This work was supported by grant 1R01CA152303-01.

Ethics Statement: Patient-derived colorectal adenocarcinoma tumor specimens were obtained from consenting patients at the University of Colorado Hospital in accordance with a protocol approved by the Colorado Multiple Institutional Review Board (08-0439). All animal work was performed under animal protocols approved by the University of Colorado Denver Institutional Animal Care and Use Committee (IACUC, Protocol # 51412(06)1E and 96813(04)1E).
1. Receiving and Preparing Patient Blood
<ol>
	<li>Collect 1 - 2 ml of blood in a blood/cell separation tube containing sodium citrate (tube phases included are plasma, lymphocyte and monocyte band, density gradient fluid, gel barrier, and erythrocytes and neutrophils). Caution, follow Blood Borne Pathogen guidelines with blood or tissue. 
	Note: PBMCs and plasma could be used for future studies that may include: comparing germline genetic variation with tumor mutations, isolating circulating tumor cells, evaluating cell free DNA (cfDNA), examining proteins and microRNAs, etc.</li>
	<li>Centrifuge the blood/cell separation tube at 2,500 x g for 15 min with no brake at RT.</li>
	<li>Collect peripheral blood mononuclear cells (PBMCs in lymphocyte and monocyte band of tube) and put into a 1.5 ml microcentrifuge tube (clear, autoclavable, DNase, RNase, and pyrogen free), and fill to the top of the tube with sterile Phosphate-Buffered Saline (PBS).
	<ol>
		<li>Centrifuge at 2,300 x g for 3 min at RT to form a pellet of PBMCs at the bottom of the tube. Next, remove the supernatant carefully. Add 1 ml of sterile PBS to wash the pellet and centrifuge at 3,000 x g for 30 sec and then remove supernatant.</li>
	</ol>
	</li>
	<li>Use a pipette to collect plasma from the blood/cell separation tube (in plasma phase of tube) in a 1.5 ml sterile cryogenic vial (DNase and RNase free, no human DNA, endotoxin free) and put the plasma and pellet of PMBC&#39;s into a -80 &#176;C freezer for storage.</li>
</ol>
2. Receiving and Preparing Patient Tumor Sample
<ol>
	<li>Prepare either RPMI or DMEM media with 1:100 (10 %) of Penicillin-Streptomycin and Non-essential Amino Acids, 1:1000 (1%) Plasmocin, and 10 % inactivated Fetal Bovine Serum (complete media) and add 20 - 25 ml to a sterile collection cup for the tumor specimen.</li>
	<li>Retrieve tumor sample in a sterile cup containing complete media on ice or keep at 4 &#176;C. 
	Note: The tumor sample is a piece of excess patient tumor tissue that is removed by a surgeon, processed by a pathologist, and placed into a sterile collection cup with complete media. Caution, follow Blood Borne Pathogen guidelines with blood or tissue. Also, ideally to inject five mice, the tumor should be approximately 1 cm&#179;.</li>
	<li>Bring the tumor sample to the animal facility for injecting into immunocompromised mice. 
	Note: It is best to process the tumor sample as soon as possible.
	<ol>
		<li>In a laminar flow hood, place the tumor sample into a sterile plastic cutting dish from the tumor cup. Use autoclaved-small scissors and forceps to cut into 10 - 12 approximately 3 x 3 x 3 mm pieces and place into an autoclaved 1.5 ml microcentrifuge tube filled with 300 &#181;l of gelatinous protein mixture solution. Keep tube on ice.</li>
		<li>As a priority inject tumor into mice, but if there is any tumor left over, collect flash frozen vials (FF), formalin fixed paraffin embedded (FFPE) cups, and a viable tumor vial.</li>
		<li>For FF, collect small pieces of tumor tissues from a sterile cryogenic vial for further analysis such as protein, RNA, or DNA isolation. Place FF immediately into a liquid nitrogen dewar and store long-term in a -80 &#176;C freezer.</li>
		<li>For FFPE, if there is enough tumor, place 3 small pieces into a 10 ml 10 % formalin cup and process into paraffin embedded blocks once tumor has been in formalin for at least 24 hr. 
		Note: 24 hr is based off our Histology Core suggestions 11.</li>
		<li>Lastly, place the remaining tumor into a cryogenic tube. Make viable media by adding 10 % Dimethyl Sulfoxide (DMSO) to complete media. Next, add 1 ml to a cryogenic tube and store on ice. Note: Do not keep on ice for long periods of time.
		<ol>
			<li>Cut any leftover tumor into small pieces that ideally will be enough for 10 tumors to be injected into 5 mice in the future. Then, place the viable tumor tube on ice until it can be placed into an Isopropyl Alcohol freezing container (slowly freezes the tumor 1&#176;C at a time) and put into -80&#160;&#176;C freezer to ensure that the tumors do not die during the freezing process. 
