Published: September 4th, 2021
Here, we present a protocol to establish versatile orthotopic and ectopic zebrafish xenograft models for ocular melanoma to assess the growth kinetics of the primary tumor, dissemination, extravasation and distant, peri-vascular metastasis formation and the effect of chemical inhibition thereon.
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.
Metastatic dissemination is considered the main cause of death of ocular melanoma; currently there is no viable treatment regime for disseminated ocular melanoma1,2. Furthermore, there are no animal models available for ocular melanoma that reflects the metastatic disease. To bridge this gap, we generated two distinct zebrafish models that recapitulate either primary tumor formation or the early stages of metastatic dissemination, thus readily allowing the study of these normally difficult to study processes 3. The micro-metastasis models allow the analysis of the last phases of metastatic spread, including homing, colonization and extravasation. Genetic or chemical interventions at this stage and beyond could potentially provide a powerful handhold in the treatment of metastatic ocular melanoma.
The use of the zebrafish larvae as a recipient of xeno- and allografts is supported by the intrinsic strengths of this species, such as its optical transparency at the early stages of development (or its entire life-cycle for casper mutants4), high fecundity and ex utero fertilization5. High transcriptional homology in vertebrates ensures the retention of core signaling mechanisms between the zebrafish and humans and therefore high potential translatability of results 6, although genetic approaches are sometimes marred or complicated due to the teleost genome duplication 7. Recent developments have underscored the importance of zebrafish xenograft models as pre-clinical "avatars" of human disease8, effectively yielding a multitude of personalized cancer therapy models for the pre-clinical evaluation of treatment strategies from a single zebrafish experiment 9.
Considering the lack of animal models and the concordant lack of treatment options for metastatic ocular melanoma, our models provide a quick and easy translational platform to screen both genetic alterations (cancer cell intrinsic) or develop chemical intervention strategies in a pre-clinical setting. Within the same model we can visualize and measure cancer cell growth kinetics, engraftment rate/metastatic potential, and cell homing on a whole animal level using low level magnification in a stereo fluorescent microscope, and make similar measurements using medium or high magnification confocal microscopic analysis to dissect different steps of ocular melanoma progression at subcellular resolution 10.
Here, we describe comprehensive and detailed protocols for: the generation of fluorescently labeled cancer cells using highly optimized lentiviral transduction11; subsequent intravenous and retro-orbital (RO) engraftments of these cells into 2 days post fertilization (dpf) zebrafish larvae to generate ectopic and orthotopic models respectively; followed by data acquisition and analysis. These methods although comprehensive for the applications described herein can be modified to engraft cells in the hind brain cavity, liver and perivitellin space when required (solely by changing the injection site, or time of injection)12,13.
As a proof-of-concept we elaborated upon the findings of Pontes et al. 2018, where we showed a dose and cell intrinsic mutation specific response of conjunctival melanoma cell lines in the zebrafish model 14. We elaborated upon these findings by showing the efficacy of BRAF V600E mutation-specific inhibitor vemurafenib in both metastatic and primary conjunctival melanoma models.
All animal experiments were approved by Animal Experiments Committee (Dier Experimenten Commissie, D.E.C.) under license AVD1060020172410. All animal were maintained in accordance with local guidelines using standard protocols (www.ZFIN.org).
NOTE: Make sure that the capillaries have been calibrated on the filament used. When switching either the filament or the capillary, determine the ramp value of the capillaries on the filament used (see needle puller manual).
3. Generation of lentiviral particles
NOTE: To prevent a waste of time and resources a quick tumorigenicity check can be performed prior to lentiviral transduction. This is done to ensure that the cell line to be used is sufficiently tumorigenic in the zebrafish model, to this end the cells can be stained with a CMdiI (or analogous tracer) as described in Liverani et al. 2017 15.
