The lipid droplet protein DHRS3 is a regulator of melanoma cell state.

Lipid droplets are fat storage organelles composed of a protein envelope and lipid rich core. Regulation of this protein envelope underlies differential lipid droplet formation and function. In melanoma, lipid droplet formation has been linked to tumor progression and metastasis, but it is unknown whether lipid droplet proteins play a role. To address this, we performed proteomic analysis of the lipid droplet envelope in melanoma. We found that lipid droplet proteins were differentially enriched in distinct melanoma states; from melanocytic to undifferentiated. DHRS3, which converts all-trans-retinal to all-trans-retinol, is upregulated in the MITFLO/undifferentiated/neural crest-like melanoma cell state and reduced in the MITFHI/melanocytic state. Increased DHRS3 expression is sufficient to drive MITFHI/melanocytic cells to a more undifferentiated/invasive state. These changes are due to retinoic acid mediated regulation of melanocytic genes. Our data demonstrate that melanoma cell state can be regulated by expression of lipid droplet proteins which affect downstream retinoid signaling.

Desmosome mutations impact the tumor microenvironment to promote melanoma proliferation.

Desmosomes are transmembrane protein complexes that contribute to cell-cell adhesion in epithelia and other tissues. Here, we report the discovery of frequent genetic alterations in the desmosome in human cancers, with the strongest signal seen in cutaneous melanoma where desmosomes are mutated in over 70% of cases. In primary but not metastatic melanoma biopsies, the burden of coding mutations on desmosome genes associates with a strong reduction in desmosome gene expression. Analysis by spatial transcriptomics suggests that these expression decreases occur in keratinocytes in the microenvironment rather than in primary melanoma tumor cells. In further support of a microenvironmental origin, we find that loss-of-function knockdowns of the desmosome in keratinocytes yield markedly increased proliferation of adjacent melanocytes in keratinocyte/melanocyte co-cultures. Thus, gradual accumulation of desmosome mutations in neighboring cells may prime melanocytes for neoplastic transformation.

GABA Regulates Electrical Activity and Tumor Initiation in Melanoma.

Oncogenes can initiate tumors only in certain cellular contexts, which is referred to as oncogenic competence. In melanoma, whether cells in the microenvironment can endow such competence remains unclear. Using a combination of zebrafish transgenesis coupled with human tissues, we demonstrate that GABAergic signaling between keratinocytes and melanocytes promotes melanoma initiation by BRAFV600E. GABA is synthesized in melanoma cells, which then acts on GABA-A receptors in keratinocytes. Electron microscopy demonstrates specialized cell-cell junctions between keratinocytes and melanoma cells, and multielectrode array analysis shows that GABA acts to inhibit electrical activity in melanoma/keratinocyte cocultures. Genetic and pharmacologic perturbation of GABA synthesis abrogates melanoma initiation in vivo. These data suggest that GABAergic signaling across the skin microenvironment regulates the ability of oncogenes to initiate melanoma. SIGNIFICANCE: This study shows evidence of GABA-mediated regulation of electrical activity between melanoma cells and keratinocytes, providing a new mechanism by which the microenvironment promotes tumor initiation. This provides insights into the role of the skin microenvironment in early melanomas while identifying GABA as a potential therapeutic target in melanoma. See related commentary by Ceol, p. 2128. This article is featured in Selected Articles from This Issue, p. 2109.

Adult zebrafish as advanced models of human disease.

Modelling adult diseases to understand their aetiology and progression, and to develop new therapies, is a major challenge for medical biology. We are excited by new efforts in the zebrafish community to develop models of adult diseases that range from cancer to heart, infectious and age-related diseases, and those that relate to toxicology and complex social behaviours. Here, we discuss some of the advances in the field of zebrafish models of adult disease, and where we see opportunities and challenges ahead.

BayesTME: An end-to-end method for multiscale spatial transcriptional profiling of the tissue microenvironment.

