We thank to Champalimaud Foundation, Congento (LISBOA-01-0145-FEDER-022170, co-financed by FCT/Lisboa2020)&#160;and FCT-PTDC/MEC-ONC/31627/ 2017&#160;for funding. FCT fellowships for VP (SFRH/BD/118252/2016), MML (PD/BD/138203/2018). All members of the Fior Lab for critical discussions; B. Costa and C. Rebelo de Almeida for sharing data; and our Lab members C. Rebelo de Almeida, M. Barroso and L. Leite for their participation in the video. We would like to thank the CF Fish Facility (C. Certal, J. Monteiro et al) and Champalimaud Communication, Events and Outreach team in particular Alexandre Azinheira for the fantastic film making and Catarina Ramos and Teresa Fernandes for their help.

<ol>
	<li>Gut, P., Reischauer, S., Stainier, D. Y. R., Arnaout, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Little+Fish,+Big+Data:+Zebrafish+as+a+Model+for+Cardiovascular+and+Metabolic+Disease.">Little Fish, Big Data: Zebrafish as a Model for Cardiovascular and Metabolic Disease.</a> Physiological Reviews. 97, 889-938 (2017).</li><li>Howe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496, 498-503 (2013).</li><li>Santoriello, C., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooked!+Modeling+human+disease+in+zebrafish.">Hooked! Modeling human disease in zebrafish.</a> Journal of Clinical Investigation. 122, 2337-2343 (2012).</li><li>Stoletov, K., Klemke, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catch+of+the+day:+zebrafish+as+a+human+cancer+model.">Catch of the day: zebrafish as a human cancer model.</a> Oncogene. 27, 4509-4520 (2008).</li><li>Weintraub, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All+eyes+on+zebrafish.">All eyes on zebrafish.</a> Lab Animals. 46, 323-326 (2017).</li><li>Novoa, B., Figueras, A., Lambris, J. D., Hajishengallis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+Model+for+the+Study+of+Inflammation+and+the+Innate+Immune+Response+to+Infectious+Diseases.">Zebrafish: Model for the Study of Inflammation and the Innate Immune Response to Infectious Diseases.</a> Current Topics in Innate Immunity II. , 253-275 (2012).</li><li>Fazio, M., Ablain, J., Chuan, Y., Langenau, D. M., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+patient+avatars+in+cancer+biology+and+precision+cancer+therapy.">Zebrafish patient avatars in cancer biology and precision cancer therapy.</a> Nature Reviews Cancer. 20, 263-273 (2020).</li><li>Fior, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+functional+and+chemosensitive+profiling+of+combinatorial+colorectal+therapy+in+zebrafish+xenografts.">Single-cell functional and chemosensitive profiling of combinatorial colorectal therapy in zebrafish xenografts.</a> Proceedings of the National Academy of Sciences of the United States of America. 114, 8234-8243 (2017).</li><li>P&#243;voa, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+evasion+revealed+in+a+colorectal+zebrafish+xenograft+model.">Innate immune evasion revealed in a colorectal zebrafish xenograft model.</a> Nature Communications. 12, 1156 (2021).</li><li>Galletti, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Macrophages+Sensitizes+Chronic+Lymphocytic+Leukemia+to+Apoptosis+and+Inhibits+Disease+Progression.">Targeting Macrophages Sensitizes Chronic Lymphocytic Leukemia to Apoptosis and Inhibits Disease Progression.</a> Cell Reports. 14, 1748-1760 (2016).</li><li>Rebelo de Almeida, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+xenografts+as+a+fast+screening+platform+for+bevacizumab+cancer+therapy.">Zebrafish xenografts as a fast screening platform for bevacizumab cancer therapy.