			Note: For long-term storage the tumors are removed (after 2 - 3 days) and placed into a large liquid nitrogen dewar. Please note that viable tubes should not be allowed to thaw unless it is being taken out to inject into mice.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Injection of Patient Derived Tumor Xenografts
<ol>
	<li>Use five 6 - 8 week old male and/or female athymic nude mice (T-cell deficient) for each separate patient derived tumor. Inject a total of 10 tumors (2 injections per mouse) subcutaneously (SQ). 
	Note: The original human tumor is designated as F0 and then once injected into mice the next generation is F1. Also,use autoclaved forceps, scissors, and trocars for each unique explant.</li>
	<li>Load pieces of tumor from the gelatinous protein mixture solution into autoclaved 12 G trocars and ensure that the tumor is completely pushed into the trocar.
	<ol>
		<li>In a sterile laminar flow hood, place 5 mice into an anesthesia box that is connected to an Isoflurane anesthesia machine (started at the initial rate of 5% Isoflurane, 3 - 4% oxygen, and 2 - 3% Isoflurane for maintenance of anesthesia) and charcoal filter (absorbs excess gas). 
		Note: An experienced technician should be able to perform the entire procedure on a cage of 5 mice within approximately 5 min total (roughly 30 sec per mouse). Therefore additional thermal support and eye lubrication is not generally used. For less experienced individuals or during training, it is highly recommended that individuals either perform the procedure on less animals at one time and/or use a warming pad/eye lubrication during procedure if animals will be anesthetized longer than 5 min. Procedure is based upon IACUC standards at our university.</li>
	</ol>
	</li>
	<li>Pinch a mouse toe to ensure the mouse is no longer responsive, then take the mouse out of the Isoflurane box. Place the mouse onto a clean field to inject tumors on the middle dorsal neck region and sliding the trocar down subcutaneously until the flank region is reached with autoclaved 12 G trocars.
	<ol>
		<li>Deliver one tumor subcutaneously to each side of the mouse&#39;s flank. Pinch the tumor when the trocar is pulled out, ensuring that the tumor stays in the desired flank region. The gelatinous protein mixture hardens with the mouse&#8217;s body temperature and encapsulates the tumor for approximately 1 week to help the tumor grow and secure it to the SQ connective tissue. The lesion made by the trocar is no larger than 4 mm and there is no need to close the skin with staples. 
		Note: Our veterinarians determined no closure is needed based on very small size of the lesion, quick healing time (middle dorsal neck region reduces any skin tension or grooming of lesion), no infections noted, and close monitoring of the animals during this time period.</li>
	</ol>
	</li>
	<li>Before the mouse is fully awake inject Meloxicam 2 mg/kg SQ (pain medication) away from the site of tumor injection. Next, place the mouse into the cage and monitor until the mouse is awake and moving. 
	Note: Do not leave recovering animals unattended until fully awake. Meloxicam dosage is based upon IACUC standards at our university.
	<ol>
		<li>Next, repeat the same procedure with the 4 remaining mice and monitor their breathing. 
		Note: Please note this is a very quick procedure and mice wake up very soon after Meloxicam injection, but adjust Isoflurane as needed. Each time a mouse is taken out, turn down the Isoflurane. The Meloxicam will last 24 hr and the lesions from the trocars will fully heal in 1 week.</li>
	</ol>
	</li>
</ol>
4. Maintenance of Patient Derived Tumor Xenograft Bank
<ol>
	<li>Monitor growth and health of mice at least once per week. Use a spreadsheet (or other tracking system) to track tumor sizes, tumor generation, date of tumor injection, and health of mice. 
	Note: If mice have lost 15% of their original body weight, low body condition scores (&#8805; 2), have tumor ulcerations, tumors reaching 2,000 mm3 or total tumors 3,000 mm3, or are sickly (hunched, cold, lethargy, etc.) in any way, mice are euthanized via CO&#8322; or anesthetized followed by cervical dislocation as a secondary method. Euthanize via anesthesia and cervical dislocation if collecting tumor, otherwise mice are euthanized via CO&#8322; and cervical dislocation.</li>
