The authors thank the Wistar Institute Animal Facility, Microscopy Facility, Histotechnology Facility, and Research Supply Center. This study was funded in part by grants from the U54 (CA224070-01), SPORE (CA174523), P01 (CA114046-07), the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, and the Melanoma Research Foundation.

The following animal protocols follow the guidelines of The Wistar Institute&#8217;s humane ethics committee and animal care guidelines.
1. Melanoma tumor tissue collection
<ol>
	<li>Collect tumor tissue (termed passage 0) from melanoma patients by one of the following surgery or biopsy methods.
	<ol>
		<li>For surgical excision tissue, maintain a minimum of 1 g of tissue (resect metastases and primary lesions) in transport storage media (RPMI 1640 + 0.1% fungizone + 0.2% gentamicin) at 4 &#176;C or on ice.</li>
		<li>For surgical biopsy tissue, maintain less than 1 g of tissue (often punch biopsies of subcutaneous [s.c.] metastases and lymph node [LN] metastases) in transport storage media at 4 &#176;C or on ice.</li>
		<li>For core biopsy tissue, wash out a cylinder (core) of tissue of approximately 10 mm x 1 mm (often, liver biopsies) into a 15 mL tube containing 5 mL of transport storage media at 4 &#176;C or on ice.</li>
		<li>For fine needle aspirate (FNAs) tissue, keep&#160; a very small amount of tissue (less than 1 mm in size) taken directly from the patient in a needle and syringe at 4 &#176;C or on ice.</li>
	</ol>
	</li>
	<li>Deliver the tissue in transport storage media at 4 &#176;C or on ice on the same day or with overnight shipping after surgical excision or biopsy. Process the tissue within 1-2 h of delivery.</li>
</ol>
2. Tumor tissue processing for mouse implantation
<ol>
	<li>Surgical excision or surgical biopsy tissue processing
	<ol>
		<li>Transfer the tissue to a sterile Petri dish and separate the tumor tissue from surrounding normal tissue as much as possible.</li>
		<li>Remove necrotic tissue (usually identified as pale-whitish tissue located centrally within the tumor) from the remaining tumor as much as possible.</li>
		<li>Use a scalpel to subdivide an initial tumor chunk into approximately equal pieces (~3 mm x 3 mm) for surgical mouse implantation (Figure 2).</li>
		<li>Optionally, if enough tumor tissue is available, snap-freeze the tissue for downstream assays (RNA sequencing [RNASeq], whole exome sequencing [WES], etc.).</li>
		<li>Make a tumor slurry by mincing the tumor tissue using a cross blade technique with two scalpel blades. Mince the tumor chunks as finely as possible to form a slurry, which is now ready for surgical mouse implantation.</li>
		<li>Alternatively, if the tumor tissue is too hard for mechanical dissociation, use a digestion dissociation procedure to form gel-like slurry and a single-cell suspension for implantation and/or injection.
		<ol>
			<li>Mince the tumor chunks as finely as possible to form slurry.</li>
			<li>Put the slurry in a 50 mL tube with cold Hank&#8217;s balanced salt solution (HBSS)-/- (without Ca++ and Mg++); then, centrifuge and pellet at 220 x g for 4 min at 4 &#176;C.</li>
			<li>Resuspend the slurry in 10 mL of warmed fresh digest media (200 U/mL collagenase IV + 5 mM CaCl2 + 50 U/mL DNase in HBSS-/-) per 1 g of tumor tissue.</li>
			<li>Place the tube in a 37 &#176;C water bath for 20 min and mix vigorously every 5 min with a disposable pipette.</li>
			<li>Wash with up to 50 mL of HBSS-/-; then, centrifuge at 220 x g for 4 min at 4 &#176;C.</li>
			<li>Add 5 mL of prewarmed TEG (0.025% trypsin + 40 &#181;g/mL ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#8217;,N&#8217;-tetraacetic acid [EGTA] + 10 &#181;g/mL polyvinyl alcohol [PVA]) per 1 g of tumor tissue, gently resuspend/shake, and place the tube at 37 &#176;C for 2 min without mixing.</li>
			<li>Add at least 1 equal volume of cold staining media (1% bovine serum albumin [BSA] + 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES] + 1x penicillin-streptomycin in L15 media) to quench the trypsin and centrifuge at 220 x g for 4 min at 4 &#176;C.</li>
			<li>Resuspend the sample in 10 mL of staining media per 1 g of tumor tissue and filter it through a 40 &#181;m cell strainer to get a single-cell suspension for mouse injection (Figure 2).</li>
			<li>Slurry remaining on top of the cell strainer can also be collected for surgical mouse implantation.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Core biopsy tissue processing
	<ol>