</a> Communications Biology. 3, 1-13 (2020).</li><li>Varanda, A. B., Martins-Logrado, A., Godinho Ferreira, M., Fior, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+Xenografts+Unveil+Sensitivity+to+Olaparib+beyond+BRCA+Status.">Zebrafish Xenografts Unveil Sensitivity to Olaparib beyond BRCA Status.</a> Cancers. 12, 1769 (2020).</li><li>Osmani, N., Goetz, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+Imaging+of+Metastasis+in+Zebrafish.">Multiscale Imaging of Metastasis in Zebrafish.</a> Trends in Cancer. 5, 766-778 (2019).</li><li>Hyenne, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+Fate+of+Tumor+Extracellular+Vesicles+at+High+Spatiotemporal+Resolution+Using+the+Zebrafish+Embryo.">Studying the Fate of Tumor Extracellular Vesicles at High Spatiotemporal Resolution Using the Zebrafish Embryo.</a> Developmental Cell. 48, 554-572 (2019).</li><li>Costa, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+in+zebrafish+avatars+as+radiotherapy+sensitivity+reporters+-+towards+personalized+medicine.">Developments in zebrafish avatars as radiotherapy sensitivity reporters - towards personalized medicine.</a> EBioMedicine. 51, 102578 (2020).</li><li>Costa, B., Estrada, M. F., Mendes, R. V., Fior, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+Avatars+towards+Personalized+Medicine-A+Comparative+Review+between+Avatar+Models.">Zebrafish Avatars towards Personalized Medicine-A Comparative Review between Avatar Models.</a> Cells. 9, 293 (2020).</li><li>TRICAINE - Protocols. ZFIN Community Wiki Available from: <a target="_blank" href="https://wiki.zfin.org/display/prot/TRICAINE">https://wiki.zfin.org/display/prot/TRICAINE</a> (2021)</li><li>Zhao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Xenograft+Model+in+Zebrafish+for+High-Resolution+Investigating+Dynamics+of+Neovascularization+in+Tumors.">A Novel Xenograft Model in Zebrafish for High-Resolution Investigating Dynamics of Neovascularization in Tumors.</a> Plos One. , (2011).</li><li>Veinotte, C. J., Dellaire, G., Berman, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooking+the+big+one:+the+potential+of+zebrafish+xenotransplantation+to+reform+cancer+drug+screening+in+the+genomic+era.">Hooking the big one: the potential of zebrafish xenotransplantation to reform cancer drug screening in the genomic era.</a> Disease Models &amp; Mechanisms. 7, 745-754 (2014).</li><li>Haldi, M., Ton, C., Seng, W. L., McGrath, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+melanoma+cells+transplanted+into+zebrafish+proliferate,+migrate,+produce+melanin,+form+masses+and+stimulate+angiogenesis+in+zebrafish.">Human melanoma cells transplanted into zebrafish proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrafish.</a> Angiogenesis. 9, 139-151 (2006).</li><li>Zon, L. I., Peterson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+New+Age+of+Chemical+Screening+in+Zebrafish.">The New Age of Chemical Screening in Zebrafish.</a> Zebrafish. 7, 1 (2010).</li><li>Mycoplasma Contamination of Cell Cultures. InvivoGen Available from: <a target="_blank" href="https://www.invivogen.com/review-mycoplasma">https://www.invivogen.com/review-mycoplasma</a> (2016)</li><li>van Zijl, F., Krupitza, G., Mikulits, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+steps+of+metastasis:+cell+invasion+and+endothelial+transmigration.">Initial steps of metastasis: cell invasion and endothelial transmigration.</a> Mutation Research. 728, 23-34 (2011).</li></ol>