Figure 1. Schematic representation of the described zebrafish engraftment system. A) The timeline of the approach, with breeding the zebrafish at day 0 (B1). The fish are harvested in the morning after crossing the fish (day 1). After 48-54 hours the fish have largely hatched (shedding their chorion) and the fish are injected (retro-orbitally or systemically, B2) after cleaning the water of the chorion debris (day 2). The larvae are subsequently screened using a stereo fluorescent microscope and all larvae displaying unwanted phenotypes are discarded (day 3). Depending on the goal of the experiment either the larvae are imaged over time (B3, engraftment kinetics, imaged at 1-, 4- and 6-days post injection (dpi)) or the fish are randomized and entered into experimental groups, treated with drugs and compared to vehicle control (drug screening, imaged at 6 dpi). Please click here to view a larger version of this figure.
4. Lentiviral transduction
5. Breeding zebrafish
6. Harvesting cells
NOTE: Proper cell preparation is key to the implantation procedure, using a superfluous amount of cells allows for easier downstream processing. The third centrifugation step is critical, as this will leave you with only the cell pellet, the remaining PBS stuck on the sides of the centrifuge tube greatly exceeds the final resuspension volume.
7. Xenograft modeling
All experiments should be performed in compliance with local animal welfare regulations.
Depending on the application two main variations in experimental design are classified as a phenotype assessment (7.1 the pre-screening stage) and secondly 7.2 a screen where either the cells have been modified prior to engraftment or 7.3 where the embryos are treated with a chemical inhibitor.
NOTE: Use a pneumatic pulse controller coupled to a compressed air line, supplying pressure in surplus of 100 psi. This allows for enough pressure to both inject (≈20 psi) and to eject possible cell aggregates (≈100 psi). The starting pressure and time should be approximately 200 ms at 20 psi. If either has to be decreased more than 50% at the start of the injection either the cell suspension is too fluid (cell or PVP40 concentration too low) or needle opening is too large.
10. Epifluorescent imaging of zebrafish larvae
11. Confocal imaging of (engrafted) zebrafish larvae
12. Setting the confocal microscope
13. Data analysis
We have provided step by step instructions for a fast and easy approach to progress from a novel cell line to its analysis. We start with the over expression of a fluorescent tracer using a lentiviral overexpression cassette (steps 3 and 4). This is followed by cell preparation to ensure the least possible dead volume while injecting, allowing to inject high cell numbers into both doC and retro-orbital space (steps 6 and 7). Subsequently, we perform semi-high throughput data acquisition using stereo-fluorescent microscopy and higher magnification confocal microscopy for qualitative analysis of whole-body cancer cell dissemination (Figure 2 and steps 10, 11 and 12). Care has to be taken when acquiring data, as to ensure the reproducibility for both stereo and confocal microscopic imaging, the generic settings and standardization are delineated (steps 11 and 12). Data analysis is discussed (using imageJ/Fiji) 16, along with standardization using imageJ macros (step 13).
In step 3 we mentioned the transient labelling of (cancer) cells to perform a quick pre-screening to assess the tumorigenic potential of a new cancer cell line. One important caveat is that although easy to use and long living, the transient stain described herein has the possibility to form artefacts (i.e., care has to be taken to ensure that cell fragments can be distinguished from whole cells as was performed extensively by Fior and colleagues 9). In our experience the formation of these artefacts is directly linked to the extreme stability of the stain and the brightness (even after cell death), where cell fragments are dispersed and taken up by immune cells, which could subsequently be falsely concluded to derive from active metastasis.
In both described models, the systemic engraftment through the doC and the localized engraftment in the retro-orbital space, thorough screening of the larvae one day after injection is of paramount importance. As shown in Figure 2B all larvae that display mechanical displacement of the engrafted cells into the head area (beyond the retro-orbital site) in the retro-orbital model and cells in the yolk sac, or displaying an edema in the doC injected pool should be removed. All negatively selected phenotypes are displayed as high-resolution confocal stitches in Figure 2, but can be readily seen and removed through stereo microscopical observation.
