We thank Dr. Leonard I. Zon in whose laboratory these techniques were initially developed; Kristen Kwan and the late Chi-Bin Chien for gifts of plasmids used in this work; James Lister and David Raible for assistance with antibody staining; and James Neiswender for the microinjection video. This work was funded by NIH grant R00AR056899-03 to C.J.C.

1. Screening for Melanoma Onset Modifiers
<ol>
	<li>Create Gateway middle entry clones by PCR amplifying the full-length open reading frame of genes of interest (GOI) and recombining into pDONR 221 using BP clonase II (Invitrogen). Use Multisite Gateway technology (Invitrogen) to recombine p5E_mitfa, pME_GOI, Tol2kit #302 p3E_SV40polyA6 and miniCoopR5 to place genes of interest under the mitfa promoter in the miniCoopR vector (Figure 1A).</li>
	<li>Inject 25 picograms of each clone along with 25 picograms Tol2 transposase mRNA into one-cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)triply homozygous zebrafish embryos as previously described7. Incubate the injected embryos at 28.5 &deg;C. Remove any dead embryos at 24 hpf.</li>
	<li>Select transgenic animals with rescued melanocytes at 72 hpf by placing the Petri dish containing injected embryos on a dissecting microscope under incident light against a white background (Figure 1A).</li>
	<li>Transfer animals to 3 L tanks in the nursery of the zebrafish facility at 4 dpf as previously described8.</li>
	<li>At 2 months, select the animals with at least one area of melanocyte rescue greater than 4 mm2 (Figure 1A). There is a strong correlation between the degree of melanocyte rescue at 72 hpf and the degree of melanocyte rescue at 2 months. When embryos with melanocyte rescue are picked at 72 hpf, the majority of them (~80%) will have at least one area of melanocyte rescue greater than 4 mm2 at 2 months.</li>
	<li>Screen the selected animals weekly for the presence of tumors (Figure 1B). There is a strong correlation between histopathologic and morphologic changes. Transition from benign to malignant is recognized as a morphologic change when lesions become raised off the surface of the animal. Isolate tumor bearing animals for study.</li>
	<li>Draw melanoma-free survival curves with age in weeks on the abscissa and percent melanoma-free survival on the ordinate. Animals with melanocytes that express a gene of interest are compared to control animals that express EGFP (enhanced green fluorescent protein) in melanocytes (Figure 1C). The median tumor onset for animals injected with miniCoopR-EGFP is approximately 18 weeks. Determine whether the two curves are statistically different using a log rank test.</li>
</ol>
2. Tumor Invasion Assay
<ol>
	<li>Select isolated zebrafish that develop tumors dorsally, in a region between the posterior boundary of the hindbrain and anterior border of the dorsal fin (Figure 2A).</li>
	<li>Two weeks after melanoma onset euthanize the fish according to guidelines specified by the American Veterinary Medical Association Panel on Euthanasia and approved by the University of Massachusetts Medical School Institutional Animal Care and Use Committee (IACUC). Specifically, a fish is placed into a dish with 0.6 mg/ml tricaine until gill movement stops.</li>
	<li>Make an incision in the peritoneal cavity of the fish with a scalpel and then place the fish in 4% paraformaldehyde (PFA) overnight at 4 &deg;C for fixation.</li>
	<li>Treat with 0.5 M EDTA overnight at 4 &deg;C to decalcify.</li>
	<li>Fix with formalin in cassette overnight, dehydrate for 1 hr, clear with xylene 3 times for 1 hr and then treat with paraffin 2 times for 2 hr each at 60 &deg;C.</li>
	<li>Embed the tissue in paraffin in a mould and allow it to solidify for 30 min to 1 hr.</li>
	<li>Make 5 &mu;m sections, one every 50 &mu;m. The sections should be transverse and through the entire lesion so that the point of deepest invasion can be identified. Follow by hematoxylin and eosin staining.</li>
	<li>Assess tumor invasion by measuring whether tumor cells remain outside the collagenous stratum compactum, invade into the dorsal musculature or invade further into the spinal column (Figure 2B,C). Determine statistical difference between the two sets of samples.</li>
</ol>
3. Antibody Staining of Scale Melanocytes
<ol>
	<li>Make up PO4 buffer with 80 ml 0.1 M Na2HPO4 and 20 ml 0.1M NaH2PO4. Make sure the pH is 7.3. Make up 2x fix buffer containing 8.0 g sucrose, 0.15 ml 0.2M CaCl2 and 90 ml 0.2M PO4 buffer, pH 7.3. If necessary adjust the pH to 7.3 with NaOH or HCl then make up to 100 ml with PO4 buffer.</li>