The increasing importance of the zebrafish as a model for cancer development and drug screening has resulted in numerous publications3,4,7,13,14,16,18,19,20,21. However, the injection of cancer cells in zebrafish embryos is a technique that requires a high level of dexterity which can be challenging for researchers. In this protocol we aim at providing practical information and some tips that can help overcome the initial challenges of setting up zebrafish embryo xenografts.
Cell handling prior injection 
This optimized protocol for the generation of zebrafish xenografts with cell lines can be adapted to various types of (cancer) cells with different morphologies. We recommend that all the cell lines used for zebrafish xenografts are mycoplasma free. Unlike other bacterial contaminations, the presence of mycoplasma in cell culture does not generate changes that can be easily detected under the microscope22. Mycoplasma contamination may affect the engraftment potential of the cell lines, their sensitivity to drugs, as well as the viability of the zebrafish embryos.
Although cells may continue to proliferate for an extended period, their phenotype and genotype can be prone to changes. It is important to become familiar with the morphology and behavior of the cell lines in culture. We recommend keeping the number of cell passages after thawing between 3-12 to get reproducible results. Thus, regular mycoplasma tests should be carried out.
Cells should be at their log phase (exponential growth phase before reaching confluence ~70%) on the day of the injection. This will enable an adequate engraftment and proper development of the distinctive hallmarks of the tumor. To prevent variation in the phenotype of cells within the xenograft, it is critical to maintain the confluence for injection constant between experiments. The number of injected cells can be adapted to the characteristics of each cell line as some may require higher densities of injection in order to thrive in the zebrafish embryos.
Cell labelling considerations 
To better visualize human tumor cells for injection and future analysis, tumor cells can be labelled with fluorescent dyes. Due to differences in cellular size, the total number of cells/cm2 in adherent cultures varies among cell lines. This will impact the efficacy of the staining protocol as well as the number of cells harvested for injection. Large cells that yield low numbers per flask (i.e., Hs578T) or grow in clusters (i.e., BFTC905) will require the pooling of several flasks for a single experiment. In this case, the staining of cells shouldn&#39;t be performed directly in the flask as this will cause the use of excessive amounts of dye (high cost). On the other hand, cells that are highly sensitive to excessive cycles of centrifugation as well as those that yield high numbers per flask (i.e., HCT116) can be stained directly in the flask and then detached with EDTA/cell scraper (For more information see Table 1).
Whenever possible, instead of using an enzymatic approach, use EDTA to detach the cells on the day of injection, so that cells recover their cell-cell junctions more rapidly and are subjected to less centrifugation steps. Nevertheless, if the cells are sensitive to EDTA, very adherent or grow in clusters - an enzymatic method can be applied. The optimization of the ideal concentration for injection as well as the injection medium is dependent on the characteristics of each cell line, thus it might need some adjustments (Table 1).
Microinjection calibration 
Contrary to the delivery of oligonucleotides or drugs into the zebrafish embryo, a graticule is not used to calibrate the needle when working with cell lines for xenografts. After some time during injection, cells will start to clog, and it is necessary to cut the tip of the needle to increase its diameter or change the needle altogether. This procedure hinders the graticule calibration.