Over time cells will both migrate and proliferate. For the retro-orbital model, we observed infiltration into neighboring tissues for CRMM1, but we observed less proliferation for CRMM2. We strikingly did observe distant metastasis arising between 2-4 dpi in some individuals (20%), where we measured a significant difference at 6 dpi, as shown in Figure 4. For both cell lines, we tested the proliferative potential when injected in both sites. For CRMM1 there was a significant (p<0.0001) increase in cancer cell number for or at the injection sites, when displayed as normalized tumor cell burden, normalizing to day one for each model (7.8-fold increase, ±3.2 for the RO model and an increase of 15-fold ±8,8 for the doC model). CRMM2 did not display significant growth when normalized to day one for each individual model (2.4-fold increase, ±1.9- and 2.3-fold increase, ±1.14 for the RO and doC). CRMM1 was found to readily proliferate in both retro-orbital tissue and the caudal hematopoietic tissue after engraftment. Cell line CRMM2 was less proliferative in both models, but interestingly was found to be capable of distant metastasis when injected in the retro-orbital space as shown in Figure3B,C.
After screening the injected larvae at 1 dpi and randomly assigning the individuals to either treatment or control groups, the fish were treated for 6 days, changing the water containing Vemurafenib (this inhibitor can readily be interchanged for any other titrated antitumor compound). We chose to elaborate upon the previously published hematogenous conjunctival melanoma dissemination model engrafting CRMM114, by testing Vemurafenib's efficacy on orthotopically engrafted CRMM1. CRMM1 showed a strong significant reduction of the Vemurafenib treated ectopically engrafted group (P<0.0001) and a stunted yet significant response for the orthotopically engrafted model (p<0.05) as shown in Figure 4.
Figure 2. Phenotypic assessment and screening after injection. A) Schematic depiction of zebrafish xenograft confocal stitch generation, yielding seamless, high resolution images after integration of subsequent confocal projection. Here zebrafish xenografts are embedded in 1% low melting agarose and mounted on a glass bottom confocal dish (as described in step 11.3). B) All possible outcomes of retro-orbital and duct of Cuvier engraftment are displayed injected in green fluorescent blood vessel reporter zebrafish (TG:fli:GFP), with cells stained through lentiviral over expression of tdTomato). We denote the correct engraftment at 1 dpi (RO panel) and the unwanted phenotypes (both brain leakage and blood vessel leakage). The latter two populations must be removed to ensure they do not confound downstream experimental findings. C) The unwanted phenotypes for the hematogenous engraftment through the duct of Cuvier (doC) are outlines where cardiac edematous larvae (Cardiac edema) and larvae with cells leaking into the yolk sac (Yolk injection) must be removed to prevent interference with downstream measurements. The correctly injected larvae are entered into experimental groups as described in step 7.1. (All images acquired at 1 dpi, using a confocal microscope, scale bars 200 µm. Yellow boxes indicate metastatic sites for both RO and doC engraftments, head region and caudal hematopoietic tissue, respectively). Please click here to view a larger version of this figure.
Figure 3. Comparative analysis of conjunctival melanoma cell lines CRMM1 and CRMM2 show differential metastatic and growth capacity. A) Schematic representation of injection models, retro-orbital model (RO) and hematogenous engraftment model (doC) the fish used are TG(fli:GFP) green blood vessel reporters, with cells over expressing tdTomato shown in red. B) Representative phenotypes of fish engrafted with CRMM1 and CRMM2, CRMM1 displays efficient engraftment (both RO and doC) and small scale invasion into the tissue surrounding the RO engraftment site (RO, yellow arrowheads). CRMM2 exhibits a remarkably lower engraftment efficiency for both engraftment models, but shows distant metastasis when injected retro-orbitally (as shown in RO, denoted by the arrowheads). (All images acquired at 6 dpi, a confocal microscope, scale bars 200 µm. Yellow arrowheads indicate metastatic sites for both RO and doC engraftments, head region and caudal hematopoietic tissue respectively). C) Kinetic engraftment plots for both CRMM1 and CRMM2, comparing both engraftment models to day 1 (normalizing to day 1), there is a significant (p<0.0001) increase in normalized tumor burden for cell line CRMM1(between 1 dpi and 6 dpi) where there is a (non-significant) upward trend for CRMM2. CRMM1 reveals a significant difference between RO and doC growth, where the doC model shows a higher tumor expansion rate (approximately 2-fold higher for the doC engrafted larvae). Graphs display the mean and standard error of the mean (SEM). All groups were normalized to 1 dpi for each individual condition. Please click here to view a larger version of this figure.