	<li>When a tumor is approximately 1,500-2,000 mm3 anesthetize the mice as described above and then perform cervical dislocation to euthanize.
	<ol>
		<li>Check that the mouse has no heartbeat. Excise the SQ tumor with autoclaved scissors and forceps. 
		NOTE: Allow tumors to grow for up to 1 year and if no tumor is seen then euthanize the mice via CO2, followed by cervical dislocation as a secondary method.</li>
		<li>Passage the best growing tumor into the next generation (aka a new set of 5 mice). Use instructions above to collect 10 - 12 tumors for passing and then collect the leftover tumor as also described above. 
		Note: Collecting numerous viable tubes is very important in the early generations (F1 - F8); therefore collect several viable tubes, FF tubes, and 1 FFPE per generation.</li>
		<li>Keep the remaining mice until a new generation of mice has tumor growth of approximately 300 mm3. When remaining mice have tumors that are large, continue to collect as described above.</li>
	</ol>
	</li>
	<li>Continue to passage tumors and collect at each stage until F15. At this point, take a viable tube of the earliest generation possible out of liquid nitrogen and let thaw on ice and follow tumor-injecting procedures described above. 
	Note: Tumors that grow from viable tubes take longer to grow in mice compared to passing from generation to generation, so keep that in mind when planning. If a particular PDTX model is no longer needed at any passage, euthanize and collect tumor, to ensure numerous viable tubes are collected for future use.</li>
</ol>
5. Developmental Therapeutics with Patient Derived Tumor Xenografts
Note: Most tumors at F3 generation have good growth kinetics (grow faster and more consistent), therefore, proceed to PDTX drug efficacy studies.
<ol>
	<li>When the desired tumor is very large (1,500 - 2,000 mm3), follow procedures above to collect tumor to expand into desired amount of mice.
	<ol>
		<li>Depending on the hypothesis being tested determine the number of tumors needed per treatment group. 
		Note: 1 ml of gelatinous protein mixture solution can approximately fit 30 small tumor pieces (depending on tumor morphology) for injecting into mice.</li>
	</ol>
	</li>
	<li>Check mice with tumors weekly and when most tumors are visibly small (approximately between 50 - 300 mm&#179;) measure the tumors with calipers to determine the tumor volume. Tumor volume = [width&#178; (smallest measurement) x length (largest measurement)] x 0.52. 
	Note: The gelatinous protein mixture solution surrounds the injected tumor pieces for one week, then after one-week tumor growth will be accurate.</li>
	<li>Use tumor volumes between 50 - 300 mm&#179; and then average the left and right tumors. Then randomize into treatment groups with 10 tumors per group and a group average within a few numbers of each other. Next, sort the mice into groups and begin the desired treatment study.</li>
	<li>Dose the mice with drug (schedule dependent on drug), weigh and measure (tumor) twice per week. At the end of study, euthanize mice via anesthesia and cervical dislocation and collect tumor for future pharmacodynamic analysis in the lab. 
	Note: The study lasts for 30 days depending on vehicle tumor size and health of mice.</li>
</ol>
6. Organization of a PDTX bank
<ol>
	<li>Keep a well-documented form to prevent repeating of research and appropriate use of animals. 
	NOTE: This is key to a successful PDTX bank.
	<ol>
		<li>Use spreadsheets to keep track of a tumor from the human patient in the clinic, to mice in the PDTX bank, treatments, data, and what is collected. Note: this includes freezer, liquid nitrogen dewars, and FFPE storage as well.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.+2015.">Cancer statistics. 2015.</a> CA Cancer J Clin. 65 (1), 5-29 (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+characterization+of+human+colon+and+rectal+cancer.">Comprehensive molecular characterization of human colon and rectal cancer.</a> Nature. 487 (7407), 330-337 (2012).</li><li>Arcaroli, J. 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=Tumours+with+elevated+levels+of+the+Notch+and+Wnt+pathways+exhibit+efficacy+to+PF-03084014,+a+gamma-secretase+inhibitor,+in+a+preclinical+colorectal+explant+model.">Tumours with elevated levels of the Notch and Wnt pathways exhibit efficacy to PF-03084014, a gamma-secretase inhibitor, in a preclinical colorectal explant model.</a> Br J Cancer. 