Spatial variation in cellular phenotypes underlies heterogeneity in immune recognition and response to therapy in cancer and many other diseases. Spatial transcriptomics holds the potential to quantify such variation, but existing analysis methods are limited by their focus on individual tasks such as spot deconvolution. We present BayesTME, an end-to-end Bayesian method for analyzing spatial transcriptomics data. BayesTME unifies several previously distinct analysis goals under a single, holistic generative model. This unified approach enables BayesTME to deconvolve spots into cell phenotypes without any need for paired single-cell RNA-seq. BayesTME then goes beyond spot deconvolution to uncover spatial expression patterns among coordinated subsets of genes within phenotypes, which we term spatial transcriptional programs. BayesTME achieves state-of-the-art performance across myriad benchmarks. On human and zebrafish melanoma tissues, BayesTME identifies spatial transcriptional programs that capture fundamental biological phenomena such as bilateral symmetry and tumor-associated fibroblast and macrophage reprogramming. BayesTME is open source.

Lipid droplets are a metabolic vulnerability in melanoma.

Melanoma exhibits numerous transcriptional cell states including neural crest-like cells as well as pigmented melanocytic cells. How these different cell states relate to distinct tumorigenic phenotypes remains unclear. Here, we use a zebrafish melanoma model to identify a transcriptional program linking the melanocytic cell state to a dependence on lipid droplets, the specialized organelle responsible for lipid storage. Single-cell RNA-sequencing of these tumors show a concordance between genes regulating pigmentation and those involved in lipid and oxidative metabolism. This state is conserved across human melanoma cell lines and patient tumors. This melanocytic state demonstrates increased fatty acid uptake, an increased number of lipid droplets, and dependence upon fatty acid oxidative metabolism. Genetic and pharmacologic suppression of lipid droplet production is sufficient to disrupt cell cycle progression and slow melanoma growth in vivo. Because the melanocytic cell state is linked to poor outcomes in patients, these data indicate a metabolic vulnerability in melanoma that depends on the lipid droplet organelle.

Identifying the Transcriptional Drivers of Metastasis Embedded within Localized Melanoma.

In melanoma, predicting which tumors will ultimately metastasize guides treatment decisions. Transcriptional signatures of primary tumors have been utilized to predict metastasis, but which among these are driver or passenger events remains unclear. We used data from the adjuvant AVAST-M trial to identify a predictive gene signature in localized tumors that ultimately metastasized. Using a zebrafish model of primary melanoma, we interrogated the top genes from the AVAST-M signature in vivo. This identified GRAMD1B, a cholesterol transfer protein, as a bona fide metastasis suppressor, with a majority of knockout animals rapidly developing metastasis. Mechanistically, excess free cholesterol or its metabolite 27-hydroxycholesterol promotes invasiveness via activation of an AP-1 program, which is associated with increased metastasis in humans. Our data demonstrate that the transcriptional seeds of metastasis are embedded within localized tumors, suggesting that early targeting of these programs can be used to prevent metastatic relapse. SIGNIFICANCE: We analyzed human melanoma transcriptomics data to identify a gene signature predictive of metastasis. To rapidly test clinical signatures, we built a genetic metastasis platform in adult zebrafish and identified GRAMD1B as a suppressor of melanoma metastasis. GRAMD1B-associated cholesterol overload activates an AP-1 program to promote melanoma invasion. This article is highlighted in the In This Issue feature, p. 1.

Modeling the effects of genetic- and diet-induced obesity on melanoma progression in zebrafish.

Obesity is a rising concern and associated with an increase in numerous cancers, often in a sex-specific manner. Preclinical models are needed to deconvolute the intersection between obesity, sex and melanoma. Here, we generated a zebrafish system that can be used as a platform for studying these factors. We studied how germline overexpression of Agrp along with a high-fat diet affects melanomas dependent on BRAFV600E and loss of p53. This revealed an increase in tumor incidence and area in male, but not female, obese fish, consistent with the clinical literature. We then determined whether this was further affected by additional somatic mutations in the clinically relevant genes rb1 or ptena/b. We found that the male obesogenic effect on melanoma was present with tumors generated with BRAF;p53;Rb1 but not BRAF;p53;Pten. These data indicate that both germline (Agrp) and somatic (BRAF, Rb1) mutations contribute to obesity-related effects in melanoma. Given the rapid genetic tools available in the zebrafish, this provides a high-throughput system to dissect the interactions of genetics, diet, sex and host factors in obesity-related cancers.

Shifting the focus of zebrafish toward a model of the tumor microenvironment.