To address this issue, the number of cells dispensed is regulated by eject pressure and time needed to reach a size similar to the embryo eye within 1-3 pulses. Then, to further control the tumor size, at 1 dpi, xenografts are sorted according to the tumor size as shown in Figure 6 B-B&#34;. As represented in Figure 6C example, this method of sorting is efficient in reducing the variation of tumor sizes: if we pool them all together (+, ++, +++) the STEV is ~double of the ++class (~906 cells to ~422 cells) and the coefficient variation is ~31.9% compared to 14,5% in the ++class. Since the reference for injection is volume, the total number of cells varies a lot between cell types - being dependent on the size and shape of the cells. For example, big cells with a lot of cytoplasm like breast cancer Hs578T, produce much smaller tumors (~600 cells). Also, each cell line requires different number of cells. For instance, the HT29 CRC and RT112 urinary bladder cancer cell lines have shown that the higher the number of cells injected, the higher the zebrafish mortality. Therefore, a period of optimization while developing the xenografts is needed to test if the cell line has toxic effects in the embryo or requires higher/lower densities of injection.
Injection site 
One of the most common discrepancies when generating zebrafish embryo xenografts is the site of injection. The yolk is usually the place of choice for injection due to its easy accessibility. However, we have observed that cells injected in the yolk have a higher tendency to die. Although technically more difficult, we recommend injecting in the PVS and as far as possible from the heart. Within the PVS, cells can aggregate, recruit vessels and immune cells, and migrate, intravasate, extravasate and form micrometastasis, if they display metastatic characteristics8,11.
Engraftment efficiency 
Differences in the engraftment efficiency and tumor size among cell lines at 4 dpi are expected due to their distinct degrees of basal cell death/survival/proliferation but also due to the innate immunogenicity that each cell line may display9.
Metastasis 
Metastasis comprises a multistep cascade of events that can be divided into two arbitrary stages. In the first stage, tumor cells have to detach from the primary site, migrate and invade adjacent tissues and then intravasate into the bloodstream. In the second stage, tumor cells must survive in circulation, extravasate from the blood or lymphatic vessels, and finally colonize at secondary sites23. To distinguish between these early and late events and address the potential/proficiency of the different tumor cells to perform these steps, we designed a simple assay.
In general, when injected into the PVS, tumor cells can enter directly into circulation and then get physically trapped in the caudal hematopoietic tissue (CHT) (tail region). However, according to the characteristics of each tumor cell - we have seen that some tumor cells remain at the CHT 4 dpi and are able to form micrometastasis while other tumor cells disappear (cleared after getting caught in the CHT).
Therefore, by comparing the micrometastasis efficiency (at 4 dpi) when cells were placed directly into circulation - CIRC (cells only have to go through the late steps of metastasis) vs. when not - NO CIRC (cells need to go through early and late steps to be able to form a micrometastasis) we can assess their early or late metastatic potential. We have observed tumor cells that can efficiently form micrometastasis in the CHT in both groups (CIRC and NO CIRC), suggesting that these cells have the capacity to undergo all steps of the metastatic cascade (SW480 and MDA-MB-468 for example)8,11. In contrast, other tumor cells have a very low metastatic potential in both groups, hardly ever making micrometastasis, even when injected into circulation (i.e., visible in the CHT at 24 hpi, but at 4 dpi they are no longer there, Hs578T for example)8. However, we have clearly found another group - one that is only able to form micrometastasis when injected into circulation (we can only observe micrometastasis in the CIRC group). This suggests that these cells have a low efficiency in performing the first steps of the metastatic cascade but on the other hand are able to survive in circulation, extravasate and colonize a distant site.
Immunostaining and imaging 
Before fixation, this injection protocol can be used for other live imaging approaches like live differential interference contrast (DIC) microscopy, spinning disk microscopy, high-resolution live confocal imaging and light sheet microscopy, etc.