		<li>Pour the core cylinder tissue in tissue-transport tube into a 5 cm Petri dish.</li>
		<li>Remove excess liquid, scrape tissue to the edge of the Petri dish, finely mince the tumor tissue, add ~100&#8211;150 &#181;L of HBSS-/- on top of the tumor tissue, and quickly draw the HBSS/tumor tissue suspension into the 1 mL syringe.</li>
		<li>Attach a 23 G needle to the 1 mL syringe, pass the HBSS/tumor tissue suspension through the needle into a 1.5 mL spin tube.</li>
		<li>Redraw the tumor suspension into the syringe with the needle still on until it can pass smoothly through the needle.</li>
		<li>Finally draw the tumor suspension back into the syringe and detach the 23 G needle.</li>
		<li>Attach a 27 G needle and draw an equal volume (~100&#8211;150 &#181;L) of artificial extracellular matrix (see Table of Materials) into the syringe.</li>
		<li>Add an equal volume of artificial extracellular matrix slowly into the 1.5 mL spin tube with the tumor/HBSS-/- suspension, carefully avoiding the formation of bubbles.</li>
		<li>Draw one last time back into the syringe and the core biopsy tumor tissue is now ready for mouse injection.</li>
	</ol>
	</li>
	<li>FNA tissue processing
	<ol>
		<li>Place the syringe containing the FNA samples (trapped in the needle) on ice.</li>
		<li>Separate the needle (containing FNA tissue) from the syringe and remove the plunger from the syringe, add ~150&#8211;200 &#181;L of HBSS-/- to the top of the syringe.</li>
		<li>Re-insert the plunger into the syringe and the needle (containing FNA tissue), and push out the HBSS-/- through the needle into a 1.5 mL spin tube. This step removes the FNA tumor from the needle.</li>
		<li>Repeat step 2.3.3 with the HBSS-/-/FNA suspension twice using the same syringe to maximize the retrieval of tumor tissue from the needle.</li>
		<li>Add an equal volume (~100&#8211;150 &#181;L) of artificial extracellular matrix to the 1.5 mL spin tube containing the HBSS/FNA suspension.</li>
		<li>Adequately mix by slowly drawing up the artificial extracellular matrix/HBSS-/-/FNA suspension back into the 23 G needle and repeating twice.</li>
		<li>Draw the entire artificial extracellular matrix/HBSS-/-/FNA volume back into the syringe, replace the 23 G needle with a 27 G needle, and the FNA sample is now ready for mouse injection.</li>
	</ol>
	</li>
</ol>
3. Tumor implantation and injection in mice
<ol>
	<li>Implantation of surgical excision or surgical biopsy tissue 
	NOTE: Ensure all surgical instruments are sterile by autoclaving or the use of pre-sterilized disposable instruments.
	<ol>
		<li>Shave hair from the lower back of NSG 6-8 week male or female mice leaving an approximately 1.5 cm x 3 cm area with no hair. Anesthetize mice using isoflurane, and confirm by gently squeezing the foot as a test of responsiveness. Use vet ointment on their eyes to prevent dryness.</li>
		<li>Place individual mice on a heat pad in the nose cone of the anesthesia machine, scrub the shaved area with chlorhexidine. Then douse with 70% ethanol and allow to evaporate.</li>
		<li>Prepare chunks or divide tumor slurry in a Petri dish into individual mounds for surgical implantation (i.e., into three equal mounds if to be implanted into 3 mice).</li>
		<li>Using the scalpel blade, make an incision of approximately 5 mm long on the center of the back of the mouse, take one pair of forceps and lift up skin on the side of the incision opposite of operator.</li>
		<li>Take the scissors into the other hand and separate the skin from the muscle layer by gently cutting the fascial membrane with small scissor cuts, thereby creating a &#34;pocket&#34;&#160;for the tumor tissue.</li>
		<li>Pick up one tumor chunk or one individual mound of tumor slurry tissue with the scalpel blade and gently place tissue into the created pocket.</li>
		<li>Administer 100 &#181;L of artificial extracellular matrix on the tumor tissue mound in the pocket.</li>
		<li>Using two pairs of forceps, pull up the incision on both ends so that the wound edges come close together, and close the wound by applying one or two wound clips.</li>
		<li>Subcutaneous inject 1-5 mg/kg meloxicam as an analgesic in mice after surgery.</li>
		<li>Take the mouse out of the nose cone and place it back into its original cage, observe the mouse while waking up. Do not return to a cage until fully recovered.</li>
		<li>Remove wound clips after approximately 7 days. If healing is not complete after 7 days, leave the wound clip in for an additional one or two days. 