	<li>Weigh up to 300 mg solid PFA and add to a microcentrifuge tube. Add 5 mM NaOH to a volume equal to 4.5 x mass, in mg, of PFA (e.g. 450 ul 5 mM NaOH to 100 mg PFA). Heat at 60-70 &deg;C with occasional shaking until the PFA is dissolved. Spin down remaining particulates and recover 20% PFA supernatant. Make fresh every time.</li>
	<li>Prepare fixation solution containing 1x fix buffer and 4% PFA.</li>
	<li>Anesthetize the fish in 0.17 mg/ml tricaine.</li>
	<li>Using incident light under a dissecting microscope pluck dorsal scales using sharp fine-tipped forceps, taking care not to damage the melanocyte-containing half of the scale (Figure 3A). Place scales directly from the forceps into 1 ml fixation solution and incubate at room temperature, while turning, for &ge; 2 hr. Transfer the fish into fish water and monitor the fish to confirm successful recovery.</li>
	<li>To bleach pigmented melanocytes wash fixed scales in PBST (1x PBS pH 7.4, 0.1% Triton X-100) 2 times for 5 min at room temperature. Add 1 ml freshly made bleach solution containing 0.4 ml 10% KOH and 0.15 ml 30% H2O2 in 5 ml with dH2O. Put on parafilm or cap locks to prevent gas from bursting the tubes open. Continue bleaching until pigment is gone (10 min is typical) (Figure 3B,C). Wash in PBST four times for 5 min at room temperature.</li>
	<li>Block for at least 30 min in block solution made up of 1x PBS pH 7.4, 0.2% Triton X-100, 2 mg/ml BSA, 1% DMSO, 0.02% NaN3 and 2% lamb serum. Leave out NaN3 if developing with HRP reaction.</li>
	<li>Incubate with primary antibody in block solution overnight at room temperature. You need to have at least 400 &mu;l of solution over the samples to keep them submerged.</li>
	<li>Wash the scales 3 times for 30 min in PBST at room temperature.</li>
	<li>Incubate with secondary antibody from 2 hr to overnight in block solution at room temperature.</li>
	<li>Stain for 10 min with PBST+ 0.1 &mu;M DAPI.</li>
	<li>Wash 3 times for 5 min with PBST.</li>
	<li>Mount the scales on a glass slide in a drop of vectashield so that the concave side of the scales faces down towards the slide and place a glass cover slip over them. Seal the edges of the slide with clear nail polish and observe the slides under a fluorescence microscope (Figure 3D,E,F,G).</li>
</ol>
4. Transplantation Assay
<ol>
	<li>Prepare recipient 2-3 month old casper9 fish by irradiating with 25 Gy of gamma irradiation one day prior to transplantation10. Allow the fish to recover in fish water.</li>
	<li>Euthanize a tumor-bearing fish as previously described (Section 2.2).</li>
	<li>Cut off the tumor and put it in a Petri dish with approximately 5 ml filter sterilized 0.9x PBS with 5% FBS. Dice the tumor with a razor while in solution.</li>
	<li>Working at room temperature, triturate with a P1000 pipette to get single cells. Make up the volume to 25 ml with filter sterilized 0.9x PBS with 5% FBS.</li>
	<li>Filter through 40 &mu;M mesh filter.</li>
	<li>Spin in an Eppendorf 5810R tabletop centrifuge at 1,500 rpm (453 rcf) for 10 min.</li>
	<li>Remove the supernatant and make cell suspension to roughly desired concentration (assume 107 cells for a 100 mm3 tumor).</li>
	<li>Calculate the exact cell number using a hemocytometer and dilute the cell suspension so that the final injection volume is approximately 5 &mu;l. For example, if 50,000 cells are to be injected, the cell suspension should be diluted to a final concentration of 10,000 cells/&mu;l. Flick the tube containing the cell suspension every few min to prevent clumping of the cells.</li>
	<li>Wash a 26s gauge (bevel tip) 701 N 10 &mu;l Hamilton syringe 2-3 times with 100% ethanol and 0.9x PBS. Load the syringe with 5 &mu;l of the cell suspension.</li>
	<li>Anesthetize the irradiated recipient fish in 0.17 mg/ml tricaine. Place fish on its side on a damp Kimwipe (Figure 4A).</li>
	<li>Stabilize the fish with one hand and insert the needle with the bevel facing up at a 45&deg; angle into the flank of the fish above the peritoneal cavity about halfway between the posterior boundary of the hindbrain and anterior border of the dorsal fin. Gently depress the plunger (Figure 4B).</li>
	<li>Allow the fish to recover in fresh fish water and observe the fish daily for tumor engraftment (Figure 4C). If engraftment has occurred, continued growth and disease development can also be observed.</li>
</ol>