Dead cells and cellular debris appear bright when observed through the fluorescent stereomicroscope and can be mistaken for live cells, especially if the aim of the study is assessing the metastatic potential of the cell lines. We would like to stress the importance of performing confocal imaging with specific viability markers and DAPI to assess the survival state of the tumor and micrometastasis. Also, it is fundamental to use specific human antibodies to detect human cells such as anti-human mitochondria or anti-human HLA. When implementing the protocol, train experimenter eyes by comparing the staining in the stereomicroscope with the confocal images. After some time, experimenters can clearly distinguish debris from live cells in the fluorescent stereomicroscope.
Although other methods to quantify tumor burden such as whole fluorescence area are widely used, we recommend performing whole mount immunostaining and confocal imaging as a more accurate method. Not only the efficiency of lipophilic dye staining is very variable (i.e., some cells are very well stained whereas others are not -probably due to the lipid content of their membranes), but also many times lipophilic dyes form aggregates, and dead cells tend to be brighter - creating several artifacts that can be mistaken for live cells.
Cells can be transduced with fluorescent proteins to aid in their tracking and to skip cell labelling. However, make sure that the transduced and non-transduced cells produce the same results in the zebrafish xenografts.
In addition, macrophages can phagocyte these fluorescent cell debris becoming fluorescently labelled and migrate, generating false positive metastatic cells. Thus, we recommend a series of analytic tools, which of course can be extended to many other readouts for a more accurate interpretation of tumor behavior:
<ul style="list-style-type: square;">
	<li>Proliferation - quantification of mitotic figures with DAPI or anti-pHH3Ser10 antibody (Merck Millipore Cat. #06-570),</li>
	<li>Cell death by apoptosis- antibody anti-activated Caspase3Asp175 (Cell Signalling Technologies Cat. #9661) or equivalent,</li>
	<li>Tumor size - DAPI counting- human tumor cells show a very distinct chromatin organization, so they are easily distinguished from the zebrafish cells and is always possible to double check with the dye (when you are training your eye),</li>
	<li>For metastatic studies, to unambiguously detect human cells, - anti-human HLA (Abcam EP1395Y Cat. #ab52922), anti-human mitochondria (Merck Millipore Cat. #MAB1273- Clone 113-1).</li>
</ul>
For a confocal acquisition with 5 &#181;m interval between slices in the Z-stack of the control cancer cells HCT116 (average nuclear size of 10-12 &#181;m of diameter) with DAPI counterstaining, we have observed that ~50% of the cells are shared between two consecutive slices. Therefore, if every slice is counted, there is a high risk of counting the same cells twice. Going back and forth between slices to avoid problems in quantification results in a time consuming and error prone technique. To ease the quantification of the total number of cells and allow for more reproducibility between researchers, we created the tumor size formula previously described in this protocol8.
We included a correction number (1.5) to account for the ~50% cells shared between slices. We found that the average error of manual counting of the whole tumor between researchers was 20%. Two researchers using the formula had a 2% error. The use of this formula has a 93% accuracy rate and 98% reproducibility rate. We also tested automated methods, but they demonstrated an error higher than 50% caused by threshold settings.
Due to the characteristics of apoptotic cells, the quantification of activated Caspase 3 cells is more difficult. To reduce the number of mistakes and variation in results, we recommend that the control and experimental samples are counted by the same researcher. Additionally, when learning this technique, a new researcher must count images that were already quantified by more experienced researchers to compare results and train.
The length of the assay can be extended if required. However, it is important to consider that zebrafish larvae require live feeding starting from ~7 days post-fertilization (5 days post-injection). Additionally, the animal welfare guidelines and regulations applied to larvae older than 6 days post-fertilization may vary.
This protocol provides useful tools to enable a single researcher to inject approximately ~200-300 zebrafish larvae per hour; and results for the complete assay, including analysis and statistical interpretation, obtained in three weeks. We hope that this protocol can help researchers become experts in generating zebrafish xenografts. It is not easy; you need to practice but you will get there. Good Luck!