		NOTE: If using a single cell suspension from surgical excision or surgical biopsy tissue processing, it will mix with artificial extracellular matrix (at 1:1 ratio) for mice injection.</li>
	</ol>
	</li>
	<li>Injection of FNA or core biopsy tissue
	<ol>
		<li>Place an NSG mouse on a steel grid rack, hold the mouse firmly by the tail, and gently pull the mouse back. It will grasp the grid with its front legs firmly. Alternatively, restrain a mouse in the non-dominant hand and let the flank area visible.</li>
		<li>Disinfect the skin of the flank with alcohol prep swabs, slowly and steadily inject the contents of the syringe under the skin of the mouse.</li>
		<li>Pull out the needle and place the mouse back into its cage.</li>
	</ol>
	</li>
</ol>
4. Monitor tumor growth
<ol>
	<li>Monitor mice once weekly to check for palpable tumors.</li>
	<li>Once tumors are at a measurable size (approximately 50 mm3), use a caliper to record tumor dimensions. Use the following formula to calculate tumor volumes: (width x width x length) / 2.</li>
	<li>Harvest tumor once the tumor volume reaches around 1.5 cm3 (approximately 4&#8211;10 weeks). The tumor is now called mouse passage 1 (MP1).</li>
</ol>
5. Harvest tumor for banking tissue, reimplantation, and experiment/characterization
<ol>
	<li>Euthanize mouse in a CO2 chamber, check vital signs to confirm death, and then submerge the mouse in a Virkon solution to sterilize the skin for 30 s in the bio-cabinet.</li>
	<li>Use curved scissors and surgical forceps to lift skin adjacent to the tumor and make a horizontal cut.</li>
	<li>Use a blunt separation technique to mobilize the skin on both sides of the tumor and over the tumor, exposing the tumor.</li>
	<li>Use scissors or a scalpel blade to separate the tumor from the fascia.</li>
	<li>Resect the tumor and transfer the tumor to a sterile Petri dish, cut the tumor into small pieces and remove necrotic tissue from the tumor.</li>
	<li>Bank tumor tissue for future implantation.
	<ol>
		<li>Take 2-3 small tumor pieces smaller than ~10 mm x 10 mm and mince them into pieces smaller than ~1 mm x 1 mm.</li>
		<li>Transfer all minced tissue to a 2 mL cryogenic vial, add 1 mL of freezing media (10% DMSO + 90% FBS).</li>
		<li>Mix well and place cryogenic vials into a pre-cooled isopropanol-based cell-freezing container on dry ice.</li>
		<li>Store container in -80 &#176;C freezer overnight and then transfer cryogenic vials to liquid nitrogen (LN2) storage.</li>
	</ol>
	</li>
	<li>Snap-freeze tissue for downstream assays (RNASeq, WES, etc.).
	<ol>
		<li>Place tumor tissue pieces (~3 mm x 3 mm) into a cryogenic vial and put the cryogenic vial in LN2 immediately. Store vials in -80 &#176;C freezer.</li>
	</ol>
	</li>
</ol>
6. PDX therapy trials
NOTE: It will take two expansion phases to grow enough tumor tissue to generate the necessary number of PDX bearing mice for the therapy trial.
<ol>
	<li>Pick up a cryovial containing banked PDX tissue from LN2, and place the cryovial in a 37 &#176;C water bath until the freezing media containing the tumor tissue is just starting to melt.</li>
	<li>Empty the contents of the cryovial into pre-warmed HBSS-/- in a 50 mL tube, and wash tissue.</li>
	<li>Pellet tissue by centrifugation for 5 min at 1,200 rpm.</li>
	<li>Remove HBSS-/- from the tumor sample by vacuum aspiration with a Pasteur pipet.</li>
	<li>Slide the PDX tissue from the 50 mL tube into a 5 cm or 10 cm Petri dish and place on wet ice.</li>
	<li>Follow the above implantation protocol to implant banked tissue from a cryogenic vial into 5 NSG mice for the first round of PDX tumor expansion.</li>
	<li>Once tumors reach ~600&#8211;800 mm3, harvest 1-2 tumors to get a single cell suspension with the above protocol for tumor tissue processing: mechanical dissociation, collagenase digestion dissociation procedure.</li>
	<li>Pellet cells and resuspend in 6 mL of HBSS-/-/artificial extracellular matrix (1:1 ratio), subcutaneous inject ~50 NSG mice, with 100 &#181;L of cell mixture per mouse for the second round of PDX tumor expansion.</li>
	<li>Measure tumor size with calipers biweekly.</li>
	<li>Wait for a tumor size of ~100 mm3 in 3-5 weeks, randomize mice into groups, and start treatment.</li>
	<li>When tumor size reaches the maximum IACUC-approved volume, stop treatment.</li>
	<li>Follow the above harvest tumor protocol to collect snap frozen tumor pieces, tumor pieces for paraffin blocks.</li>
</ol>