Cancer cells exist in a complex ecosystem with numerous other cell types in the tumor microenvironment (TME). The composition of this tumor/TME ecosystem will vary at each anatomic site and affects phenotypes such as initiation, metastasis, and drug resistance. A mechanistic understanding of the large number of cell-cell interactions between tumor and TME requires models that allow us to both characterize as well as genetically perturb this complexity. Zebrafish are a model system optimized for this problem, because of the large number of existing cell-type-specific drivers that can label nearly any cell in the TME. These include stromal cells, immune cells, and tissue resident normal cells. These cell-type-specific promoters/enhancers can be used to drive fluorophores to facilitate imaging and also CRISPR cassettes to facilitate perturbations. A major advantage of the zebrafish is the ease by which large numbers of TME cell types can be studied at once, within the same animal. While these features make the zebrafish well suited to investigate the TME, the model has important limitations, which we also discuss. In this review, we describe the existing toolset for studying the TME using zebrafish models of cancer and highlight unique biological insights that can be gained by leveraging this powerful resource.

Drug-Eluting Rubber Bands for Tissue Ligation.

Rubber band ligation is a commonly used method for the removal of tissue abnormalities. Most often, rubber band ligation is performed to remove internal hemorrhoids unresponsive to first line treatments to avoid surgery. While the procedure is considered safe, patients experience mild to significant pain and discomfort until the tissue sloughs off. As patients often require multiple bandings and sessions, reducing these side effects can have a considerable effect on patient adherence and quality of life. To reduce pain and discomfort, we developed drug-eluting rubber bands for ligation procedures. We investigated the potential for a band to elute anesthetics and drug combinations to durably manage pain for a period of up to 5 days while exhibiting similar mechanical properties to conventional rubber bands. We show that the rubber bands retain their mechanical properties despite significant drug loading. Lidocaine, released from the bands, successfully altered the calcium dynamics of cardiomyocytes in vitro and modulated heart rate in zebrafish embryos, while the bands exhibited lower cytotoxicity than conventional bands. Ex vivo studies demonstrated substantial local drug release in enteric tissues. These latex-free bands exhibited sufficient mechanical and drug-eluting properties to serve both ligation and local analgesic functions, potentially enabling pain reduction for multiple indications.

Bioluminescent Zebrafish Transplantation Model for Drug Discovery.

In the last decade, zebrafish have accompanied the mouse as a robust animal model for cancer research. The possibility of screening small-molecule inhibitors in a large number of zebrafish embryos makes this model particularly valuable. However, the dynamic visualization of fluorescently labeled tumor cells needs to be complemented by a more sensitive, easy, and rapid mode for evaluating tumor growth in vivo to enable high-throughput screening of clinically relevant drugs. In this study we proposed and validated a pre-clinical screening model for drug discovery by utilizing bioluminescence as our readout for the determination of transplanted cancer cell growth and inhibition in zebrafish embryos. For this purpose, we used NanoLuc luciferase, which ensured rapid cancer cell growth quantification in vivo with high sensitivity and low background when compared to conventional fluorescence measurements. This allowed us large-scale evaluation of in vivo drug responses of 180 kinase inhibitors in zebrafish. Our bioluminescent screening platform could facilitate identification of new small-molecules for targeted cancer therapy as well as for drug repurposing.

Anatomic position determines oncogenic specificity in melanoma.

Oncogenic alterations to DNA are not transforming in all cellular contexts1,2. This may be due to pre-existing transcriptional programmes in the cell of origin. Here we define anatomic position as a major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the palms of the hands, soles of the feet or under the nails3. We sequenced the DNA of cutaneous and acral melanomas from a large cohort of human patients and found a specific enrichment for BRAF mutations in cutaneous melanoma and enrichment for CRKL amplifications in acral melanoma. We modelled these changes in transgenic zebrafish models and found that CRKL-driven tumours formed predominantly in the fins of the fish. The fins are the evolutionary precursors to tetrapod limbs, indicating that melanocytes in these acral locations may be uniquely susceptible to CRKL. RNA profiling of these fin and limb melanocytes, when compared with body melanocytes, revealed a positional identity gene programme typified by posterior HOX13 genes. This positional gene programme synergized with CRKL to amplify insulin-like growth factor (IGF) signalling and drive tumours at acral sites. Abrogation of this CRKL-driven programme eliminated the anatomic specificity of acral melanoma. These data suggest that the anatomic position of the cell of origin endows it with a unique transcriptional state that makes it susceptible to only certain oncogenic insults.