Zebrafish (Danio rerio) is emerging as a powerful vertebrate model organism to study development and disease. Zebrafish shares highly conserved genetic (~70% genetic homology and ~84% disease-related genes) and basic organ morphological features with humans1,2. This conservation allows the use of zebrafish to model several human diseases, including cancer3,4.
The handling and maintenance of zebrafish is much easier and more cost effective than mice due to their small size, high fecundity all year round and external fertilization3,5. Zebrafish embryos do not require live feeding during their first 5-7 days of life and have been used as an effective model for development, infection, and cancer1,4,6,7. Zebrafish embryos hatch at 48 hours post-fertilization (hpf) and are free-swimming animals with all organs formed, a beating heart and functional circulatory system, liver, brain, kidney marrow, etc.1,3. Also, at this stage of development only innate immunity is at play, adaptive immunity is still developing, allowing a general efficient engraftment of human cells with no need for use of immunocompromised mutants7,8. Nevertheless, it is important to note that not all human cells engraft equally9 and that, for instance, for leukemia cells it was shown that phagocytes (neutrophils and macrophages) need to be depleted for efficient engraftment10.
The zebrafish genetic tractability and the optical transparency of its early embryonic stages allow for single cell intravital imaging at a high resolution and thus, for the establishment of state-of-the art imaging techniques in diverse fields of biology. Furthermore, in the context of cancer, these features are useful for real-time studies of the earliest stages of host-tumor interactions, like studying angiogenic and metastatic potential, as well as interactions with the innate immune system8,9,11,12,13.
Although in the short xenograft assay there is no time for metastatic &#34;evolution&#34;- it is possible to analyze the metastatic capacity of the tumor cells (i.e., their efficiency to go through metastatic steps like invasion, intravasation, survival in circulation, extravasation, and colonization, and therefore study these processes in vivo and in real-time8,11,13,14).
The characteristics of its life cycle place the zebrafish as a unique model for personalized medicine in cancer. Assays can be performed in a shorter time span and results obtained in a few weeks7,8,9,11,12,15,16. The celerity and feasibility of these assays provide doctors and researchers the possibility of obtaining translational results that can be useful for cancer patients, for whom time is an essential need.
Despite the increasing attempts at generating successful zebrafish embryo xenografts, there is still a need for the standardization of the injection procedure as well as the evaluation of cell viability and tumor behavior after injection.
In this protocol we provide researchers with a clear and detailed step-by-step guide for the injection of human cancer cell lines in zebrafish embryos and subsequent fixation, immunostaining, imaging, and quantification of tumor cell behavior.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar for bacteriology</td>
			<td>VWR</td>
			<td>97064-336</td>
			<td>Agar plate</td>
		</tr>
		<tr>
			<td>anti-Caspase3Asp175 (Rabbit monoclonal)</td>
			<td>Cell Signalling Technologies</td>
			<td>9661</td>
			<td>Primary antibody for whole mount immuno staining (Dilution 1:100)</td>
		</tr>
		<tr>
			<td>anti-human HLA (Rabbit monoclonal)</td>
			<td>Abcam EP1395Y</td>
			<td>ab52922</td>
			<td>Primary antibody for whole mount immuno staining (Dilution 1:100)</td>
		</tr>
		<tr>
			<td>anti- 488 (Rabbit monoclonal)</td>
			<td>ThermoFisher Scientific</td>
			<td>35552</td>
			<td>Secondary antibody for whole mount immuno staining (Dilution 1:200)</td>
		</tr>
		<tr>
			<td>anti- 594 (Rabbit monoclonal)</td>
			<td>ThermoFisher Scientific</td>
			<td>35560</td>
			<td>Secondary antibody for whole mount immuno staining (Dilution 1:200)</td>
		</tr>
		<tr>
			<td>CellTracker Deep Red Dye</td>
			<td>ThermoFisher Scientific</td>
			<td>C34565</td>
			<td>Lipophilic dye (Dilution 1:1000)</td>
		</tr>
		<tr>
			<td>CellTracker Green CMFDA Dye</td>
			<td>ThermoFisher Scientific</td>
			<td>C2925</td>
			<td>Lipophilic dye (Dilution 1:1000)</td>
		</tr>
		<tr>
			<td>Conical Centrifuge tube 50mL</td>
			<td>VWR</td>
			<td>525-0610</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Centrifuge tube 15mL</td>
			<td>VWR</td>
			<td>525-0604</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td></td>
			<td></td>
			<td>Nuclear and chromosome counterstain</td>
		</tr>
		<tr>
			<td>Laser-Based Micropipette Puller P-2000</td>
			<td>Sutter-Instrument</td>
			<td></td>
			<td>Micropipette Puller</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube 1.5mL</td>
			<td>Abdos</td>
			<td>P10202</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides, cut edge</td>
			<td>RS France</td>
			<td>BPB016</td>
			<td>Slides for mounting</td>
		</tr>
		<tr>
			<td>Mowiol</td>
			<td>Sigma-Aldrich</td>
			<td>81381</td>
			<td>Mounting medium</td>
		</tr>
		<tr>
			<td>Pneumatic Picopump</td>
			<td>World Precision Instruments</td>
			<td>PV820</td>
			<td>Microinjector</td>
		</tr>
		<tr>
			<td>Rectangular cover glasses, Menzel Gl&#228;ser</td>
			<td>ThermoFisher Scientific</td>
			<td>631-9430</td>
			<td>Coverslips for mounting</td>
		</tr>
		<tr>
			<td>SeaKem LE Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td>Agar plate</td>
		</tr>
		<tr>
			<td>Thin Wall Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>TW100-4</td>
			<td>Borosilicate capillaries</td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Gibco</td>
			<td>12605036</td>
			<td>Enzymatic detachment solution</td>
		</tr>
		<tr>
			<td>Vaseline</td>
			<td></td>
			<td></td>
			<td>Petroleum jelly for slide sealing</td>
		</tr>
		<tr>
			<td>Vybrant CM-DiI Dye</td>
			<td>ThermoFisher Scientific</td>
			<td>V22888</td>
			<td>Lipophilic dye (Dilution 1:1000)</td>
		</tr>
		<tr>
			<td>Vybrant DiO Cell-Labeling Solution</td>
			<td>ThermoFisher Scientific</td>
			<td>V22886</td>
			<td>Lipophilic dye (Dilution 1:1000)</td>
		</tr>
		<tr>
			<td>ZEISS Axio Zoom.V16 for Biology</td>
			<td>ZEISS</td>
			<td></td>
			<td>&#160;Fluorescence Stereo Zoom Microscope</td>
		</tr>
		<tr>
			<td>Zeiss LSM 710</td>
			<td>ZEISS</td>
			<td></td>
			<td>Confocal microscope</td>
		</tr>
	</tbody>
</table>