<ol>
	<li>Garman, 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=Genetic+and+Genomic+Characterization+of+462+Melanoma+Patient-Derived+Xenografts,+Tumor+Biopsies,+and+Cell+Lines.">Genetic and Genomic Characterization of 462 Melanoma Patient-Derived Xenografts, Tumor Biopsies, and Cell Lines.</a> Cell Reports. 21 (7), 1936-1952 (2017).</li><li>Krepler, 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+Comprehensive+Patient-Derived+Xenograft+Collection+Representing+the+Heterogeneity+of+Melanoma.">A Comprehensive Patient-Derived Xenograft Collection Representing the Heterogeneity of Melanoma.</a> Cell Reports. 21 (7), 1953-1967 (2017).</li><li>Davies, H., 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=Mutations+of+the+BRAF+gene+in+human+cancer.">Mutations of the BRAF gene in human cancer.</a> Nature. 417 (6892), 949-954 (2002).</li><li>Paraiso, K. H., 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=Recovery+of+phospho-ERK+activity+allows+melanoma+cells+to+escape+from+BRAF+inhibitor+therapy.">Recovery of phospho-ERK activity allows melanoma cells to escape from BRAF inhibitor therapy.</a> British Journal Of Cancer. 102 (12), 1724-1730 (2010).</li><li>Long, G. 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=Long-Term+Outcomes+in+Patients+With+BRAF+V600-Mutant+Metastatic+Melanoma+Who+Received+Dabrafenib+Combined+With+Trametinib.">Long-Term Outcomes in Patients With BRAF V600-Mutant Metastatic Melanoma Who Received Dabrafenib Combined With Trametinib.</a> Journal of Clinical Oncology. 36 (7), 667-673 (2018).</li><li>Hidalgo, 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=Patient-derived+xenograft+models:+an+emerging+platform+for+translational+cancer+research.">Patient-derived xenograft models: an emerging platform for translational cancer research.</a> Cancer Discovery. 4 (9), 998-1013 (2014).</li><li>Hausser, H. J., Brenner, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+instability+of+Saos-2+cells+in+long-term+culture.">Phenotypic instability of Saos-2 cells in long-term culture.</a> Biochemical and Biophysical Research Communications. 333 (1), 216-222 (2005).</li><li>Fiebig, H. H., 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=Development+of+three+human+small+cell+lung+cancer+models+in+nude+mice.">Development of three human small cell lung cancer models in nude mice.</a> Recent Results In Cancer Research. 97, 77-86 (1985).</li><li>Izumchenko, 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=Patient-derived+xenografts+effectively+capture+responses+to+oncology+therapy+in+a+heterogeneous+cohort+of+patients+with+solid+tumors.">Patient-derived xenografts effectively capture responses to oncology therapy in a heterogeneous cohort of patients with solid tumors.</a> Annals of Oncology. 28 (10), 2595-2605 (2017).</li><li>Shi, H., 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=Acquired+resistance+and+clonal+evolution+in+melanoma+during+BRAF+inhibitor+therapy.">Acquired resistance and clonal evolution in melanoma during BRAF inhibitor therapy.</a> Cancer Discovery. 4 (1), 80-93 (2014).</li><li>Monsma, D. 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=Melanoma+patient+derived+xenografts+acquire+distinct+Vemurafenib+resistance+mechanisms.">Melanoma patient derived xenografts acquire distinct Vemurafenib resistance mechanisms.</a> American Journal of Cancer Research. 5 (4), 1507-1518 (2015).</li><li>Das Thakur, 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=Modelling+vemurafenib+resistance+in+melanoma+reveals+a+strategy+to+forestall+drug+resistance.">Modelling vemurafenib resistance in melanoma reveals a strategy to forestall drug resistance.</a> Nature. 494 (7436), 251-255 (2013).</li><li>Meehan, T. 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The authors have nothing to disclose.