109 (3), 667-675 (2013).</li><li>Hubbard, J., Grothey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiangiogenesis+agents+in+colorectal+cancer.">Antiangiogenesis agents in colorectal cancer.</a> Curr Opin Oncol. 22 (4), 374-380 (2010).</li><li>van Es, J. 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=Notch/gamma-secretase+inhibition+turns+proliferative+cells+in+intestinal+crypts+and+adenomas+into+goblet+cells.">Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells.</a> Nature. 435 (7044), 959-963 (2005).</li><li>Cassidy, J. W., Caldas, C., Bruna, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintaining+Tumor+Heterogeneity+in+Patient-Derived+Tumor+Xenografts.">Maintaining Tumor Heterogeneity in Patient-Derived Tumor Xenografts.</a> Cancer Res. 75 (15), 2963-2968 (2015).</li><li>Jin, 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=Patient-derived+human+tumour+tissue+xenografts+in+immunodeficient+mice:+a+systematic+review.">Patient-derived human tumour tissue xenografts in immunodeficient mice: a systematic review.</a> Clin Transl Oncol. 12 (7), 473-480 (2010).</li><li>Julien, S., 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=Characterization+of+a+large+panel+of+patient-derived+tumor+xenografts+representing+the+clinical+heterogeneity+of+human+colorectal+cancer.">Characterization of a large panel of patient-derived tumor xenografts representing the clinical heterogeneity of human colorectal cancer.</a> Clin Cancer Res. 18 (19), 5314-5328 (2012).</li><li>Siolas, D., Hannon, G. J. <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+tumor+xenografts:+transforming+clinical+samples+into+mouse+models.">Patient-derived tumor xenografts: transforming clinical samples into mouse models.</a> Cancer Res. 73 (17), 5315-5319 (2013).</li><li>Tentler, J. 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=Patient-derived+tumour+xenografts+as+models+for+oncology+drug+development.">Patient-derived tumour xenografts as models for oncology drug development.</a> Nat Rev Clin Oncol. 9 (6), 338-350 (2012).</li><li>Carson, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Histotechnology: A Self-Assessment Workbook. , (1996).</li><li>Arcaroli, J. 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=Common+PIK3CA+mutants+and+a+novel+3'+UTR+mutation+are+associated+with+increased+sensitivity+to+saracatinib.">Common PIK3CA mutants and a novel 3' UTR mutation are associated with increased sensitivity to saracatinib.</a> Clin Cancer Res. 18 (9), 2704-2714 (2012).</li><li>Arcaroli, J. 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=A+NOTCH1+gene+copy+number+gain+is+a+prognostic+indicator+of+worse+survival+and+a+predictive+biomarker+to+a+Notch1+targeting+antibody+in+colorectal+cancer.">A NOTCH1 gene copy number gain is a prognostic indicator of worse survival and a predictive biomarker to a Notch1 targeting antibody in colorectal cancer.</a> Int J Cancer. 138 (1), 195-205 (2016).</li><li>Arcaroli, J. 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=Gene+array+and+fluorescence+in+situ+hybridization+biomarkers+of+activity+of+saracatinib+(AZD0530),+a+Src+inhibitor,+in+a+preclinical+model+of+colorectal+cancer.">Gene array and fluorescence in situ hybridization biomarkers of activity of saracatinib (AZD0530), a Src inhibitor, in a preclinical model of colorectal cancer.</a> Clin Cancer Res. 16 (16), 4165-4177 (2010).</li><li>Lieu, C. 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=Antitumor+activity+of+a+potent+MEK+inhibitor,+TAK-733,+against+colorectal+cancer+cell+lines+and+patient+derived+xenografts.">Antitumor activity of a potent MEK inhibitor, TAK-733, against colorectal cancer cell lines and patient derived xenografts.</a> Oncotarget. 6 (33), 34561-34572 (2015).</li><li>Pitts, T. 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=Association+of+the+epithelial-to-mesenchymal+transition+phenotype+with+responsiveness+to+the+p21-activated+kinase+inhibitor,+PF-3758309,+in+colon+cancer+models.">Association of the epithelial-to-mesenchymal transition phenotype with responsiveness to the p21-activated kinase inhibitor, PF-3758309, in colon cancer models.</a> Front Pharmacol. 4, 35 (2013).</li><li>Song, E. 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=Potent+antitumor+activity+of+cabozantinib,+a+c-MET+and+VEGFR2+inhibitor,+in+a+colorectal+cancer+patient-derived+tumor+explant+model.">Potent antitumor activity of cabozantinib, a c-MET and VEGFR2 inhibitor, in a colorectal cancer patient-derived tumor explant model.