Cancer stem cells: advances in biology and clinical translation-a Keystone Symposia report.

The test for the cancer stem cell (CSC) hypothesis is to find a target expressed on all, and only CSCs in a patient tumor, then eliminate all cells with that target that eliminates the cancer. That test has not yet been achieved, but CSC diagnostics and targets found on CSCs and some other cells have resulted in a few clinically relevant therapies. However, it has become apparent that eliminating the subset of tumor cells characterized by self-renewal properties is essential for long-term tumor control. CSCs are able to regenerate and initiate tumor growth, recapitulating the heterogeneity present in the tumor before treatment. As great progress has been made in identifying and elucidating the biology of CSCs as well as their interactions with the tumor microenvironment, the time seems ripe for novel therapeutic strategies that target CSCs to find clinical applicability. On May 19-21, 2021, researchers in cancer stem cells met virtually for the Keystone eSymposium "Cancer Stem Cells: Advances in Biology and Clinical Translation" to discuss recent advances in the understanding of CSCs as well as clinical efforts to target these populations.

Spatially resolved transcriptomics reveals the architecture of the tumor-microenvironment interface.

During tumor progression, cancer cells come into contact with various non-tumor cell types, but it is unclear how tumors adapt to these new environments. Here, we integrate spatially resolved transcriptomics, single-cell RNA-seq, and single-nucleus RNA-seq to characterize tumor-microenvironment interactions at the tumor boundary. Using a zebrafish model of melanoma, we identify a distinct "interface" cell state where the tumor contacts neighboring tissues. This interface is composed of specialized tumor and microenvironment cells that upregulate a common set of cilia genes, and cilia proteins are enriched only where the tumor contacts the microenvironment. Cilia gene expression is regulated by ETS-family transcription factors, which normally act to suppress cilia genes outside of the interface. A cilia-enriched interface is conserved in human patient samples, suggesting it is a conserved feature of human melanoma. Our results demonstrate the power of spatially resolved transcriptomics in uncovering mechanisms that allow tumors to adapt to new environments.

Cooperation between melanoma cell states promotes metastasis through heterotypic cluster formation.

Melanomas can have multiple coexisting cell states, including proliferative (PRO) versus invasive (INV) subpopulations that represent a "go or grow" trade-off; however, how these populations interact is poorly understood. Using a combination of zebrafish modeling and analysis of patient samples, we show that INV and PRO cells form spatially structured heterotypic clusters and cooperate in the seeding of metastasis, maintaining cell state heterogeneity. INV cells adhere tightly to each other and form clusters with a rim of PRO cells. Intravital imaging demonstrated cooperation in which INV cells facilitate dissemination of less metastatic PRO cells. We identified the TFAP2 neural crest transcription factor as a master regulator of clustering and PRO/INV states. Isolation of clusters from patients with metastatic melanoma revealed a subset with heterotypic PRO-INV clusters. Our data suggest a framework for the co-existence of these two divergent cell populations, in which heterotypic clusters promote metastasis via cell-cell cooperation.

Developmental chromatin programs determine oncogenic competence in melanoma.

Oncogenes only transform cells under certain cellular contexts, a phenomenon called oncogenic competence. Using a combination of a human pluripotent stem cell­derived cancer model along with zebrafish transgenesis, we demonstrate that the transforming ability of BRAFV600E along with additional mutations depends on the intrinsic transcriptional program present in the cell of origin. In both systems, melanocytes are less responsive to mutations, whereas both neural crest and melanoblast populations are readily transformed. Profiling reveals that progenitors have higher expression of chromatin-modifying enzymes such as ATAD2, a melanoma competence factor that forms a complex with SOX10 and allows for expression of downstream oncogenic and neural crest programs. These data suggest that oncogenic competence is mediated by regulation of developmental chromatin factors, which then allow for proper response to those oncogenes.

Zebrafish as a New Model to Study the Crosstalk between Tumor and Host Metabolism.

The mechanism of metabolic crosstalk between different tissues in the host remains poorly understood but is likely to play important roles in tumor growth and cachexia. A recent study from Naser, Jackstadt, et al. utilizes isotope tracing in a zebrafish melanoma model and finds that tumors secrete alanine into circulation, which is taken up by the liver and used for gluconeogenesis.

An in vivo reporter for tracking lipid droplet dynamics in transparent zebrafish.

Lipid droplets are lipid storage organelles found in nearly all cell types from adipocytes to cancer cells. Although increasingly implicated in disease, current methods to study lipid droplets in vertebrate models rely on static imaging or the use of fluorescent dyes, limiting investigation of their rapid in vivo dynamics. To address this, we created a lipid droplet transgenic reporter in whole animals and cell culture by fusing tdTOMATO to Perilipin-2 (PLIN2), a lipid droplet structural protein. Expression of this transgene in transparent casper zebrafish enabled in vivo imaging of adipose depots responsive to nutrient deprivation and high-fat diet. Simultaneously, we performed a large-scale in vitro chemical screen of 1280 compounds and identified several novel regulators of lipolysis in adipocytes. Using our Tg(-3.5ubb:plin2-tdTomato) zebrafish line, we validated several of these novel regulators and revealed an unexpected role for nitric oxide in modulating adipocyte lipid droplets. Similarly, we expressed the PLIN2-tdTOMATO transgene in melanoma cells and found that the nitric oxide pathway also regulated lipid droplets in cancer. This model offers a tractable imaging platform to study lipid droplets across cell types and disease contexts using chemical, dietary, or genetic perturbations.

Organisms need fat molecules as a source of energy and as building blocks, but these 'lipids' can also damage cells if they are present in large amounts. Cells guard against such toxicity by safely sequestering lipids in specialized droplets that participate in a range of biological processes. For instance, these structures can quickly change size to store or release lipids depending on the energy demands of a cell. It is possible to image lipid droplets ­ using, for example, dyes that preferentially stain fat ­ but often these methods can only yield a snapshot: tracking lipid droplet dynamics over time remains difficult. Lumaquin, Johns et al. therefore set out to develop a new method that could label lipid droplets and monitor their behaviour 'live' in the cells of small, transparent zebrafish larvae. First, the fish were genetically manipulated so that a key protein found in lipid droplets would carry a fluorescent tag: this made the structures strongly fluorescent and easy to track over time. And indeed, Lumaquin, Johns et al. could monitor changes in the droplets depending on the fish diet, with the structures getting bigger when the animal received rich food, and shrinking when resources were scarce. Finally, experiments were conducted to screen for compounds that could lead to lipids being released in fat cells. The new imaging technique was then used to confirm the effect of these molecules in live cells, revealing an unexpected role for a signalling molecule known as nitric oxide, which also turned out to be regulating lipid droplets in cancerous cells. Further work then showed that drugs affecting nitric oxide could modulate lipid droplet size in both normal and tumor cells. This work has validated a new method to study the real-time behavior of lipid droplets and their responses to different stimuli in living cells. In the future, Lumaquin, Johns et al. hope that the technique will help to shed new light on how lipids are involved in both healthy and abnormal biological processes.

SATB2 induction of a neural crest mesenchyme-like program drives melanoma invasion and drug resistance.

Recent genomic and scRNA-seq analyses of melanoma demonstrated a lack of recurrent genetic drivers of metastasis, while identifying common transcriptional states correlating with invasion or drug resistance. To test whether transcriptional adaptation can drive melanoma progression, we made use of a zebrafish mitfa:BRAFV600E;tp53-/- model, in which malignant progression is characterized by minimal genetic evolution. We undertook an overexpression-screen of 80 epigenetic/transcriptional regulators and found neural crest-mesenchyme developmental regulator SATB2 to accelerate aggressive melanoma development. Its overexpression induces invadopodia formation and invasion in zebrafish tumors and human melanoma cell lines. SATB2 binds and activates neural crest-regulators, including pdgfab and snai2. The transcriptional program induced by SATB2 overlaps with known MITFlowAXLhigh and AQP1+NGFR1high drug-resistant states and functionally drives enhanced tumor propagation and resistance to Vemurafenib in vivo. In summary, we show that melanoma transcriptional rewiring by SATB2 to a neural crest mesenchyme-like program can drive invasion and drug resistance in autochthonous tumors.