generation of zebrafish larval xenografts and tumor behavior analysis

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly visualizing the response to anti-cancer therapies (chemo, radiotherapy, and biologicals), angiogenesis and metastasis with single cell resolution, places the zebrafish xenograft model as a top choice to develop preclinical studies.

The zebrafish larval xenograft assay presents several experimental advantages compared to other models, but probably the most striking is the reduction of size scale and consequently time. This reduction of scale allows single cell imaging, the use of a relatively low number of human cells (compatible with biopsies), medium-high-throughput drug screenings, but most importantly enables a significant reduction of the time of the assay. All these advantages make the zebrafish xenograft assay extremely attractive for future personalized medicine applications.

Many zebrafish xenograft protocols have been developed with a wide diversity of human tumors; however, a general and standardized protocol to efficiently generate zebrafish larval xenografts is still lacking. Here we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Watch this Scientific Journal Video about Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis at JoVE.com

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...

generation-of-zebrafish-larval-xenografts-and-tumor-behavior-analysis

Research

JoVE Journal

Cancer Research

11.4K Views.  Champalimaud Centre for the Unknown, Champalimaud Foundation. Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Video: Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines respond to therapeutic compounds in 2D (dimensional) monolayer cell cultures, 3D culture systems were developed to understand the efficacy of drugs in more physiologically relevant models. In recent years, a paradigm shift was observed in pre-clinical research to validate the potency of new molecules in 3D culture systems, more precisely mimicking the tumor microenvironment. These systems characterize the disease state in a more physiologically relevant manner and help to gain better mechanistic insight and understanding of the pharmacological potency of a given molecule. Moreover, with the current trend in improving in vivo cancer models, zebrafish has emerged as an important vertebrate model to assess in vivo tumor formation and study the effect of therapeutic agents. Here, we investigated the therapeutic efficacy of hydroxycoumarin OT48 alone or in combination with BH3 mimetics in lung cancer cell line A549 by using three different 3D culture systems including colony formation assays (CFA), spheroid formation assay (SFA) and in vivo zebrafish xenografts.

Here, we present the preclinical screening of anticancer coumarins using 3D culture and zebrafish.

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Generation and Expansion of Primary, Malignant Pleural Mesothelioma Tumor Lines

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary tumor cells from surgically resected, malignant pleural mesothelioma (MPM) by providing a complete overview of the process from digestion to enrichment, expansion, cryopreservation, and phenotypic characterization. In addition, this protocol introduces concepts for tumor generation that may be useful for multiple tumor types such as differential trypsinization and the impact of dissociation methods on the detection of cell surface markers for phenotypic characterization. The major limitation of this study is the selection of tumor cells that will expand in a two-dimensional (2D) culture system. Variations to this protocol, including three-dimensional (3D) culture systems, media supplements, plate coating to improve adhesion, and alternate disaggregation methods, could improve this technique and the overall success rate of establishing a tumor line. Overall, this protocol provides a base method for establishing and characterizing tumor cells from this rare tumor.

The goal of this method paper is to demonstrate a robust and reproducible methodology for the enrichment, generation, and expansion of primary tumor cell lines from surgically resected pleural mesothelioma.

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary ...