maintain the original phenotype of ex vivo cells, and highlights the potential of the zebrafish model to provide a versatile in vivo system for studies with limited biological material. Such models could be used in translational research studies for biomolecular analyses, drug screenings and tumor aggressiveness assays.

Development of a Patient-Derived Xenograft (PDX) of Breast Cancer Bone Metastasis in a Zebrafish Model.

Bone metastasis is a complex process that needs to be better understood in order to help clinicians prevent and treat it. Xenografts using patient-derived material (PDX) rather than cancer cell lines are a novel approach that guarantees more clinically realistic results. A primary culture of bone metastasis derived from a 67-year-old patient with breast cancer was cultured and then injected into zebrafish (ZF) embryos to study its metastatic potential. In vivo behavior and results of gene expression analyses of the primary culture were compared with those of cancer cell lines with different metastatic potential (MCF7 and MDA-MB-231). The MCF7 cell line, which has the same hormonal receptor status as the bone metastasis primary culture, did not survive in the in vivo model. Conversely, MDA-MB-231 disseminated and colonized different parts of the ZF, including caudal hematopoietic tissues (CHT), revealing a migratory phenotype. Primary culture cells disseminated and in later stages extravasated from the vessels, engrafting into ZF tissues and reaching the CHT. Primary cell behavior reflected the clinical course of the patient's medical history. Our results underline the potential for using PDX models in bone metastasis research and outline new methods for the clinical application of this in vivo model.

Imaging of Human Cancer Cell Proliferation, Invasion, and Micrometastasis in a Zebrafish Xenogeneic Engraftment Model.

The xenograft model, using the early life stages of the zebrafish, allows imaging of tumor cell behavior both on a single cell and whole organism level, over time, within a week. This robust and reproducible assay can be used as an intermediate step between in vitro techniques and the expensive, and time consuming, murine models of cancer invasion and metastasis.In this chapter, a detailed protocol to inject human cancer cells into the blood circulation of a zebrafish embryo is described; the engraftment procedure is then followed by visualization and quantification methods of tumor cell proliferation, invasion, and micrometastasis formation during subsequent larval development. Interaction with the host microenvironment is also considered.

Automation of Technology for Cancer Research.

Zebrafish embryos can be obtained for research purposes in large numbers at low cost and embryos develop externally in limited space, making them highly suitable for high-throughput cancer studies and drug screens. Non-invasive live imaging of various processes within the larvae is possible due to their transparency during development, and a multitude of available fluorescent transgenic reporter lines.To perform high-throughput studies, handling large amounts of embryos and larvae is required. With such high number of individuals, even minute tasks may become time-consuming and arduous. In this chapter, an overview is given of the developments in the automation of various steps of large scale zebrafish cancer research for discovering important cancer pathways and drugs for the treatment of human disease. The focus lies on various tools developed for cancer cell implantation, embryo handling and sorting, microfluidic systems for imaging and drug treatment, and image acquisition and analysis. Examples will be given of employment of these technologies within the fields of toxicology research and cancer research.