</a> Int J Cancer. 136 (8), 1967-1975 (2015).</li><li>Tentler, J. 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=Identification+of+predictive+markers+of+response+to+the+MEK1/2+inhibitor+selumetinib+(AZD6244)+in+K-ras-mutated+colorectal+cancer.">Identification of predictive markers of response to the MEK1/2 inhibitor selumetinib (AZD6244) in K-ras-mutated colorectal cancer.</a> Mol Cancer Ther. 9 (12), 3351-3362 (2010).</li><li>Bardelli, 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=Amplification+of+the+MET+receptor+drives+resistance+to+anti-EGFR+therapies+in+colorectal+cancer.">Amplification of the MET receptor drives resistance to anti-EGFR therapies in colorectal cancer.</a> Cancer Discov. 3 (6), 658-673 (2013).</li><li>Bertotti, 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=A+molecularly+annotated+platform+of+patient-derived+xenografts+("xenopatients")+identifies+HER2+as+an+effective+therapeutic+target+in+cetuximab-resistant+colorectal+cancer.">A molecularly annotated platform of patient-derived xenografts ("xenopatients") identifies HER2 as an effective therapeutic target in cetuximab-resistant colorectal cancer.</a> Cancer Discov. 1 (6), 508-523 (2011).</li><li>Davis, S. L., 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=Combined+inhibition+of+MEK+and+Aurora+A+kinase+in+KRAS/PIK3CA+double-mutant+colorectal+cancer+models.">Combined inhibition of MEK and Aurora A kinase in KRAS/PIK3CA double-mutant colorectal cancer models.</a> Front Pharmacol. 6, 120 (2015).</li><li>Morelli, M. P., 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=Preclinical+activity+of+the+rational+combination+of+selumetinib+(AZD6244)+in+combination+with+vorinostat+in+KRAS-mutant+colorectal+cancer+models.">Preclinical activity of the rational combination of selumetinib (AZD6244) in combination with vorinostat in KRAS-mutant colorectal cancer models.</a> Clin Cancer Res. 18 (4), 1051-1062 (2012).</li><li>Pitts, T. 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=Dual+pharmacological+targeting+of+the+MAP+kinase+and+PI3K/mTOR+pathway+in+preclinical+models+of+colorectal+cancer.">Dual pharmacological targeting of the MAP kinase and PI3K/mTOR pathway in preclinical models of colorectal cancer.</a> PLoS One. 9 (11), e113037 (2014).</li><li>Spreafico, 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=Rational+combination+of+a+MEK+inhibitor,+selumetinib,+and+the+Wnt/calcium+pathway+modulator,+cyclosporin+A,+in+preclinical+models+of+colorectal+cancer.">Rational combination of a MEK inhibitor, selumetinib, and the Wnt/calcium pathway modulator, cyclosporin A, in preclinical models of colorectal cancer.</a> Clin Cancer Res. 19 (15), 4149-4162 (2013).</li><li>Arcaroli, J. 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=ALDH++tumor-initiating+cells+exhibiting+gain+in+NOTCH1+gene+copy+number+have+enhanced+regrowth+sensitivity+to+a+gamma-secretase+inhibitor+and+irinotecan+in+colorectal+cancer.">ALDH+ tumor-initiating cells exhibiting gain in NOTCH1 gene copy number have enhanced regrowth sensitivity to a gamma-secretase inhibitor and irinotecan in colorectal cancer.</a> Mol Oncol. 6 (3), 370-381 (2012).</li><li>Hoey, T., 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=DLL4+blockade+inhibits+tumor+growth+and+reduces+tumor-initiating+cell+frequency.">DLL4 blockade inhibits tumor growth and reduces tumor-initiating cell frequency.</a> Cell Stem Cell. 5 (2), 168-177 (2009).</li><li>Ikebuchi, F., 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=Dissociation+of+c-Met+phosphotyrosine+sites+in+human+cells+in+response+to+mouse+hepatocyte+growth+factor+but+not+human+hepatocyte+growth+factor:+the+possible+roles+of+different+amino+acids+in+different+species.">Dissociation of c-Met phosphotyrosine sites in human cells in response to mouse hepatocyte growth factor but not human hepatocyte growth factor: the possible roles of different amino acids in different species.</a> Cell Biochem Funct. 31 (4), 298-304 (2013).</li><li>Zhang, Y. W., 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=Enhanced+growth+of+human+met-expressing+xenografts+in+a+new+strain+of+immunocompromised+mice+transgenic+for+human+hepatocyte+growth+factor/scatter+factor.">Enhanced growth of human met-expressing xenografts in a new strain of immunocompromised mice transgenic for human hepatocyte growth factor/scatter factor.</a> Oncogene. 24 (1), 101-106 (2005).</li></ol>