We have herein described generating PDX models of melanoma with patient tissue derived from primary and metastatic tumors, core biopsies, and FNAs. When directly engrafted into NSG mice, tumors present similar morphologic, genomic, and biologic properties to those observed in the patient. In the case when only a small quantity of tissue is available to investigators, as often occurs with FNAs, the PDX technique allows for the expansion of the tumor tissue for DNA, RNA, and protein characterization, as well as for therapy...

Preclinical models are critical for all aspects of translational cancer research, including disease characterization, discovery of actionable vulnerabilities unique to cancer versus normal cells, and the development of efficacious therapies that exploit these vulnerabilities to increase the overall survival of patients. In the melanoma field, tens of thousands of cell line models have been heavily utilized for drug screening, with &#62;4,000 contributed by our group alone (WMXXX series). These cell line models were derived from melanoma patients with various forms of cutaneous melanoma (i.e., acral, uveal, and superficial spreading) and diverse genotypes (i.e., BRAFV600-mutant and neuroblastoma RAS viral oncogene homolog [NRASQ61R-mutant]), which span the spectrum of disease present in the clinic1,2.
Unequivocally, the most successful, targeted therapy strategy in the melanoma field has emerged from 1) the genomic characterization of patients&#8217; tumors identifying BRAF mutations in ~50% of melanomas3 and from 2) preclinical investigation leveraging melanoma cell line models4. The BRAF/MEK inhibitor combination was Food and Drug Administration (FDA)-approved in 2014 for the treatment of patients whose melanomas harbor activating BRAFV600E/K mutations and boasts a &#62;75% response rate5. Despite this initial efficacy, resistance rapidly arises in nearly every case due to multifarious intrinsic and acquired resistance mechanisms and intratumoral heterogeneity. Unfortunately, cell line models do not recapitulate representative biological heterogeneity when grown in two-dimensional culture in plastic vessels, which masks their clinically predictive potential when investigators attempt to experimentally determine therapies that might be effective in patients with a specific form or genotype of melanoma6. Understanding how to best model patient intratumoral heterogeneity will allow investigators to better develop therapeutic modalities that can kill therapy-resistant subpopulations that drive failure to current standard-of-care therapies.
Paramount to the limited predictive value of cell line models is how they are initially established. Irreversible alterations occur in the tumor clonal landscape when a single-cell suspension of a patients&#8217; tumor is grown on two-dimensional, plastic tissue culture vessels, including changes in proliferative and invasive potential, the elimination of specific subpopulations, and the alteration of genetic information7. Xenografts into mice of these melanoma cell line models represent the most frequently used in vivo platform for preclinical studies; however, this strategy also suffers from the poor recapitulation of complex tumor heterogeneity observed clinically. To overcome this shortcoming, there has been a growing interest in incorporating more sophisticated preclinical models of melanoma, including the PDX model. PDX models have been utilized for &#62;30 years, with seminal studies in lung cancer patients demonstrating concordance between the patients&#39;&#160;response to cytotoxic agents and the response of the PDX model derived from the same patient8. Recently, there has been a drive to utilize PDX models as the tool of choice for preclinical investigations both in the industry and in academic centers. PDX models, because of their superior recapitulation of tumor heterogeneity in human patients, are more clinically relevant to use in therapy optimization efforts than cell line xenografts9. In melanoma, there are immense hurdles that blunt the therapeutic management of advanced disease10. Clinically relevant PDX models have been used to model clinical resistance and identify therapeutic strategies with clinically available agents to treat therapy-resistant tumors11,12. Briefly, the protocol presented here to generate PDX models requires the subcutaneous implantation of fresh tissue from primary or metastatic melanomas (collected by biopsy or surgery) into NOD/scid/IL2-receptor null (NSG) mice. Different variations in methodological approach are used by different groups; however, a fundamental core exists13.