Figure 4. BRAF V600E inhibitor Vemurafenib significantly inhibits both RO and doC conjunctival melanoma engrafted zebrafish larvae. A) Schematic representation of zebrafish phenotypes, RO and doC models. B) Both RO and doC engrafted larvae, injected with conjunctival melanoma cell line CRMM1 display a significant reduction of normalized tumor burden (p<0.05 and P<0.001 respectively). The doC engrafted zebrafish models indicate an enhanced drug response and a dose independent relationship to drug inhibition, indicating a possible saturation of inhibition). Graphs show the mean and standard error of the mean (SEM), All groups were normalized to control for each individual cell line. Please click here to view a larger version of this figure.
|1.71 pmol (12.14µg)
|0.94 pmol (3.66µg)
|1.64 pmol (calculate exact volume)
Here, we have defined a meticulous approach to model primary and metastatic ocular melanoma in zebrafish xenografts. By combining both a localized, orthotopic injection and a systemic, ectopic injection models we have recapitulated the etiology of carcinogenesis for a cancer where no animal models were previously available. The inherent transparency of the early zebrafish larva allows the tracking of fluorescently labelled cancer cells on a whole animal level, ensuring the easy visualization of potential metastatic sites17. Moreover high magnification confocal microscopical analysis allows us to track cells at a subcellular resolution10.
We have provided step by step instructions for a fast and easy approach to progress from a novel cell line to establishment of the xenograft and its analysis. We start with the overexpression of a fluorescent tracer using a lentiviral overexpression cassette (step 3 and 4) followed by cell preparation to ensure the least possible dead volume while injecting. This enables the injection of high cell numbers into both doC and retro-orbital space (step 7 and 8). Then we perform semi-high throughput data acquisition using stereo-fluorescent microscopy and higher magnification confocal microscopy for qualitative analysis of whole-body cancer cell dissemination (Figure 2 and step 9 and 10). Care has to be taken when acquiring data, as to ensure the reproducibility for both stereo and confocal microscopic imaging, the generic settings and standardization are delineated (steps 11 and 12). Data analysis is discussed (using imageJ/Fiji) 16, along with standardization using ImageJ macros (step 13).
In step 3 we mention the transient labelling of (cancer) cells to perform a quick pre-screen to assess the tumorigenic potential of a new cancer cell line. One important caveat is that although easy to use and long living, the transient stain described herein has the possibility to form artefacts (e.g., care has to be taken to ensure that cell fragments can be distinguished from whole cells as was performed extensively by Fior and colleagues 9). In our experience the formation of these artefacts is directly linked to the extreme stability of the stain and the brightness (even after cell death), where cell fragments are dispersed and taken up by immune cells, which could subsequently be falsely concluded to derive from active metastasis.
Using these models, we simulated primary tumor development by physically confining the engrafted cells within the retro-orbital interstice. Subsequent thorough screening at 1 day post engraftment ensures that cells found at distant site later in the experiment have actively metastasized (intravasated and disseminated, ultimately to extravasate at the metastatic niche). Engraftment through the doC, the embryonic common cardinal vein, allows for easy and highly reproducible implantation of large quantities for cells (at a surplus of 600 cells when properly concentrated), effectively circumventing the primary stages of the metastatic cascade (intravasation) and allowing us to focus on the later stages of the metastatic cascade (adhesion, extravasation and outgrowth). Although powerful tools when used properly, both models should be monitored extensively during the first day post engraftment to ensure that no false positive conclusions are drawn during the later stages of the experiment.
In line with previous publications we have shown that conjunctival melanoma lines readily form metastatic colonies after dissemination throughout the zebrafish blood circulation system14. Here we report the expanding of the engraftment repertoire with the retro-orbital injection as an orthotopic model, and the subsequent active metastasis to the caudal hematopoietic tissue of the cell line CRMM2. Subsequently we report the efficacy of BRAF V600E specific inhibitor Vemurafenib also on the primary form of conjunctival melanoma when modelled in zebrafish larvae.