The authors have nothing to disclose.

The PDTX drug discovery platform offers an improved model to the shortcomings of other preclinical models that are unreliable in predicting clinical activity of novel compounds. Importantly, tumors in this model are biologically stable, retain metastatic potential, and exhibit similar drug responsiveness from generation to generation. In this model, patient derived tumors are injected into athymic nude mice, passaged, and subsequently used in therapeutic evaluation. There are several critical steps for a successful PDTX ...

Colorectal cancer (CRC) is a significant contributor to cancer deaths in the United States. In 2015, there were an estimated 132,700 new cases of CRC with 49,700 deaths 1. Although the prognosis in patients with localized disease is excellent, patients with advanced disease have poor outcomes, making this a major priority in the development of novel therapies. Despite standard of care chemotherapeutic regimens and newer biologics that are deployed against this disease, there has been only an incremental increase in overall survival. Accordingly, there is a significant effort in understanding the driver pathways involved in facilitating tumor growth in this disease. The Cancer Genome Atlas Network has recently identified numerous main pathways that are implicated in CRC dysregulation and include: WNT, phosphoinositide 3-kinase (PI3K), RAS, transforming growth factor-&#946; (TGF- &#946;) and TP53 2. Together, with investigations describing other pathways that potentiate growth in CRC have ignited the development of newer therapies aimed at significantly improving the survival in this patient population 3-5. Utilizing preclinical models in oncology drug development have been essential in this process in predicting the clinical activity of these novel compounds.
Various preclinical models have been utilized in the drug development process. Considering that preclinical transgenic animal models and immortalized cell lines have been unsuccessful in determining the clinical activity of novel oncology therapies, largely due to their inability to reflect the complexity of human tumors, patient-derived tumor xenograft (PDTX) models have been established. The greatest advantage of this model is that tumor heterogeneity remains intact and closely reflects the molecular characteristics and clonality of the originating patient tumor 6-9. PDTX models provide an excellent in vivo preclinical platform to study novel agents, drug resistance pathways, combinational strategies, and cancer stem cell biology 10.
A general overview of the PDTX process is illustrated in Figure 1. It begins in the clinic, consenting patients to allow some of their excess tumor tissue to be used for this research. Next, at surgery, a piece of the tumor is grossed by a pathologist and put into media to be transported to research personnel. Immediately after this, a section of the tumor is cut into small pieces and transplanted into immunodeficient mice subcutaneously. Once the tumor grows, it is passaged into different generations of mice in order to maintain the tumor10. Typically, after the F3 generation the tumor can be expanded into a treatment study where novel compounds and/or combinational therapies are evaluated. Utilizing Next Gen Seq (Exome Seq, RNA Seq and SNP array) potential predictive biomarkers are discovered that assist in the selection of patients that may derive benefit from a particular treatment.
The overarching goals of using PDTX models are to: 1) evaluate the efficacy of novel therapies as single agent or in combination and 2) identify predictive biomarkers of sensitivity or resistance prior to clinical investigation. In this manuscript, we provide the methodology in the initiation and maintenance of a CRC PDTX bank and provide the advantages and limitations of this model in drug development discovery.
<img alt="Figure 1" src="/files/ftp_upload/54393/54393fig1.jpg" /> 
Figure 1. Overview of the CRC PDTX Model Protocol. A patient derived tumor is received from surgery and immediately injected into athymic nude mice subcutaneously. Once the tumor grows it is expanded into subsequent generations and eventually expanded for treatment studies. Treatment responses are assessed and predictive biomarkers are identified that may aid in patient selection. <a href="https://www.jove.com/files/ftp_upload/54393/54393fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="915">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI or DMEM</td>
			<td style="width:81px;">Corning</td>
			<td style="width:119px;">10-040-CV</td>
			<td style="width:499px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin-Streptomycin</td>
			<td style="width:81px;">Corning</td>
			<td style="width:119px;">30-002-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Non-essential Amino Acids</td>
			<td style="width:81px;">Corning</td>
			<td style="width:119px;">25-025-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:81px;">Corning</td>
			<td style="width:119px;">35-010-CV</td>
			<td>Thaw in -4 &#176;C, then activate for 30 minutes at 60 &#176;C water bath</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CPT blood tube</td>
			<td style="width:81px;">BD vacutainer</td>
			<td style="width:119px;">362761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge tube</td>
			<td style="width:81px;">Surelock</td>
			<td style="width:119px;">A-7002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phospate-Buffered Saline</td>
			<td style="width:81px;">Corning</td>
			<td style="width:119px;">21-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cyrogenic vials</td>
			<td style="width:81px;">Cyroking</td>
			<td style="width:119px;">C0732901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic tumor cutting dish</td>
			<td style="width:81px;">Trueline</td>
			<td style="width:119px;">TR4001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scissors</td>
			<td style="width:81px;">Roboz</td>
			<td style="width:119px;">RS-5881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps</td>
			<td style="width:81px;">Roboz</td>
			<td style="width:119px;">RS-5135</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Matrigel (gelatinous protein mixture)</td>
			<td style="width:81px;">Corning</td>
			<td style="width:119px;">354234</td>
			<td>Store at -20 or -80 &#176;C, then thaw on ice, do not leave at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10% Formalin cups</td>
			<td style="width:81px;">Protocol</td>
			<td style="width:119px;">032-059</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Liquid Nitrogen Dewar Storage</td>
			<td style="width:81px;">Thermolyne</td>
			<td style="width:119px;">CY50900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Portable liquid nitrogen dewar</td>
			<td style="width:81px;">Nalgene</td>
			<td style="width:119px;">4150-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethyl Sulfoxide</td>
			<td style="width:81px;">Fischer</td>
			<td style="width:119px;">67-68-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Freezing container: Mr Frosty</td>
			<td style="width:81px;">Nalgene</td>
			<td style="width:119px;">5100-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isopropyl Alcohol</td>
			<td style="width:81px;">Decon</td>
			<td style="width:119px;">64-17-5</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Trocars</td>
			<td style="width:81px;">Innovative Research of America</td>
			<td style="width:119px;">MP-182</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anesthesia machine</td>
			<td style="width:81px;">Patterson Veterinary</td>
			<td style="width:119px;">none</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anesthesia box</td>
			<td style="width:81px;">Patterson Veterinary</td>
			<td style="width:119px;">none</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane</td>
			<td style="width:81px;">Vet one</td>
			<td style="width:119px;">1038005</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">F-Air Canister</td>
			<td style="width:81px;">Bickford Omnicon</td>
			<td style="width:119px;">80120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Meloxicam</td>
			<td style="width:81px;">Vet one</td>
			<td style="width:119px;">5182-90C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calipers</td>
			<td style="width:81px;">Fowler</td>
			<td style="width:119px;">54-100-167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Weight scale</td>
			<td style="width:81px;">Ohaus</td>
			<td style="width:119px;">Scout Pro SP601</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Similarities of Common Mutations in the CRC PDTX Models and the TCGA
We investigated whether the percentage of common mutations (KRAS, NRAS, BRAF, PIK3CA, APC, CTNNB1 and TP53) in the CRC PDTX bank were representative to the mutation frequency seen in the CRC patient population. As shown in Figure 2A (TCGA) and B (CRC PDTX bank), the frequency of mutations in these gene...

Watch this Scientific Journal Video about Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies at JoVE.com

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

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 ...

development-maintenance-preclinical-patient-derived-tumor-xenograft

Research

JoVE Journal

Cancer Research

13.6K Views.  University of Colorado Denver Anschutz Medical Campus. 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.

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

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 ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

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 ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

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 ...

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.

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 ...

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. ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...