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			<td>Invitrogen</td>
			<td>Cat # 15140163</td>
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			<td>SIGMA-ALDRICH CORPORATION</td>
			<td>Cat # T4549-100ML</td>
			<td>10 mL aliquots stored at &#8211;20oC</td>
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			<td>SIGMA-ALDRICH CORPORATION</td>
			<td>Cat # A9418-500G</td>
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			<td>Fisher Scientific</td>
			<td>Cat# 50-118-0313</td>
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			<td height="40" style="height:40px;">Collagenase IV (2,000 u/mL)</td>
			<td>Worthington&#160;</td>
			<td>Cat #4189</td>
			<td>make up in HBSS-/- from Collagenase IV powder stock (Worthington #4189, u/mg indicated on bottle and varies with each lot); freeze 1</td>
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			<td height="40" style="height:40px;">DMSO</td>
			<td>SIGMA-ALDRICH CORPORATION</td>
			<td>Cat # C6295-50ML</td>
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			<td>SIGMA-ALDRICH CORPORATION</td>
			<td>Cat # D4527</td>
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			<td>Cat # 324626.25</td>
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			<td>INVITROGEN LIFE TECHNOLOGIES</td>
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			<td>INVITROGEN LIFE TECHNOLOGIES</td>
			<td>Cat # 15290-018</td>
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			<td>FISHER SCIENTIFIC</td>
			<td>Cat # BW17518Z</td>
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			<td>HENRY SCHEIN ANIMAL HEALTH</td>
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			<td height="40" style="height:40px;">Leibovitz&#39;s L15 media&#160;</td>
			<td>Invitrogen</td>
			<td>Cat # 21083027</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Matrigel</td>
			<td>Corning</td>
			<td>Cat # 354230</td>
			<td>Artificial extracellular matrix</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Meloxicam</td>
			<td>HENRY SCHEIN ANIMAL HEALTHRequisition # ::Henry Schein</td>
			<td>Cat # 025115</td>
			<td>1-5mg/kg, as painkiller</td>
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		<tr height="21">
			<td height="21" style="height:21px;">NOD/SCID/IL2-receptor null (NSG) Mice</td>
			<td>The Wistar Institute, animal facility</td>
			<td></td>
			<td>breeding</td>
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		<tr height="21">
			<td height="21" style="height:21px;">PVA (polyvinyl alcohol)</td>
			<td>SIGMA-ALDRICH CORPORATION</td>
			<td>Cat # P8136-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium (Mod.) 1X with L-Glutamine</td>
			<td>Fisher Scientific</td>
			<td>Cat# MT10041CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel</td>
			<td>Feather</td>
			<td>Cat # 2976-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Virkon</td>
			<td>GALLARD-SCHLESINGER IND</td>
			<td>Cat # 222-01-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wound clips</td>
			<td>MikRon</td>
			<td>Cat #427631</td>
			<td></td>
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a melanoma patient-derived xenograft model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Tumor tissue for melanoma PDX models can come from a variety of different sources and can also be processed per the growth dynamics of individual models and the desired use of the PDX tissue. The priority when establishing a PDX model is to have sufficient material to bank for future use and DNA for characterization (Figure 1).
Once sufficient material is banked, tumor tissue can be expanded in one of three main methods to grow enough tumor to perform a formal the...

