Name: development and maintenance of a preclinical patient derived tumor xenograft model for the investigation of novel anti-cancer therapies
Uploaded: 2016-09-30T13:00:00
Description: 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

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
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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. 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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 ...

The overall goal of this subcutaneous Patient Derived Tumor Xenograft procedure is to the study the efficacy of novel therapies predictive biomarker discovery and drug resistant pathways in tumors. This method can help answer key questions in the oncology field about whether a particular drug exhibits activity against a specific tumor type. The main advantage of this technique is that Patient Derived Tumor Xenographs maintain the molecular, genetic and histologist heterogeneity typical of the tumors of origin, making the model more clinically relevant.Demonstrating the procedure will be Stacey Bagby, a professional research assistant and certified veterinary technician from our laboratory. After collecting one to two milliliters of blood in a blood cell separation tube containing sodium citrate, centrifuge the samples and transfer the peripheral blood mononuclear cell layer into a 1.5 milliliter micro-centrifuge tube. Fill the tube to the top with sterile PBS.Then, spin down the cells again. Followed by a quick wash with one milliliter of PBS, being careful not to disturb the PMBC pellet. Then, use a pipette to transfer the plasma from the blood cell collection tube to a 1.5 milliliter sterile cryogenic vial, and store the plasma and the PBMC pellet at negative 80 degrees Celsius.Transfer the human tumor tissue to the animal facility in a sterile cup containing complete medium and place the cup in a laminar flow hood. As soon as possible, after harvesting, transfer the tissue into a sterile plastic cutting dish in a small amount of the retrieval medium. Then, using autoclave sterilized small scissors and forceps, cut the tissue into approximately three by three by three milliliter pieces and place 10 to 12 pieces into an autoclave sterilized 1.5 milliliter micro-centrifuge tube containing 300 microliters of gelatinous protein mixture solution on ice for the implantation.For flash frozen storage, transfer the appropriate number of tumor pieces into a new 1.5 milliliter micro-centrifuge tube and place the vial in a liquid nitrogen doer. Then, place in a minus 80 degrees Celsius freezer. For formal infixing and paraffin embedding, place three small tumor pieces into a 10 milliliter 10%formalin cup for 24 hours.After the tissues are processed into paraffin embedded blocks, transfer any remaining tissue pieces into a cryogenic tube. Then, add one milliliter of complete medium supplemented with 10%DMSO to the tissue and store the tubes on ice until they can be transferred into an isopropyl alcohol freezing container. And put that container in a minus 80 degrees Celsius freezer.To inject the tumor tissue, load the pieces of tumor stored in the gelatinous protein mixture solution into autoclaved 12-gauge trocars, taking care that each tumor piece is completely inserted into the trocar. Next, confirm a lack of response to toe pinch in a six to eight week old athymic, nude mouse and transfer the mouse onto a clean field in a sterile laminar flow hood. Deliver the F0 tumor subcutaneously to the dorsal neck region and to each side of the mouse's flank, followed by the subcutaneous administration of analgesia distal to one of the flank sites of tumor injection.Then, place the mouse back in its cage with monitoring until it is fully recovered. Monitor the growth and health of the xenograph-implanted mice at least once per week, recording the tumor sizes, tumor generation, date of tumor injection, and health of the mice in an appropriate tracking system. When a tumor reaches approximately 1, 500 to 2, 000 cubic millimeters in size, use autoclaved scissors and forceps to excise the tumor and passage the F1 tumor into the next generation of five animals, as just demonstrated, taking care to collect flash frozen formalin and viable tumor samples as demonstrated for each tumor.When the remaining mice from the F0 tumor group exhibit tumors approximately 1, 500 to 2, 000 cubic millimeters in size, collect these tumors as just demonstrated for collection. When the stage F15 tumor generation is reached, use the earliest generation of viable tumor pieces available in liquid nitrogen storage for the next series of tumor injections. When the desired tumor is very large, 1, 500 to 2, 000 cubic millimeters, follow the procedures above to collect the tumor to expand it into the desired amount of mice.Dose the mice with the drug and weigh and measure them twice per week. Randomize the mice into treatment groups with 10 tumors per group, and a group tumor volume average of 100 to 300 cubic millimeters within a few standard deviations of each other. Then, sort the mice into the appropriate groups for treatment.Dual color fluorescence in situ hybridization for the human and mouse caught one DNA demonstrates that the human stromal cells in the F0 tumor are replaced by the mouse stromal cells in the F1 tumor. Whereas, all of the human-derived hepatocyte growth factor in the F0 generation is replaced with mouse hepatocyte growth factor in F1 generation, the vascular endothelial growth factor ligand consists of both human and mouse protein in the F1 generation. The treatment of different generations of the same tumor does not change the treatment response.With the resistance or sensitivity of the tumor appearing to be tumor strain rather than tumor generation specific. Further, the frequency of mutations in these commonly mutated genes is very similar between the cancer genome atlas and the colorectal patient-derived tumor xenograph bank. Suggesting that the common mutations observed in the colorectal patient population are well represented in the colorectal patient-derived xenotransplant model.Once mastered, this technique can be completed in one to two hours, if performed properly. While attempting this procedure, it is important to have a cohesive clinical team for identifying and obtaining consent from the cancer patients and for the removal and grossing of the tumor tissue. Following this procedure, other methods, like the investigation of anti-cancer compounds as single or combination agents, can be performed to answer additional questions about the viability of these strategies for patients with a particular tumor type.After its development, this technique paved the way for researchers in the field of oncology to explore novel cancer therapeutics, tumor heterogeneity, treatment resistant mechanisms, cancer stem cell biology, and tumor stromal interactions. After watching this video, you should have a good understanding on how to develop and maintain a patient derived tumor xenograph model for evaluating novel cancer therapeutics.

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Research

JoVE Journal

Cancer Research

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 ...

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 ...

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. ...