Using the aforementioned methods, a skilled researcher is capable of generating in excess of hundreds of engrafted larvae per day (approximately 200 per hour) of either model proposed. In a timescale of two weeks a drug can be both titrated for maximum tolerated dose, and screened on established xenograft model. From start to finish, using a non-transduced cell line, to having a drug sensitivity profile in the zebrafish model can be achieved within a month (given that the injected cell line is tumorigenic within the zebrafish model). In our hands as little as 20 larvae per experiments and two biological repeats have reproducibly yielded robust drug inhibition, when two individual experiments conflict (or do not yield statistically significant growth inhibition) a third biological repeat can be conducted.
Through minor adjustments, these models have allowed us to quickly adapt these implantation strategies for glioblastoma (hind brain cavity injection), breast cancer (doC injection) and osteo sarcoma (doC) among others 18,19,20,21. These models can subsequently be utilized for both basic research and pre-clinical screening of both single drugs and combinatorial drug strategies. Recently, we described different administration regimes of drugs and their photo activation using these models 13.
This work was supported by funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 667787 (UM Cure 2020 project, www.umcure2020.org). The Chinese Scholarship Council is kindly acknowledged for a PhD grants to J.Y.
|2.5mm box filament
|for pulling micro injection needles using a Sutter P97 or P1000
|3mL transfer pipettes
|for transfer and selection of zebrafish embryos
|1.5% (w/v) in eggwater, 1.5 g in 100 mL DPBS, microwave to dissolve, for injecting and stereofluorescence imaging of zebrafish larvae
|Capillaries: borosilicate glass outer
|World precision instruments
|Borosilicate glass capillaries used for needle preparation
|Often used as solvent in drug treatments, should be stored at 2-8°C the dark.
|Thermo Fischer Scientific
|Dulbecco’s phosphate buffered saline, without Mg2+ and Ca2+ for washing the cells, lack of Ca2+ impairs cell-cell adhesion through cadherins and prevents cell aggregation during injection
|0.6 mg/L final concentration sea salt in demineralized water
|GFP encoding lentiviral transfer plasmid
|Generated in Snaar lab, available at Addgene
|Stable cell line for generating lentiviral particles, contains SV40-T antigen required for the generation of lentiviral particles
|Leica sp8 confocal
|Leica TCS SP8
|automated stage confocal microscope with 405/488/514/635nm lasers
|Highly efficient HEK293t optimized transfection reagent
|1% (w/v) in eggwater 1.5 g in 100 mL DPBS, microwave to dissolve, for embedding zebrafish larvae for confocal imaging
|Micro loader tips
|flexible microloader tips
|World precision instruments
|x/y/z manual micro manipulator for microinjection
|Needle puller: P-97 or P-1000
|needle puller used for generating standardized micro engraftment needles
|Nr.5 watchmakers forceps
|fine watchmakers forceps used for breaking back needles
|World precision instruments
|pulse controller supplying pressure for microinjection
|Gifted by Didier Trono, 2nd generation lentiviral virulence plasmid
|Gifted by Didier Trono, 2nd generation lentiviral packaging plasmid
|Polyvinylpyrrolidone average mol wt 40,000) PVP40 2% (w/v) in DPBS, 1 g PVP40 in 50 mL DPBS. Vortex and incubate at 37°C to facilitate dissolving. Store at room temperature.
|tdTomato encoding lentiviral transfer plasmid
|Generated in Snaar lab, available at Addgene
|transmitted light stereo microscope
|leica M50 with (MDG33 base)
|leica transmitted light microscope with mirror adjustable illumination.
|Ethyl 3-aminobenzoate methanesulfonate or MS-222
|Thermo Fischer Scientific
|Synthetic trypsine replacement, less damaging to the cells and allows for the gentle dispersion of strongly adherent cells. (Thermo-
|50mm glass bottom dishes, allow for the embedding of up to 20 zebrafish larvae, enabling the imaging of multiple conditions in one dish due to its large optical glass surfac
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