<ol>
	<li>Beroukhim, 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=The+landscape+of+somatic+copy-number+alteration+across+human+cancers.">The landscape of somatic copy-number alteration across human cancers.</a> Nature. 463, 899-905 (2010).</li><li>Curtin, J. A., 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=Distinct+sets+of+genetic+alterations+in+melanoma.">Distinct sets of genetic alterations in melanoma.</a> N. Engl. J. Med. 353, 2135-2147 (2005).</li><li>Lin, W. M., 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=Modeling+genomic+diversity+and+tumor+dependency+in+malignant+melanoma.">Modeling genomic diversity and tumor dependency in malignant melanoma.</a> Cancer Res. 68, 664-673 (2008).</li><li>Patton, E. E., 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=BRAF+mutations+are+sufficient+to+promote+nevi+formation+and+cooperate+with+p53+in+the+genesis+of+melanoma.">BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.</a> Curr. Biol. 15, 249-254 (2005).</li><li>Ceol, C. J., 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+histone+methyltransferase+SETDB1+is+recurrently+amplified+in+melanoma+and+accelerates+its+onset.">The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset.</a> Nature. 471, 513-517 (2011).</li><li>Kwan, K. M., 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+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Dev. Dyn. 236, 3088-3099 (2007).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide of the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>White, R. M., 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=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2, 183-189 (2008).</li><li>Traver, D., 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=Transplantation+and+in+vivo+imaging+of+multilineage+engraftment+in+zebrafish+bloodless+mutants.">Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.</a> Nat. Immunol. 4, 1238-1246 (2003).</li></ol>

No conflicts of interest declared.

The miniCoopR method enables expression of genes of interest in zebrafish melanocytes. This approach takes advantage of the fact that the zebrafish mitfa gene acts cell-autonomously. For this reason, melanocytes rescued by the miniCoopR vector are certain to contain the minigene and any gene of interest to which it is physically coupled. Rescued melanocytes are clearly visible and can be obtained in animals that were injected as single-cell embryos. A specified degree of chimerism is selected, and animals surpas...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Gateway recombination reagents</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>miniCoopR</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reference5</td>
		</tr>
		<tr>
			<td>Mitfa antibody</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reference5</td>
		</tr>
		<tr>
			<td>FITC goat anti-rabbit IgG antibody</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Labs</td>
			<td>H-1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>casper Zebrafish</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reference9</td>
		</tr>
		<tr>
			<td>701N 10 &mu;l Syringe</td>
			<td>Hamilton/Fisher</td>
			<td>14-824</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>40 &mu;M filter</td>
			<td>BD Falcon/Fisher</td>
			<td>352340</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Invitrogen</td>
			<td>26140079</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

screening for melanoma modifiers using a zebrafish autochthonous tumor model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

One-cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos were injected with the miniCoopR vector containing the melanoma oncogene SETDB15 or EGFP, each under the control of the mitfa promoter. Embryos with melanocyte rescue were selected and allowed to mature. At 2 months of age animals with melanocyte rescue greater than 4 mm2 were selected. The animals were screened weekly for melanomas. Tumor incidence curves for the adults showed that the S...

Watch this Scientific Journal Video about Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model at JoVE.com

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

screening-for-melanoma-modifiers-using-zebrafish-autochthonous-tumor

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13.6K Views.  University of Massachusetts Medical School. A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Video: Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...