Watch this Scientific Journal Video about A Melanoma Patient-Derived Xenograft Model at JoVE.com

A Melanoma Patient-Derived Xenograft Model

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

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Research

JoVE Journal

Cancer Research

12.2K Views.  Wistar Institute. Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Video: A Melanoma Patient-Derived Xenograft Model

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful cancer research platform, providing insights into tumor biology and drug therapy. However, liver orthotopic tumor implantation is generally complicated. Here we describe our protocols for the orthotopic implantation of patient-derived liver-metastatic uveal melanoma tumors. We cultured human liver metastatic uveal melanoma cell lines into immunodeficient mice. The protocols can result in consistently high technical success rates using either a surgical orthotopic implantation technique with chunks of patient-derived uveal melanoma tumor or a needle injection technique with cultured human cell line. We also describe protocols for CT scanning to detect interior liver tumors and for re-implantation techniques using cryopreserved tumors to achieve re-engraftment. Together, these protocols provide a better platform for liver orthotopic tumor mouse models of liver metastatic uveal melanoma in translational research.

Orthotopic human liver metastatic uveal melanoma xenograft mouse models were created using surgical orthotopic implantation techniques with patient-derived tumor chunk and needle injection techniques with cultured human uveal melanoma cell lines.

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Central nervous system metastasis PDX models represent the phenotypic and molecular characteristics of human metastasis, making them excellent models for preclinical studies. Described here is how to establish PDX models and the inoculation routes that are best used for preclinical studies.

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to the recent FDA approval of several CD19-targeted CART (CART19) cell therapies. However, CART cell therapy is associated with a unique set of toxicities that carry their own morbidity and mortality. This includes cytokine release syndrome (CRS) and neuroinflammation (NI). The use of preclinical mouse models has been crucial in the research and development of CART technology for assessing both CART efficacy and CART toxicity. The available preclinical models to test this adoptive cellular immunotherapy include syngeneic, xenograft, transgenic, and humanized mouse models. There is no single model that seamlessly mirrors the human immune system, and each model has strengths and weaknesses. This methods paper aims to describe a patient-derived xenograft model using leukemic blasts from patients with acute lymphoblastic leukemia as a strategy to assess CART19-associated toxicities, CRS, and NI. This model has been shown to recapitulate CART19-associated toxicities as well as therapeutic efficacy as seen in the clinic.

Here, we describe a protocol in which an acute lymphoblastic leukemia patient-derived xenograft model is used as a strategy to assess and monitor CD19-targeted chimeric antigen receptor T cell-associated toxicities.

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to ...